1. Peripheral Induced Regulatory T-Cells Dysfunction in High-Risk Corneal Transplantation Takenori Inomata, Jing Hua, Sang-Mok Lee, Homer Chang, Tina Shiang, Qiang Zhang, and Reza Dana Schepens Eye Research Institute Massachusetts Eye & Ear Infirmary Harvard Medical School There is no conflict of interest in this research

2. Background Cornea: Immune privileged site High-Risk Corneal Transplantation – Neovascularization, Retransplantation and Infection – 50% rejection, BUT 90% acceptance in uninflamed low-risk hosts Acute rejection primarily by CD4 + helper T cells Regulatory T cells (Tregs; CD4 + CD25 + Foxp3 + ) – Suppress T effector cell function – Induce and maintain immune tolerance – Peripherally induced Neuropillin-1 - Tregs (pTregs) – Thymus-generated naturally occurring Neuropillin-1 + Tregs (tTregs)

3. Purpose Determine the kinetics and function of regulatory T cells in high-risk transplantation Assess differential frequencies of pTregs and tTregs in high-risk vs. low-risk (LR) recipients 2015 Asia-ARVO Yokohama

4. Day 1.Corneal Suture Placement (14 days before) 2. Penetrating keratoplasty (C57BL6 to BALB/c) Materials & Methods Low-risk (LR) Group -14 0 Harvest (Day 14) High-risk (HR) Group 3. Lymph node cells ・ Flow Cytometry ・ Treg Suppression Assay ・ ELISA ・ IHC vascularization Corneal Transplantation 2.

5. Treg frequencies are slightly decreased in the draining lymph nodes of high-risk recipients LN Low-riskHigh-risk Day14 CD25 Foxp3 11.5 10.3 CD4 Gated

6. The frequencies of IFN-γ + CD4 T cells are significantly increased in high-risk recipients LR HR CD4IFNγ Day14 CD4 Gated Isotype

7. High-risk recipients express modestly lower frequencies of pTregs in the draining lymph nodes Nrp-1 PeCy7 LRHR CD4 + CD25 + Foxp3 + Tregs Gated Day14 3.6 3.1 6.8 6.6 pTregs; Nrp-1 - Tregs tTregs; Nrp-1 + Tregs

8. The suppressive function of Tregs and pTregs from HR hosts is significantly reduced Day14 Post-Transplantation

9. pTregs from high-risk hosts express less co- inhibitory molecules Day14 CTLA4 MFI Isotype:3 LR:2349 HR:2229 CD4+CD25+Foxp3 Gated

10. pTregs from high-risk recipients produce lower levels of inhibitory cytokines and higher levels of inflammatory cytokines InhibitoryInflammatory Day14

11. Impaired pTreg induction and suppressive function is associated with the loss of graft tolerance in high-risk recipients. Increased inflammatory and decreased inhibitory molecules and cytokines mediate the loss of immune privilege in high-risk recipients. Summary