The Role of Tissue-Resident Memory T Cells in Cutaneous … · 2016-07-07 · CTL Cytotoxic T...

The Role of Tissue-Resident

Memory T Cells in Cutaneous

Metastatic Melanoma

_______________________________________

Samantha Winter

BSc Biomedical Science

This thesis is presented for the Honours Degree in Biomedical Science

at Murdoch University, Western Australia

School of Veterinary and Life Sciences

Murdoch University, Western Australia

October 2014

i

Declaration

I, Samantha Jade Winter, declare this thesis is my own account of my

research and contains as its main content, work that has not been

previously submitted for a degree at any tertiary educational institution.

________________________________

Samantha Jade Winter

ii

Word Count

Abstract 392

Introduction 7,069

Materials and Methods 2,866

Results 4,597

Discussion 3,916

Conclusion 330

Total (excluding references): 19,170

Formatting

Document styled as specified by John Wiley and Sons author guide

Referencing style follows format of journal ‘Immunology’

iii

Abstract

Metastatic melanoma is a highly aggressive form of cancer, with poor prognosis

when diagnosed during late stages. Modern cancer immunotherapies that exploit the

immune system were praised as the science ‘breakthrough of the year’ in 2013 and

are exhibiting improved outcomes in the treatment of advanced melanoma. Two of

these immunotherapies currently approved for clinical use are known as anti-CTLA-

4 and anti-PD-1, which respectively target the cell surface markers CTLA-4 and PD-

1 on CD8 T cells. CD8 T cell subsets play a superior role in inflammation and have

the ability to destroy cancer cells. Following resolution of inflammation, a small

number of CD8 T cells contract to form a stable pool of memory T cells. These

memory CD8 T cells have the ability to mount a faster and stronger immune

response compared to their short-lived predecessors. Due to their superior function

and the immunogenicity of melanoma, these memory CD8 T cells play a pivotal role

in the development of successful immunotherapeutic treatments. Recently, a subset

of non-migratory memory CD8 T cells that reside at peripheral sites has been

described, known as tissue-resident memory T cells (TRM). TRM cells are found

primarily at barrier sites, such as the epidermis of the skin. Current research

surrounding TRM cells has centred primarily on their role in viral infections. This

project explores the presence and phenotype of TRM cells at the site of cutaneous

murine melanoma. A novel model of melanoma engraftment was utilised that

resembles the human disease with increased accuracy due to epidermal/dermal

infiltration, which is not seen in traditional murine melanoma models. TRM cells were

able to be identified, enumerated and phenotyped at different stages of tumour

control, by designing a protocol which employed cutaneous melanoma, traceable

iv

tumour-specific gBT.I CD8 T cells and HSV-1 infection. TRM cells were found to be

present at the site of tumour at numbers similar to unmanipulated control skin. In

contrast, elevated numbers were seen in HSV-1 infected skin. At the site of

cutaneous melanoma, TRM cells were found to have a KLRG1lo, CTLA-4hi and PD-

1lo phenotype at each time point analysed, identical to TRM cells isolated from control

and HSV-1 infected skin. These findings suggest the need for further research into

TRM cell function during immunotherapy, as expression of CTLA-4 and PD-1 surface

markers render TRM cells as potential targets for anti-CTLA-4 and anti-PD-1

monoclonal antibody treatment.

v

Table of Contents

Declaration _________________________________________________________ i

Word Count _______________________________________________________ ii

Abstract __________________________________________________________ iii

Table of Contents ___________________________________________________ v

List of Figures and Tables ___________________________________________ ix

List of Abbreviations _______________________________________________ xi

Awards and Presentations __________________________________________ xiii

CHAPTER 1: INTRODUCTION ______________________________________ 1

1.1 MELANOMA ........................................................................................... 1

1.1.1 Melanoma Biology ............................................................................. 2

1.1.2 Epidemiology ..................................................................................... 3

1.1.3 Prognosis ............................................................................................ 4

1.1.4 Diagnosis ............................................................................................ 5

1.1.5 Current Treatments............................................................................. 6

1.1.5.1 Surgical Excision and Radiotherapy ............................ 6

1.1.5.2 Chemotherapy ............................................................... 6

1.1.5.3 Interleukin-2 .................................................................. 7

1.1.5.4 Kinase Inhibitors ........................................................... 7

1.1.5.5 Interferon-α2 Adjuvant Therapy ................................... 8

1.1.5.6 Adoptive Cell Therapy .................................................. 8

1.1.5.7 Anti-CTLA-4 Therapy ................................................... 9

1.1.5.8 Anti-PD-1Therapy ...................................................... 10

1.2 T CELLS ................................................................................................. 13

1.2.1 CD4 T Cells ..................................................................................... 14

1.2.2 CD8 T Cells ..................................................................................... 15

1.2.3 CD8 T Cell Response ....................................................................... 15

1.2.4 Effector CD8 T Cells ....................................................................... 18

1.2.5 Central Memory T Cells................................................................... 19

1.2.6 Effector Memory T Cells ................................................................. 19

vi

1.2.7 Tissue-Resident Memory T Cells .................................................... 20

1.3 TISSUE RESIDENT MEMORY ............................................................ 20

1.3.1 Lodgment in Peripheral Tissues ....................................................... 22

1.3.2 Molecular Mediators of TRM Retention ............................................ 22

1.3.3 Local Survival and Proliferation ...................................................... 23

1.3.4 Requirements for Reactivation ......................................................... 24

1.3.5 Evidence of Effector Activity .......................................................... 25

1.3.6 Potential Functions ........................................................................... 25

1.3.7 Implications and Significance .......................................................... 26

1.4 AIMS AND OBJECTIVES ..................................................................... 27

1.4.1 Summary .......................................................................................... 27

1.4.2 Hypotheses ....................................................................................... 28

1.4.3 Aims ................................................................................................. 28

CHAPTER 2: MATERIALS AND METHODS _________________________ 29

2.1 EQUIPMENT AND REAGENTS .......................................................... 29

2.2 MONOCLONAL ANTIBODIES ........................................................... 32

2.3 PREPARED BUFFERS AND SOLUTIONS ......................................... 33

2.4 CELL LINES........................................................................................... 34

2.5 VIRUSES ................................................................................................ 34

2.6 ANIMALS ............................................................................................... 35

2.6.1 Ethics ................................................................................................ 35

2.6.2 Animal Handling .............................................................................. 35

2.6.3 Mouse Strains ................................................................................... 35

2.6.4 Phenotyping ..................................................................................... 36

2.6.5 Anesthesia ........................................................................................ 37

2.6.6 Cutaneous Melanoma Inoculation.................................................... 37

2.6.7 Intravenous Tail Vein Injection ....................................................... 38

2.6.8 HSV-1 Flank Infection ..................................................................... 38

2.6.9 Skin Perfusion .................................................................................. 38

2.7 MELANOMA CELLS ............................................................................ 39

2.7.1 B16-F1-gB Cell Line ....................................................................... 39

2.7.2 Thawing Cells .................................................................................. 39

vii

2.7.3 Culturing and Harvesting ................................................................. 39

2.7.4 Cryopreservation .............................................................................. 40

2.7.5 Centrifuging and Counting ............................................................... 40

2.7.6 MatrigelTM Preparation..................................................................... 41

2.8 IMMUNOLOGICAL METHODS .......................................................... 41

2.8.1 T Cell Harvesting and Purification .................................................. 41

2.8.2 T Cell Isolation ................................................................................. 43

2.9 FLOW CYTOMETRY ............................................................................ 43

2.9.1 Cell Surface Staining........................................................................ 43

2.9.2 Enumeration ..................................................................................... 44

2.10 SOFTWARE AND STATISTICS .......................................................... 46

CHAPTER 3: RESULTS ____________________________________________ 47

3.1 gBT.I TRANSGENIC T CELLS ............................................................ 47

3.1.1 Differentiation of B6 and gBT.I T Cells .......................................... 47

3.1.2 gBT.I T Cell Expansion in B6 Spleen .............................................. 47

3.2 DEVELOPING A MODEL OF TUMOUR CONTROL ........................ 50

3.2.1 Cutaneous Melanoma Model ........................................................... 50

3.2.2 Experimental Model Design ............................................................ 50

3.2.3 HSV-1 Infection ............................................................................... 51

3.2.4 Tumour Growth Kinetics ................................................................. 53

3.3 ISOLATION OF T CELLS FROM THE SKIN ..................................... 55

3.3.1 Staining Panel Optimisation ............................................................. 55

3.4 CHARACTERISING gBT.I T CELLS IN THE SKIN .......................... 57

3.4.1 Gating Strategy and Background Fluorescence ............................... 57

3.4.2 Gating Strategy for Spleen and TILs ............................................... 60

3.4.3 Expression of Epidermal TRM Surface Markers on Transferred gBT.I

Cells ................................................................................................. 63

3.4.4 Enumerating gBT.I TRM Cells in the Skin ....................................... 65

3.4.5 Phenotyping gBT.I TRM cells ........................................................... 67

3.4.5.1 KLRG1 Expression ..................................................... 67

3.4.5.2 CTLA-1 Expression ..................................................... 70

3.4.5.3 PD-1 Expression ......................................................... 72

viii

CHAPTER 4: DISCUSSION _________________________________________ 74

4.1 PROJECT SUMMARY .......................................................................... 74

4.2 DEVELOPING A MODEL OF TUMOUR CONTROL ........................ 76

4.2.1 HSV-1 Infection ............................................................................... 76

4.2.2 Transgenic gBT.I CD8 T Cells ........................................................ 77

4.2.3 Model Optimisation ......................................................................... 77

4.2.4 gBT.I Expansion in B6 Spleen ......................................................... 79

4.3 ISOLATING T CELLS FROM THE SKIN ........................................... 80

4.4 IDENTIFYING AND ENUMERATING TRM CELLS .......................... 80

4.5 PHENOTYPING TRM CELLS ................................................................ 82

4.5.1 KLRG1 Expression .......................................................................... 82

4.5.2 CTLA-4 Expression ......................................................................... 83

4.5.3 PD-1 Expression .............................................................................. 84

4.6 GENERAL DISCUSSION ...................................................................... 85

4.7 AIMS AND LIMITATIONS .................................................................. 87

CHAPTER 5:CONCLUSION ________________________________________ 89

CHAPTER 6: REFERENCES ________________________________________ 91

ix

List of Figures and Tables

CHAPTER 1: INTRODUCTION

Figure 1.1: Clark model of melanoma diagnosis .......................................... 3

Figure 1.2: PD-1’s role in T cell inhibition ................................................ 12

CHAPTER 2: MATERIALS AND METHODS

Table 2.1: Antibodies used, dilution and manufacturer .............................. 32

Table 2.2: Constitutes of prepared buffers .................................................. 33

Figure 2.1: Harvesting naïve gBT.I CD8 T cells for intravenous

injection ....................................................................................................... 42

Table 2.3: Experimental staining panel....................................................... 44

Figure 2.2: Enumeration using Sphero BeadsTM ........................................ 45

CHAPTER 3: RESULTS

Figure 3.1: gBT.I CD8 T cell phenotype and splenic expansion ................ 49

Figure 3.2: Model of tumour control .......................................................... 52

Figure 3.3: B16-gB tumour growth kinetics ............................................... 54

Figure 3.4: Enzymatic modification of lymphocyte surface molecules. .... 56

Figure 3.5: Gating strategy to identify transferred gBT.I TRM cells in the

skin and analysis of background fluorescence ............................................. 59

Figure 3.6: Isolating and phenotyping gBT.I CD8 T cells from the

spleen .......................................................................................................... 61

Figure 3.7: Isolating and phenotyping gBT.I CD8 TILs ............................ 62

Figure 3.8: CD69 and CD103 expression on transferred gBT.I CD8 T ..... 64

x

Figure 3.9: Enumeration of transferred gBT.I TRM cells............................. 66

Figure 3.10: Analysis of KLRG1 expression on the surface of TRM cells as

compared to TILs and splenic gBT.I CD8 T cells ....................................... 69

Figure 3.11: Analysis of CTLA-4 expression on the surface of TRM cells as


Figure 3.12: Analysis of PD-1 expression on the surface of TRM cells as


xi

List of Abbreviations

B16-F1-gB B16 F1 gB B10M murine melanoma cell line

B6 C57BL/6J mice

BSA Bovine Serum Albumin

CD103 Integrin αEβ7

CD69 C-type lectin 69

CTL Cytotoxic T Lymphocyte

CTLA-4 Cytotoxic T Lymphocyte Antigen 4

D-PBS Dulbecco's Phosphate-Buffered Saline

EDTA Ethylenediaminetetraacetate

FACS Fluorescent Activated Cell Sorting

FCS Fetal Calf Serum

FSC-A Forward Scatter-Area

gB glycoprotein B

HSV-1 Herpes Simplex Virus type-I

IFN-γ Interferon -γ

IL Interleukin

KLRG1 Killer cell Lectin-like Receptor subfamily G member 1

mAb Monoclonal Antibody

MHC Major Histocompatibility Complex

PBS Phosphate Buffered Saline

PD-1 Programmed Death Receptor 1

PD-L1 Programmed Death Ligand-1

PI Propidium Iodide

xii

SSC-A Side Scatter-Area

TCM Central Memory T cells

TCR T Cell Receptor

TEFF Effector T cells

TEM Effector Memory T cells

TGF-β Tumour Growth Factor-β

TIL Tumour Infiltrating Lymphocyte

TNF-α Tumour Necrosis Factor-α

TRM Tissue-Resident Memory T cells

xiii

Presentations and Awards

ORAL PRESENTATIONS

Winter S, Endersby R, Gebhardt T, Waithman J. The role of tissue-resident memory

T cells in cutaneous melanoma. The Australian Society for Medical Research

Western Australia Scientific Symposium; 2014 June 4; Edith Cowen

University, Western Australia.


T cells in cutaneous melanoma. Telethon Kids Institute Student Symposium;

2014 August 25; Telethon Kids Institute Subiaco, Western Australia.

POSTER PRESENTATIONS


T cells in cutaneous melanoma. 24th Annual Combined Biological Sciences

Meeting; 2014 August 29; University of Western Australia, Western Australia.

AWARDS

Winter S. Australasian Society for Immunology Student Poster Presentation Award.

24th Annual Combined Biological Sciences Meeting; 2014 August 29;

University of Western Australia, Western Australia.

Introduction

1

Chapter 1: Introduction

1.1 MELANOMA

Melanoma is the deadliest form of skin cancer and incidence rates are steadily

increasing throughout Western countries (Perlis and Herlyn 2004). Advanced

metastatic melanoma is highly resilient to treatment, resulting in a remarkably poor

prognosis. Metastatic patients have a median survival time of 6-9 months, with fewer

than 5% surviving after 5 years (Cummins, Cummins et al. 2006). Surgical excision

is currently the gold standard of treatment, but does not significantly alter

progression-free survival in late stage patients (Perlis and Herlyn 2004). New

treatment approaches are emerging that are exhibiting improved outcomes, but are

limited by low response rates, severe toxicities and relatively short duration of

effects. Some of the most promising breakthroughs involve immunomodulation with

both anti-CTLA-4 and anti-PD-1 monoclonal antibodies (mAb) and are currently

undergoing development and clinical trials. These mAb’s act as immune checkpoint

inhibitors, blocking the localised immune suppression mechanisms commonly

associated with tumours (Phan, Yang et al. 2003, Durgan, Ali et al. 2011). Recent

studies have shown that combinational therapy with anti-CTLA-4 and anti-PD-1

mAb’s has a synergistic effect, improving the clinical response rate and decreasing

adverse events (Curran, Montalvo et al. 2010, Duraiswamy, Kaluza et al. 2013).

Introduction

2

1.1.1 Melanoma Biology

Cutaneous malignant melanoma (hereinafter referred to as ‘melanoma’) is a tumour

that arises from the malignant transformation of melanocytes. Melanocytes are

specialised pigment-producing cells, located in all epithelial layers. Their primary

function is known as melanogenesis and this involves the production of melanins, the

pigments responsible for colouring of the hair and skin (Slominski, Tobin et al.

2004). Skin-derived melanocytes reside in the basal lamina and hair follicles of the

epidermis, where they closely interact with keratinocytes. Upon ultraviolet

irradiation, keratinocytes secrete α-melanocyte stimulating hormone as well as other

proopiomelanocortico-derived peptides. These peptides bind to the melanocortin 1

receptor on melanocytes, regulating melanocyte homeostasis by governing

melanocyte survival, differentiation, proliferation, motility and stimulating melanin

production (Valyi-Nagy, Hirka et al. 1993, Slominski, Tobin et al. 2004). Melanin

production is pivotal in protecting the skin from ultraviolet induced damage,

preventing the occurrence of skin cancers. In the event of mutations in critical

growth regulatory genes, the production of autocrine growth factors and the loss of

adhesion receptors, such as E-cadherin, disrupts the tight regulation of melanocytes

(Haass, Smalley et al. 2004). This disruption promotes proliferation and spreading of

melanocytes, leading to a mole or ‘naevus’. Naevi are generally benign, but have

dysplastic potential and the ability to enter into the radial-growth phase,

characterised by an intra-epidermal melanocytic lesion (Gray-Schopfer, Wellbrock et

al. 2007). Following the radial-growth phase, the intra-epidermal melanoma can

transition into vertical-growth phase by invading the dermis. Once the tumour has

reached the vascularised dermis, it has the capacity to metastasise to regional lymph

nodes and distal secondary sites such as the lungs, liver and brain (Figure 1.1)

(Miller and Mihm 2006). In addition to this, melanocytes can escape the regulation

Introduction

3

of keratinocytes through the loss of the adhesion receptor E-cadherin. E-cadherin

down-regulation followed by N-cadherin up-regulation corresponds with melanoma

development. N-cadherin assists the communication between melanoma cells

causing smooth migration through the dermal layer (Haass, Smalley et al. 2004).

1.1.2 Epidemiology

The incidence of melanoma is rising faster than any other solid tumour and has been

consistently rising in Western countries over the past four decades. In 2010, 11,405

new cases of melanoma were diagnosed and 2011 saw 1544 melanoma related deaths

in Australia (Australian Institute of Health and Welfare 2014). Highest incidence

rates worldwide appear in Australia and New Zealand, where it is documented to be

the third most common cancer and the most common cancer in people aged 15-44.

Figure 1.1: Clark model of melanoma diagnosis. The stages of melanoma

growth according to the Clark model of diagnosis. Benign naevi become

dysplastic and enter into the radial-growth phase. Subsequently, melanoma cells

invade the dermis, known as the vertical-growth phase, from which they then

metastasise to lymph nodes and distal sites, such as the brain, liver and lungs.

Adapted from: (Miller and Mihm 2006).

Introduction

4

Queensland tops the global charts for highest recorded melanoma prevalence, in

2001 displaying 60 cases per 100,000 inhabitants diagnosed annually, with males

shown to be significantly more effected than females (Garbe and Blum 2001).

Current studies suggest that the increases in incidence are not followed by an

increase in mortality rates. Since the 1980’s, mortality rates have remained static,

likely attributed to the increased awareness and early detection of highly curable

early stage melanomas (stage I and II) (Garbe and Leiter 2009).

1.1.3 Prognosis

Melanoma is a typically curable disease when detected in the early stages.

Conversely, advanced stages (stage III and IV) are refractory to treatment and late

stage melanoma is almost always incurable with traditional therapies, having a

median survival time of only 6-9 months (Cummins, Cummins et al. 2006).

Prognostic outcomes of melanoma patients are predicted using a staging system.

Localised melanomas (stage I and II) are contained to the epidermis and dermis,

largely exhibiting positive outcomes and a survival rate of almost 100% upon

surgical excision. Stage III melanoma is characterised by invasion of the highly

vascularised dermis and the presence of regional nodal metastases. Distant

metastases in areas such as the brain, lungs and liver, defines the most advanced

stage of melanoma (stage IV) (Balch, Gershenwald et al. 2009). Sentinel lymph node

biopsies have also been used to provide precise staging, but has not been reported to

affect survival (Phan, Messina et al. 2009). Interestingly, greater survival outcomes

are reported in females rather than males and this has been ascribed to earlier

diagnosis (Sanlorenzo, Ribero et al. 2014).

Introduction

5

1.1.4 Diagnosis

Early diagnosis of melanoma is essential in improving survival outcomes and

primarily involves a straightforward visual examination. During the visual exam a set

of common guidelines, known as ‘ABCDE’ (asymmetry, border, colour, diameter

and evolving) are employed to visually identify features of potential superficial

spreading melanomas (Salopek, Slade et al. 1995). Patients with large numbers of

atypical naevi may also be subject to additional ongoing total body photography to

assist in early diagnosis. Proceeding visual inspection, any areas that are visually

thought to be displaying some of these characteristics are excised and undergo

histological examination. The current histological approach to diagnosis is the Clark

model (Figure 1.1). The Clark model describes the morphological changes that occur

during the progression from normal melanocytes to malignant melanoma. The first

phenotypic change, as identified by the Clark model is the development of benign

naevi due to proliferation of naeval melanocytes (Clark, Elder et al. 1984). Between

20-40% of melanoma cases develop from benign naevi that undergo aberrant growth,

with the remaining occurring in de novo locations (Salopek, Slade et al. 1995).

Dysplastic naevi can be identified by their morphologically dysplastic cells and

random atypia of the lesion. Radial-growth phase is classified via intraepidermal

growth, whilst vertical-growth phase can be characterised by dermal invasion. Once

the melanoma has become metastatic the distal tumour metastases can also be

biopsied and examined via histology to confirm diagnosis (Miller and Mihm 2006).

Introduction

6

1.1.5 Current Treatments

1.1.5.1 Surgical Excision and Radiotherapy

As previously discussed, early detection of melanoma results in the best possible

prognostic outcomes. Tumours diagnosed in either stage I or II are surgically excised

shortly after diagnosis leading to an almost 100% survival rate (Testori, Rutkowski

et al. 2009, Kunishige, Brodland et al. 2012). However, melanomas that have

invaded the lymphatics and/or distal sites (stage III and IV) are beyond the scope of

effective surgical excision, which is often limited to the primary tumour site and

nodal resection. With surgical excision currently being the most effective method for

treating melanoma, late diagnosis markedly decreases the chance of survival. Stage

III and IV melanomas treated with the surgical removal exclusively have an average

survival time of only 6-9 months (Cummins, Cummins et al. 2006). In conjunction

with surgical treatment radiotherapy is commonly administered post-operatively.

Studies have demonstrated that high dose radiotherapy following surgical removal

increases life expectancy in only a small number of melanoma cases. Typically,

melanoma is highly aggressive and resistant to radiation therapy, requiring the need

for alternate treatments for patients with metastatic and unresectable tumours

(Testori, Rutkowski et al. 2009).

1.1.5.2 Chemotherapy

Metastatic melanoma patients are commonly treated with the chemotherapeutic drug

decarbazine. Regardless, melanoma is known to be notoriously resistant to

chemotherapy regimes with only 7-20% of patients showing objective tumour

remission, and a median survival rate of 9 months. Decarbazine is also known to

have debilitating side effects including, but not limited to nausea, vomiting,

Introduction

7

gastrointestinal disease, leukopenia and thrombocytopenia. Hence, the benefits of

using decarbazine in the treatment of metastatic melanoma must be carefully

weighed up against the side effects (Chapman, Einhorn et al. 1999, Middleton, Grob

et al. 2000).

1.1.5.3 Interleukin-2

One therapy currently approved for the treatment of melanoma is high-dose

interleukin-2 (IL-2). Lymphocytes produce IL-2, which is a potent T cell growth

factor and activator of natural killer cells. IL-2 is secreted predominantly by

activated CD4 T cells, but is also shown to be manufactured by activated CD8,

natural killer and dendritic cells (Rosenberg 2014). In particular, IL-2 has the ability

to stimulate cell proliferation as well as differentiation into effector T cells

(Waithman, Gebhardt et al. 2008). High dose administration is required to yield

objective responses in roughly 15% of patients. Treatment is reserved to the young

and physically fit due to the severity of side effects which are en masse not well

tolerated (Rosenberg, Mule et al. 1985, Atkins, Lotze et al. 1999).

1.1.5.4 Kinase Inhibitors

Kinase inhibitors are currently the mainstay treatment for melanomas that harbour

key mutations contributing to melanoma pathogenesis, namely BRAF mutations.

BRAF mutations are present in 40-60% of melanoma patients and these mutations

lead to the activation of kinase activity. Activation of kinase activity leads to

increased intracellular signalling which acts to direct cell growth (Davies, Bignell et

al. 2002). Inhibitors of these BRAF mutations, such as vemurafenib and dabrafenib,

have shown response rates of 48-53% and have revolutionised personalised

Introduction

8

melanoma treatment, but are limited to treating melanomas that harbour a BRAF

mutation (Sosman, Kim et al. 2012). Additionally, the majority of patients treated

with BRAF inhibitors go on to relapse within 6-12 months (Chapman, Hauschild et

al. 2011, Sosman, Kim et al. 2012). Side effects are cutaneous-orientated and include

hand-and-foot skin reaction, alopecia, stomatitis, verrucous keratosis, eruptive

squamous cell carcinoma, eruptive naevi and the development of new primary

atypical melanocytic lesions and melanoma (Chapman, Hauschild et al. 2011).

1.1.5.5 Interferon-α2 Adjuvant Therapy

Currently, the most commonly used immunotherapy for treating late stage melanoma

patients is high dose interferon-α2. Used as an adjuvant therapy, interferon-α2 has

high clinical success due to decreased tumour burden and extended time to relapse

(approximately 9 months). However, an overall increase in survival has only been

shown in 1 out of 11 comparable trials, with ongoing relapse-free survival occurring

in very rare cases (Cameron, Cornbleet et al. 2001, Cascinelli, Belli et al. 2001,

Eggermont, Suciu et al. 2008, Bottomley, Coens et al. 2009).

1.1.5.6 Adoptive Cell Therapy

Adoptive Cell Therapy is an autologous live T cell treatment, derived from patients’

endogenously circulating T cells. Several types of cancer, including melanoma, have

shown to exhibit anti-tumour T cell responses (Letsch, Keilholz et al. 2000).

Adoptive cell therapy works to mimic and expand these T cell responses, aiding the

immune system in its fight against the tumour. Adoptive cell therapy involves

acquiring T cells from an excised tumour or blood. These cells are then cultured with

IL-2, which promotes T cell proliferation, and left to grow for roughly 2 weeks or

Introduction

9

until 1 x 106 T cells are present in culture. Tumour antigens from the patients’

melanoma are then used to find the T cells that produce the highest amounts of

interferon-γ (IFN-γ). These clones are isolated and cultured further in the presence of

IL-2 until enough cells are available for autologous transfusion (Topalian, Muul et al.

1987, Rosenberg, Yannelli et al. 1994, Dudley, Wunderlich et al. 2002). A major

limitation of this therapy is that it can take up to 6 weeks. However, it has been

largely successful in human trials in pre-treated metastatic melanoma patients with

objective clinical response rates reported between 40% and 72%. Amongst these

patients, up to 40% experienced a complete response lasting up to 7 years (ongoing)

(Phan and Rosenberg 2013). Additionally, many patients do not meet the selective

requirements to produce activated lymphocytes, with only 45% of metastatic

melanoma patients being eligible for adoptive cell therapy (Yee, Thompson et al.

2002, Rosenberg, Yang et al. 2011).

1.1.5.6.1 Anti-CTLA-4 Therapy

The first immunotherapy approved by the US Food and Drug Administration for

treatment of advanced metastatic melanoma was ipilimumab, a mAb that targets

cytotoxic T-lymphocyte antigen-4 (CTLA4). CTLA-4 is a glycoprotein on the

surface of T cells that acts as an inhibitory receptor and when expressed suppresses

the proliferation of T cells. Ipilimumab, an anti-CTLA-4 mAb, therefore works to

block this receptor, preventing the inactivation of T cells. Consequently the effector

functions of tumour-specific T cells are enhanced. Early and late-phase ipilimumab

clinical trials have demonstrated a survival benefit when administered to late stage

melanoma patients (Phan, Yang et al. 2003, Lipson and Drake 2011). Median

survival rates have increased to 10 months with a small number of patients surviving

2-3 years post-immunotherapy. However, 10-15% of patients reported serious (grade

Introduction

10

3-5) adverse events, requiring careful monitoring of patients and the need for further

studies into predictive biomarkers (Lipson and Drake 2011).

1.1.5.6.2 Anti-PD-1 Therapy

Immunomodulation targeting an alternative mechanism of immune suppression

involving the immune checkpoint Programmed Death-1 (PD-1) receptor and its

programmed Death Ligand-1 is currently undergoing clinical trials for the treatment

of advanced melanoma. An immune escape mechanism observed during melanoma

spread is the activation of the PD-1 receptor on infiltrating T cells. PD-1 activation

inhibits the immune response via T cell downregulation and induction of tolerance.

This results in the inhibition of T cells directed against melanoma antigens, blocking

the immune system’s ability to effectively combat the malignancy. PD-1 is an

inhibitory receptor that can terminate T cell immune responses when interacting with

PDL-1. When the PD-1 receptor is blocked via anti-PD-1 mAb’s such a nivolumab,

T cells remain uninhibited by tumour expressed PDL-1 and are free to attack

melanoma cells (Durgan, Ali et al. 2011). A large phase I study of nivolumab

showed a 28% objective response rate in melanoma patients. Many participants

experienced adverse events with suspected immune-related etiology, most of which

were reversible by standard treatment protocols (Topalian, Hodi et al. 2012).

Recent studies have shown that immune checkpoint combination therapies

demonstrate beneficial results for the treatment of advanced melanoma. Preclinical

studies on B16 murine melanoma models have shown synergy when combining anti-

CTLA-4 with anti-PD-1 mAb immunotherapies (Curran, Montalvo et al. 2010). A

phase I clinical trial involving the combination of ipilimumab (anti-CTLA4) and

nivolumab (anti-PD1) exhibited greater response rates compared to monotherapy

Introduction

11

with either of the drugs. 40% of participants showed a rapid objective clinical

response, with some patients having more than 80% tumour reduction at 12 weeks.

This synergy between the two therapeutic treatments also reported more manageable

toxicity and had fewer adverse events (Wolchok, Kluger et al. 2013).

Introduction

12

Figure 2: PD-1’s role in T cell inhibition. Tumour cells present antigens via the

major histocompatibility complex (MHC) which can be recognised through

interaction with the T cell receptor (TCR) on T cells. When programmed death

ligand-1 (PD-L1) on tumour cells interacts with the PD-1 receptor on T cells,

inhibition of the T cell response occurs. (A) PD-L1 expressing tumour cells evade

immune responses by binding to PD-1 on T cells, downregulating T cell activity.

(B) Blocking the PD-1 receptor with anti-PD-1 mAb’s, so that PD-L1 cannot bind

results in T cells no longer being inhibited, upregulating anti-tumour immunity

(Mamalis, Garcha et al. 2014).

Introduction

13

1.2 T CELLS

T lymphocytes, otherwise known as T cells, play an integral role in the adaptive

immune response. T cells originate from bone marrow-derived progenitors that

migrate to the thymus, where they become committed to the T cell lineage and

undergo thymic selection (Takahama 2006). Following entry into the thymus, early T

cell progenitors progress into CD4+CD8+ double positive thymocytes (Kisielow,

Bluthmann et al. 1988, Takahama 2006). Double positive thymocytes undergo

positive and negative selection completing thymocyte development and ensuring

central tolerance (Kisielow, Bluthmann et al. 1988). During positive selection, T

cells that recognise self-MHC in conjunction with self-peptide are expanded (Hu,

Nicol et al. 2012). Among these positively selected T cells a large percentage will be

auto-reactive having very high affinities for self-peptides (Stritesky, Jameson et al.

2012). Autoreactive T cells are purged from the T cell pool in the consecutive

process known as negative selection (Dzhagalov, Chen et al. 2013). During the early

phase of differentiation, early T cell progenitors are double positive for the CD4 and

CD8 coreceptors. These cells then undergo reactions with class I and II MHC

molecules resulting in the respective selection and restriction of CD8 and CD4 T

cells based upon their MHC molecule interaction. MHC Class II restricted CD4 T

cells are involved in the regulation of the cellular and humoral immune responses

and MHC Class I restricted CD8 T cells provide the foundations of cell-mediated

immunity (Kisielow, Bluthmann et al. 1988, Takahama 2006, Farber, Yudanin et al.

2014).

Introduction

14

1.2.1 CD4 T Cells

CD4 T cells are a subpopulation of αβ T cells that acquire the CD4 phenotype during

thymic development. Aside from being a phenotypic marker, CD4 is also a

coreceptor in the T cell-antigen presenting cell interaction. The CD4 molecule on the

surface of the T cells is responsible for stabilising the interaction between the cell

and the APC. This occurs via the CD4 interaction between the membrane-proximal

β2 domain of the antigen-presenting class II molecule (Seder and Paul 1994,

Woodland and Dutton 2003). CD4 T cells can be classified as either helper T cells

(TH) or regulatory cells (TREG) (Seder and Paul 1994). TH cells can be divided into

several subsets according to their cytokine profile and effector functions, namely TH1

and TH2. During TH development, naïve CD4 T cells are activated by the secretion of

IL-12 from APCs. Upon IL-12 secretion, naïve CD4 T cells are differentiated into

IFN-γ and IL-2 producing effector cells, known as TH1 cells. TH2 cells develop from

naïve CD4 T cells that are activated by IL-4 stimulated dendritic cells (DC) that

express CD86, causing the CD4 cells to secrete IL-4, IL-5, IL-9 and IL-13. TH1 cells

promote effective cell-mediated immune responses against intracellular pathogens

and TH2 responses support humoral, anti-helminthic and allergic responses (Seder

and Paul 1994, Woodland and Dutton 2003).

TREG cells on the other hand are involved in the down regulation and termination of

immune responses (Adeegbe, Bayer et al. 2006). TREG cells can suppress over-

shooting immune responses and autoreactive cells via mechanisms that are not yet

completely understood (Samanta, Li et al. 2008). These methods can occur either

indirectly through the secretion of cytokines such as IL-10 and TGF-β or directly

through inhibition due to CTLA-4 contact (Samanta, Li et al. 2008, Kolar, Knieke et

al. 2009). As a consequence, suppressive abilities of TREG’s appear to be a crucial

Introduction

15

player in preventing the development of autoimmunity and allergy (Adeegbe, Bayer

et al. 2006).

1.2.2 CD8 T cells

CD8 T cells, otherwise known as cytotoxic T lymphocytes (CTL), are primarily

involved in the cell-mediated killing of pathogen infected cells. CD8, like CD4,

serves as a coreceptor in T cell-APC interaction (Mittrucker, Visekruna et al. 2014).

The CD8 molecule acts to stabilise the interaction between the CTL and its target by

binding to the membrane-proximal α3 domain of peptide-loaded MHC Class I

molecules on the target cell (Salter, Benjamin et al. 1990). CTLs are responsible for

direct elimination of pathogen infected cells, before transitioning to memory cells

that have long term survival and are capable of conferring immediate protection

against pathogen re-challenge (Opferman, Ober et al. 1999). CD8 T cells express a

range of effector molecules that mediate defence against invading pathogens,

including perforin and granzyme release (Kagi, Vignaux et al. 1994). CD8 T cells

also play an important role in anti-microbial defence by releasing cytokines such as

tumour necrosis factor-α (TNF-α) and IFN-γ. Additionally CD8 T cells also play a

role in inflammation, as CTLs express chemokines that attract inflammatory cells to

the site of pathogen infection (Harty, Tvinnereim et al. 2000, Wong and Pamer

2003).

1.2.3 CD8 T Cell Response

In response to pathogen infection, antigen specific naïve T cells become activated

and undergo clonal expansion, followed by contraction and the formation of a stable

pool of memory cells (Shrikant, Rao et al. 2010). Prior to infection, precursor

Introduction

16

frequency of CD8 T cells for each epitope is estimated to be 100 to 200 cells per

person (Blattman, Antia et al. 2002). When CD8 T cells discern their specific peptide

bound to the Class I-MHC ligands on antigen presenting cells, the TCR signals

adhesion molecules that strengthen and prolong T cell-antigen presenting cell

contact. During this extended period of interaction a second event is needed to

trigger activation and proliferation. This event, known as ‘signal 2’, involves a

costimulatory signal from CD28 on T cells, binding to B7 molecules on antigen

presenting cells (Sepulveda, Cerwenka et al. 1999, Lanzavecchia and Sallusto 2001).

Lastly a third event involving IL-12 stimulation, known as ‘signal 3’ is required to

trigger activation or ‘priming’ of CD8 T cells (Curtsinger, Johnson et al. 2003).

Following activation, distinct transcriptional programs result in modifications of

gene expression, chromatin structure and membrane organisation within CD8 T cells.

These cells then enter into the cycle of cell division and undergo expansion.

Expansion is required to produce sufficient effector CD8 T cells with the migratory

and effector functions needed to defend the host against rapidly multiplying

pathogens. This cellular division occurs rapidly and exponentially and is referred to

as clonal expansion (Kaech and Wherry 2007). T cells resulting from this clonal

expansion are effectively known as CTLs and their primary mechanism is to limit

pathogen infection by killing infected cells before they have the opportunity to

release infectious progeny. This cytotoxic function occurs through the secretion of

perforin and granzyme molecules, and the expression of the Fas ligand which causes

apoptosis through the granular exocytosis pathway of the targeted cell. Perforin aids

granzymes by creating pores in the target cellular or endosomal membrane, which

enables granzymes to access the cytosol. Granzyme B in particular is capable of

granular killing by initiating the apoptotic cascade via either the cleavage of target

Introduction

17

cell caspases or the release of cytochrome c from mitochondria (Kagi, Vignaux et al.

1994, Dobbs, Strasser et al. 2005). Alongside cytolytic activity, activated CD8 T

cells utilise anti-microbial and inflammatory cytokines such as IFN-γ and TNF-α to

confer pathogen control (Hamilton and Jameson 2012). IFN-γ acts to decrease host

cell permissiveness to viral replication and also causes mononuclear phagocytes to

produce nitrous oxide (Karupiah, Xie et al. 1993). TNF-α has the ability to

upregualte MHC expression and adhesion molecules on target cells, stimulating the

release of inflammatory cytokines (Ljunggren and Anderson 1998). This array of

effector functions results in CD8 T cells having the ability to eliminate pathogen

infected cells and allowing endogenous cells to resist viral replication (Shrikant, Rao

et al. 2010).

Ensuing the effector phase, CD8 T cells enter into the contraction phase. Contraction

is the downsizing of the circulating T cell pool which often occurs between one and

two weeks post-infection. Contraction onset is suggested to occur due to pre-

conceived hardwiring during the priming phase of the immune response and results

in the reduction of CD8 T cells by approximately 90%. The process of contraction is

attributable to two mechanisms (Badovinac, Porter et al. 2002, Kaech and Wherry

2007). The first involves the exodus of activated T cells into peripheral tissues, and

the second involves T cell apoptosis due to activation induced cell death, the

mechanism of which remains unclear (Badovinac, Porter et al. 2002, Shrikant, Rao et

al. 2010).

After contraction the formation of memory T cells occurs. The origin and

differentiation of memory T cells is not yet defined and several different models for

this process are proposed. The linear differentiation model hypothesises that memory

Introduction

18

cells progress through the contraction phase, with the remaining effector cells

converting to long-lived memory cells. Conversely, a less favoured hypothesis,

known as the decreasing potential model, hypothesises that during invasion a subset

of T cells remains at an intermediate stage of activation in consequence to attenuated

priming. This model depends on variable signal strengths during the priming phase.

As a consequence of variable signal strengths, it is thought that T cells are forced

into hierarchical thresholds of differentiation, ultimately defining their phenotype

and fate. Therefore, the decreasing potential model places a crucial emphasis on

lineage commitment events in the priming phase. These memory cells have an

increased frequency of antigen-specific precursors (100 to 1000-fold greater than the

naïve host) and accelerated responsiveness as well as rapid effector molecule

acquisition upon encounter with their cognate antigen. Memory cells are also

dependent on IL-7 and IL-15 for antigen-independent homeostatic proliferation.

Several subsets of memory T cells have been described, namely central and effector

memory T cells as well as the recently described tissue-resident memory T cells

(TRM) (Kaech and Cui 2012, Farber, Yudanin et al. 2014).

1.2.4 Effector CD8 T cells

Effector CD8 T cells (TEFF) are a distinct subset of short lived CD8 T cells. TEFF’s

exhibit immediate effector functions following expansion and migrate to peripheral

areas of inflammation (Reinhardt, Khoruts et al. 2001). Some TEFF cells have a very

restricted yet highly functional repertoire, whilst other ‘multifunctional’ TEFF cells

can yield multiple effector functions at the same time (Seder, Darrah et al. 2008).

TEFF cells recognise their target antigens on MHC class I molecules that are present

on most nucleated cells. Following antigen recognition, TEFF cells are able to fulfil

their programmed cytotoxic functions. Characteristically, TEFF cells are identified by

Introduction

19

a CD44hi, CD62Llo, CD27lo, KLRG1hi and IL7-Rαlo phenotype. Upon resolution of

infection, the majority of TEFF cells die via apoptotic pathways, whilst a small pool

remains as long-lived memory CD8 T cells. Upon re-exposure to their cognate

antigen, these memory CD8 T cells can effectively respond again with rapid

conversion into TEFF cells (Kaech and Cui 2012, Mittrucker, Visekruna et al. 2014).

1.2.5 Central Memory T Cells

Central memory CD8 T cells (TCM) are a subpopulation of long lived memory cells.

TCM cells circulate through and home to secondary lymphoid organs and are

commonly found in the lymph nodes, spleen (white pulp), blood and bone marrow

(Mueller, Gebhardt et al. 2013). Upon antigen re-encounter TCM cells proliferate

extensively and are known to produce large quantities of IL-2 (Sallusto, Lenig et al.

1999, Huster, Busch et al. 2004, Sallusto, Geginat et al. 2004). The surface

molecules CD62L and CCR7 are constitutively expressed on TCM cells and play a

role in cellular extravasation in high endothelial venules (Wherry, Teichgraber et al.

2003, Klebanoff, Gattinoni et al. 2005). TCM cells also display a CD44hi, CD127+,

CD69- and CD103- phenotype (Mueller, Gebhardt et al. 2013).

1.2.6 Effector Memory T Cells

Another subset of memory CD8 T cells are known as effector memory CD8 T cells

(TEM). TEM cells have been described as preferentially homing to peripheral tissues

where they have immediate effector functions upon antigen exposure (Masopust,

Vezys et al. 2001, Huster, Busch et al. 2004). Unlike their TCM counterparts, TEM

cells have a much lower proliferative potential and produce effector cytokines such

as IFN-γ (Reinhardt, Khoruts et al. 2001, Gebhardt, Wakim et al. 2009, Masopust,

Introduction

20

Choo et al. 2010). TEM cells are characterised by having no CD62L or CCR7 surface

molecule expression as well as being CD44hi, CD127+, CD69- and CD103-. Common

sites of TEM cells include the spleen (red pulp), blood, lungs, liver, intestinal tract,

reproductive tract, kidneys, adipose tissue and heart (Mueller, Gebhardt et al. 2013).

1.2.7 Tissue-Resident Memory T Cells

Recently described TRM cells are another highly function subset of memory CD8 T

cells. These TRM cell populations permanently reside in peripheral tissues after the

clearance of an infection (Gebhardt, Wakim et al. 2009, Masopust, Choo et al. 2010).

Unlike the other memory CD8 T cell subsets, TRM cells are non-migratory and are

not found in the circulation. TRM cells have so far been located in the epithelial layers

of skin, gut and vagina as well as salivary glands, lungs, brain and ganglia. They

constitutively express the surface markers CD69 and CD103 and can further be

characterised as CD44hi, CD62L-, CCR7- and CD11ahi (Mueller, Gebhardt et al.

2013). The remainder of this literature review will be centred on the characteristics

and functions of these recently described TRM cells.

1.3 TISSUE RESIDENT MEMORY

A progressive loss of circulating effector T cells is seen following pathogen

clearance (von Andrian and Mackay 2000). In contrast, notable sites such as the skin,

brain, lungs, intestinal, vaginal mucosa, salivary glands, thymus and secondary

lymphoid organs retain a unique population of long-lived pathogen-specific CD8 T

cells (Masopust, Vezys et al. 2006, Gebhardt, Wakim et al. 2009, Wakim,

Woodward-Davis et al. 2010, Teijaro, Turner et al. 2011, Mackay and Gebhardt

Introduction

21

2013, Schenkel, Fraser et al. 2014). These CD8 T cells are consistent with the TRM

phenotype, where they uniquely act to elicit protective defences and immune

surveillance in response to pathogen infection. TRM cells are commonly localised to

epithelial or neuronal tissue, therefore having restricted access to lymphatic vessels

(Schenkel, Fraser et al. 2014). Due to the context of this project, the remainder of

this literature review will be focused on but not limited to, epidermal-dwelling TRM

cells. Aside from their localisation, TRM cells can be phenotypically identified by the

expression of specific surface receptors. Integrin αEβ7, otherwise known as CD103

has high expression (CD103hi) on skin-derived TRM cells, in contrast to its low or

negative expression on circulating memory CD8 T cells (Hofmann and Pircher 2011,

Jiang, Clark et al. 2012, Mackay, Rahimpour et al. 2013). Another molecule

constitutively expressed by TRM cells is C-type lectin 69, or more commonly known

as CD69. Typically, CD69 is thought to be down-regulated on circulating effector T

cells after brief expression during activation, although with constant high expression

on TRM cells this conjecture is now questionable. Sustained high levels of CD103+

and CD69+ on TRM cells are suggested to be the result of microenvironmental factors

in the epidermal niche (Jiang, Clark et al. 2012, Mackay, Rahimpour et al. 2013).

Studies have shown that TRM cells are a non-migratory population, located primarily

at barrier sites and areas of recrudescence, that are maintained without replenishment

from the pool of circulating memory CD8 T cells (Gebhardt, Wakim et al. 2009,

Wakim, Woodward-Davis et al. 2010). To date, many mechanisms and roles of skin-

derived TRM cells are yet to be elucidated and will be discussed in further detail.

Introduction

22

1.3.1 Lodgement in Peripheral Tissues

Circulating TEM cells have access to the majority of tissues throughout the body, but

some peripheral sites are not permissive unless subjected to an inflammatory T cell

response. Conversely, TRM cells are located at peripheral tissue sites, with skin

resident TRM cells found in the avascular epidermis. In mice, TRM cells coalesce in

the basal layers of the epidermis, having regular contact with the basement

membrane (Sheridan and Lefrancois 2011). Normally, HSV infected and psoriatic

skin in humans has also shown to gather CD8 TRM cells at the dermis/epidermis

border (Zhu, Hladik et al. 2009). Epidermal-residing CD8 T cells have a unique

dynamic dendritic morphology, a characteristic not observed on T cells within the

dermis. T cells present in the dermis possess an amoeboid shape, a characteristic

shared by all other circulating T cells (Ariotti, Beltman et al. 2012). TRM cells in the

epidermis exhibit limited mobility and have shown to be non-functional when

removed from their site of lodgement and experimentally returning to the circulation

(Wakim, Woodward-Davis et al. 2010, Gebhardt, Whitney et al. 2011).

1.3.2 Molecular Mediators of TRM Retention

T cell retention in peripheral tissues is likely to be associated with a number of

phenotypic changes. Expression of the αEβ7 integrin CD103, is well documented in

memory T cells that reside within the skin, unlike their circulating counterparts

(Gebhardt, Wakim et al. 2009, Wakim, Woodward-Davis et al. 2010). Other

epidermal-resident immune cell types, such as γδT cells, are shown to be diminished

in the epidermis of CD103-deficient mice (Schon, Arya et al. 1999). Studies

conducted by Pauls et al, demonstrate that CD103 is involved in long-term retention

Introduction

23

of TRM cells in the skin (Pauls, Schon et al. 2001). Hadley et al established that

CD103 surface expression on CD8 T cells increases upon tumour growth factor-β

(TGF-β) exposure. TGF-β production is increased during wound healing when

dermal fibroblasts migrate towards the site of inflammation (Hadley, Rostapshova et

al. 1999). An increase in E-cadherin expression is also seen in TRM cells. E-cadherin

is the ligand for intergrin αEβ7 and α1β1 that binds collagen and laminin, which are

both major components of the basement membrane. E-cadherin has a suspected role

in the tethering of TRM cells to the epidermal compartment (Cepek, Shaw et al.

1994). Additionally, TRM cells in the skin have increased expression of the

chemokine receptor CCR8. CCR8 expression is programmed in the epidermis,

suggesting that αβT cell residence at this site involves the expression of this receptor.

Together, adhesion, morphology and survival of T cells in the epidermis are

potentially influenced by these receptors (McCully, Ladell et al. 2012, Mackay,

Rahimpour et al. 2013). It is also plausible that other molecular mediators also

influence the retention of skin-dwelling TRM cells, such as increased expression of

the α1β1 integrin, which is capable of binding type I and IV collagen (Hemler 1990).

1.3.3 Local Survival and Proliferation

Currently TRM cells have been recovered from skin, lungs, brain, intestinal and

vaginal mucosa, salivary glands, thymus and secondary lymphoid organs, weeks

proceeding antigen encounter (Masopust, Vezys et al. 2006, Gebhardt, Wakim et al.

2009, Wakim, Woodward-Davis et al. 2010, Teijaro, Turner et al. 2011, Mackay and

Gebhardt 2013, Schenkel, Fraser et al. 2014). In the skin, TRM cells can be dispersed

throughout the epidermis but also appear to cluster around sites of infection or

inflammation. These clusters of TRM cells have been shown to persist in mouse skin

Introduction

24

for over 1 year post-infection. Studies have shown that TRM cells remain within the

immediate microenvironment in which they were formed, providing robust site-

specific immunity. Skin TRM cells randomly migrate, which is thought to facilitate

the surveillance of skin against reinfection or the recrudescence of latent viruses such

as herpes simplex virus type-1 (HSV-1) (Gebhardt, Wakim et al. 2009, Gebhardt and

Mackay 2012). Homeostatic maintenance of the systemic T cell memory pool

depends on cytokines, such as IL-7 and IL-15, which act to stimulate survival and

proliferation. IL-15 is expressed by keratinocytes, thus potentially promoting the

survival of TRM cells in the epidermis (Johnson and Jameson 2012).

1.3.4 Requirements for Reactivation

The presence of a local population of long-lived CD8 T cells seems only of value if

these T cells undergo reactivation and perform either effector functions or in situ

proliferation at the peripheral site. Current data on TRM cell reactivation comes from

Carbone and colleagues who demonstrated that increases in cell number of latently

infected ganglia of HSV-1 mice, was antigen driven with strong evidence to suggest

that T cell proliferation took place in situ (Gebhardt, Whitney et al. 2011).

Reactivation has shown to be independent of CD4 T cell help, as the elimination of

CD4 T cells had no effect on TRM cell activity after TRM cells had been established

(Masopust, Choo et al. 2010). The role of local antigen presenting cells, such as

Langerhan’s cells, in the reactivation of skin residing TRM cells is still yet to be

established. Evidence suggests that differentiation and maintenance of TRM cells

residing in tissues is antigen-independent. However, CD103 expression driven by

TGF-β has shown to be required for TRM cell maintenance within intestinal

epithelium (Casey, Fraser et al. 2012).

Introduction

25

1.3.5 Evidence of Effector Activity

TRM cells are highly efficient in mediating protective immunity in non-lymphoid

tissues. Evidence of this effector activity has been gathered through several

approaches, such as parabiosis and selective depletion strategies (Hofmann and

Pircher 2011, Jiang, Clark et al. 2012, Mackay, Stock et al. 2012). Gebhardt and

colleagues have studied effector functions of TRM cells in the skin. Mice deficient in

B cells were depleted of CD8 T cells proceeding HSV-1 infection. Following

antibody depletion it was observed that only TRM cells remained. These mice were

then reinfected at the primary site and on the contralateral flank, following which

TRM cells caused rapid and efficient clearance of HSV-1 (Gebhardt, Wakim et al.

2009). Further antibody depletion experiments demonstrated that at epithelial

surfaces, TRM cells are functionally superior to central memory or naïve CD8 T cells,

in the event of a viral rechallenge (Hamilton and Jameson 2012).

1.3.6 Potential Functions

Previously described TCM and TEM cell populations have demonstrated effector and

cytolytic properties, thus playing distinct roles in protective immunity. TRM cells

typically reside at barrier sites where they provide rapid control against invading

pathogens (Hamilton and Jameson 2012). For example, following skin infection with

HSV-1, a pool of circulating HSV-1 specific TEM cells remain as well as a population

of TRM cells at the site of both primary and latent infection, where they provide more

rapid viral control in secondary infection compared to an unmanipulated site

(Gebhardt, Wakim et al. 2009). The unique features of TRM cells provide an

immediate response against infection because of their localisation and potentially due

to their activation status. TRM cells exhibit high expression of CD69, a marker

Introduction

26

typically associated with lymphocyte activation (Mackay, Rahimpour et al. 2013).

TRM cells have also been reported having high expression levels of granzyme B, a

cytolytic granule that is released by CTLs and natural killer cells to kill pathogen

infected cells (Schenkel, Fraser et al. 2014). Therefore it is hypothesized that this

increase in the expression of effector molecules such as granzyme B may aid TRM

cells in immediately limiting the spread of pathogens at the site of infection.

1.3.7 Implications and Significance

Many of the diseases with the greatest burden on society, such as HIV, influenza and

HSV, begin as infections at peripheral or barrier sites. Vaccines designed to protect

against such invading pathogens are of the up-most importance to society and the

establishment of TRM cells at the portal of pathogen entry via vaccination has the

potential to provide swift immune responses that are necessary for optimal protective

immunity.

Introduction

27

1.4 AIMS AND OBJECTIVES

1.4.1 Summary

Metastatic melanoma is an advanced malignancy of melanocytes. Incidence and

mortality rates of melanoma are steadily increasing worldwide, with Australia having

the highest prevalence. Prognosis is poor, with only few patients responding to

conventional therapies. Modern immunotherapies that exploit the immune system to

treat metastatic melanoma are exhibiting improved outcomes. Many of these

immunotherapies utilise T cells, namely CD8 T cells due to their superior role in

inflammation and ability to effectively destroy cancer cells. Following resolution of

inflammation, a small amount of CD8 T cells contract to form a stable pool of

memory T cells. These memory T cells can mount a faster and stronger immune

response compared to their predecessors, thus playing a pivotal role in the

development of successful immunotherapeutic treatments.

Traditionally, memory T cells are classified as a heterogeneous population,

comprised of two subsets: TCM and TEM CD8 T cells. TCM cells circulate within

secondary lymphoid organs, where they lack immediate effector function, instead

having the capability to proliferate and rapidly differentiate into CTLs. TEM cells are

excluded from the lymph nodes and unlike TCM cells, have immediate effector

functions. Recently, another subset of memory T cells has been recognised, known

as TRM cells. TRM cells are found primarily at barrier sites, such as the epidermis of

the skin. Research on TRM cells has centred on their role in viral infections, with

cutaneous HSV-1 infection a classic example. It has been observed that post-viral

infection, the TRM subset remains resident at the infected site where they can

efficiently provide enhanced control during secondary challenge.

Introduction

28

We would like to investigate whether these TRM cells are present during tumour

control and if they are of a similar phenotype to those observed in viral infection. We

will use an immunotherapy protocol reliant on HSV-1 infection. This will be applied

in a preclinical novel cutaneous melanoma model, where growth of the melanoma is

within the epidermis and dermis. Utilising this model will effectively allow us to

assess TRM cell development in a cancer setting.

1.4.2 Hypotheses

We hypothesise that after cutaneous inoculation of B16 melanoma cells, effector T

cells control the tumour to the point of remission and transition to a TRM cell

population.

1.4.3 Aims

1. To develop an in vivo murine model where immunity against melanoma

controls tumour outgrowth for a minimum of 14 days.

2. To identify and enumerate TRM cells at the site of HSV-1 infection, control

and tumour skin.

3. To phenotype TRM cells and tumour infiltrating lymphocytes (TILs) by

determining surface expression of KLRG1, CTLA-4 and PD-1.

Materials and Methods

29

Chapter 2: Materials and Methods

2.1 EQUIPMENT AND REAGENTS

0.25% Trypsin-EDTA, phenol red Life Technologies, USA

2-Mercaptoethanol Life Technologies, USA

5810R Centrifuge Eppendorf, Germany

Absolute ethanol Fronine, Australia

Ammonium Chloride Sigma, USA

BD LSRFortessa flow cytometer BD Biosciences, USA

Bovine Serum Albumin (BSA) Sigma, USA

Calipers Kinchrome, Australia

Collagenase Type III Worthington, USA

CoolCell® Biocision, USA

Cryovials (1.5mL) Fisher-Biotech, Canada

Dispase II Roche Life Science, Australia

Dimethyl sulfoxide (DMSO) Merck, Darmstadt, Germany

DNAse Sigma Aldrich, USA

DremelTM and fine grinding stone S-B Powertools, USA

Dulbecco's Phosphate-Buffered Saline (D-

PBS)

Life Technologies, UK

Eppendorf Tubes Eppendorf, Germany

Ethylenediaminetetraacetate (EDTA) Sigma, USA

Ferric chloride Pharm. Australia, Australia

Fetal Calf Serum (FCS) Serana, WA, Australia


30

GlutaMAX Life Technologies, USA

Hemacytometer Neubauer, Germany

Heparin Thermo Fisher Scientific, USA

HSV-1 Made in house using KOS

laboratory strain

Ilium Xylazil (20mg/ml) xylazine in

hydrochloride

Parnell Laboratories, Australia

Insulin syringe (1mL) BD Falcon, USA

Ketamine (100 mg/ml in hydrochloride) Troy Laboratories, Australia

Lacri-lube® Allergan, Australia

L-Glutamine Life Technologies, USA

Light microscope BX41 Olympus, Japan

LSRFortessaTM flow cytometer BD Falcon, USA

MatrigelTM BD Falcon, USA

Metal Mesh (0.25 mm thick) Metal Mesh Pty Ltd, Australia

Micropore TapeTM 3M Health Care, Germany

Needles (26-gauge) Terumo, Philippines

Nylon mesh (70µm, 30µm) Madison Filter Pty Ltd, Australia

Op-site FlexigridTM Smith and Nephew, UK

Oster® 2-speed Clipper WAHL, Australia

Penicillin (1000µg/ml)/Streptomycin

(10000µg/ml)

Life Technologies, USA

Polypropylene round-bottom FACS tubes

(5ml)

BD Falcon, USA

Potassium Bicarbonate Sigma, USA


31

Propidium Iodide (50µg/mL) Sigma, USA

RPMI – 1640 Life Technologies, USA

Scalpel blades Intergra LifeSciences, USA

Sphero BeadsTM BD Falcon, USA

Syringe (1mL, 3mL, 10mL) Terumo, Philippines

Tissue culture flasks (T175) Greiner Bio-one, Germany

Tissue culture plates BD Falcon, USA

Transpore TapeTM 3M Health Care, USA

Trypan Blue 0.1% in PBS Life Technologies, USA

Tubes (10ml, 15mL, 50mL) BD Falcon, USA

Trypl-E Life Technologies, USA

VeetTM Reckitt Benckiser, Australia


32

2.2 MONOCLONAL ANTIBODIES

Table 2.1: Antibodies used, dilution and manufacturer

Antibody Dilution Manufacturer

Anti-mouse CD45.1 V450

1:200

BD Biosciences, USA

Anti-mouse Vα2 APC-Cy7 1:200 BD Biosciences, USA

Anti-mouse TCR-β PE-CF594 1:200 BD Biosciences, USA

Anti-mouse CD69 FITC 1:200 e-Bioscience, USA

Anti-mouse CD103 PerCP-Cy5.5 1:100 BioLegend, USA

Anti-mouse KLRG1 APC 1:200 BD Biosciences, USA

Anti-mouse CTLA-4 PE 1:200 BD Biosciences, USA

Anti-mouse PD-1 PE-Cy7 1:200 BioLegend, USA

Anti-mouse CD45 BV421 1:200 BioLegend, USA

Anti-mouse CD45.1 PE-Cy7 1:200 BD Biosciences, USA

Anti-mouse CD8 PE-CF594 1:200 BD Biosciences, USA

Anti-mouse CD8 APC 1:200 BD Biosciences, USA

Anti-mouse Vα2 PE 1:200 e-Bioscience, USA

Anti-mouse CD69 PerCP-Cy5.5 1:200 BD Biosciences, USA

Anti-mouse CD103 FITC 1:100 BD Biosciences, USA

Anti-mouse PD-1 APC 1:200 BioLegend, USA

Anti-mouse CCR7 APC 1:200 BioLegend, USA

Anti-mouse CD62L PE 1:200 BD Biosciences, USA


33

2.3 PREPARED BUFFERS AND SOLUTIONS

Table 2.2: Constitutes of prepared buffers

RPMI-10 RPMI - 1640 (RPMI-0) (Life technologies, USA)

supplemented with:

- 10% FCS (Serana, WA, Australia)

- 1% 2-mercaptoethanol (1000x) (Life

Technologies, USA)

- 1% GlutaMAX (100x) (Life Technologies, USA)

- 1% Penicillin/Streptomycin

(10000µg/mL/10000µg/mL) (Life Technologies,

USA)

3mg/mL Collagenase Type

III in RPMI-10 + 5µg/mg

DNAse

333.3mL sterile RPMI-10 (Life Technologies, USA),

supplemented with:

- 1g Collagenase Type III (1mg/mL)

(Worthington, USA)

- 0.119µL DNAse (14mg/mL) (Sigma Aldrich,

USA)

2.5mg/mL Dispase II 25% Dispase II (10mg/mL) (Roche Life Science,

Australia)

75% Sterile 1 x D-PBS (Life Technologies, UK)


34

FACS Wash 1 x D-PBS (Life Technologies, UK) supplemented

with:

- 1% BSA (Sigma, USA)

- 5mM EDTA (Sigma, USA)

Freeze Media 10% DMSO (Merck, Darmstadt, Germany)

90% FCS (Serana, WA, Australia)

Ketamine/Xylazine Solution 8.25mL of 1 x D-PBS (Life Technologies, UK)

supplemented with:

- 1mL Ketamine (100mg/mL) (Troy

Laboratories, Australia)

- 0.75mL Ilium Xylazil (20mg/mL) (Parnell

Laboratories, Australia)

RBC Lysis Buffer 500mL distilled H20 supplemented with:

- 4.23g Ammonium chloride (Sigma, USA)

- 0.5g Potassium Bicarbonate (Sigma, USA)

- 0.0185g EDTA (Sigma, USA)

2.4 CELL LINES

B16 F1 gB B10M (B16-gB) murine

melanoma cells

Dr Jason Waithman, Telethon Kids

Institute, Perth WA

2.5 VIRUSES

HSV-1 KOS Dr Jason Waithman, Telethon Kids

Institute, Perth WA


35

2.6 ANIMALS

2.6.1 Ethics

All animal experiments performed throughout this project were approved by the

Telethon Kids Institute Animal Ethic Committee (AEC #254 and #B021).

2.6.2 Animal Handling

Mice were obtained from the Animal Resources Centre WA (ARC) or bred in house

at the Telethon Kids Institute Bioresource Centre. Mice were housed within the

Telethon Kids Institute Bioresource Centre in accordance to AEC guidelines and

maintained under strict living conditions in individually ventilated cages with

controlled temperature and lighting. Lighting was set to a 12 hour light/dark cycle to

mimic day and night living. Mice had free access to food and water. Tissue paper and

plastic tubes were provided for environmental enrichment. Euthanasia was

performed by CO2 asphyxiation in compliance with the Telethon Kids Institute and

AEC guidelines.

2.6.3 Mouse Strains

C57BL/6J (B6) is a strain of wild-type inbred mice with the H-2b haplotype,

inclusive of the Kb restricting element. B6 lymphocytes express the CD45

alloantigen CD45.2 (or Ly5.2) and were obtained from the ARC from 7 to 9 weeks

of age, undergoing experimental procedures between the ages of 8-12 weeks. gBT.I x

CD45.1 mice were bred in house and express the CD45 alloantigen CD45.1 (or

Ly5.1). The CD8 T cell receptors of gBT.I x CD45.1 mice are transgenic, with the


36

V2 and V8 TCR chains derived from a H-2Kb-resticted HSV-1 glycoprotein B

(gB) specific cytotoxic lymphocyte clone (Mueller, Heath et al. 2002).

2.6.4 Phenotyping

gBT.I x B6.CD45.1 mice were bred and the offspring were phenotyped to determine

gBT.1 transgene expression. gBT.I mice have transgenic CD8 T cells that express a

Vα2 TCR and CD45.1, which are used to confirm their phenotype. Once offspring

reached the age of 6 weeks, blood was collected via tail vein bleeding for phenotypic

analysis. Initially, mice were warmed under a heat lamp and a small incision was

made in the tail vein. From the incision, approximately 100µL of blood was collected

into a 1.5mL tube containing 20µL of heparin (Thermo Fisher Scientific, USA). To

cease bleeding, pressure was applied to the wound, followed by administration of

ferric chloride (Pharm. Australia, Australia). The red blood cells in the samples were

then lysed by adding RBC lysis buffer (Table 2.2) to the blood and incubated at room

temperature for 5 minutes. Blood samples were then washed in PBS by

centrifugation for 5 minutes at 1600rpm and the supernatant was discarded. An

antibody cocktail of Vα2-PE, CD45.1-V450 and CD8-APC was added to samples for

30 minutes and kept at 4ºC in the dark (see section 2.9.1 for cell staining). Following

cell staining protocol, samples were analysed using flow cytometry. The phenotype

of a B6 mouse in comparison to a gBT.I x CD45.1 mouse is shown in Figure 3.1A-

D.


37

2.6.5 Anaesthesia

Mice were anaesthetised using a Ketamine/Xylazine solution (Table 2.2).

Anaesthesia was performed at a dosage of 10µL/g body weight via intraperitoneal

injection, using a 1mL syringe and a 26-gauge needle (Terumo, Phillipines). Once

anaesthetised, the eyes of the mice were liberally coated with Lacri-Lube®

lubricating eye ointment (Allergan, Australia) to prevent drying and possible

blindness. Following experimental procedures, mice were returned to cages and

placed on heat pads to maintain a stable core body temperature during

unconsciousness. Once all mice had regained consciousness they were removed from

heat pads and were monitored until full recovery.

2.6.6 Cutaneous Melanoma Inoculation

B16 F1 gB B10M (B16-F1-gB) melanoma cells were grown in RPMI-10 (Table 2.2)

to 70%-80% confluency and harvested using Trypl-E (life Technologies, USA).

Cells were then washed 3 times with RPMI-0 (Life Technologies, USA) to remove

all traces of fetal calf serum and resuspended in MatrigelTM (105 cells/10µL) (BD

Falcon, USA) and kept on ice. Mice were anaesthetised (see section 2.6.5) with

ketamine/xylazine (Table 2.2) and the upper left flank was depilated. A DremelTM

was then used for scarification on the skin at the dorsal spleen tip. 10µL of

MatrigelTM/B16-gB solution was then placed on top of scarified site and the mouse

torso was bandaged for 5 days following tumour inoculation using FlexigridTM,

MicroporeTM and TransporeTM tape. Mice were monitored daily until the removal of

bandages and tumour volume was recorded every other day until tumours reached a

size of 8mm x 8mm, in which daily monitoring resumed.


38

2.6.7 Intravenous Tail Vein Injection

Prior to injection, mice were warmed under a heat lamp and the tail was doused with

80% ethanol. Subsequently, 200µL of cells suspended in RPMI-0 (Life

Technologies, USA) were intravenously injected into the tail vein using a 1mL

insulin syringe. Following injection, mice were returned to their cages and monitored

until full recovery.

2.6.8 HSV-1 Flank Infection

1 x 106 PFU/10µL KOS HSV-1 was prepared in D-PBS (Life Technologies, UK).

Mice were anesthetised according to section 2.6.5 and hair was depilated from the

lower right flank. Mice were then scarified using a DremelTM and 10µL of HSV-

1/PBS solution was placed directly onto the wound. The torso of the mice, including

inoculation site, were then bandaged using FlexigridTM, MicroporeTM and

TransporeTM tape. Bandages were removed 2 days post-HSV-1 infection. Mice were

monitored daily for 7 days.

2.6.9 Skin Perfusion

Mice were euthanased according to section 2.6.2. Immediately after sacrifice, the

chest cavity was opened with scissors and the heart exposed. An incision was made

in the right atrium to allow for drainage and 10mL of D-PBS (Life Technologies,

UK) was injected into the left ventricle to flush blood from the circulatory system.

This allowed for the removal of effector and memory T cells from the circulation.

Following perfusion skin samples were surgically excised.


39

2.7 MELANOMA CELLS

2.7.1 B16-F1-gB Cell Line

The B16 F1 gB B10M clone, referred to as B16-F1-gB, was provided by Dr Jason

Waithman (Telethon Kids Institute, WA). The B16-F1-gB cell line was initially

derived from an induced B6 murine melanoma. The B16-F1-gB clones were then

genetically engineered to express both HSV-derived gB and a green fluorescent

protein. The B10M nomenclature arose from the clones’ coordinates on the plate

from which it grew (B10) and its in vitro growth kinetics (M=medium).

2.7.2 Thawing Cells

Cells were removed from liquid nitrogen storage and promptly placed in a 37ºC

water bath to thaw. Once defrosted cell suspension was added to 5mL of RPMI-10

(Table 2.2) and centrifuged at 1600rpm for 5 minutes at 4ºC. Supernatant was then

aspirated and pellet was resuspended in RPMI-10 (Table 2.2) in a 10mL culture

plate. Plates were then kept at 37ºC in a 5% CO2 incubator for cell growth.

2.7.3 Culturing and Harvesting

Cell cultures were maintained at 37ºC in a 5% CO2 environment. Cells were grown in

RPMI-10 media (Table 2.2) and were passaged when plates reached 80%

confluency. When harvesting for experimental use, cells were required to be

approximately 70%-80% confluent. Media was aspirated and 2mL of Trypl-E (Life

Technologies, USA) (enough to cover the cell monolayer) was added to the culture

to plate for 5 minutes at 37ºC. After 5 minutes, adherent cells became suspended in


40

Trypl-E solution (Life Technologies, USA) and cell suspensions were then added to

RPMI-10 (Table 2.2) at which they were centrifuged at 1600rpm for 5 minutes at

4ºC. Following centrifugation, supernatant was aspirated and cell pellet was used in

accordance to protocol.

2.7.4 Cryopreservation

Cultured cells were washed, pelleted and resuspended in freeze media (Table 2.2).

Cell suspension was aliquoted into cryovials at a concentration of 1 x 106cells/mL

and placed into a CoolCell® which was stored in a -80ºC freezer overnight. The

following day cryovials were then transferred to liquid nitrogen.

2.7.5 Centrifuging and Counting

Cultured cells were washed and pelleted by centrifugation at 1600rpm for 5 minutes.

Cell counts were performed by thoroughly vortexing cell suspensions in a known

amount of RPMI-10 media (Table 2.2). A 10µL aliquot was removed and mixed with

40µL of Trypan Blue (0.4% in D-PBS) (Life Technologies, NY, USA). 10µL of

cell/Trypan Blue solution was then place on a haemocytometer and counted under a

microscope. Several grids were counted and the average number of cells in each grid

was determined. The following formula was then used to determine overall cell

number:

#cells/mL = Average no. of cells per grid × dilution factor × 104


41

2.7.6 MatrigelTM Preparation

Cells were harvested according to section 2.7.3. Cell pellet was washed a further 3

times in RPMI-0 (Life Technologies, USA) to remove all traces of FCS. On the last

wash, cells were counted according to section 2.7.5 and carefully resuspended in

MatrigelTM (BD Falcon, USA) at a concentration of 1x105 cells per 10µL of

MatrigelTM (BD Falcon, USA). Cell suspension was then kept on ice to prevent

MatrigelTM (BD Falcon, USA) from solidifying.

2.8 IMMUNOLOGICAL METHODS

2.8.1 T Cell Harvesting and Purification

T cells were harvested from the lymph nodes of a gBT.I x CD45.1 mouse for

intravenous transfer. Dissected lymph nodes were passed through a sterile metal

mesh to create a single cell suspension in RPMI-0 (Life Technologies, USA). Single

cell suspension was then filtered through a 70µm nylon mesh and stained for

characteristic gBT.I CD8 T cells surface markers; CD8, Vα2 and CD45.1 (see cell

surface staining section 2.9.1). The percentage of CD8 T cells was then determined

using flow cytometry (Figure 2.1). Lymph node samples were then washed 3 times in

RPMI-0 (Life Technologies, USA) and on the last wash a cell count was performed

(see section 2.7.5). Naïve gBT.I CD8 T cells were then resuspended in RPMI-0 (Life

Technologies, USA) at a concentration of 1 x 105 cells /200µL and kept on ice.


42

Figure 2.1: Harvesting naïve gBT.I CD8 T cells for intravenous injection.

Lymph nodes from gBT.I x CD45.1 mice were harvested, homogenised into a

single cell suspension and stained for characteristic gBT.I CD8 T cells surface

markers; CD8, Vα2 and CD45.1. (A) Lymphocytes were isolated. (B) The

percentage of CD8+Vα2+ cells present (61.1%) was used to calculate 1 x 105 CD8

T cells in 200µL of RPMI-0 (Life Technologies, USA) media, per mouse. (C)

Confirmation of the presence of the CD45.1 allele. Representative plot from

multiple experiments shown.


43

2.8.2 T Cell Isolation

Skin was harvested and the subcutaneous tissue was removed with a scalpel. Skin

was then floated dermis side down on Dispase II (2.5mg/mL) for 90 minutes at 37°C,

followed by the separation of the epidermis and dermis. Epidermal sheets were then

incubated in trypsin/EDTA (0.25%/0.1%) for 30 minutes at 37°C. The remaining

dermal tissue was manually chopped into fragments and incubated at 37°C in

Collagenase/DNase Type III (3mg/mL) for 30 minutes. Following incubation

epidermal and dermal samples were pooled together in 3mL of RPMI-10 and

centrifuged for 5 minutes at 4°C. Samples were then filtered through a 70µm nylon

mesh and then subsequently through a 30µm nylon mesh. Samples were

subsequently stained with antibodies (see section 2.9.1) and analysed via flow

cytometry.

2.9 FLOW CYTOMETRY

2.9.1 Cell Surface Staining

Antibody staining for extracellular markers was conducted in polypropylene round-

bottom FACS tubes. Cells were washed with FACS wash (Table 2.2) and pelleted

via centrifugation. Supernatant was discarded and antibody cocktails were added to

pelleted cells. Cells were then resuspended and kept in the dark at 4ºC for 30

minutes. Following staining incubation, cells were washed with FACS wash and

centrifuged. Prior to flow cytometric analysis, cells were resuspended in 100-200µL

of FACS wash and immediately before analysis 10µL of propidium iodide (PI)

(50µg/mL) (Sigma, USA) was added to discriminate live (PI-) and dead (PI+) cells.

Sample data was collected using an LSRFortessaTM (BD Falcon, USA) flow


44

cytometer and FACSDIVATM (v.6.2) software. Subsequent analysis was performed

using FlowJoTM (TreeStar, USA) software. Table 2.3 summarises the antibodies used

to identify and phenotype gBT.I CD8 TRM cells:

Table 2.3: Experimental staining panel.

Antibody

V450 CD45.1

APC-Cy7 Vα2

PE-CF594 TCR-β

FITC CD69

PerCP-Cy5.5 CD103

APC KLRG1

PE CTLA-4 (CD152)

PE-Cy7 PD-1 (CD279)

2.9.2 Enumeration

SpheroTM calibration beads (BD Falcon, USA) were used to allow calculation of cell

numbers. Immediately before flow cytometric analysis, each sample received 20,000

SpheroTM beads (BD Falcon, USA) and was thoroughly vortexed. Samples were then

run on an LSRFortessaTM (BD Falcon, USA) and beads were visible at a certain

forward scatter/side scatter (450V/225V). Forward scatter (FSC-A) is a measure of

cell surface area or size and side scatter (SSC-A) measures granularity or internal

complexity. FSC-A and SSC-A measurements can allow for differentiation of cell

types in a heterogeneous cell population, in our case calibration beads and

lymphocytes. Beads were gated and number of recovered beads equated to the

percentage of sample analysed (Figure 2.2).


45

Figure 2.2: Enumeration using Sphero BeadsTM. Sphero BeadsTM were utilised

to enumerate flow cytometry samples by adding a known amount of beads to a

sample. Bead recovery was then used to establish the percentage of sample

analysed. Firstly PI- cells were gated on (A) followed by the Sphero BeadsTM

which are evident in a FSC-A vs. SSC-A plot (B). (C) Number of beads

recovered, which is then used for enumeration.


46

2.10 SOFTWARE AND STATISTICS

Flow cytometry analysis, tables and graphs were produced using FlowJoTM software

(TreeStar, USA). Column graphs and statistical analysis were created and performed

in GraphPad Prism. A Grubb’s test was used to exclude outliers and a Students T-test

was conducted to analyse the percentage expansion of transferred gBT.I T cells in a

B6 spleen, enumeration of TRM cells and quantification of KLRG1, CTLA-4 and PD-

1 positive cells. Significance was reached when p≤0.05 and was signified as follows:

*=p≤0.05, **=p≤0.01, ***=p≤0.001 and ****=p≤0.0001.

Results

47

Chapter 3: Results

3.1 gBT.I TRANSGENIC T CELLS

3.1.1 Differentiation of B6 and gBT.I T Cells

gBT.I mice are transgenic mice that have been genetically engineered to have HSV-

derived gB-specific and MHC class I-restricted CD8 T cells (Mueller, Heath et al.

2002). Transgenic gBT.I mice were utilised as a main component of our

immunotherapeutic model due to the gB-specific properties of gBT.I CD8 T cells.

gBT.I CD8 T cells express a range of markers on their cell surface, including CD8,

Vα2, TCR-β and CD45.1. CD45.1 expression allows gBT.I cells to be tracked in B6

mice that express CD45.2. Figure 3.1A-D shows the blood phenotype of B6 (B) and

gBT.I x CD45.1 (C-D) mice. Blood samples were taken from both B6 and gBT.I

mice and red blood cells were lysed. Lymphocytes were stained for gBT.I surface

markers CD8, Vα2 and CD45.1 and analysed via flow cytometry. Figure 3.1B shows

only a small population of CD8+Vα2+, showing that B6 mice lack the gBT.I

transgene. Figure 3.1C shows a strong skew towards a CD8+Vα2+ population of cells

when compared to the B6 mouse, indicative of a gBT.I mouse phenotype. The

presence of the CD45.1 allele is confirmed in Figure 3.1D.

3.1.2 gBT.I T Cell Expansion in B6 Spleen

Expansion of transferred gBT.I CD8 T cells in the spleen was measured to determine

successful HSV-1 infection as well as systemic expansion at different time points

throughout the immune response. 1 x 105 naïve gBT.I T cells were intravenously

Results

48

transferred into the tail vein of B16-F1-gB tumour bearing B6 mice at day -1.

Spleens were harvested at day 7, 14 and 30 and were stained for transgenic gBT.I T

cell markers CD8, CD45.1 and Vα2 and analysed via flow cytometry. Figure 3.1E-F

demonstrates how the percentage of gBT.I CD8 T cell expansion was determined in

each mouse. The expansion within each animal was quantified and compared across

the different time points (Figure 3.1G). Our results show that gBT.I CD8 expansion

percentage peaks between day 14 and then contracts by day 30 to a memory

population. The percentage of gBT.I CD8 expansion in each mouse was also

compared to TRM cells numbers at each site (HSV-1, control and tumour skin). When

analysed using a linear regression, no correlation was found (data not shown).

Results

49

Figure 3.1: gBT.I CD8 T cell phenotype and splenic expansion. Transgenic gBT.I

T cells are routinely identified by their expression of the markers CD8, Vα2 and

CD45.1. Figure A-D shows the phenotype of a B6 alongside a gBT.I x CD45.1

transgenic mouse. (A) Isolation of live lymphocytes from the blood. (B) Phenotype of

a B6 mouse. (C) Phenotype of a transgenic gBT.I mouse. (D) Confirmation of

CD45.1 allele in gBT.I mouse. (E-F) The percentage expansion of CD8 gBT.I T cells

in the spleen of a B6 mouse following intravenous gBT.I transfer. (G) Quantification

of gBT.I CD8 T cell expansion in B6 spleens at day 7 (n=9), 14 (n=8) and 30 (n=7)

post intravenous transfer. Results were analysed using a Student’s t-test and depicted

as mean±SEM. A Grubb’s test was used to exclude outliers. No significant difference

was shown between samples (p≥0.05).

Results

50

3.2 DEVELOPING A MODEL OF TUMOUR CONTROL

3.2.1 Cutaneous Melanoma Model

Traditional methods of murine melanoma inoculation involve injecting B16 cells

directly underneath the skin into the subcutaneous tissue, resulting in subdermal

tumour growth (Overwijk and Restifo 2001). Developed in house, the cutaneous

melanoma inoculation method involves grafting B16 cells directly within the dermis

and epidermis. This model more accurately resembles the human disease as it allows

intraepidermal growth that spreads to the dermis. It also reliably forms metastases in

tumour draining lymph nodes (brachial and less commonly, the axillary) (Figure

3.2A-D). This is crucial to examine the relationship between TRM cells and advanced

melanoma growth as TRM cells are found in the epidermis.

3.2.2 Experimental Model Design

To assess the presence and phenotype of epidermal TRM cells at the site of tumour

development, an immunotherapy protocol that utilised CD8 T cells and HSV-1

infection to target cutaneous murine melanoma was optimised. B6 mice were

inoculated with B16-F1-gB cutaneous tumours on the upper left flank (Figure 3.2E)

and allowed to grow until tumours became palpable, generally between days 11-13

(following optimisation). Once palpable, tumour-bearing mice were intravenously

injected with 1 x 105 naïve gBT.I CD8 T cells into the tail vein. The following day,

mice underwent a 1 x 106 PFU HSV-1 infection on the lower contralateral flank

(Figure 3.2F). Tumour kinetics were mapped and skin samples were taken at day 7,

14 and 30 for analysis (Figure 3.2H-I).

Results

51

3.2.3 HSV-1 Infection

The B16-gB cell line utilised in this model was genetically engineered to express a

HSV-derived gB epitope, allowing it to be recognised by gB-specific T cells.

Transferred naïve gBT.I CD8 T cells are gB-specific and have been shown to be

activated in skin draining lymph nodes following HSV-1 infection (Mueller, Heath et

al. 2002). During HSV-1 infection, virus enters through the primary scarification site

and progresses down the axons to the dorsal root ganglia. Subsequently, the HSV-1

viral particles migrate distally, infecting the whole dermatome. This is seen

macroscopically as a ‘band-like’ or zosteriform lesion (Figure 3.2F-G).

Results

52

Figure 3.2: Model of tumour control. The components of an immunotherapeutic

model used to control cutaneous metastatic melanoma. (A) Subcutaneous tumour. (B)

Metastases-free subcutaneous tumour draining brachial lymph node. (C) Cutaneous

tumour that reliably forms metastases in the tumour draining brachial lymph nodes

(D). (E) Cutaneous melanoma site. (F) HSV-1 infection site. (G) Cutaneous tumour

and contralateral HSV-1 zosteriform lesion at day 7 post HSV-1 infection. (H)

Diagrammatic representation of immunotherapeutic model. (I) Experimental model

timeline including days of harvest.

Results

53

3.2.4 Tumour Growth Kinetics

Simultaneous HSV-1 infection with gB-expressing B16 melanoma engraftment was

hypothesised to prime a strong immune response from transferred gB-specific gBT.I

CD8 T cells, resulting in tumour clearance and stabilisation (remission) for a

minimum of 14 days prior to relapse. Following optimisation, it was shown that the

transfer of naïve gBT.I CD8 T cells and consequent HSV-1 infection was optimal at

day 13 and 14 respectively, post tumour inoculation. Infecting with HSV-1 14 days

after tumour inoculation resulted in several tumours being controlled past day 30

post-HSV-1 infection. After intervention, tumours appeared to enter into a remission

phase at day 6-7 post-HSV-1 infection, stabilising between days 7-17 before

relapsing at approximately day 17-21 (Figure 3.3). Mice shown in Figure 3.3 were

pooled from 3 separate experimental cohorts (n=25) to demonstrate the optimised

immunotherapeutic model. It is important to note that tumours in these cohorts were

not left to outgrow to accurately map tumour progression (compared to initial

optimisation cohorts). Instead, these mice were used experimentally and harvested at

day 7, 14 and 30 for TRM cell analysis.

Results

54

Figure 3.3: B16-gB tumour growth kinetics. Cutaneous tumour progression in mice

before and after receiving HSV-1 and gBT.I naïve CD8 T cell intervention. At day -

14, B6 mice were cutaneously inoculated with B16-F1-gB melanoma cells. Tumours

became visible at approximately day -1, following which 1 x 105 naïve gBT.I T cells

were intravenously injected into the tail vein. The following day, day 0, mice were

infected with HSV-1 on their lower flank, contralateral to the tumour site. Tumour

volume was recorded throughout, and mice were harvested at day 7, 14 and 30

represented by the dotted lines. 3 pooled cohorts of mice are shown (n=25) and each

line represents an individual mouse. Lines that do not continue to harvest time points

were euthanized in accordance to ethical guidelines.

Results

55

3.3 ISOLATION OF T CELLS FROM THE SKIN

3.3.1 Staining Panel Optimisation

The proteolytic enzymes trypsin, dispase II and collagenase III were required to

isolate T cells from the skin. These enzymes have previously been shown to modify

cell surface receptors and were therefore tested against several surface markers used

to optimise an appropriate antibody staining panel (Abuzakouk, Feighery et al. 1996,

Huang, Hsing et al. 2010). Samples shown in grey in Figure 3.4 are untreated

compared to unshaded samples which underwent either trypsin, dispase II or

collagenase III enzymatic digestion (for digestion temperature and duration see

section 2.8.2). The markers CD8 and CD62L were almost completely cleaved by

0.25% trypsin (Figure 3.4A and B), CD45 was modified by 2.5mg/mL dispase II

(Figure 3.4C) and the Vα2 marker endured minor stripping in response to 3mg/mL

collagenase III digestion (Figure 3.4D). CD8, CD62L and CD45 were therefore

excluded from surface staining analysis. CD45.1 was included to demonstrate an

important surface receptor that was not modified by enzymatic digestion. Other

surface molecules tested (see Table 2.1) were not affected by enzymatic activity

(data not shown).

Results

56

Figure 3.4: Enzymatic modification of lymphocyte surface molecules. Enzymes

ultilised to isolate T cells from the skin (trypsin, disapse II and collagenase III) were

tested for their potential to modify lymphocyte surface receptors. Lymph nodes were

harvested from gBT.I x CD45.1 transgenic mice and were treated with different

enzymes for times and temperatures adherent to the T cell isolation protocol shown in

section 2.8.2. Following enzymatic digestion, treated and untreated control samples

were stained with markers listed in Table 2.3 and analysed via flow cytometry. (A-B)

CD8 and CD62L cleavage by trypsin. (C) Modification of CD45 by dispase II. (D)

Minor modification of Vα2 by collagenase III enzyme. (E) Lack of modification

caused by enzymatic digestion on the CD45.1 lymphocyte marker.

Results

57

3.4 CHARACTERISING gBT.I TRM CELLS IN THE SKIN

3.4.1 Gating Strategy and Background Fluorescence

Sites of HSV-1 infected skin are reported to have elevated numbers of epidermal

residing TRM cells post-infection, when compared to unmanipulated control skin

(Gebhardt, Wakim et al. 2009). HSV-1 was therefore used as a positive control to

optimise a gating strategy to identify and phenotype gBT.I TRM cells and to also

determine background fluorescence. B6 mice received HSV-1 infection at day 0,

following gBT.I T cell transfer on day -1 and were harvested at day 30+. Animals

were perfused immediately post-mortem to remove effector and memory T cells

found in the circulation. T cells were then isolated from pooled HSV-1 skin and split

into 4 different samples, each receiving a different staining panel. The gating

strategy shown in Figure 3.5 is utilised to isolate transferred gBT.I CD8 T cells and

to also determine their expression of CD69 and CD103. gBT.I T cells isolated from

the skin that were double positive for CD69 and CD103 were indicative of TRM cells.

This double positive TRM population was then isolated and analysed for phenotypic

expression of the markers KLRG1, CTLA-4 and PD-1. PI was used to exclude dead

cells from analysis (data not shown).

Various staining panels were used to identify background fluorescence. Panels began

with a completely unstained sample (Figure 3.5A), a core gBT.I CD8 T cell stain

including CD45.1 Vα2 and TCR-β (Figure 3.5B) and a core gBT.I CD8 T cell stain

with the addition of reported epidermal TRM cell markers, CD69 and CD103 (Figure

3.5C). The last sample received a full stain, with the inclusion of KLRG1, CTLA-4

and PD-1 phenotypic markers, as seen in Table 2.3 (Figure 3.5D). This step-wise

Results

58

staining allowed a comparison between unstained populations and stained

populations to assess background fluorescence. Unstained CD103, KLRG1 and PD-1

all exhibit a moderate autofluorescence when compared to their stained counterparts

(Figure 3.5B-C). Mean background fluorescence intensity in later sections are

calculated from background fluorescence shown in Figure 3.5.

Results

59

Fig

ure

3.5

: G

ati

ng

stra

teg

y

to

iden

tify

tr

an

sfer

red

gB

T.I

T

RM

cel

ls

in

the

skin

an

d

an

aly

sis

of

back

gro

un

d

flu

ore

scen

ce.

Day 3

0+

HS

V-1

sk

in s

ample

s w

ere

po

ole

d f

rom

sev

eral

mic

e an

d s

tain

ed w

ith d

iffe

rent

anti

bod

y c

ock

tail

s to

dem

onst

rate

bac

kgro

und

flu

ore

scen

ce.

(A)

Unst

ained

sam

ple

. (B

) gB

T.I

CD

8 T

cel

l co

re s

tain

(C

D45.1

, V

α2 a

nd T

CR

-β).

(C)

gB

T.I

CD

8 T

cel

l co

re s

tain

wit

h a

ddit

ional

TR

M c

ell

mar

ker

s, C

D69 a

nd C

D103

. (D

) F

ull

sta

in i

ncl

ud

ing t

he

ph

eno

typ

ic

mar

ker

s, K

LR

G1,

CT

LA

-4 a

nd P

D-1

. T

he

gat

ing s

trat

egy s

how

n w

as u

sed o

n a

ll s

am

ple

s to

iden

tify

and p

hen

oty

pe

TR

M c

ells

in t

he

skin

. R

epea

t ex

per

imen

ts w

ere

condu

cted

to d

eter

min

e m

ean b

ack

gro

und f

luore

scen

ce a

nd

pro

vid

ed s

imil

ar r

esult

s.

n=

2 f

or

each

sta

inin

g p

anel

(A

-D).

Results

60

3.4.2 Gating Strategy for Spleen and TILs

Lymphocytes from the spleen were analysed to provide an insight into the systemic

CD8 T cell response and to compare to analysed TIL and skin samples. TILs were

analysed to show the phenotypes of infiltrating CD8 T cells within the tumour. These

are important to investigate alongside skin samples for comparison of local

epidermal residing TRM cells and those present within the tumour microenvironment.

Splenocytes and tumour cells were harvested from B16-F1-gB tumour-bearing B6

mice that harboured transferred gBT.I T cells and were infected with HSV-1,

alongside skin samples at day 7, 14 and 30. These samples were homogenised into a

single cell suspension and then stained and analysed identical to skin samples (see

Table 2.3). This allowed for the comparison of CD69, CD103, KLRG1, CTLA-4 and

PD-1 markers on transferred gBT.I CD8 T cells at anatomical sites other than the

skin.

A representative gating strategy is shown in Figure 3.6A which shows the isolation

of gBT.I CD8 T cells in the spleen. To identify transferred gBT.I CD8 T cells, the

markers CD45.1, Vα2 and TCR-β were used. The gBT.I cells were then analysed for

CD69 and CD103 expression as well as the phenotypic markers KLRG1, CTLA-4

and PD-1 (Figure 3.6B). Figure 3.7A shows the gating strategy used for the isolation

of gBT.I CD8 T cells from tumours. These gBT.I cells were also analysed for CD69,

CD103, KLRG1, CTLA-4 and PD-1 expression (Figure 3.7A-B).

Results

61

Figure 3.6: Isolating and phenotyping gBT.I CD8 T cells from the spleen. Spleens

were harvested from B16-F1-gB tumour bearing B6 mice that received naïve gBT.I T

cells followed by HSV-1 infection. Splenocytes were stained and flow cytometric

analysis to identify and phenotype gBT.I CD8 T cells present in the spleen. (A) The

process of identifying gBT.I cells within the spleen by CD45.1, Vα2 and TCR-β

markers as well as the expression of epidermal TRM cell markers CD69 and CD103 is

shown. (B) All gBT.I cells were gated on, regardless of CD69 and CD103 expression

to assess phenotypic expression of KLRG1, CTLA-4 and PD-1 on transferred CD8 T

cells in the spleen.

Results

62

Figure 3.7: Isolating and phenotyping gBT.I CD8 TILs. Tumour cells were

harvested from B16-F1-gB tumour bearing B6 mice that received naïve gBT.I T cells

followed by HSV-1 infection. Tumour cells were stained and analysed to identify and

phenotype TILs present in the tumour. (A) The process of identifying gBT.I TILs

within the tumour by CD45.1, Vα2 and TCR-β markers as well as the expression of

epidermal TRM cell markers CD69 and CD103 is shown. (B) All gBT.I TILs were

gated on, regardless of CD69 and CD103 expression to assess phenotypic expression

of KLRG1, CTLA-4 and PD-1 on transferred CD8 T cells in the tumour.

Results

63

3.4.3 Expression of Epidermal TRM Surface Markers on Transferred gBT.I

Cells.

The expression of CD69 and CD103 cell surface markers were examined to

determine TRM cell presence at different sites. Tumour-bearing B6 mice receiving

gBT.I T cells prior to HSV-1 infection were harvested at day 7, 14 and 30 post-HSV-

1 infection. Mice were euthanised, perfused with PBS to remove circulating T cells

and various tissues were harvested. Samples were homogenised and stained with a

full staining panel (see Table 2.3) to identify transferred gBT.I CD8 T cells and to

analyse surface expression of CD69, CD103, KLRG1, CTLA-4 and PD-1. Tissue

samples from the skin (HSV-1, control and tumour) all showed a strong

CD69+CD103+ population at each time point. Samples isolated from other sites, such

as the spleen and tumour, did not have a strong double positive CD69 and CD103

population. TILs showed a spectrum of CD69 expression, ranging from CD69- to

CD69lo. CD103 expression on the surfaces of TILs was also variable, with most cells

appearing to be CD103- and a small amount with CD103lo, when compared to the

unstained sample in Figure 3.5C. Spleen samples had a CD69-CD103- population

when compared to background fluorescence demonstrated in Figure 3.5C. Samples

taken from the spleen were only present for days 14 and 30 due to time constraints.

Only a small amount of TRM cell numbers were recovered in the control and tumour

skin at day 7, resulting in the appearance of disorderly contour plots.

Results

64

Figure 3.8: CD69 and CD103 expression on transferred gBT.I CD8 T. B6 tumour

bearing mice that received gBT.I T cell transfer and HSV-1 infection were harvested

at day 7, 14 and 30 post HSV-1 infection and analysed for gBT.I TRM cells at different

sites. The plots shown were derived from one animal and are representative of the

total population at day 7 (n=9), day 14 (n=8) and day 30 (n=7). Rows are indicative of

spleen, TILs, HSV-1, control and tumour skin.

Results

65

3.4.4 Enumerating gBT.I TRM Cells in the Skin

Transferred gBT.I TRM cells at sites of HSV-1, control and tumour skin were

isolated, enumerated, quantified and summarised in Figure 3.10. Known areas of skin

were harvested from B16-F1-gB tumour bearing B6 mice that received gBT.I T cells

and HSV-1 infection. Samples were stained with gBT.I markers and the reported

TRM markers: CD69 and CD103. CD69+CD103+ TRM cells were identified (Figure

3.5) and enumerated using Sphero BeadsTM (see section 2.9.2). Figure 3.10A

compares the three skin sites within each time point, to enumerate TRM cells at the

different skin sites. Figure 3.10B shows the same data as Figure 3.10A, instead

comparing the same skin sites across the different time points investigated. Statistical

significance was shown between HSV-1/control (day 14: p=0.0042, day 30:

p=0.0065) and HSV-1/tumour skin sites (day 14: p=0.0054, day 30: p=0.0079) on

day 14 and 30, with tumour and control skin showing lower levels of TRM cell

numbers. Although not significant (HSV-1/control: p=0.0813, HSV-1/tumour

p=0.085), the same trend was observed at day 7 (Figure 3.10A). TRM cell number

differences were also evident within the same sites across different time points.

HSV-1 showed a significant difference between day 7 and 14 (p=0.006) as well as

day 7 and 30 (p=0.0164). Control skin showed a significant rise in TRM cell numbers

from both day 7 to 14 (p=0.0033) and day 7 to 30 (p=0.0048). Tumour skin had

significantly higher numbers at day 14 when compared to the day 7 samples

(p=0.0254). The control and tumour skin samples appear to contain similar TRM cell

numbers as compared to the elevated numbers seen in the HSV-1 samples. TRM cells

numbers appear to peak at day 14 at all sites analysed.

Results

66

Figure 3.9: Enumeration of transferred gBT.I TRM cells. Skin samples were

harvested from HSV-1, control and tumour skin sites on days 7, 14 and 30 post-HSV-

1 infection and naïve gBT.I CD8 T cells transfer. Samples were then processed and

stained with reported markers for epidermal residing TRM cells, CD103 and CD69.

Numbers of gBT.I TRM cells in each cm2 of skin were then quantified using Sphero

BeadsTM (see section 2.9.3). (A) The number of TRM cells at different sites at each

time point. (B) The number of TRM cells at the same site over different time points.

Day 7 (n=9), day 14 (n=8) and day 30 (n=7). Y-axis follows a log-10 scale.

Significance was assessed using a Student’s T-test and depicted as mean±SEM. A

Grubb’s test was used to exclude outliers. Significance between samples is indicated

by *=p≤0.05, **=p≤0.01 and ***=p≤0.001.

Results

67

3.4.5 Phenotyping gBT.I TRM cells

With limited literature surrounding the phenotype of epidermal TRM cells, the

expression of the cell surface markers KLRG1, CTLA-4 and PD-1 was analysed to

determine the phenotype of gBT.I CD8 T cells at different sites, over several time

points. Spleen cells and TILs were also analysed for KLRG1, CTLA-4 and PD-1

expression to gain an insight into the phenotype of systemic gBT.I CD8 T cells and

those present within the tumour microenvironment.

B6 tumour-bearing mice that received 1 x 105 naïve gBT.I CD8 T cells, followed by

HSV-1 infection 24 hours later were analysed (Figure 3.10-3.12). Samples of HSV-1,

control and tumour skin were harvested as well as TILs at day 7, 14 and 30. Each

row represents skin sites taken from a single animal and are representative of the

whole population analysed. A spleen sample from day 7 was not analysed due to

time constraints. Spleen samples had a population of n=1 for days 14 and 30.

3.4.5.1 KLRG1 Expression

KLRG1 is typically expressed on mature or senescent T cells. Expression levels of

KLRG1 were analysed on the gBT.I TRM cells found in epidermal areas of HSV-1

infection, control and tumour skin as well as gBT.I T cells in the tumour (TILs) and

spleen. HSV-1, control and tumour skin samples show a KLRG1lo expression at each

time point, when compared to mean background fluorescence. TILs appear to have

bimodal KLRG1 expression levels at day 7, with a KLRG1hi population reducing to a

small tail at day 14 and 30. The results show that a population transitions from

Results

68

KLRG1lo TILs to KLRG1- from day 14 to 30. Splenic gBT.I cells show a distinctly

bimodal KLRG1- and KLRG1hi population at both days 14 and 30.

The percentage of KLRG1hi gBT.I cells is quantified in Figure 3.10B. Each skin site

(HSV-1 infected, control and tumour) has a small amount of KLRG1 positive TRM

cells. A significant increase in the percentage of KLRG1hi TRM cells in tumour skin

(p=0.0013) and TILs (p=0.0003) compared to TRM cells at site of HSV-1 infection, is

shown at day 7. Samples analysed at day 30 also showed a significant increase in

KLRG1hi TILs in relation to TRM cells in the tumour skin (p=0.0246).

Results

69

Figure 3.10: Analysis of KLRG1 expression on the surface of TRM cells as

compared to TILs and splenic gBT.I CD8 T cells. HSV-1, control and tumour skin

samples were harvested alongside the spleen and tumour from individual mice at day

7, 14 and 30 post-HSV-1 infection. (A) TRM cells were isolated (see Figure 3.5) and

stained for KLRG1 surface molecules. Samples were then compared to mean

background KLRG1 fluorescence to provide an insight into TRM cell phenotype

(dashed line). Each row shows a single animal, representative of the whole

population; day 7 (n=9), day 14 (n=8) and day 30 (n=7). (B) Quantification of

percentage of KLRG1hi cells at different sites, 7 days post-HSV-1 infection. (C)

Quantification of percentage of KLRG1hi cells at different sites, 14 days post-HSV-1

infection. (D) Quantification of percentage of KLRG1hi cells at different sites, 30 days

post-HSV-1 infection. Results were analysed using a Student’s t-test and depicted as

mean±SEM. A Grubb’s test was used to exclude outliers. Significance between

samples is indicated by *=p≤0.05, **=p≤0.01 and ***=p≤0.001.

Results

70

3.4.5.2 CTLA-4 Expression

The surface receptor CTLA-4 acts as a negative regulatory molecule on activated T

cells, making it an attractive target for cancer immunotherapies. The expression

levels of CTLA-4 were examined on epidermal TRM cells and compared to TILs and

splenic gBT.I CD8 T cells. Dashed lines are representative of mean background

fluorescence intensity and are indicative of a true negative population. HSV-1,

control and tumour skin gBT.I TRM cells on average all displayed high expression of

CTLA-4. TILs at day 7 were mainly CTLA-4lo with a small proportion appearing

CTLA-4hi. At day 14, TILs lack a CTLA-4hi population with the majority of cells

appearing to be CTLA-4lo/-. By day 30 most TILs no longer express the CTLA-4

surface receptor, with only a small number of cells displaying a CTLA-4lo

phenotype. Spleen residing gBT.I CD8 T cells show a distinctly CTLA-4- population

at both day 14 and 30 (Figure 3.11A).

Figure 3.11B-D shows the quantification of CTLA-4hi gBT.I CD8 T cells at each

time point. CTLA-4hi cells were most prevalent at day 7 (Figure 3.11B) with a

decrease of this population observed at later time points (day 14 and 30) (Figure

3.11C-D). At each time point analysed TILs had a significantly lower proportion of

CTLA-4hi expressing cells when compared to HSV-1, control or tumour skin sites

(each p-value was <0.0001). Each of the skin sites showed similar CTLA-4hi

percentages at all time points, apart from tumour skin at day 7 which shows a

significantly higher (p=0.0252) percentage of CTLA-4hi cells than HSV-1 skin

(Figure 3.11B).

Results

71

Figure 3.11: Analysis of CTLA-4 expression on the surface of TRM cells as

compared to TILs and splenic gBT.I CD8 T cells. HSV-1, control and tumour skin

samples were harvested alongside the spleen and tumour from individual mice at day

7, 14 and 30 post-HSV-1 infection. (A) TRM cells were isolated (see Figure 3.5) and

stained for CTLA-4 surface molecules. Samples were then compared to mean

background CTLA-4 fluorescence to provide an insight into TRM cell phenotype

(dashed line). Each row shows a single animal, representative of the whole

population; day 7 (n=9), day 14 (n=8) and day 30 (n=7). (B) Quantification of

percentage of CTLA-4hi cells at different sites, 7 days post-HSV-1 infection. (C)

Quantification of percentage of CTLA-4hi cells at different sites, 14 days post-HSV-1

infection. (D) Quantification of percentage of CTLA-4hi cells at different sites, 30

days post-HSV-1 infection. Results were analysed using a Student’s t-test and

depicted as mean±SEM. A Grubb’s test was used to exclude outliers. Significance

between samples is indicated by *=p≤0.05, **=p≤0.01, ***=p≤0.001 and

****=p≤0.0001.

Results

72

3.4.5.3 PD-1 Expression

PD-1 plays an inhibitory role in T cell expansion and is associated with T cell

exhaustion. Like CTLA-4, PD-1 is an attractive target for new cancer

immunotherapies. The expression levels of PD-1 were examined to aid in the

phenotyping of epidermal TRM cells in comparison to TILs and splenic gBT.I CD8 T

cells. Dashed lines in Figure 3.12A represent the mean background fluorescence

intensity of PD-1, indicative of a true negative population. At each skin site

examined (HSV-1, control and tumour), the majority of gBT.I TRM cells appear to be

PD-1lo from day 7 through to day 30. TILs also duplicated this PD-1lo expression on

day 7 and 14 but appear to transition to a PD-1hi phenotype by day 30. Splenic gBT.I

CD8 T cells at day 14 and 30 are similar to background fluorescence and are

therefore mainly PD-1- (Figure 3.12A).

Figure 3.12B-D shows the quantification of the percentage of PD-1+ gBT.I cells

across the different sites at each time point. At day 7, PD-1+ expression is

consistently high amongst each skin site, including TILs (Figure 3.12B). By day 14,

PD-1 expression decreases fractionally at each skin site, but significantly increases in

the TILs (TILs/HSV-1: p=0.0014, TILs/control: P=<0.0001, TILs/tumour: p=

0.0002) (Figure 3.12C). Day 30 shows similar PD-1+ expression levels at each site,

with skin samples remaining consistent to previous time points and TILs having a

higher percentage of PD-1+ cells (TILs/HSV-1: p=0.0007, TILs/control: P=<0.0014,

TILs/tumour: p= 0.021) (Figure 3.12D).

Results

73

Figure 3.12: Analysis of PD-1 expression on the surface of TRM cells as compared

to TILs and splenic gBT.I CD8 T cells. HSV-1, control and tumour skin samples

were harvested alongside the spleen and tumour from individual mice at day 7, 14 and

30 post HSV-1 infection. (A) TRM cells were isolated (see Figure 3.5) and stained for

the PD-1 surface molecules. Samples were then compared to mean background PD-1

fluorescence to provide an insight into TRM cell phenotype (dashed line). Each row

shows a single animal, representative of the whole population; day 7 (n=9), day 14

(n=8) and day 30 (n=7). (B) Quantification of percentage of PD-1+ cells at different

sites, 7 days post-HSV-1 infection. (C) Quantification of percentage of PD-1+ cells at

different sites, 14 days post-HSV-1 infection. (D) Quantification of percentage of PD-

1+ cells at different sites, 30 days post-HSV-1 infection. Results were analysed using a

Student’s t-test and depicted as mean±SEM. A Grubb’s test was used to exclude

outliers. Significance between samples is indicated by *=p≤0.05, **=p≤0.01,

***=p≤0.001 and ****=p≤0.0001.

Discussion

74

Chapter 4: Discussion

4.1 PROJECT SUMMARY

The aim of this project was to investigate the presence and phenotype of TRM cells in

murine cutaneous melanoma. To date, murine models involving the transplant of

melanoma cells have been limited to subcutaneous administration that does not

accurately replicate the pathogenesis of the human disease. Fortunately, our lab has

developed a cutaneous model of murine melanoma, which was essential for

addressing the experimental objectives of this project. Cutaneous inoculation

replicates the pathological process of intraepidermal/dermal growth of human

melanoma (stage I and II) and also reliably metastasises to the draining lymph node,

replicating properties of stage III clinical disease. Intraepidermal/dermal growth

allows for appropriate interactions between the tumour and immune cells present

within the skin, which would not occur in a subcutaneous model where growth is

beyond these outer skin layers.

Melanoma is known as an immunogenic cancer and the most current effective

treatment options are immunotherapeutic of origin (Anichini, Molla et al. 2010).

Two promising therapeutic treatments available utilise anti-CTLA-4 and anti-PD-1

mAb’s to enhance anti-tumour immune responses. With a recently described highly

functional population of epidermal memory CD8 T cells (TRM cells) and a novel

model of epidermal-invading murine melanoma, we decided to investigate the

presence and phenotype of these TRM cells with a special focus on CTLA-4 and PD-1

surface expression.

Discussion

75

To begin, a model by which tumour outgrowth was control for an appropriate period

of time to allow for TRM cell formation was optimised. Mice were cutaneously

inoculated with B16-F1-gB expressing melanoma cells. Once tumours were palpable,

mice were intravenously administered naïve gBT.I CD8 T cells and infected with

HSV-1 24 hours later. gBT.I CD8 T cells are genetically engineered to target HSV-

derived gB and it was therefore hypothesised that with the aid of HSV-1 infection,

these gB-specific T cells would undergo massive expansion, eliciting effector

functions not only towards the gB-expressing HSV-1 infected areas, but also the gB-

expressing tumour cells. We managed to optimise this model with many tumours

successfully being controlled to day 30 post HSV-1 infection. This allowed ample

time for gBT.I CD8 T cells to transition to memory, allowing us to analyse tissue

samples at a range of time points (day 7, 14 and 30).

Following model optimisation, exogenously transferred gBT.I CD8 T cells were

isolated from the skin, from which TRM cells were then identified, enumerated and

phenotyped. We found that TRM cells are present at the site of tumour, although at

numbers similar to unmanipulated control skin, unlike the elevated number seen in

HSV-1 infected skin. Analysis of samples at day 7 revealed a relatively low number

of TRM cells compared to later time points, indicating a progressive transition to

memory cells. TILs analysed also showed a difference in CD69 and CD103

expression, indicating a true TRM population could be identified in the skin in

comparison to the TEFF cells found inside the tumour. TRM cells were also

phenotyped and were found to exhibit the same expression of KLRG-1, CTLA-4 and

PD-1 markers at sites of HSV-1, control and tumour skin. TRM cells displayed a

Discussion

76

KLRG-1lo, CTLA-4hi and PD-1lo phenotype whilst TILs were variable in their

surface molecule expression at different time points.

4.2 DEVELOPING A MODEL OF TUMOUR CONTROL

4.2.1 HSV-1 Infection

HSV-1 is a αherpes virus that readily infects epithelial surfaces, following which it

establishes latency within sensory neurons (Gebhardt and Halford 2005). HSV-1

pathogenesis begins with a lytic phase where the virus enters at a primary epithelial

site and travels down the axons to the dorsal root ganglia. From the dorsal root

ganglia, the HSV-1 viral particles are then transported via anterograde axonal flow,

infecting the whole dermatome and shedding at the periphery. This presents as band-

like or ‘zosteriform’ lesions that spread ventrally from the primary inoculation site

(Simmons and Nash 1984, Khanna, Lepisto et al. 2004, van Lint, Ayers et al. 2004).

Unlike the recurrent human HSV-1 infection, viral reactivation does not regularly

occur in mouse models, instead remaining latent after initial infection (Gebhardt and

Halford 2005). Prior studies have demonstrated that T cells of the adaptive immune

response are essential in the elimination of replicating HSV-1 and establishment of

latency (Sciammas, Kodukula et al. 1997, Lang and Nikolich-Zugich 2005).

Gebhardt et al observed that within HSV-1 infected skin, a population of non-

migratory virus specific CD8 T cells persisted long after clearance of primary

infection (Gebhardt, Wakim et al. 2009). Since then it has been widely accepted that

this non-migratory population are in fact TRM cells, which present in elevated

numbers at sites of HSV-1 infection (Mackay, Rahimpour et al. 2013).

Discussion

77

4.2.2 Transgenic gBT.I CD8 T Cells

Transgenic gBT.I x CD45.1 CD8 T cells harvested from the lymph nodes of gBT.I x

CD45.1 mice, were also utilised as a component of our tumour control model. gBT.I

CD8 T cells are specific for gB, a HSV-derived epitope (Mueller, Heath et al. 2002).

gBT.I CD8 T cells have previously been used alongside HSV-1 flank infection in

studies conducted by Van Lint et al. Activated gBT.I CD8 T cells were adoptively

transferred 24 hours post HSV-1 flank infection, following which they were first

detected in the dorsal root ganglia at day 5 post infection, with infiltration peaking at

day 6. Exogenously transferred gBT.I’s were also shown to dominate the endogenous

CD8 T cells response (van Lint, Ayers et al. 2004).

4.2.3 Model Optimisation

Melanoma is an immunogenic type of cancer that produces many antigens. These

antigens provoke antigen-specific CD8 T cells, resulting in an anti-tumour

immunological response (Dunn, Bruce et al. 2002). The B16-F1-gB melanoma cell

line utilised in conjunction with gB-specific transferred gBT.I CD8 T cells, provided

a powerful model to track the anti-tumour immune response. These gBT.I CD8 T

cells were present in elevated numbers due to HSV-1 infection therefore eliciting

effective tumour control for a short period of time.

When developing our model of tumour control, we used a HSV-1 zosteriform flank

infection protocol derived from Simmons and Nash (Simmons and Nash 1984) and

described by Van Lint et al (van Lint, Ayers et al. 2004). Infection involved localised

mechanical scarification resulting in HSV-1 infection initially confined to the

Discussion

78

epidermal cells immediately adjacent to the site of skin damage. Utilising this HSV-1

flank infection allowed for the development of previously described TRM cells in the

epidermis at both sites of primary infection and recrudescence. These sites of

secondary infection acted as a positive control for TRM cell recovery in later T cell

isolation experiments. HSV-1 was also used due to its ability to prime a large

immune response from transgenic gBT.I CD8 T cells. These T cells are specific for

the HSV-derived gB epitope, therefore specifically targeting not only gB-expressing

viral particles, but also the B16-F10-gB melanoma cell line.

The optimised model of tumour control demonstrated a peak in tumour growth at day

6 after which, tumours entered into an equilibrium-like phase before they begun

escaping immune control. Following HSV-1 infection, gB-specific T cells are primed

and expanded in the draining lymph nodes until they are released at day 5 post

infection (Coles, Mueller et al. 2002). Van Lint et al adoptively transferred activated

gBT.I CD8 T cells, 24 hours prior to flank infection resulting in a peak immune

response at day 6 after infection (van Lint, Ayers et al. 2004). Therefore a correlation

between peak CD8 T cell infiltration in HSV-1 and the beginning of tumour

remission can be elucidated.

Tumours became palpable at various times, (roughly day 12-13), attributable to

human inconsistency in the cutaneous inoculation procedure. It was observed that the

larger the tumours were at the time of HSV-1 infection the faster they escaped from

immune control. During model optimisation, gBT.I and HSV-1 administration times

were varied and therapy times later than the optimised day 13/14, led to an increase

in tumour size and a decrease in time to immune evasion and therefore outgrowth.

Discussion

79

These shorter periods of time to immune evasion is most likely due to the contraction

and therefore decrease in short-lived gBT.I TEFF cells as HSV-1 infection was

controlled to latency. Tumour burden could have eventually outbalanced the

declining tumour-specific T cell numbers, resulting in tumour escape. Overall,

experimental optimisation resulted in a model of tumour control where tumours were

stabilised for a period of time long enough to allow for the transition to TRM cell

memory. Some mice survived until day 30 post infection, allowing for the collection

of samples at day 7, 14 and 30 time points.

4.2.4 gBT.I Expansion in B6 Spleen

Expansion seen in the spleen of B6 mice that received naïve gBT.I T cells and HSV-

1 infection did not mimic expected results. Many studies have shown that CD8 T

cells reach their peak response to HSV-1 infection at day 6-7 post infection (Khanna,

Bonneau et al. 2003, van Lint, Ayers et al. 2004). Spleens harvested at day 7, 14 and

30 show a peak in transferred gBT.I CD8 T cells expansion between day 7-14. To

assess this further, spleen samples could be taken from a wide array of days, such as

day 6, 8, 10 and 12, to determine a more accurate peak in splenic CD8 expansion.

The error bar on day 7 was also sizable, so repeat experiments should be conducted

to get a more normally distributed set of results. A control experiment with B6 mice

receiving gBT.I T cell transfer and HSV-1 infection, without B16-F1-gB tumours,

would be beneficial to further determine a normal immune response.

Discussion

80

4.3 ISOLATING T CELLS FROM THE SKIN

The process of isolating T cells from the skin required the use of harsh proteolytic

enzymes. Dispase II, trypsin/EDTA and collagenase III were used on each skin

sample to detach the basement membrane, degrade proteins and dissociate tissues,

respectively. These enzymes have the ability to modify lymphocyte surface markers.

Decreased expression and complete cleavage of a variety of cell surface markers has

been documented following dispase II, trypsin/EDTA and collagenase III treatment

(Abuzakouk, Feighery et al. 1996, Huang, Hsing et al. 2010).

To create an optimal staining panel, lymphoid cells obtained from lymph nodes, were

subjected to enzymatic digestion and stained with a range of appropriate fluorescent

antibodies. Several surface molecules were cleaved or modified by particular

enzymes and could not be used as antibody staining targets. CD8 was completely

stripped by the trypsin/EDTA enzyme and therefore a staining panel had to be

created that enabled identification of CD8 T cells without physically staining for

CD8. This was achieved by using the antibodies TCR-β, Vα2 and CD45.1, all of

which targeted surface receptors that underwent either minor or no modification and

were present on the surface of transferred gBT.I CD8 T cells.

4.4 IDENTIFYING AND ENUMERATING TRM CELLS

To identify TRM cells, transferred gBT.I CD8 T cells need to be isolated. Vα2 and

TCR-β are markers expressed on the surface of gBT.I CD8 T cells (Brooks, Balk et

al. 1993, Mueller, Heath et al. 2002). Transgenic gBT.I mice were bred on a CD45.1

(or ly5.1) background, whilst B6 mice are on a CD45.2 background (or ly5.2). This

Discussion

81

allowed for the detection of gBT.I T cells via CD45.1 antibody staining. Once gBT.I

CD8 T cells were isolated, CD69 and CD103 expression was analysed. A unique

surface marker signature has been identified for epidermal TRM cells, which are

CD69+CD103+ (Masopust, Vezys et al. 2006, Wakim, Woodward-Davis et al. 2010,

Gebhardt, Whitney et al. 2011, Casey, Fraser et al. 2012). CD103 binds to E-

cadherin, which is expressed by keratinocytes in the skin. It has been elucidated that

the CD103 role on the surface of TRM cells has involvement in T cell survival and

possible tethering of the TRM cells to the epidermal compartment (Cepek, Shaw et al.

1994, Schon, Arya et al. 1999, Wakim, Woodward-Davis et al. 2010). The CD69

molecule is commonly associated with early activation, although it has been shown

that ongoing stimulation is not required for TRM cells, which constitutively express

the marker (van Lint, Kleinert et al. 2005, Mackay, Stock et al. 2012). Distinct

CD69+CD103+ were shown at each skin site, with populations becoming tighter as

time progressed, most likely due to the increased contraction to a TRM cell

population. TILs have previously been described to show a range of CD69

expression, but do not mimic the distinct CD69hiCD103hi populations observed in the

skin. It has been described that TRM cells are not present in the spleen during HSV-1

infection, consistent with our findings (Gebhardt, Wakim et al. 2009, Casey, Fraser

et al. 2012).

The enumeration protocol carried out revealed that an elevated number of antigen-

specific TRM cells exist at the site of HSV-1 infected skin, compared to

unmanipulated control skin. This follows previously documented work done by

Gebhardt et al, who demonstrated a higher number of TRM cells in HSV-1 infected

skin, as well as no change in endogenous, non-antigen specific TRM cell population

Discussion

82

numbers (Gebhardt, Wakim et al. 2009). Upon further inspection the TRM cell

population at the site of cutaneous melanoma did not replicate the elevated

expression as seen in HSV-1 infected skin, instead showing a similar cell count to

unmanipulated control skin. This could suggest that a presence of a cutaneous

tumour alone may not necessarily trigger the formation of TRM cells and that TRM cell

presence at the tumour site could be a normal physiological process. However, it is

important to note that TRM cells are present at the tumour site in substantial enough

numbers to potentially be a target for immune therapies. TRM cell numbers at all skin

sites were lowest at day 7 with only a few cells beginning to transition to memory.

This peaks at day 14 and then decreases by day 30, at which a stable pool of non-

migratory TRM cells is established. This flux in number in the control skin could

potentially mean unspecific TRM cell skin infiltration is caused by the systemic

immune response to HSV-1 infection or simply a normal physiological process.

4.5 PHENOTYPING TRM CELLS

4.5.1 KLRG1 Expression

KLRG1 or killer cell lectin-like receptor subfamily G member 1, expression is

associated with replicative senescence and impaired proliferative potential on CD8 T

cells (Voehringer, Blaser et al. 2001, Voehringer, Koschella et al. 2002, Ye, Turner

et al. 2012). KLRG1 may also be involved in the dampening of cytokine production

and killing, but current roles of KLRG1+ memory cells in non-lymphoid tissues are

still being debated (Ye, Turner et al. 2012). KLRG1+ cells give rise to effector-like

memory populations or short-lived TEFF cells. In contrast, KLRG1- cells are

precursors for long-lived circulating memory cells (Kaech and Wherry 2007, Obar

Discussion

83

and Lefrancois 2010, Sheridan and Lefrancois 2011). TRM cells have recently been

found to originate from long-lived KLRG1- precursor cells (Mackay, Rahimpour et

al. 2013).

In attempt to identify the phenotype of TRM cells, we analysed cells at different sites

for KLRG1 expression. Mackay et al demonstrated that one week following gBT.I

transfer and HSV-1 infection, KLRG1 expression on skin TRM cells was high

followed by a reduction to a population with low expression at week 2 and then an

almost completely negative population by week 4 (Mackay, Rahimpour et al. 2013).

Conversely each site of HSV-1 infected, control and tumour skin constantly

expressed low levels of KLRG1, different to what has been previously described.

This suggests that there is a shift in expression following transition from KLRG1-

precursor cells to a potentially constitutively expressed KLRG1lo TRM phenotype.

TILs were also analysed for KLRG1 and a bimodal population was seen with

KLRG1 expression shifting from high to low/negative expression as time progressed.

This is potentially the result of short-lived KLRG1+ TEFF cells becoming exhausted

and moving to the spleen, which consistently shows an increased number of

KLRG1hi cells as time progresses.

4.5.2 CTLA-4 Expression

CTLA-4, or cytotoxic T lymphocyte-associated antigen 4, is an inhibitory receptor

expressed on activated T cells and is involved in the downregulation of the T cell

response during immunological homeostasis (Hodi, O'Day et al. 2010). Due to its

immunosuppressive functions, CTLA-4 has become a target for immune checkpoint

Discussion

84

inhibition therapies. TILs are abundant inside cutaneous melanomas, yet they easily

undergo T cell exhaustion, causing the expression of inhibitory receptors, such as

CTLA-4. This expression of CTLA-4 aids in tumour escape and is used as a target

for anti-CTLA-4 mAb therapy. Anti-CTLA-4 mAb therapy has shown to enhance the

anti-tumour functions of T cells, by increasing the effectiveness of the CD8 T cell

response and decreasing regulatory T cell numbers. Most patients show modest or

minor clinical benefits to anti-CTLA-4 mAb therapy, yet a small handful exhibit

durable clinical responses (Allard, Pommey et al. 2013).

Due to the success of anti-CTLA-4 therapy for melanoma treatment, we decided to

identify CTLA-4 expression on the surface of TRM cells at different skin sites.

Interestingly, CTLA-4 was shown to be highly expressed on TRM cells across each

time point at sites of HSV-1 infected, control and tumour skin. This could therefore

mean that these cells, although reportedly having highly efficient effector functions,

could potentially be dampened by CTLA-4 expression, which could benefit from

anti-CTLA-4 therapy. TILs analysed showed a low expression of CTLA-4 at day 7

and 14, which transitioned to a CTLA-4- population by day 30. Popular belief would

render this confusing, but studies have shown that CTLA-4 is highly expressed

intracellularly in TILs and can be variably expressed on the cell surface (Wang,

Zheng et al. 2001).

4.5.3 PD-1 Expression

PD-1, or programmed death ligand-1, is a molecule expressed on activated T cells.

An increased expression of PD-1 is seen on TILs and is associated with poor patient

Discussion

85

prognosis (Gao, Wang et al. 2009, Gadiot, Hooijkaas et al. 2011). Targeted blockade

of PD-1 has shown to elicit objective clinical functions in 6% to 28% of metastatic

melanoma patients (Topalian, Hodi et al. 2012). This targeted PD-1 blockade is

achieved by using anti-PD-1 mAb’s and acts to enhance anti-tumour immunity by

preventing T cell exhaustion and promoting homing by T cells to tumour sites

(Curran, Montalvo et al. 2010, Peng, Liu et al. 2012). We decided to investigate PD-

1 expression on TRM cells at different skin sites, due to the immunotherapeutic

potential of targeting the PD-1 receptor. PD-1 was found to be consistently expressed

at low levels across all time points in HSV-1 infected, control and tumour skin-

derived TRM cells. This was similar to the expression of PD-1 shown on TILs which

varied from PD-1lo to PD-1hi as time progressed. This PD-1 expression is in

accordance to the literature and is attributable to the promising responses seen in

anti-PD-1 immunotherapies. The PD-1lo expression seen on TRM cells, may also

benefit by targeting with anti-PD-1 mAb’s to prevent T cell exhaustion and promote

the CD8 T cell responses that surrounds the tumour within the epidermal layer.

4.6 GENERAL DISCUSSION

CTLA-4 and PD-1 targeted immunotherapy is currently the most promising

treatment in regards to late stage melanoma. With TRM cells analysed displaying a

KLRG1lo, CTLA-4hi and PD-1lo phenotype, it is thought that these cells could

potentially have implications in the overall anti-tumour T cell response. TRM cells,

although not as prevalent at the site of tumour compared to HSV-1 infected skin, are

still present in significant numbers and express the same phenotype as the highly

functional population found following HSV-1 infection. These TRM cell could

therefore be used as a target for checkpoint blockade immunotherapies. The

Discussion

86

differences in local or systemic mAb delivery and its effect on TRM cells is also an

area worth exploring. Currently there is a lack of literature on TRM cell function

during cancer, further suggesting more research to be conducted in this area.

To further investigate the role of TRM cells several additional experiments could be

conducted. Assessing the spatiotemporal arrangement of TRM cells at the site of

cutaneous melanoma is important to determine tumour-TRM interactions. One way to

assess this would be by immunohistochemistry staining and confocal analysis. The

same model of tumour control used throughout this project could be utilised, instead

with skin and tumour samples being harvested, cryo-embedded, sectioned and

stained for the CD45.1 marker to identify gBT.I CD8 T cells and nuclei to show skin

morphology. The B16-F1-gB cell line used has also been genetically engineered to

express a green fluorescent protein, enabling tumour cells to be identified via

fluorescent microscopy. Z-stack technology can then be performed using a confocal

microscope and the TRM cell-tumour interactions in the epidermis can be assessed.

Another future direction would be to analyse whether pre-existing antigen-specific

TRM cells can confer protection against cutaneous melanoma. B6 mice receiving an

intravenous injection of naïve gBT.I CD8 T cells 24 hours prior to HSV-1 infection

on the left flank. These mice could be left for 30+ days to allow ample time for TRM

cell formation at the site of HSV-1 infection. After day 30+, a dual cutaneous

melanoma graft could be placed on opposite flanks of the mouse, with the tumour on

the left flank inoculated at the site of HSV-1 recrudescence. Tumour growth could

then be mapped to determine whether or not kinetics of both tumours are equal. We

Discussion

87

predict that the previously infected site would promote enhance protection. Thus,

identifying a potentially important role of TRM cells.

Lastly, the effect of the administration of anti-CTLA-4 and anti-PD-1 mAb’s on TRM

cells could be assessed. Utilising our model of tumour control, anti-CTLA-4 and

anti-PD-1 mAb’s could be administered. Sites of HSV-1 infected, control and tumour

skin samples could be harvested and the TRM cells isolated. The TRM cells could then

undergo flow cytometric analysis for enumeration and phenotyping of CTLA-4 and

PD-1 expression to determine whether these checkpoint inhibition therapies are

having an impact on TRM cells at the site of cutaneous melanoma. Circulating CD8 T

cells could also be depleted before mAb administration. This could potentially

pinpoint a role for TRM cells within mAb therapy.

4.7 AIMS AND LIMITATIONS

The aim of this project was to investigate the role of TRM cells in cutaneous

melanoma by identifying, enumerating and phenotyping at different stages of tumour

control. These aims were successfully achieved, firstly with the establishment of a

model of tumour control that allowed stabilisation of tumours to day 30 post HSV-1

infection. Utilising the model, skin samples from HSV-1 infected, control and

tumour sites were harvested and TRM cells were identified and enumerated. TRM cells

residing at the site of HSV-1 infection were elevated in number, in contrast to TRM

cells residing at unmanipulated control and tumour skin sites which had comparable

TRM cell presence. TRM cells were phenotyped and constitutively displayed a

KLRG1lo, CTLA-4hi, PD-1lo phenotype, across all time points and skin sites.

Discussion

88

The adequate use of control experiments was a limitation of this project. Future

research would require several controls to be conducted. Tumour kinetics in B6 mice

that have do not receive gBT.I CD8 T cell transfer and HSV-1 infection, would be

required to compare the tumour growth kinetics to tumour specific gBT.I CD8 T

cells and HSV-1 infection. Another experiment replicating the model without HSV-1

infection would be useful to demonstrate the anti-tumour immune response from

antigen-specific CD8 T cells without the aid of expansion from HSV-1 infection.

Lastly, another useful control experiment would be to replicate the model, instead

using non-tumour specific CD8 T cells that are still specific for HSV-derived

epitopes. This could be done by either utilising the same tumour cell line and instead

intravenously transferring naïve gDT.II CD8 T cells from gDT.II mice, which are

specific for HSV-derived glycoprotein D (not expressed by B16-F1-gB) or utilising

the same gBT.I CD8 T cells, instead changing the melanoma cell line used to one

which does not express the gB epitope.

References

91

Chapter 6: References

Abuzakouk, M., C. Feighery and C. O'Farrelly (1996). "Collagenase and Dispase

enzymes disrupt lymphocyte surface molecules." J Immunol Methods 194(2):

211-216.

Adeegbe, D., A. L. Bayer, R. B. Levy and T. R. Malek (2006). Cutting edge: allogeneic

CD4+CD25+Foxp3+ T regulatory cells suppress autoimmunity while

establishing transplantation tolerance. J Immunol. 176: 7149-7153.

Allard, B., S. Pommey, M. J. Smyth and J. Stagg (2013). "Targeting CD73 enhances the

antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs." Clin Cancer Res

19(20): 5626-5635.

Anichini, A., A. Molla, C. Vegetti, I. Bersani, R. Zappasodi, F. Arienti, F. Ravagnani,

A. Maurichi, R. Patuzzo, M. Santinami, H. Pircher, M. Di Nicola and R.

Mortarini (2010). "Tumor-reactive CD8+ early effector T cells identified at

tumor site in primary and metastatic melanoma." Cancer Res 70(21): 8378-

8387.

Ariotti, S., J. B. Beltman, G. Chodaczek, M. E. Hoekstra, A. E. van Beek, R. Gomez-

Eerland, L. Ritsma, J. van Rheenen, A. F. Maree, T. Zal, R. J. de Boer, J. B.

Haanen and T. N. Schumacher (2012). "Tissue-resident memory CD8+ T cells

continuously patrol skin epithelia to quickly recognize local antigen." Proc Natl

Acad Sci USA 109(48): 19739-19744.

Atkins, M. B., M. T. Lotze, J. P. Dutcher, R. I. Fisher, G. Weiss, K. Margolin, J.

Abrams, M. Sznol, D. Parkinson, M. Hawkins, C. Paradise, L. Kunkel and S.

A. Rosenberg (1999). "High-dose recombinant interleukin 2 therapy for

patients with metastatic melanoma: analysis of 270 patients treated between

1985 and 1993." J Clin Oncol 17(7): 2105-2116.

Australian Institute of Health and Welfare (2014). ACIM (Australian Cancer Incidence

and Mortality) Books. Canberra, AIHW.

Badovinac, V. P., B. B. Porter and J. T. Harty (2002). "Programmed contraction of

CD8(+) T cells after infection." Nat Immunol 3(7): 619-626.

Balch, C. M., J. E. Gershenwald, S. J. Soong, J. F. Thompson, M. B. Atkins, D. R.

Byrd, A. C. Buzaid, A. J. Cochran, D. G. Coit, S. Ding, A. M. Eggermont, K.

T. Flaherty, P. A. Gimotty, J. M. Kirkwood, K. M. McMasters, M. C. Mihm,

Jr., D. L. Morton, M. I. Ross, A. J. Sober and V. K. Sondak (2009). "Final

version of 2009 AJCC melanoma staging and classification." J Clin Oncol

27(36): 6199-6206.

Blattman, J. N., R. Antia, D. J. Sourdive, X. Wang, S. M. Kaech, K. Murali-Krishna, J.

D. Altman and R. Ahmed (2002). "Estimating the precursor frequency of naive

antigen-specific CD8 T cells." J Exp Med 195(5): 657-664.

References

92

Bottomley, A., C. Coens, S. Suciu, M. Santinami, W. Kruit, A. Testori, J. Marsden, C.

Punt, F. Sales, M. Gore, R. Mackie, Z. Kusic, R. Dummer, P. Patel, D.

Schadendorf, A. Spatz, U. Keilholz and A. Eggermont (2009). "Adjuvant

therapy with pegylated interferon alfa-2b versus observation in resected stage

III melanoma: a phase III randomized controlled trial of health-related quality

of life and symptoms by the European Organisation for Research and

Treatment of Cancer Melanoma Group." J Clin Oncol 27(18): 2916-2923.

Brooks, E. G., S. P. Balk, K. Aupeix, M. Colonna, J. L. Strominger and V. Groh-Spies

(1993). "Human T-cell receptor (TCR) alpha/beta + CD4-CD8- T cells express

oligoclonal TCRs, share junctional motifs across TCR V beta-gene families,

and phenotypically resemble memory T cells." Proc Natl Acad Sci USA

90(24): 11787-11791.

Cameron, D. A., M. C. Cornbleet, R. M. Mackie, J. A. Hunter, M. Gore, B. Hancock

and J. F. Smyth (2001). "Adjuvant interferon alpha 2b in high risk melanoma -

the Scottish study." Br J Cancer 84(9): 1146-1149.

Cascinelli, N., F. Belli, R. M. MacKie, M. Santinami, R. Bufalino and A. Morabito

(2001). "Effect of long-term adjuvant therapy with interferon alpha-2a in

patients with regional node metastases from cutaneous melanoma: a

randomised trial." Lancet 358(9285): 866-869.

Casey, K. A., K. A. Fraser, J. M. Schenkel, A. Moran, M. C. Abt, L. K. Beura, P. J.

Lucas, D. Artis, E. J. Wherry, K. Hogquist, V. Vezys and D. Masopust (2012).

"Antigen-independent differentiation and maintenance of effector-like resident

memory T cells in tissues." J Immunol 188(10): 4866-4875.

Cepek, K. L., S. K. Shaw, C. M. Parker, G. J. Russell, J. S. Morrow, D. L. Rimm and

M. B. Brenner (1994). "Adhesion between epithelial cells and T lymphocytes

mediated by E-cadherin and the alpha E beta 7 integrin." Nature 372(6502):

190-193.

Chapman, P. B., L. H. Einhorn, M. L. Meyers, S. Saxman, A. N. Destro, K. S.

Panageas, C. B. Begg, S. S. Agarwala, L. M. Schuchter, M. S. Ernstoff, A. N.

Houghton and J. M. Kirkwood (1999). "Phase III multicenter randomized trial

of the Dartmouth regimen versus dacarbazine in patients with metastatic

melanoma." J Clin Oncol 17(9): 2745-2751.

Chapman, P. B., A. Hauschild, C. Robert, J. B. Haanen, P. Ascierto, J. Larkin, R.

Dummer, C. Garbe, A. Testori, M. Maio, D. Hogg, P. Lorigan, C. Lebbe, T.

Jouary, D. Schadendorf, A. Ribas, S. J. O'Day, J. A. Sosman, J. M. Kirkwood,

A. M. Eggermont, B. Dreno, K. Nolop, J. Li, B. Nelson, J. Hou, R. J. Lee, K.

T. Flaherty and G. A. McArthur (2011). "Improved survival with vemurafenib

in melanoma with BRAF V600E mutation." N Engl J Med 364(26): 2507-

2516.

Clark, W. H., Jr., D. E. Elder, D. t. Guerry, M. N. Epstein, M. H. Greene and M. Van

Horn (1984). "A study of tumor progression: the precursor lesions of

superficial spreading and nodular melanoma." Hum Pathol 15(12): 1147-1165.

References

93

Coles, R. M., S. N. Mueller, W. R. Heath, F. R. Carbone and A. G. Brooks (2002).

"Progression of armed CTL from draining lymph node to spleen shortly after

localized infection with herpes simplex virus 1." J Immunol 168(2): 834-838.

Cummins, D. L., J. M. Cummins, H. Pantle, M. A. Silverman, A. L. Leonard and A.

Chanmugam (2006). "Cutaneous malignant melanoma." Mayo Clin Proc 81(4):

500-507.

Curran, M. A., W. Montalvo, H. Yagita and J. P. Allison (2010). "PD-1 and CTLA-4

combination blockade expands infiltrating T cells and reduces regulatory T and

myeloid cells within B16 melanoma tumors." Proc Natl Acad Sci USA 107(9):

4275-4280.

Curtsinger, J. M., C. M. Johnson and M. F. Mescher (2003). "CD8 T cell clonal

expansion and development of effector function require prolonged exposure to

antigen, costimulation, and signal 3 cytokine." J Immunol 171(10): 5165-5171.

Davies, H., G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H.

Woffendin, M. J. Garnett, W. Bottomley, N. Davis, E. Dicks, R. Ewing, Y.

Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes, V. Kosmidou, A. Menzies, C.

Mould, A. Parker, C. Stevens, S. Watt, S. Hooper, R. Wilson, H. Jayatilake, B.

A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K. Pritchard-Jones, N.

Maitland, G. Chenevix-Trench, G. J. Riggins, D. D. Bigner, G. Palmieri, A.

Cossu, A. Flanagan, A. Nicholson, J. W. Ho, S. Y. Leung, S. T. Yuen, B. L.

Weber, H. F. Seigler, T. L. Darrow, H. Paterson, R. Marais, C. J. Marshall, R.

Wooster, M. R. Stratton and P. A. Futreal (2002). "Mutations of the BRAF

gene in human cancer." Nature 417(6892): 949-954.

Dobbs, M. E., J. E. Strasser, C. F. Chu, C. Chalk and G. N. Milligan (2005). "Clearance

of herpes simplex virus type 2 by CD8+ T cells requires gamma interferon and

either perforin- or Fas-mediated cytolytic mechanisms." J Virol 79(23): 14546-

14554.

Dudley, M. E., J. R. Wunderlich, P. F. Robbins, J. C. Yang, P. Hwu, D. J.

Schwartzentruber, S. L. Topalian, R. Sherry, N. P. Restifo, A. M. Hubicki, M.

R. Robinson, M. Raffeld, P. Duray, C. A. Seipp, L. Rogers-Freezer, K. E.

Morton, S. A. Mavroukakis, D. E. White and S. A. Rosenberg (2002). "Cancer

regression and autoimmunity in patients after clonal repopulation with

antitumor lymphocytes." Science 298(5594): 850-854.

Dunn, G. P., A. T. Bruce, H. Ikeda, L. J. Old and R. D. Schreiber (2002). "Cancer

immunoediting: from immunosurveillance to tumor escape." Nat Immunol

3(11): 991-998.

Duraiswamy, J., K. M. Kaluza, G. J. Freeman and G. Coukos (2013). "Dual blockade of

PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell

rejection function in tumors." Cancer Res 73(12): 3591-3603.

Durgan, K., M. Ali, P. Warner and Y. E. Latchman (2011). "Targeting NKT cells and

PD-L1 pathway results in augmented anti-tumor responses in a melanoma

model." Cancer Immunol Immunother 60(4): 547-558.

References

94

Dzhagalov, I. L., K. G. Chen, P. Herzmark and E. A. Robey (2013). "Elimination of

self-reactive T cells in the thymus: a timeline for negative selection." PLoS

Biol 11(5): e1001566.

Eggermont, A. M., S. Suciu, M. Santinami, A. Testori, W. H. Kruit, J. Marsden, C. J.

Punt, F. Sales, M. Gore, R. Mackie, Z. Kusic, R. Dummer, A. Hauschild, E.

Musat, A. Spatz and U. Keilholz (2008). "Adjuvant therapy with pegylated

interferon alfa-2b versus observation alone in resected stage III melanoma:

final results of EORTC 18991, a randomised phase III trial." Lancet 372(9633):

117-126.

Farber, D. L., N. A. Yudanin and N. P. Restifo (2014). "Human memory T cells:

generation, compartmentalization and homeostasis." Nat Rev Immunol 14(1):

24-35.

Gadiot, J., A. I. Hooijkaas, A. D. Kaiser, H. van Tinteren, H. van Boven and C. Blank

(2011). "Overall survival and PD-L1 expression in metastasized malignant

melanoma." Cancer 117(10): 2192-2201.

Gao, Q., X. Y. Wang, S. J. Qiu, I. Yamato, M. Sho, Y. Nakajima, J. Zhou, B. Z. Li, Y.

H. Shi, Y. S. Xiao, Y. Xu and J. Fan (2009). "Overexpression of PD-L1

significantly associates with tumor aggressiveness and postoperative

recurrence in human hepatocellular carcinoma." Clin Cancer Res 15(3): 971-

979.

Garbe, C. and A. Blum (2001). "Epidemiology of cutaneous melanoma in Germany and

worldwide." Skin Pharmacol Appl Skin Physiol 14(5): 280-290.

Garbe, C. and U. Leiter (2009). "Melanoma epidemiology and trends." Clin Dermatol

27(1): 3-9.

Gebhardt, B. M. and W. P. Halford (2005). "Evidence that spontaneous reactivation of

herpes virus does not occur in mice." Virol J 2: 67.

Gebhardt, T. and L. K. Mackay (2012). "Local immunity by tissue-resident CD8(+)

memory T cells." Front Immunol 3: 340.

Gebhardt, T., L. M. Wakim, L. Eidsmo, P. C. Reading, W. R. Heath and F. R. Carbone

(2009). "Memory T cells in nonlymphoid tissue that provide enhanced local

immunity during infection with herpes simplex virus." Nat Immunol 10(5):

524-530.

Gebhardt, T., P. G. Whitney, A. Zaid, L. K. Mackay, A. G. Brooks, W. R. Heath, F. R.

Carbone and S. N. Mueller (2011). "Different patterns of peripheral migration

by memory CD4+ and CD8+ T cells." Nature 477(7363): 216-219.

Gray-Schopfer, V., C. Wellbrock and R. Marais (2007). "Melanoma biology and new

targeted therapy." Nature 445(7130): 851-857.

Haass, N. K., K. S. Smalley and M. Herlyn (2004). "The role of altered cell-cell

communication in melanoma progression." J Mol Histol 35(3): 309-318.

References

95

Hadley, G. A., E. A. Rostapshova, D. M. Gomolka, B. M. Taylor, S. T. Bartlett, C. I.

Drachenberg and M. R. Weir (1999). "Regulation of the epithelial cell-specific

integrin, CD103, by human CD8+ cytolytic T lymphocytes." Transplantation

67(11): 1418-1425.

Hamilton, S. E. and S. C. Jameson (2012). "CD8 T cell memory: it takes all kinds."

Front Immunol 3: 353.

Harty, J. T., A. R. Tvinnereim and D. W. White (2000). "CD8+ T cell effector

mechanisms in resistance to infection." Annu Rev Immunol 18: 275-308.

Hemler, M. E. (1990). "VLA proteins in the integrin family: structures, functions, and

their role on leukocytes." Annu Rev Immunol 8: 365-400.

Hodi, F. S., S. J. O'Day, D. F. McDermott, R. W. Weber, J. A. Sosman, J. B. Haanen, R.

Gonzalez, C. Robert, D. Schadendorf, J. C. Hassel, W. Akerley, A. J. van den

Eertwegh, J. Lutzky, P. Lorigan, J. M. Vaubel, G. P. Linette, D. Hogg, C. H.

Ottensmeier, C. Lebbe, C. Peschel, I. Quirt, J. I. Clark, J. D. Wolchok, J. S.

Weber, J. Tian, M. J. Yellin, G. M. Nichol, A. Hoos and W. J. Urba (2010).

"Improved survival with ipilimumab in patients with metastatic melanoma." N

Engl J Med 363(8): 711-723.

Hofmann, M. and H. Pircher (2011). "E-cadherin promotes accumulation of a unique

memory CD8 T-cell population in murine salivary glands." Proc Natl Acad Sci

USA 108(40): 16741-16746.

Hu, Q., S. A. Nicol, A. Y. Suen and T. A. Baldwin (2012). "Examination of thymic

positive and negative selection by flow cytometry." J Vis Exp(68).

Huang, H. L., H. W. Hsing, T. C. Lai, Y. W. Chen, T. R. Lee, H. T. Chan, P. C. Lyu, C.

L. Wu, Y. C. Lu, S. T. Lin, C. W. Lin, C. H. Lai, H. T. Chang, H. C. Chou and

H. L. Chan (2010). "Trypsin-induced proteome alteration during cell

subculture in mammalian cells." J Biomed Sci 17: 36.

Huster, K. M., V. Busch, M. Schiemann, K. Linkemann, K. M. Kerksiek, H. Wagner

and D. H. Busch (2004). "Selective expression of IL-7 receptor on memory T

cells identifies early CD40L-dependent generation of distinct CD8+ memory T

cell subsets." Proc Natl Acad Sci USA 101(15): 5610-5615.

Jiang, X., R. A. Clark, L. Liu, A. J. Wagers, R. C. Fuhlbrigge and T. S. Kupper (2012).

"Skin infection generates non-migratory memory CD8+ T(RM) cells providing

global skin immunity." Nature 483(7388): 227-231.

Johnson, L. D. and S. C. Jameson (2012). "TGF-beta sensitivity restrains CD8+ T cell

homeostatic proliferation by enforcing sensitivity to IL-7 and IL-15." PLoS

One 7(8): e42268.

Kaech, S. M. and W. Cui (2012). "Transcriptional control of effector and memory

CD8+ T cell differentiation." Nat Rev Immunol 12(11): 749-761.

Kaech, S. M. and E. J. Wherry (2007). "Heterogeneity and cell-fate decisions in effector

and memory CD8+ T cell differentiation during viral infection." Immunity

27(3): 393-405.

References

96

Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner

and P. Golstein (1994). "Fas and perforin pathways as major mechanisms of T

cell-mediated cytotoxicity." Science 265(5171): 528-530.

Karupiah, G., Q. W. Xie, R. M. Buller, C. Nathan, C. Duarte and J. D. MacMicking

(1993). "Inhibition of viral replication by interferon-gamma-induced nitric

oxide synthase." Science 261(5127): 1445-1448.

Khanna, K. M., R. H. Bonneau, P. R. Kinchington and R. L. Hendricks (2003). "Herpes

simplex virus-specific memory CD8+ T cells are selectively activated and

retained in latently infected sensory ganglia." Immunity 18(5): 593-603.

Khanna, K. M., A. J. Lepisto, V. Decman and R. L. Hendricks (2004). "Immune control

of herpes simplex virus during latency." Curr Opin Immunol 16(4): 463-469.

Kisielow, P., H. Bluthmann, U. D. Staerz, M. Steinmetz and H. von Boehmer (1988).

"Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature

CD4+8+ thymocytes." Nature 333(6175): 742-746.

Klebanoff, C. A., L. Gattinoni, P. Torabi-Parizi, K. Kerstann, A. R. Cardones, S. E.

Finkelstein, D. C. Palmer, P. A. Antony, S. T. Hwang, S. A. Rosenberg, T. A.

Waldmann and N. P. Restifo (2005). "Central memory self/tumor-reactive

CD8+ T cells confer superior antitumor immunity compared with effector

memory T cells." Proc Natl Acad Sci U S A 102(27): 9571-9576.

Kolar, P., K. Knieke, J. K. Hegel, D. Quandt, G. R. Burmester, H. Hoff and M. C.

Brunner-Weinzierl (2009). "CTLA-4 (CD152) controls homeostasis and

suppressive capacity of regulatory T cells in mice." Arthritis Rheum 60(1):

123-132.

Kunishige, J. H., D. G. Brodland and J. A. Zitelli (2012). "Surgical margins for

melanoma in situ." J Am Acad Dermatol 66(3): 438-444.

Lang, A. and J. Nikolich-Zugich (2005). "Development and migration of protective

CD8+ T cells into the nervous system following ocular herpes simplex virus-1

infection." J Immunol 174(5): 2919-2925.

Lanzavecchia, A. and F. Sallusto (2001). "Antigen decoding by T lymphocytes: from

synapses to fate determination." Nat Immunol 2(6): 487-492.

Letsch, A., U. Keilholz, D. Schadendorf, D. Nagorsen, A. Schmittel, E. Thiel and C.

Scheibenbogen (2000). "High frequencies of circulating melanoma-reactive

CD8+ T cells in patients with advanced melanoma." Int J Cancer 87(5): 659-

664.

Lipson, E. J. and C. G. Drake (2011). "Ipilimumab: an anti-CTLA-4 antibody for

metastatic melanoma." Clin Cancer Res 17(22): 6958-6962.

Ljunggren, G. and D. J. Anderson (1998). "Cytokine induced modulation of MHC class

I and class II molecules on human cervical epithelial cells." J Reprod Immunol

38(2): 123-138.

References

97

Mackay, L. K. and T. Gebhardt (2013). "Tissue-resident memory T cells: local guards

of the thymus." Eur J Immunol 43(9): 2259-2262.

Mackay, L. K., A. Rahimpour, J. Z. Ma, N. Collins, A. T. Stock, M. L. Hafon, J. Vega-

Ramos, P. Lauzurica, S. N. Mueller, T. Stefanovic, D. C. Tscharke, W. R.

Heath, M. Inouye, F. R. Carbone and T. Gebhardt (2013). "The developmental

pathway for CD103(+)CD8+ tissue-resident memory T cells of skin." Nat

Immunol 14(12): 1294-1301.

Mackay, L. K., A. T. Stock, J. Z. Ma, C. M. Jones, S. J. Kent, S. N. Mueller, W. R.

Heath, F. R. Carbone and T. Gebhardt (2012). "Long-lived epithelial immunity

by tissue-resident memory T (TRM) cells in the absence of persisting local

antigen presentation." Proc Natl Acad Sci USA 109(18): 7037-7042.

Mamalis, A., M. Garcha and J. Jagdeo (2014). "Targeting the PD-1 pathway: a

promising future for the treatment of melanoma." Arch Dermatol Res.

Masopust, D., D. Choo, V. Vezys, E. J. Wherry, J. Duraiswamy, R. Akondy, J. Wang,

K. A. Casey, D. L. Barber, K. S. Kawamura, K. A. Fraser, R. J. Webby, V.

Brinkmann, E. C. Butcher, K. A. Newell and R. Ahmed (2010). "Dynamic T

cell migration program provides resident memory within intestinal epithelium."

J Exp Med 207(3): 553-564.

Masopust, D., V. Vezys, A. L. Marzo and L. Lefrancois (2001). "Preferential

localization of effector memory cells in nonlymphoid tissue." Science

291(5512): 2413-2417.

Masopust, D., V. Vezys, E. J. Wherry, D. L. Barber and R. Ahmed (2006). "Cutting

edge: gut microenvironment promotes differentiation of a unique memory CD8

T cell population." J Immunol 176(4): 2079-2083.

McCully, M. L., K. Ladell, S. Hakobyan, R. E. Mansel, D. A. Price and B. Moser

(2012). "Epidermis instructs skin homing receptor expression in human T

cells." Blood 120(23): 4591-4598.

Middleton, M. R., J. J. Grob, N. Aaronson, G. Fierlbeck, W. Tilgen, S. Seiter, M. Gore,

S. Aamdal, J. Cebon, A. Coates, B. Dreno, M. Henz, D. Schadendorf, A. Kapp,

J. Weiss, U. Fraass, P. Statkevich, M. Muller and N. Thatcher (2000).

"Randomized phase III study of temozolomide versus dacarbazine in the

treatment of patients with advanced metastatic malignant melanoma." J Clin

Oncol 18(1): 158-166.

Miller, A. J. and M. C. Mihm, Jr. (2006). "Melanoma." N Engl J Med 355(1): 51-65.

Mittrucker, H. W., A. Visekruna and M. Huber (2014). "Heterogeneity in the

Differentiation and Function of CD8 T Cells." Arch Immunol Ther Exp

(Warsz).

Mueller, S. N., T. Gebhardt, F. R. Carbone and W. R. Heath (2013). "Memory T cell

subsets, migration patterns, and tissue residence." Annu Rev Immunol 31: 137-

161.

References

98

Mueller, S. N., W. Heath, J. D. McLain, F. R. Carbone and C. M. Jones (2002).

"Characterization of two TCR transgenic mouse lines specific for herpes

simplex virus." Immunol Cell Biol 80(2): 156-163.

Obar, J. J. and L. Lefrancois (2010). "Early events governing memory CD8+ T-cell

differentiation." Int Immunol 22(8): 619-625.

Opferman, J. T., B. T. Ober and P. G. Ashton-Rickardt (1999). "Linear differentiation

of cytotoxic effectors into memory T lymphocytes." Science 283(5408): 1745-

1748.

Overwijk, W. W. and N. P. Restifo (2001). "B16 as a mouse model for human

melanoma." Curr Protoc Immunol Chapter 20: Unit 20 21.

Pauls, K., M. Schon, R. C. Kubitza, B. Homey, A. Wiesenborn, P. Lehmann, T.

Ruzicka, C. M. Parker and M. P. Schon (2001). "Role of integrin

alphaE(CD103)beta7 for tissue-specific epidermal localization of CD8+ T

lymphocytes." J Invest Dermatol 117(3): 569-575.

Peng, W., C. Liu, C. Xu, Y. Lou, J. Chen, Y. Yang, H. Yagita, W. W. Overwijk, G.

Lizee, L. Radvanyi and P. Hwu (2012). "PD-1 blockade enhances T-cell

migration to tumors by elevating IFN-gamma inducible chemokines." Cancer

Res 72(20): 5209-5218.

Perlis, C. and M. Herlyn (2004). "Recent advances in melanoma biology." Oncologist

9(2): 182-187.

Phan, G. Q., J. L. Messina, V. K. Sondak and J. S. Zager (2009). "Sentinel lymph node

biopsy for melanoma: indications and rationale." Cancer Control 16(3): 234-

239.

Phan, G. Q. and S. A. Rosenberg (2013). "Adoptive cell transfer for patients with

metastatic melanoma: the potential and promise of cancer immunotherapy."

Cancer Control 20(4): 289-297.

Phan, G. Q., J. C. Yang, R. M. Sherry, P. Hwu, S. L. Topalian, D. J. Schwartzentruber,

N. P. Restifo, L. R. Haworth, C. A. Seipp, L. J. Freezer, K. E. Morton, S. A.

Mavroukakis, P. H. Duray, S. M. Steinberg, J. P. Allison, T. A. Davis and S. A.

Rosenberg (2003). "Cancer regression and autoimmunity induced by cytotoxic

T lymphocyte-associated antigen 4 blockade in patients with metastatic

melanoma." Proc Natl Acad Sci USA 100(14): 8372-8377.

Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell and M. K. Jenkins (2001). "Visualizing

the generation of memory CD4 T cells in the whole body." Nature 410(6824):

101-105.

Rosenberg, S. A. (2014). "IL-2: the first effective immunotherapy for human cancer." J

Immunol 192(12): 5451-5458.

Rosenberg, S. A., J. J. Mule, P. J. Spiess, C. M. Reichert and S. L. Schwarz (1985).

"Regression of established pulmonary metastases and subcutaneous tumor

mediated by the systemic administration of high-dose recombinant interleukin

2." J Exp Med 161(5): 1169-1188.

References

99

Rosenberg, S. A., J. C. Yang, R. M. Sherry, U. S. Kammula, M. S. Hughes, G. Q. Phan,

D. E. Citrin, N. P. Restifo, P. F. Robbins, J. R. Wunderlich, K. E. Morton, C.

M. Laurencot, S. M. Steinberg, D. E. White and M. E. Dudley (2011).

"Durable complete responses in heavily pretreated patients with metastatic

melanoma using T-cell transfer immunotherapy." Clin Cancer Res 17(13):

4550-4557.

Rosenberg, S. A., J. R. Yannelli, J. C. Yang, S. L. Topalian, D. J. Schwartzentruber, J.

S. Weber, D. R. Parkinson, C. A. Seipp, J. H. Einhorn and D. E. White (1994).

"Treatment of patients with metastatic melanoma with autologous tumor-

infiltrating lymphocytes and interleukin 2." J Natl Cancer Inst 86(15): 1159-

1166.

Sallusto, F., J. Geginat and A. Lanzavecchia (2004). "Central memory and effector

memory T cell subsets: function, generation, and maintenance." Annu Rev

Immunol 22: 745-763.

Sallusto, F., D. Lenig, R. Forster, M. Lipp and A. Lanzavecchia (1999). "Two subsets of

memory T lymphocytes with distinct homing potentials and effector

functions." Nature 401(6754): 708-712.

Salopek, T. G., J. Slade, A. A. Marghoob, D. S. Rigel, A. W. Kopf, R. S. Bart and R. J.

Friedman (1995). "Management of cutaneous malignant melanoma by

dermatologists of the American Academy of Dermatology. I. Survey of biopsy

practices of pigmented lesions suspected as melanoma." J Am Acad Dermatol

33(3): 441-450.

Salter, R. D., R. J. Benjamin, P. K. Wesley, S. E. Buxton, T. P. Garrett, C. Clayberger,

A. M. Krensky, A. M. Norment, D. R. Littman and P. Parham (1990). "A

binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-

A2." Nature 345(6270): 41-46.

Samanta, A., B. Li, X. Song, K. Bembas, G. Zhang, M. Katsumata, S. J. Saouaf, Q.

Wang, W. W. Hancock, Y. Shen and M. I. Greene (2008). "TGF-beta and IL-6

signals modulate chromatin binding and promoter occupancy by acetylated

FOXP3." Proc Natl Acad Sci USA 105(37): 14023-14027.

Sanlorenzo, M., S. Ribero, S. Osella-Abate, D. Zugna, F. Marenco, G. Macripo, M. T.

Fierro, M. G. Bernengo and P. Quaglino (2014). "Prognostic differences across

sexes in melanoma patients: what has changed from the past?" Melanoma Res.

Schenkel, J. M., K. A. Fraser, L. K. Beura, K. E. Pauken, V. Vezys and D. Masopust

(2014). "T cell memory. Resident memory CD8 T cells trigger protective

innate and adaptive immune responses." Science 346(6205): 98-101.

Schenkel, J. M., K. A. Fraser and D. Masopust (2014). "Cutting edge: resident memory

CD8 T cells occupy frontline niches in secondary lymphoid organs." J

Immunol 192(7): 2961-2964.

Schon, M. P., A. Arya, E. A. Murphy, C. M. Adams, U. G. Strauch, W. W. Agace, J.

Marsal, J. P. Donohue, H. Her, D. R. Beier, S. Olson, L. Lefrancois, M. B.

Brenner, M. J. Grusby and C. M. Parker (1999). "Mucosal T lymphocyte

References

100

numbers are selectively reduced in integrin alpha E (CD103)-deficient mice." J

Immunol 162(11): 6641-6649.

Sciammas, R., P. Kodukula, Q. Tang, R. L. Hendricks and J. A. Bluestone (1997). "T

cell receptor-gamma/delta cells protect mice from herpes simplex virus type 1-

induced lethal encephalitis." J Exp Med 185(11): 1969-1975.

Seder, R. A., P. A. Darrah and M. Roederer (2008). "T-cell quality in memory and

protection: implications for vaccine design." Nat Rev Immunol 8(4): 247-258.

Seder, R. A. and W. E. Paul (1994). "Acquisition of lymphokine-producing phenotype

by CD4+ T cells." Annu Rev Immunol 12: 635-673.

Sepulveda, H., A. Cerwenka, T. Morgan and R. W. Dutton (1999). "CD28, IL-2-

independent costimulatory pathways for CD8 T lymphocyte activation." J

Immunol 163(3): 1133-1142.

Sheridan, B. S. and L. Lefrancois (2011). "Regional and mucosal memory T cells." Nat

Immunol 12(6): 485-491.

Shrikant, P. A., R. Rao, Q. Li, J. Kesterson, C. Eppolito, A. Mischo and P. Singhal

(2010). "Regulating functional cell fates in CD8 T cells." Immunol Res 46(1-

3): 12-22.

Simmons, A. and A. A. Nash (1984). "Zosteriform spread of herpes simplex virus as a

model of recrudescence and its use to investigate the role of immune cells in

prevention of recurrent disease." J Virol 52(3): 816-821.

Slominski, A., D. J. Tobin, S. Shibahara and J. Wortsman (2004). "Melanin

pigmentation in mammalian skin and its hormonal regulation." Physiol Rev

84(4): 1155-1228.

Sosman, J. A., K. B. Kim, L. Schuchter, R. Gonzalez, A. C. Pavlick, J. S. Weber, G. A.

McArthur, T. E. Hutson, S. J. Moschos, K. T. Flaherty, P. Hersey, R. Kefford,

D. Lawrence, I. Puzanov, K. D. Lewis, R. K. Amaravadi, B. Chmielowski, H.

J. Lawrence, Y. Shyr, F. Ye, J. Li, K. B. Nolop, R. J. Lee, A. K. Joe and A.

Ribas (2012). "Survival in BRAF V600-mutant advanced melanoma treated

with vemurafenib." N Engl J Med 366(8): 707-714.

Stritesky, G. L., S. C. Jameson and K. A. Hogquist (2012). "Selection of self-reactive T

cells in the thymus." Annu Rev Immunol 30: 95-114.

Takahama, Y. (2006). "Journey through the thymus: stromal guides for T-cell

development and selection." Nat Rev Immunol 6(2): 127-135.

Teijaro, J. R., D. Turner, Q. Pham, E. J. Wherry, L. Lefrancois and D. L. Farber (2011).

"Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal

protection to respiratory virus infection." J Immunol 187(11): 5510-5514.

Testori, A., P. Rutkowski, J. Marsden, L. Bastholt, V. Chiarion-Sileni, A. Hauschild and

A. M. Eggermont (2009). "Surgery and radiotherapy in the treatment of

cutaneous melanoma." Ann Oncol 20 (6): 22-29.

References

101

Topalian, S. L., F. S. Hodi, J. R. Brahmer, S. N. Gettinger, D. C. Smith, D. F.

McDermott, J. D. Powderly, R. D. Carvajal, J. A. Sosman, M. B. Atkins, P. D.

Leming, D. R. Spigel, S. J. Antonia, L. Horn, C. G. Drake, D. M. Pardoll, L.

Chen, W. H. Sharfman, R. A. Anders, J. M. Taube, T. L. McMiller, H. Xu, A.

J. Korman, M. Jure-Kunkel, S. Agrawal, D. McDonald, G. D. Kollia, A. Gupta,

J. M. Wigginton and M. Sznol (2012). "Safety, activity, and immune correlates

of anti-PD-1 antibody in cancer." N Engl J Med 366(26): 2443-2454.

Topalian, S. L., L. M. Muul, D. Solomon and S. A. Rosenberg (1987). "Expansion of

human tumor infiltrating lymphocytes for use in immunotherapy trials." J

Immunol Methods 102(1): 127-141.

Valyi-Nagy, I. T., G. Hirka, P. J. Jensen, I. M. Shih, I. Juhasz and M. Herlyn (1993).

"Undifferentiated keratinocytes control growth, morphology, and antigen

expression of normal melanocytes through cell-cell contact." Lab Invest 69(2):

152-159.

van Lint, A., M. Ayers, A. G. Brooks, R. M. Coles, W. R. Heath and F. R. Carbone

(2004). "Herpes simplex virus-specific CD8+ T cells can clear established lytic

infections from skin and nerves and can partially limit the early spread of virus

after cutaneous inoculation." J Immunol 172(1): 392-397.

van Lint, A. L., L. Kleinert, S. R. Clarke, A. Stock, W. R. Heath and F. R. Carbone

(2005). "Latent infection with herpes simplex virus is associated with ongoing

CD8+ T-cell stimulation by parenchymal cells within sensory ganglia." J Virol

79(23): 14843-14851.

Voehringer, D., C. Blaser, P. Brawand, D. H. Raulet, T. Hanke and H. Pircher (2001).

"Viral infections induce abundant numbers of senescent CD8 T cells." J

Immunol 167(9): 4838-4843.

Voehringer, D., M. Koschella and H. Pircher (2002). "Lack of proliferative capacity of

human effector and memory T cells expressing killer cell lectinlike receptor G1

(KLRG1)." Blood 100(10): 3698-3702.

von Andrian, U. H. and C. R. Mackay (2000). "T-cell function and migration. Two sides

of the same coin." N Engl J Med 343(14): 1020-1034.

Waithman, J., T. Gebhardt, G. M. Davey, W. R. Heath and F. R. Carbone (2008).

"Cutting edge: Enhanced IL-2 signaling can convert self-specific T cell

response from tolerance to autoimmunity." J Immunol 180(9): 5789-5793.

Wakim, L. M., A. Woodward-Davis and M. J. Bevan (2010). "Memory T cells

persisting within the brain after local infection show functional adaptations to

their tissue of residence." Proc Natl Acad Sci USA 107(42): 17872-17879.

Wang, X. B., C. Y. Zheng, R. Giscombe and A. K. Lefvert (2001). "Regulation of

surface and intracellular expression of CTLA-4 on human peripheral T cells."

Scand J Immunol 54(5): 453-458.

References

102

Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H.

von Andrian and R. Ahmed (2003). "Lineage relationship and protective

immunity of memory CD8 T cell subsets." Nat Immunol 4(3): 225-234.

Wolchok, J. D., H. Kluger, M. K. Callahan, M. A. Postow, N. A. Rizvi, A. M.

Lesokhin, N. H. Segal, C. E. Ariyan, R. A. Gordon, K. Reed, M. M. Burke, A.

Caldwell, S. A. Kronenberg, B. U. Agunwamba, X. Zhang, I. Lowy, H. D.

Inzunza, W. Feely, C. E. Horak, Q. Hong, A. J. Korman, J. M. Wigginton, A.

Gupta and M. Sznol (2013). "Nivolumab plus ipilimumab in advanced

melanoma." N Engl J Med 369(2): 122-133.

Wong, P. and E. G. Pamer (2003). "CD8 T cell responses to infectious pathogens."

Annu Rev Immunol 21: 29-70.

Woodland, D. L. and R. W. Dutton (2003). "Heterogeneity of CD4(+) and CD8(+) T

cells." Curr Opin Immunol 15(3): 336-342.

Ye, F., J. Turner and E. Flano (2012). "Contribution of pulmonary KLRG1(high) and

KLRG1(low) CD8 T cells to effector and memory responses during influenza

virus infection." J Immunol 189(11): 5206-5211.

Yee, C., J. A. Thompson, D. Byrd, S. R. Riddell, P. Roche, E. Celis and P. D.

Greenberg (2002). "Adoptive T cell therapy using antigen-specific CD8+ T cell

clones for the treatment of patients with metastatic melanoma: in vivo

persistence, migration, and antitumor effect of transferred T cells." Proc Natl

Acad Sci USA 99(25): 16168-16173.

Zhu, J., F. Hladik, A. Woodward, A. Klock, T. Peng, C. Johnston, M. Remington, A.

Magaret, D. M. Koelle, A. Wald and L. Corey (2009). "Persistence of HIV-1

receptor-positive cells after HSV-2 reactivation is a potential mechanism for

increased HIV-1 acquisition." Nat Med 15(8): 886-892.

The Role of Tissue-Resident Memory T Cells in Cutaneous … · 2016-07-07 · CTL Cytotoxic T...

Documents

Transcript of The Role of Tissue-Resident Memory T Cells in Cutaneous … · 2016-07-07 · CTL Cytotoxic T...