Multiple roles for CD4+ T cells in anti-tumor immune responses

Multiple roles for CD41 T cells inanti-tumor immune responses

Summary: Our understanding of the importance of CD41 T cells inorchestrating immune responses has grown dramatically over the pastdecade. This lymphocyte family consists of diverse subsets ranging frominterferon-g (IFN-g)-producing T-helper 1 (Th1) cells to transforminggrowth factor-b (TGF-b)-secreting T-regulatory cells, which have oppositeroles in modulating immune responses to pathogens, tumor cells, andself-antigens. This review briefly addresses the various T-cell subsetswithin the CD41 T-cell family and discusses recent research efforts aimedat elucidating the nature of the ‘T-cell help’ that has been shown to beessential for optimal immune function. Particular attention is paid to therole of Th cells in tumor immunotherapy. We review some of our ownwork in the field describing how CD41 Th cells can enhance anti-tumorcytotoxic T-lymphocyte (CTL) responses by enhancing clonal expansion atthe tumor site, preventing activation-induced cell death and functioning asantigen-presenting cells for CTLs to preferentially generate immunememory cells. These unconventional roles for Th lymphocytes, whichrequire direct cell-to-cell communication with CTLs, are clear examples ofhow versatile these immunoregulatory cells are.

Keywords: CD41 T lymphocyte, T-helper cells, tumor immunotherapy, cancer vaccines

CD41 T lymphocytes come in many colors and flavors

The CD4 cell surface marker has come to be associated with a

varied group of lymphocytes that orchestrate both innate and

adaptive immune responses to pathogens and tumors through

a variety of mechanisms. The prototypic member of this group

is the CD41 T-helper (Th) lymphocyte subset, which augments

both humoral and cellular immune responses (1, 2). Th cells

recognize antigen as peptide epitopes of approximately 12–20

residues long, presented by major histocompatibility complex

class II (MHC-II) molecules typically found on specialized

antigen-presenting cells (APCs) such as dendritic cells (DCs),

macrophages, and B cells (3). In some instances, Th cells can

directly recognize antigen on MHC-II-expressing tumor cells,

resulting in the production of lymphokines that hinder tumor

growth or inducing tumor cell death (4–6).

Naive CD41 Th lymphocytes develop in the thymus

following a controlled developmental path involving both

positive and negative selection to cull potentially autoreactive

cells from the repertoire while maintaining the ability to

Richard Kennedy

Esteban Celis

Immunological Reviews 2008

Vol. 222: 129–144

Printed in Singapore. All rights reserved

r 2008 The Authors

Journal compilation r 2008 Blackwell Munksgaard

Immunological Reviews0105-2896

Authors’ address

Richard Kennedy1, Esteban Celis2

1Mayo Vaccine Research Group, Mayo Clinic College of

Medicine, Rochester, MN, USA.2Immunology Program, H. Lee Moffitt Cancer Center &

Research institute, University of South Florida, Tampa, FL,

USA.

Correspondence to:

Esteban Celis

H. Lee Moffitt Cancer Center & Research Institute

University of South Florida

12902 Magnolia Drive, SRB-2

Tampa, FL 33612, USA

Tel.: 813 745 1925

Fax: 813 979 7262

e-mail: [email protected]

Acknowledgements

This work was supported in part by NIH grants

P50CA91956, R01CA80782 and R01CA103921

(to E. Celis).

129

recognize a broad range of pathogen-associated peptides

presented by self MHC-II molecules. During an immune

response, recognition of the cognate antigen presented on the

surface of an APC by the T-cell receptor for antigen (TCR)

(Signal 1) along with interaction between appropriate

costimulatory molecules such as the CD28 co-receptor with

CD80/CD86 (Signal 2) initiates activation of the naive CD41 T

cell. These activated T cells undergo a phase of robust clonal

expansion and differentiation into either effector or memory

cells. CD41 memory Th cells can be classified into two main

groups based on cell surface markers and functional capacities.

Central memory Th cells (ThCM) express high levels of CCR7

and CD62L, lack CD45RA, and traffic through the lymphoid

organs (7–9). Effector memory T cells (ThEM) are CCR7

negative and reside mostly in the blood, spleen, and in

non-lymphoid tissues (10). Long-term survival of memory Th

cells relies on the participation of costimulatory molecules

(OX40/OX40L) and the availability cytokines such as

interleukin-7 (IL-7) (11–13).

The fate and function of the activated Th cells depends in

large part upon the microenvironment present at the time of

the initial antigen encounter. The composition of the local

cytokine milieu will bias development toward one of several

alternative differentiation pathways. Likewise, the nature of the

antigen acquired by DCs will affect the expression of different

sets of costimulatory molecules, which will also dictate the

developmental path of the antigen-stimulated Th cells (14).

This additional polarizing costimulation has been termed

‘Signal 3’ and is initiated by various innate pathogen-

associated molecular pattern receptors triggered by the

various antigens (15–18). For example, DC exposure to

intracellular pathogens programs these APCs to promote

Th1-type responses, whereas exposure to helminthes drives

DCs to promote Th2 development. A similar situation may exist

for the other various regulatory subsets of Th cells (19).

CD41 T lymphocytes can be grouped into different functional

subsets based on function and cytokine secretion patterns (Fig.

1). Originally, CD41 T cells were simply classified as Type 1

effector Th cells (Th1) that produce high levels of interferon-g(IFN-g) and tumor necrosis factor-a (TNF-a) upon antigen

stimulation and being responsible for regulating delayed type

hypersensitivity (DTH) reactions and cell-mediated immunity to

intracellular pathogens and tumor cells. The Th1 developmental

pathway is driven by IL-12 activation of signal transducer and

activator of transcription 4 (Stat4) and T-bet during immune

activation of naive T cells (20). Alternatively, Th2 are

characterized by the production IL-4, IL-5, and IL-13 and are

responsible for coordinating humoral immunity, eosinophilic

inflammation, and controlling helminthic infections. IL-4 is

primarily accountable for the differentiation of Th2 cells

through Stat6 and GATA (21). The Th1 and Th2 developmental

pathways are controlled by a delicate balance of positive

feedback loops, as IFN-g enhances further Th1 development

and IL-4 supports continued Th2 differentiation. At the same

time, cross-regulation by IFN-g and IL-4 suppresses Th2 and

Th1 differentiation, respectively.

In addition to Th1 and Th2 cells, several other subsets of

CD41 T cells participate in the development of immune

responses. In many instances, these cells act to control/

suppress immune responses and play an important role in the

prevention of autoimmune diseases. The best-studied group is

the naturally occurring CD41CD251 T-regulatory cells (Tregs)

(22–24). Approximately 5–6% of the CD41 T cells exiting

from the thymus express high levels of CD25, glucocorticoid-

induced TNF receptor (GITR), and the transcription factor

forkhead box protein 3 (Foxp3) (25–27). These Tregs mediate

Fig. 1. Diversity of CD4-expressing cell subsets. The naturally occur-ring Tregs and NKT cells both develop in an antigen-independent fashionand exit the thymus fully functional. The remaining CD41 T-cell subsetsdevelop from naive CD41 T cells after antigen-dependent T-cell activa-tion. The microenvironment present during priming, including antigendose, APC type, cytokines, and costimulatory signals, all influence thedevelopmental pathway taken by the responding T cell. The majorcytokines and secreted factors contributing to the respective functions ofthe Th subsets are listed to the right of each cell type.

130 Immunological Reviews 222/2008

Kennedy & Celis � CD41 T cells modulate tumor immunity

immune suppression through a cell-to-cell contact-dependent

mechanism that does not require antigenic stimulation (28).

While important for the prevention of autoimmunity, in some

circumstances Tregs hinder desirable immune responses, for

example against tumor-associated antigens. Depletion of this

subset in vivo, for example with anti-CD25 monoclonal

antibodies, enhances anti-tumor immunity in mice (29–32),

specially when the targeted tumor antigens are expressed to

some extent by normal cells (e.g. tissue differentiation antigens

or products of overexpressed genes). Antigen-experienced

CD41 T cells can also develop into Tregs that express CD25,

Foxp3, and GITR. Although the origins of these adaptively

induced Tregs is unclear, they have similar immune suppressive

effects as their naturally occurring counterparts. Other

populations of CD41 T cells that develop into functionally

active Tregs after antigen stimulation include Tr1 cells, which

produce large quantities of IL-10, and Th3 cells, which secrete

copious amounts of transforming growth factor-b (TGF-b)

(33). While Tr1 and Th3 cells utilize both secreted factors and

cell-to-cell contact to suppress immune responses, defining

specific markers for these CD41 T-cell subsets has been a

challenge (34).

Another subset of regulatory CD41 T cells called Th17 has

been described recently (35). The evidence suggests that Th17

cells develop independently from either Th1 or Th2 cells and

represent a distinct lineage (36). Th7 cells produce IL-17A,

IL-17F, TNF-a and IL-6 and coordinate tissue inflammation

and autoimmunity (37, 38). Th17 differentiation is thought to

require TGF-b and IL-6 (39). In addition, IL-23 (and perhaps

IL-1) serves as a growth/maintenance factor to this population.

Yet another recent addition to the regulatory CD41 T-cell

family is the ThFH subset (33). ThFH cells express CD200,

Bcl-6, CD84, and CXCR5 and migrate to the B-cell follicles after

activation (40). These cells are thought to play a role in both

antibody responses and autoimmunity; however, a definitive

function and lineage have yet to be determined (41). Last but

not least are the type-II natural killer T (NKT) cells, which

express the CD4 marker, have a limited TCR repertoire, and

recognize lipid epitopes presented by the CD1d molecule

(42–44). Type-II NKT cells secrete IL-4 and its related

cytokine IL-13, which induce myeloid derived suppressor

cells to secrete the inhibitory cytokine TGF-b. Defects in

Type-II NKT cell function result in overactive Th1 responses

and increased autoimmune pathology (45–47). In addition,

type-II NKT cells can suppress tumor immunosurveillance

through multiple mechanisms (48–50). Directed modulation

of type-II NKT cell activity is a promising avenue of increasing

tumor immunotherapy (51–53).

CD41 T cells comprise a large and growing body of distinct

cell subsets that carry out specialized immunoregulatory

functions to either enhance or inhibit immune responses. The

delicate balance of CD4-expressing lymphocytes ensures the

development of appropriate and protective immune responses

while limiting potentially damaging autoimmune disorders.

Conventional roles of Th cells in cytotoxicT-lymphocyte responses

The original concept of T-cell ‘help’ originated in the 1970s

with the description of the ‘carrier effect,’ where it was

determined that B-cell activation leading to the production of

high affinity antibody responses required two signals (54). The

first signal was provided by antigen recognition by the B cells

through cell surface immunoglobulin (Ig) receptors for antigen,

and the second signal came from interactions of the B cells

with CD41 Th cells reactive toward the same or a physically

linked (carrier) antigenic determinant. A similar helper effect

for CD81 cytotoxic T-lymphocyte (CTL) responses was

described one decade later (1). Initially thought of as primarily

cytokine producers, the role of Th cells in providing help for B

cells and CTLs is now recognized as being more complex,

involving a myriad of mechanisms. Cellular immunity against

tumors initially focused on CTLs, but in recent years it became

evident that CD41 T cells also play a critical role in the

development of effective anti-tumor immunity. Next we

describe some of the conventional mechanisms of T-cell help

that have been reported in the literature, focusing on the role of

CD41 T cells for CTL responses. In addition, we also address

some of the unconventional roles that CD41 T cells may play in

regulating CTL responses.

Before regulating anti-tumor CTL responses, both Th1 and

Th2 subsets can activate innate immune mechanisms that may

have activity against tumor cells. IFN-g production by Th1 cells

stimulates production of reactive oxygen/nitrogen species by

macrophages, which have been implicated in tumor cell

destruction. Th2 cells recruit eosinophils and stimulate

production of eosinophil cationic protein (ECP) and major

basic protein (MBP) (55). Th cells also recruit macrophages,

granulocytes, and NK cells to the tumor site (56–58). Anti-

tumor responses mediated by NK cells have also shown a

crucial need for CD41 T-cell help (59). While Th cells can have

direct anti-tumor effects through secretion of cytokines such as

TNF-a or by directly exerting tumor cell death through the

TNF-related apoptosis-inducing ligand (TRAIL) or Fas/Fas

ligand (FasL) pathways (60, 61), their major contribution for

anti-tumor effects is thought to be by providing the required

Immunological Reviews 222/2008 131


T-cell help for generating and augmenting tumor-specific CTL

responses. T-cell help can be directly provided to the CTL and/

or indirectly through accessory cells (APCs, NK cells) and can

have a profound influence not only on primary CTL responses

but also CD81 memory T-cell formation.

One major form of T-cell help comes from the transient

expression of CD40L on activated Th cells, which interacts with

CD40 on the surface of DCs, activating these APCs, enhancing

the expression of both MHC and costimulatory molecules, as

well as stimulating the production of cytokines (e.g. IL-12) that

are indispensable for CTL responses (62–64). A more direct

type of help for CTLs comes in the form of cytokines such as

IL-2 that function as growth factors for CTLs. Recently the

presence of IL-2 during priming has been shown to be

essential for the secondary expansion of memory CD81 T cells

(65). IL-2 secretion by Th cells can also function to recruit and

retain CTLs at the tumor site. IFN-g production by CD41 Th1

cells results in the upregulation of MHC molecules on tumor

cells leading to enhanced T cell (CTL and Th) recognition.

Various model systems show differing requirements for

CD41 Th cells during primary CTL responses have led to

the classification of CTL responses as Th-dependent or

Th-independent responses (66–68). It is possible that strong

CTL epitopes (e.g. high MHC-binding peptides from foreign

antigens) may induce CTLs capable of producing their own

IL-2, while weak epitopes (e.g. low MHC-binding peptides

from tumor-associated antigens) will require local IL-2

production by Th cells. Other costimulatory signals may play a

role in determining whether or not primary CTL responses will

require direct T-cell help, as high levels of CD70 expression on

DCs has been shown to circumvent the need for CD41 T cells.

However, the expression of CD70 on DCs is the result of CD40

crosslinking by Th cells (69).

In addition to their role in supporting optimal primary CTL

responses, a number of studies have shown a critical need for

Th cells in the generation and maintenance of memory CD81 T

cells, a requirement which extends to both Th-dependent and

Th-independent CTL responses (67, 70–73). It has also been

shown that the CD81 T cells that acquire a memory-like

phenotype during homeostatic division are also dependent

upon the presence of CD41 Th cells (74). Other in vitro studies

have shown, under some conditions such as with the provision

of agonistic anti-CD40 antibodies or Toll-like receptor (TLR)

stimulation, that it is possible to circumvent the need for T-cell

help to generate memory CTL responses (62, 64, 69, 75, 76).

However, it is not clear what the exact role of the Th cell is in

allowing the persistence of memory CTLs once these cells have

been generated. The emerging picture is that CD41 T cells

provide conditioning signals (direct and indirect via APCs) to

naive CD81 T cells during their initial activation. Without these

signals, CD81 memory T-cell differentiation is impaired (70).

In support of this hypothesis, it has been shown that Th cells

regulate the expression of TRAIL on activated CTL. ‘Helped’

CD81 T cells are less susceptible to activation-induced cell

death (AICD) upon secondary stimulation than their ‘helpless’

counterparts (77), which may help to explain part of the role

of CD41 T cells in generating and maintaining CD81 T-cell

memory (78).

Role of Th cells during the effector phase of theimmune response

The longevity of T-cell responses during viral infections or anti-

tumor immunity is a crucial factor in determining whether a

virus or a tumor will be eliminated. Given the terminally

differentiated nature of effector CTLs and their susceptibility to

AICD and antigen-induced non-responsiveness (AINR), it

would seem that CTL responses must race against the clock to

eliminate the pathogen or tumor before natural regulatory

mechanisms curb the response and CTL clonal contraction

ensues (79–81). Typically, antigen-specific CTLs are difficult to

maintain for long periods of time, even with appropriate APCs

and growth factors such as IL-2. However, under certain

conditions in vitro, it has been possible to expand and maintain

CTLs for extended periods of time (82–84). The massive CTL

expansion (approximately 1000-fold) in tissue cultures sup-

plemented with IL-2, TCR stimulation, and feeder cells, known

as the rapid expansion method (REM), seems at odds with the

multiple regulatory mechanisms designed to check CTL expan-

sion in vivo. Notably, depletion of CD41 T cells from the feeder

cell population in the REM cultures decreased both the expan-

sion and lytic activity of the CTLs by approximately one half

(85). The CTL expanding activity of the CD41 T cells required

direct cell-to-cell contact between CTLs and Th cells and could

not be substituted simply by providing CD41 T-cell-condi-

tioned media (85). Given that CTLs and Th cells express a large

variety of costimulatory receptors and their corresponding

ligands, these molecules became attractive targets to examine

the mechanism of this effect. For example, CD28 is expressed

on most naive and early-activated T cells, while CD80 and

CD86 have been found on activated Th cells (86, 87). A similar

situation exists with several other costimulatory molecules

including the following: CD2, CD27, CD30, 4-1BB, and

cytotoxic T-lymphocyte antigen-4 (CTLA-4). Activated, human

CTLs express MHC-II, and crosslinking of this molecule on

CTLs provides a costimulatory signal to the T cell (88–91).



Thus, direct interaction between MHC-II on the CTL and the Th

cell TCR could be another possible source of costimulation for

CTLs. The involvement of several molecules was first tested

using plate-bound antibodies in order to crosslink the costi-

mulatory CTL receptors, and the results showed that activation

of CD27, CD137, and MHC-II could significantly enhance the

proliferative response of CTLs to TCR stimulation (85). Further

confirmation of the role of these molecules in the Th cell-

derived proliferation signal was provided by the use of soluble

blocking antibodies to these molecules, which abrogated the

enhancement of CTL proliferation by Th cells (85). This

additional type of T-cell help may help to explain the fact that

many CTL responses are short lived and are unable to contain

chronic infections and tumor growth in the absence of Th cells

(92, 93). Although this form of T-cell help may occur in the

lymph node during the initial activation and expansion of

naive Th cells and CTLs, it is probably most effective at the

tumor or infection site between previously activated CTLs and

Th cells. One may envision the following situation: (i) antigen-

specific Th cells are stimulated at the tumor site by resident

APCs expressing tumor antigen (peptides derived from tumor

antigens presented in the context of MHC-II), alternatively the

Th cells may recognize tumor antigens presented by MHC-II-

expressing tumor cells; and (ii) the activated Th cells expressing

costimulatory molecules and secreting lymphokines would

provide local or direct growth and survival signals to the

tumor-specific CTLs, allowing them to expand and/or avoid

AICD. Thus, this type of T-cell help bolsters CTL responses

during the effector phase of the immune response, allowing it

to be better able to eliminate tumors and infectious agents.

Requirement for CD41 T cells in anti-tumorpeptide vaccines

Another line of research in our laboratory involves examining

the different roles that CD41 and CD81 T lymphocytes play in

immune responses to tumors following peptide vaccination.

One tumor model for these experiments is the B16 mouse

melanoma, a poorly immunogenic tumor, which, like many

of its human counterparts, expresses the tumor-associated

antigen tyrosinase-related protein-2 (TRP-2). The vaccination

protocol consisted of a peptide representing the CTL epitope

TRP-2188–188 administered in incomplete Freund’s adjuvant (IFA)

along with immunostimulatory CpG-containing oligodeoxy-

nucleotides (a TLR9 agonist) and anti-CTLA-4 blockade as

adjuncts. Vaccination of the mice prophylactically (before

tumor challenge) or therapeutically against the B16 melanoma

resulted in equally robust CTL responses; however, the

prophylactic vaccine had no effect on animal survival after the

lethal tumor challenge. In contrast, therapeutic administration

of the vaccine significantly delayed tumor growth, and double

vaccination (administered both before and after challenge)

resulted in significant protection against the tumors, where

80% of mice survived (94). This survival advantage conferred

by the double vaccine administration was abrogated in CD81

or CD41 T-cell deficient mice, indicating that both cell types

were required for protective immunity. While mice vaccinated

prophylactically or therapeutically exhibited CD81 T-cell

responses of similar magnitude shortly after tumor challenge,

the CTL responses steadily decreased in mice immunized

before tumor challenge, while animals vaccinated after tumor

implantation maintained high levels of CTL activity. This CTL

activity correlated with the presence of tumor-specific Th

responses in the animals. These results indicated that antigen-

specific Th cells were not necessary for the induction of the

CD81 T-cell response but in turn facilitated the persistence of

CTL effector function in the tumor-bearing mice.

The role of Th cells in maintaining CTL responses

In other studies, we utilized the B16-ovalbumin (OVA) mela-

noma line expressing OVA as a surrogate tumor-specific anti-

gen to further address the role of T-cell help in the efficacy of

peptide-based vaccines for tumor immunotherapy (95). Simi-

lar to other reports showing that CD41 T-cell help was

dispensable for primary CTL responses (67, 96, 97), we found

that peptide OVA257–263 (or SIINFEKL, the immunodominant

CTL epitope of OVA) immunization could induce robust,

protective CTL responses that were able to clear tumors in

60% of lethally challenged mice. In this model system, we

investigated the effect of supplementing the CTL peptide

vaccine with MHC-II-restricted peptides representing Th epi-

topes. In one vaccine type, we utilized a Th epitope expressed

by the tumor model antigen (OVA323–339), while in the other

vaccine type contained an irrelevant Th epitope [Pan-DR

epitope (PADRE)] that was not expressed by the tumor. While

the addition of either one of the Th peptide epitopes to the

vaccine led to greater CTL activity, only the use of Th peptide

epitope expressed by the tumor (OVA323–339) led to an

increased effect on survival. In these studies, we found that Th

cells had no direct activity against B16-OVA, nor did immuni-

zation with the Th epitopes alone have a significant effect on

either tumor growth or mouse survival. Interestingly, delaying

tumor challenge by as little as 2 weeks after vaccination

decreased survival in the group receiving CTL peptide alone

from 60% to 20%, indicating that in the absence of Th cell



stimulation the CTL responses were short lived. Given the

similarities in the levels of detectable CTL of the mice from

each vaccine group and given the fact that non-specific

(irrelevant) T-cell help had no beneficial effect on survival

against the tumor, we hypothesized that tumor-specific CD41

Th cells were providing ‘help’ directly at the tumor site,

perhaps by increasing CTL survival during the effector phase

of the immune response. To test this possibility, we developed

an in vitro model to measure antigen-specific CTL AICD using

peptide/MHC monomers to activate the TCR. This model

system allowed us to test the effects that the presence of Th

cells would have on CTL AICD when previously activated CTLs

reencountered antigen. Our results clearly showed a significant

decrease in CTL death (approximately 50% reduction) in the

presence of Th cells (95). Further experimentation showed that

protection was not associated with decreased CTL activation (in

fact ‘protected’ CTLs had significantly higher lytic activity) and

that the protective effect required direct cell-to-cell contact

between Th cells and CTLs. Although the exact mechanism of

this protection remains to be determined, we observed that Th

cells and CTLs interact with each other via CD2/CD48 associa-

tions, leading to lipid raft aggregation and recruitment of the

CTL TCR into the lipid rafts, which could optimize the signals

received by the CTL through its TCR upon interaction with the

antigen-expressing tumor cell. Under normal circumstances,

CTL-target interactions lead to two signals in the CTL, one

signal to release perforin/granzymes onto the target cell and a

second signal leading to a self-death inducing stimulus. The

bottom line is that in many instances, CTLs are only able to kill

a limited number of target cells before they succumb to AICD.

Nevertheless, in the presence of Th cells, CTLs receive a

protective stimulus that increases resistance to AICD and allows

survival and continued killing. We postulate that these ‘helped’

CTLs would not only be better able to clear infection/tumor

but may also be more likely to contribute to the CD81 T-cell

memory pool (Fig. 2). Decreased CTL susceptibility to AICD

may account for the greater protection seen in our experi-

mental tumor model when mice were primed with both CTLs

and Th epitopes (95) as well as in the rapid CTL expansion

experiments (85). The anti-apoptotic effect conferred by

Th cells may also be at work in viral infections and in

autoimmunity. In some viral infections, CTLs expand and

transiently control infection, but the CTL response is eventually

lost and viremia remains uncontrolled in a chronic state. It is

possible that inappropriate Th responses could be responsible

for the lack of a robust, long-lasting CTL response needed for

the elimination of the viral infection. In the male antigen (HY)

model system, it has been shown that the survival of TCR

transgenic CD81 T cells is increased with concurrent CD41 T-

cell stimulation (98). A similar result has been observed in the

OVA system using the rat insulin promoter (RIP)-OVA mice,

which express OVA in the pancreatic islet cells (99). OVA-specific

TCR transgenic CTL (OT-1) T cells, transferred into RIP-OVA

mice in large numbers, expand, become activated, and destroy

the b pancreatic islet cells and induce diabetes. However, low

numbers of OT-1 cells expand briefly in the draining lymph

node and are subsequently deleted before islet destruction

can occur. Concurrent transfer of CD4, OVA-specific Th cells

(OT-II) reduced the number of OT-1 cells required for induc-

tion of diabetes by preventing the deletion of OT-1 cells in the

RIP-OVA animals (99). While the importance of CD41 Th cells

in CTL responses is well recognized, not many mechanisms

have been put forth to explain their dramatic effects on CTL

responses during the effector phase of the immune response.

By providing survival signals to CTLs, Th cells block AICD and

increase the expansion of CTLs and their functional lifespan. By

allowing more CTLs to escape deletion, Th cells will also

promote greater CTL memory generation.

Fig. 2. Th cell-mediated protection of CTLs from AICD. (A) Uponsecondary encounter with APCs, CTLs release cytotoxic components(perforin, granzyme B) towards the target cell and in turn receive signalsthrough death receptors (FasL, TRAIL, TNFR), which initiate apoptoticsignaling cascades within the CTL. (B) The close proximity of Th cellsallows for costimulatory signals that reduce CTL susceptibility to AICDand may also increase proliferative capacity. This protective effect may bemediated by direct anti-apoptotic signals or a qualitative change insignaling through the TCR. Protected CTLs exhibit enhanced effectorfunction.



Th cells as APCs?

The interaction between Th cells and CTLs leading to increased

proliferation (85) and reduced AICD (95) requires cell-to-cell

contact and it is mediated through the binding of costimula-

tory molecules with their respective ligands and perhaps also

via Th cell-derived cytokines. One can easily envision that cell-

to-cell communication between Th cells and CTLs either in

secondary lymphoid organs or at the tumor site would be more

efficient if these cells interacted with each other in an antigen-

specific manner, for example by Th cells presenting MHC-I/

peptide complexes to the CTLs. Nevertheless, it is not so easy to

imagine how Th cells would gain access to antigens and process

them into MHC-I-binding peptides, because these cells are not

considered professional APCs. In the early 1980s, several

reports indicated that murine T cells in mixed lymphocyte

cultures acquired MHC molecules from the professional APCs

during the activation process (100–105). More recent reports

have confirmed this phenomenon and have shown that T cells

can capture cell surface proteins including MHC products from

APCs in a TCR-dependent manner (106–115). In addition to

MHC-I and II interchanges, a variety of other costimulatory

(B7.1 and B7.2) and adhesion (intercellular adhesion

molecule-1) molecules (108, 116, 117) or even extensive

membrane fragments can be acquired by the T cells (110). Most

significantly, not only are these molecules transferred to T cells

but also they are functional on the T-cell surface (106, 109, 110,

112, 114, 115, 118, 119). To study this phenomenon, our

laboratory utilized CD81 T cells from patients with bare lym-

phocyte syndrome, which lack surface expression of MHC-II due

to a mutation in the RFX5 gene. Our results show that while cell-

to-cell contact between the T cell and APC resulted in optimal

transfer of MHC-II, there was also a low level of cell surface

material transferred by exosomes shed from the APCs (120). The

transfer of MHC-II from APCs to MHC-II-deficient T cells was

mediated by various molecule pairs including CD8/MHC-I and

CD2/CD58. Interestingly, depletion of cholesterol from the

plasma membrane of the T cells resulted in a significant reduction

in the amount of acquired MHC-II, indicating that the choles-

terol-rich lipid rafts may play a vital role in the transfer

mechanism. Thus, these observations indicate that one

mechanism for Th cells to generate MHC-I/peptide complexes

to be presented to CTLs would be simply to grab them from

APCs during the antigen recognition process.

Another mechanism by which Th cells may gain access to

MHC-I-binding peptides could be when the Th cell’s TCR

interacts with MHC-II/peptide complexes, when the particular

peptide also includes an adjoining CTL epitope. Supporting this

possibility is the fact that CTLs and Th cell epitopes are frequently

found in close proximity or even overlapping one another (Table

1). In most of the cases presented in Table 1, the peptides were

found to stimulate both MHC-I and II T-cell responses in the

same individual. Furthermore, the CTL epitopes situated

within or near Th cell epitopes tend to be the strongest

immunodominant epitopes. With this information on hand, we

have proposed a model where the Th cell, while interacting with

an APC, would retain and internalize the MHC-II/peptide

complex and that somehow, through limited antigen processing

in the endosomal compartments, the CTL epitope contained

within the MHC-II-binding peptide, would be generated. The

CTL peptide epitope would bind to those MHC-I molecules that

recirculate through the early endosomal compartment.

Supporting this model, there are a number of reports showing

that MHC-I molecules recycle through the endosomal

compartment are able to efficiently pick up new peptides,

including some derived from degraded MHC-II molecules, and

carry them to the cell surface, a process that is facilitated by the

low pH (pH approximately 5.0) of the early endosomal

compartment (121–125). It has been suggested that this

exogenous loading pathway amplifies the repertoire of peptides

that can be presented by MHC-I molecules. In the case of MHC-II

epitopes that overlap or are linked with CTL epitopes, the MHC-I

binding peptide would have to be generated by proteases

residing in the endosomes. It should be noted that both the

acquired MHC-II/peptide complexes and the endogenous MHC

class I molecules are present in an environment well suited to

MHC-I peptide loading (124, 126), so it is not far fetched to

propose that the antigenic peptides contained by the acquired

peptide/MHC-II complexes can be processed and loaded onto

the recycling, endogenous MHC-I molecules and be transported

to the cell surface to become accessible for CTL presentation. In

support of this theory, it has been shown that proteins targeted to

the surface of CD41 T cells, such as antibodies or viral proteins,

are internalized, processed, and able to generate MHC-I and II-

binding peptides that find their way back onto the cell surface.

Subsequently, both MHC-I and II epitopes from these proteins

are presented by the CD41 T cells to other T lymphocytes

(127–130). This is an attractive situation from an immunologic

standpoint, as it would provide not only antigen stimulation but

also immediate T-cell help through both direct costimulation and

cytokine secretion to the CTLs.

To study some of the possible mechanisms by which MHC-I/

peptide complexes that are specific for nearby CTLs can be

generated by Th cells, we developed a model system that

allowed us to modify the MHC-I molecules on both the APC

and/or the Th cells. The multiple mechanisms of antigen



acquisition described below are depicted in Fig. 3. This model

system utilized the 25.D1.16 monoclonal antibody (mAb) that

recognizes the H-2Kb/SIINFEKL complex (131) and the OT-1,

OT-II, and DO11.10 TCR transgenic mice whose T cells

recognize two OVA peptides, OVA257–263 (restricted by

H-2Kb) and OVA323–339 (restricted by I-Ab or I-Ad). The first

scenario explored the simple shedding of CTL peptide epitopes

from inside the APC or from the APC surface, which would

simply bind to cell surface MHC-I on the Th cell (Fig. 3, #1).

A second mechanism would be the above-described acquisition

of MHC-I molecules from the APC (Fig. 3, #2). For these

experiments, we used the LB27.4 cell line, which expresses

MHC-I and II from the H-2b and H-2d haplotypes, as the APCs.

CD41 Th cells specific for the OVA323–339 were obtained from

either DO11.10 (H-2d) or OT-II (H-2b). The use of antigen-

experienced, pre-activated Th cell lines showed that the Th cell

could directly pick up MHC-I/peptide complexes from the APC

and that the transfer of these molecules did not require the

presence of the Th-specific antigen (Fig. 4A). When resting

(naive) Th cells were used, antigen presentation via MHC-II/

OVA323–339 by the APC was required for MHC-I acquisition to

take place. A third mechanism involves the acquisition of MHC

I/peptide complexes from the APC followed by the transfer of

the epitope to the self (endogenous) Th MHC-I molecules (Fig.

3, #3). To distinguish between the acquired MHC-I and the

endogenous MHC-I of the Th cell, we utilized several mutants

Table 1. Proximal T-helper and CTL epitopes

Antigen

MHC-II (Th) MHC-I (CTL)

Epitope Restriction Epitope Restriction Reference

Tumor antigensCEA 653–667 HLA-DR4, 7, 9 652–660 HLA-A24 (152)HTLV-1 env 196–210 HLA-DR9 175–183 HLA-A2 (4)HTLV-1 env 196–210 HLA-DR9 182–190 HLA-A2 (4)HTLV-1 env 384–398 HLA-DR15 395–403 HLA-A2 (4)HTLV-1 tax 191–205 DR1, DR9 181–195 HLA-B14 (5)HTLV-1 tax 305–319 DR15, DQ9 301–309 HLA-A24 (5)Melanoma gp100 175–189 HLA-DR53, DQ6 177–186 HLA-A2 (152)NY-ESO-1 87–98 Promiscuous, HLA-DR7 80–88 HLA-Cw6 (153, 154)NY-ESO-1 87–98 Promiscuous, HLA-DR7 92–100 HLA-Cw3 (153, 154)NY-ESO-1 87–98 Promiscuous, HLA-DR7 84–102 HLA-B51 (153, 154)NY-ESO-1 80–109 Promiscuous, HLA-DR7 80–88 HLA-Cw6 (153, 154)NY-ESO-1 80–109 Promiscuous, HLA-DR7 92–100 HLA-Cw3 (153, 154)NY-ESO-1 80–109 Promiscuous, HLA-DR7 84–102 HLA-B51 (153, 154)P1A 33–44 I-Ad 35–43 Ld (155, 156)p53 234–242 H-2d MHC II 234–242 Kd (157)Ras oncogene 4–16 I-Ad 4–12 Kd (158, 159)WT1 124–138 HLA-DR53 126–134 HLA-A2 (6)WT1 247–261 HLA-DR53 235–243 HLA-A24 (6)

AutoantigensGAD 206–220 I-Ag7 206–214 Kd (160)GAD 509–527 I-Ag7 505–513 Kd (160)GAD 524–543 I-Ag7 546–554 Kd (160)Insulin 9–23 I-Ag7 15–23 Kd (161, 162)

Viral antigensHBV env 10–19 HLA-DQ5 10–17 HLA-A11 (163)HBV env 182–191 HLA-DPw3 183–191 HLA-A2 (163)HBV env 182–196 HLA-DR2w15, DPw4 183–191 HLA-A2 (163)HIV-1 gp160 315–327 I-Ad 318–327 Dd (164)HIV-1 RT 36–52 H-2k MHC II 38–52 H-2k (165)Influenza B NP 335–349 HLA-DQw5 335–349 HLA-B37 (166)Rubella Capsid 263–275 HLA-DR 264–272 HLA-A3, 11 (167)Vaccinia A18R 49–63 I-Ab 57–64 Kb (168, 169)

Other antigensHEL 91–105 I-Ag7 91–99 Kd (160)HEL 106–116 I-Ed 116–124 Kd (160)OVA 265–280 I-Ab 257–264 Kb (170)

The first column indicates the type of antigen and source of the defined T-cell epitopes. The location of the MHC-II epitope within the protein and itsrestriction element are listed in columns 2 and 3. Columns 4 and 5 contain the same information for the proximal or overlapping MHC-I epitopes.Column 6 contains the bibliographical reference.MHC, major histocompatibility complex.



of the Kb molecule. The mutant Kbm3 also binds the OVA257–263

epitope, and this complex is also recognized by the 25.D1-16

Ab. An additional mAb, B8-24-3, binds to Kb but not Kbm3,

allowing for distinguishing Kb/OVA257–263 from Kbm3/

OVA257–263 complexes. The results showed that incubation of

activated OT-II Th cells with OVA257–263-pulsed Kbm3 APCs

resulted in the surface expression of both Kb/OVA257–263 and

Kbm3/OVA257–263 complexes on the Th cell surface. This effect

was enhanced in the presence of the Th-specific MHC-II

peptide OVA323� 339. Resting (naive) OT-II Th cells, however,

required the presence of their cognate MHC-II peptide

OVA323–339 to produce MHC-I/OVA257–263 complexes. More

importantly, the majority of the OVA257–263 on the naive Th

cells was presented in the context of the Th cell endogenous Kb,

not with the acquired Kbm3 from the APC. These results

demonstrated that not only could CD41 Th cells acquire MHC

I/peptide complexes from a nearby APC but they could also

re-present the acquired peptide on their own MHC-I molecules.

As previously mentioned, a fourth mechanism of antigen

transfer from APC to Th cells involves the presence of Th cell

epitopes that contain within their sequence CTL epitopes (Fig.

3, #4). To test this possibility, APCs loaded with linked

peptides containing both MHC-I and II epitopes were

co-incubated with antigen-specific Th cells. After a short

incubation, MHC-I/peptide complexes could be detected on

the surface of the Th cell, indicating that after recognition of

the MHC class II-peptide complex by the Th cell, the complex

was internalized, the full length peptide was processed, and the

MHC-I binding fragment was loaded onto the recirculating

MHC-I molecules (118, 119, 126) (Fig. 4B). Overall, these

experiments indicate that Th cells possess sophisticated

antigen-processing capabilities similar to other professional

APCs, such as cross-presentation. In some ways this process is

similar to B-cell internalization/processing of antigen through

the B-cell receptor for antigen (surface Ig) (132, 133).

Our work also reinforced a consistent theme: the need for Th

cell activation before the antigen acquisition can occur. This

requirement for TCR involvement would limit MHC and

antigen uptake to the CD41 T cells that are being activated in

response to a pathogen or tumor-derived antigens. It is evident

that the APC presenting MHC-II epitopes will also possess

MHC-I epitopes from said pathogen or tumor, so the require-

ment for linkage between CTL and Th cell epitopes would not

appear to be necessary. However, presentation of MHC-I/peptide

epitopes by Th cells could further facilitate the expansion of

DC-initiated CTL responses, because growth factors (IL-2)

would be directly delivered to the CTL via an immunological

synapse. In addition, as described below, presentation of

antigen by Th cells to naive CTLs may preferentially generate

specific functional subsets of CTLs. Our observations have been

validated by a report from another group that Th cells that have

acquired antigen and that MHC-I complexes from DCs can

stimulate effective protective tumor-specific CTL responses

in vivo (134).

Direct priming of CD81 T-cell memory by Th cellsfunctioning as APCs

Having demonstrated that Th cells are capable of acquiring and

presenting antigen to activate CTLs, we examined the func-

tional consequences of this phenomenon. Specifically, we first

addressed whether Th cells had the capability of stimulating

naive CTLs. In the past, presentation of antigen by T cells to

Fig. 3. Varied mechanisms of antigen acquisition by Th cells. (1) CTLpeptide is shed by APCs and binds to MHC-I on a nearby Th cell. (2) Cell-to-cell contact results in the direct transfer of APC MHC-I to the surface ofthe T cell. This may or may not require antigen recognition by the Th cell.(3) Acquired MHC-II molecules may travel to endosomes and transferantigen to endogenous Th cell MHC-I. (4) Large epitopes presented byMHC-II can contain both MHC-I and II T-cell epitopes (Table 1). The MHC-II/peptide complexes recognized by Th cells can be internalized andprocessed by in endosomal compartments. Proteolytic cleavage releasesthe CTL epitope, which then binds to endogenous recycling MHC-Imolecules also present in the endosome of Th cells.



other T cells has been reported in some circumstances to lead to

T-cell activation, proliferation, and function (119, 127,

135–138), while in other cases it leads to tolerogenic or

suppressive effects (127, 139–142). In addition, CD81 T-cell

presentation of antigen to other activated CTLs results in

fratricide (143, 144). Most of the evaluations of T cells as APCs

have been done using pre-activated (antigen-experienced) T

cells as responders, since it was assumed that naive T cells

require professional APCs to initiate a response. Our initial

experiments comparing the ability of activated Th cells and DCs

to prime highly purified naive CD81 T cells showed two

surprising findings: (i) CTLs activated by either peptide-pulsed

DCs or Th cells progressed through an identical number of cell

divisions, and (ii) CTLs activated by Th cells showed signifi-

cantly greater initial expansion than those primed by DCs,

suggesting that Th cells as APCs lead to a reduced rate of AICD

(145). Cell surface phenotype analysis by flow cytometry was

performed on CTLs stimulated by the two APC populations

showed that both DC- and Th-primed CTLs expressed high

levels of the activation markers CD44, CD25, and CD69 (145).

However, while DC-primed CTLs rapidly lost CD62L expres-

sion, the CTLs primed by Th cells retained high levels of

CD62L, a marker found expressed by CD81 memory T cells.

Additionally, the CD44/CD62L-expressing CTLs also expressed

high levels of Ly6C (another memory cell marker). Thus, the

Th-primed CTLs bore a striking resemblance to the central

memory T-cell phenotype described earlier. In addition to the

phenotypic characteristics of this memory-like population of

CTLs, these cells behaved as conventional memory T cells: (i)

they demonstrated fully competent cytokine production but

had reduced cytolytic activity, (ii) they exhibited long-term

survival (4 2 months in vivo), and (iii) they possessed the

ability to mount recall responses to subsequent antigenic

challenge in vivo and in vitro. Several key characteristics of Th

and DC-primed CTLs are shown in Table 2. Although the exact

mechanism by which Th cells contribute to the establishment

of CD81 T-cell memory is not known, it has been proposed

that Th cells act through conditioning APCs, which then prime

CD81 T cells to generate an effector response and a CD81 T-cell

memory subset (62–64). Th cells may be the primary source of

IL-2, which is necessary for the generation of memory T cells,

during the priming of CTL responses (65). Additionally, it has

been suggested that the presence of CD41 T cells decreases

TRAIL expression in activated CTLs, increasing their survival

and capacity to differentiate into memory T cells (77). Here we

present yet another possible role for CD41 cells in CD81 T-cell

Fig. 4. Acquisition of MHC-I determinants by Th cells. (A) LB27.4 APCs were pulsed with OVA257� 264 and incubated with resting and activatedCD41 T cells from DO11.10 (DO11.10� B6)F1 and OT-II mice. (B) CD41 T cells from OT-II mice were cultured with and without T1 APCs(expressing I-Ab) in the presence of the indicated peptides. Surface expression of Kb/OVA257–264 complexes on the Th cells is indicated by the stainingintensity of the 25.D1.16 mAb.



memory generation. We propose that Th cells can present antigen

directly to CD81 T cells and preferentially drive memory cell

formation (Fig. 5). This hypothesis provides a simple yet elegant

explanation for the required presence of CD41 T cells in the

generation of CD81 T-cell memory.

Fine-tuning CD41 T-cell responses in cancer vaccines togenerate effective CTL responses

The work done by our group and others has clearly shown that

CD41 T-cell responses are an important element of effective

vaccines for cancer or infectious agents. The provision of T-cell

help not only boosts CTL priming efficiency, it also serves to

program CD81 T-cell memory responses and allows the

persistence of these secondary responses and is therefore a

critical component of protective immunity. In addition to the

positive influence Th cells can have on developing immune

responses, the participation of CD4/CD25 Tregs can lead to the

opposite effect. Several studies have shown that the removal of

Tregs before vaccination is advantageous (146–148). Likewise,

the presence of Treg cells within tumors correlates with

reduced survival (149). We have tested the efficacy of tumor-

specific peptide vaccines administered with TLR ligands using

the BALB-neuT mouse tumor model. These animals express the

activated form of the rat neu oncogene (RNEU) and sponta-

neously develop breast tumors at 4–5 months of age (150).

Vaccination of wildtype BALB/c mice (parental strain of BALB-

neuT but not expressing RNEU) with a RNEU CTL peptide and

TLR9-L (CpG) induced strong CTL responses, which recognize

RNEU-expressing tumor cell lines. These tumor-specific CD81

T cells were sufficient to protect BALB/c mice from trans-

planted RNEU-expressing tumors but do not protect the BALB-

neuT mice (151). This lack of protection in the BALB-neuT

mice was due to immune tolerance, as the mice express RNEU

in breast tissues at 5–6 weeks of age. Nevertheless, when the

BALB-neuT mice were pretreated with antibodies to CD4 or

CD25 to deplete Treg cells, the magnitude of the CTL responses

Table 2. CTL characteristics after priming by professional and T-helper APCs

DC APC T-helper APC

Proliferative capacity

CFSE celldivision�

Phenotypew

CD251 95% 94%CD441 93% 97%CD62L1 11% 72%CD691 93% 62%

Effector functionCytolytic

activityz23.4% 6.0%

IFN-gproduction‰

89.2% 82.4%

Significant differences between the two CTL subsets are indicated in bold text.�CTLs were stained with CFSE before in vitro culture with peptide-pulsedAPCs (DCs or Th cells). The filled histograms represent the dividing CTLs inthe presence of the indicated antigen-pulsed APCs. The open histogramsare the same cells, incubated with APCs in the absence of antigen.wNumbers represent the % of CTLs expressing the listed surface marker72 h after priming by the indicated APC.zDC- or Th-primed CTLs were incubated with radiolabeled, antigen-expressing tumor cells. The number listed is the % specific killing at aneffector:target ratio of 30:1.‰The numbers indicate the % of DC- or Th-primed CTLs producing IFN-gafter restimulation with antigen-expressing tumor cells, as measured byintracellular staining flow cytometry.APC, antigen-presenting cells; CTL, cytotoxic T lymphocyte; IFN, interferon.

Fig. 5. Model of CD81 T-cell memory development. In this model, theeventual fate of a naive CD81 T cell depends upon the nature of the antigenpresentation. Certain factors such as high antigen density, high levels of B7,prolonged antigen presentation by professional APCs, and IL-2 predisposeto the development of effector and effector memory type T cells, whilelower antigen densities, shorter duration of antigen presentation or antigenpresentation by non-professional APCs, non-B7-mediated costimulation(4-1BB, OX-40, CD70), and the presence of cytokines such as IL-15 andTGF-b will skew development towards the central memory phenotype.Several surface molecules have been proposed as markers for effector andmemory T-cell subsets and are listed in the phenotype panel. The ‘function’panel at the bottom of the figure shows the reported effector functionalityand proliferative capacity of the various T-cell subsets.



increased fivefold, with an accompanying increase in cytolytic

activity against RNEU-expressing tumor lines (151). Impor-

tantly, depletion of Treg cells using anti-CD25 mAb signifi-

cantly increased anti-tumor effects in the BALB-neuT mice to

transplanted tumors. However, administration of anti-CD4

mAb to deplete Treg cells, although previously shown to

increase CTL induction, resulted in decreased protection. These

results were likely due to the concurrent depletion of conven-

tional CD41 Th cells, which, as discussed previously, are

critical for the persistence of CTLs. The effect of peptide

vaccination on the outgrowth of spontaneously developing

tumors was also analyzed. Immunization with CTL peptide and

CpG alone could only delay tumor growth and prolonged

survival by approximately 5 weeks (151). In contrast, animals

pretreated with anti-CD25 mAb, to deplete Treg cells before

vaccination, remained tumor free 15–20 weeks after the

control animals had succumbed. These results further reinforce

the concept that CD41 T-cell responses need to be carefully

manipulated to generate the optimal effect of cancer vaccines,

allowing the development of tumor-specific T-cell help while

eliminating the detrimental pressures that Tregs exert during

immune priming.

Final thoughts

Immune responses are a result of complex interactions

between the cells of the immune system. At the heart of most

immune responses are CD41 T lymphocytes. Early studies of

these T cells showed their important role in supplying help,

mostly in the form of cytokines to other lymphocytes (B cells,

CTLs) for the generation of antibodies and mature killer cells,

hence their original name, T-helper cells. It is clear now that

lymphocytes expressing the CD4 marker belong to a large

family of cells that have one function in common: the regula-

tion and fine tuning, either upwards or downwards, of

immune responses. The function of CD41 T cells in either

enhancing or suppressing immune responses is not only

through the action of lymphokines that either stimulate or

inhibit the function of other lymphocytes but also through

cell-to-cell interactions leading to costimulation, direct inhibi-

tion, and sometimes antigen presentation. It is clear that

further research into these and other potential mechanisms of

T-cell immunoregulation will be necessary to develop vaccine

strategies to induce robust primary and optimal memory

responses that will ultimately benefit cancer patients.

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Multiple roles for CD4+ T cells in anti-tumor immune responses

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