The effect of TLR on The effect of TLR on

transplantation and tolerance of transplantation and tolerance of

the corneathe corneaMee Kum Kim M.D1, Jinho Jeong M.D 1, Won Ryang Wee M.D 1, Jin Hak Lee M.D 2

1Department of ophthalmology, Seoul National University Hospital, Seoul, Korea

2Department of ophthalmology, Seoul National University Bundang Hospital, Bundang, Korea

The authors have no financial interest in the subject matter of this poster.

PurposePurpose

To investigate the role of toll-like receptor (TLR)

On the MHC mismatched allograft rejection in corneal transplantation

On the immune tolerance of the cornea

Materials and MethodsMaterials and Methods

Mouse cell culture Group

Group K : keratocytes only : 1x104 cells

Group D : dendritic cells only (DC2.4*) : 1x104 cells

Group M : macrophages only (Raw264.7*) : 1x104 cells

Group K+D : keratocytes + dendritic cells : 0.5x104 + 0.5x104 cells

Group K+M : keratocytes + macrophages : 0.5x104 + 0.5x104 cells

* Kindly provided by Kim TJ

Mouse primary corneal keratocyte

DMEM/F12 (1:1) with 10% FBS and 1%PS

Mouse DC cell line: DC2.4*

RPMI1640 with 10%FBS and 1%PS

Mouse macrophage cell line: Raw264.7*

DMEM with 10%FBS and 1%PS

TLR stimulation with agonist LPS (TLR4 agonist) Pam3csk4 (TLR2 agonist)

Change in TLR expression and its products of TLR signaling pathway TLR2 and TLR4 expression

RT-PCR Stimulation for 6 and 24 hours with LPS or Pam3csk4

Phospho-IkappaB-alpha (p-IκB-α) Western blot Stimulation for 2, 6, and 24 hours with LPS or Pam3csk4

Cytokine assay ELISA Candidate cytokines : IL-6, TNF-α, TGF-ß, IL-10 Stimulation for 24 hours with various concentration

LPS : 1, 10, 100, 1000, 10000 ng/ml Pam3csk4 : 1, 5, 10, 50, 100 ng/ml

TLR

Group (Donor Recipeint) C57BL/6 (B6) B6 : Autograft (n=10) C3H/HeN (C3H) B6 : MHC mismatched allograft (n=11) C3H TLR4 knockout (TLR4KO) : (n=13)

Transplantation procedure Donor /recipient : 3.0 / 2.0 mm trephine 8 to 10 interrupted 10-0 Nylon sutures

Clinical evaluation Analysis of graft survival using Slit-lamp biomicroscopy

Definition of rejection (end point) : 100% graft edema Kaplan-Meier survival analysis (Log rank (Mantel-Cox) test)

Immune cell assay Cervical lymph node and spleen 12 days & 4 weeks after transplantation Flow cytometry (FACSCalibur) and FlowJo software

CD11c, CD80/CD86: CD4/8, CD44/69: CD4/8, IFN

TLR4 knock-out mice on a C57BL6 (B6 background) were bred at a SPF animal facility and were kindly provided by Dr. S-Y Seong at the Department of Microbiology and Immunology, Seoul National University College of Medicine

Corneal transplantation

ResultsResults

TLR expression after agonist stimulation

P-IκB-α expression after agonist stimulation

TLR4 in M, D, and K

TLR2 in D and K

P-IκB-α in M, D, and K

P-IκB-α in M,and D

Cytokine secretion after agonist stimulation After LPS stimulation

Coculture : K + D

Coculture : K + M

K : B6 KeratocyteD : Mouse dendritic cell line (DC 2.4)M : Mouse macrophage cell line (Raw 264.7)

Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition

After Pam3csk4 stimulation Coculture : K + D

Coculture : K + M

K : B6 KeratocyteD : Mouse dendritic cell line (DC 2.4)M : Mouse macrophage cell line (Raw 264.7)

Markedly increased anti-inflammatory cytokine IL-10 and TGF-β1 in coculture condition

Corneal allograft survival and immunologic profiling

Median survival time

: C3H B6 > C3H TLR4KO

IFN-γ secreting CD8 T cell

: C3H B6 < C3H TLR4KO

CD8 IFN- in L/N at 12 days

P<0.05, Mann Whitney U testP<0.05, Log Rank test

CD86

CD80

CD 11c

C3H B6 C3H TLR4KOC3H B6 C3H TLR4KO

Lymph nodes

Spleens

1.90 3.53 1.15 5.49

1.81 2.84 2.70 10.64

CD80

CD86

CD 11c

CD 11c CD 11c

2.27 1.572.77 1.75

3.48 4.13 3.17 1.95

At 12 days At 4 weeks

0.27 0.31 0.74

0.29 0.29 0.60

0.34 0.52 0.93

C3H B6 C3H TLR4KOC3H B6

ConclusionsConclusions

TLR4 is likely to be involved in immune modulation of MHC mismatched corneal allograft rejection

Fibroblast seems to be involved in immune modulation through TLR signaling