1. The effect of TLR on transplantation and tolerance of the cornea Mee Kum Kim M.D 1, Jinho Jeong M.D 1, Won Ryang Wee M.D 1, Jin Hak Lee M.D 2 1 Department of ophthalmology, Seoul National University Hospital, Seoul, Korea 2 Department of ophthalmology, Seoul National University Bundang Hospital, Bundang, Korea The authors have no financial interest in the subject matter of this poster.

2. Purpose 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

3. Materials and Methods Mouse cell culture  Group Group K : keratocytes only : 1x10 4 cells Group D : dendritic cells only (DC2.4*): 1x10 4 cells Group M : macrophages only (Raw264.7*): 1x10 4 cells Group K+D : keratocytes + dendritic cells: 0.5x10 4 + 0.5x10 4 cells Group K+M : keratocytes + macrophages: 0.5x10 4 + 0.5x10 4 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

4.  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

5.  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

6. Results 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

7. Cytokine secretion after agonist stimulation  After LPS stimulation Coculture : K + D Coculture : K + M K : B6 Keratocyte D : 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

8.  After Pam3csk4 stimulation Coculture : K + D Coculture : K + M K : B6 Keratocyte D : 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

9. 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

10. CD86 CD80 CD 11c C3H  B6 C3H  TLR4KO C3H  B6 C3H  TLR4KO Lymph nodes Spleens 1.90 3.53 1.155.49 1.812.84 2.7010.64 CD80 CD86 CD 11c 2.27 1.57 2.771.75 3.484.13 3.171.95 At 12 daysAt 4 weeks

11. 0.270.310.74 0.29 0.60 0.340.520.93 C3H  B6 C3H  TLR4KO C3H  B6

12. Conclusions 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