Summary of Parent R01 Award (EY024602) Renowned for promoting tissue regeneration and immune quiescence, studies have demonstrated the therapeutic potential of MSCs in human disease. Nevertheless, fundamental questions remain unanswered regarding their immunomodulatory mechanisms. This proposal employs a well-characterized transplant model of the murine cornea to systematically investigate how MSCs directly interact with T cells to suppress alloimmunity. Over recent years the work of several laboratories, including our own, has made substantial progress in understanding how MSCs regulate ocular inflammation. With respect to corneal transplantation, we and others have shown that exogenous administration of MSCs suppresses alloimmunity and promotes graft survival. Reports from our lab provide evidence that MSCs: (i) specifically home to the ocular surface following corneal transplantation, where they act to (ii) limit antigen-presenting cell (APC) maturation, and (iii) decrease graft-destroying IFNγ + T helper-1 (Th1) cell responses. Moreover, our preliminary data and reports from other groups indicate that administration of MSCs following transplantation induces Foxp3+ regulatory T cells (Tregs). Despite these observations, the exact mechanisms by which MSCs suppress Th1 generation and induce Tregs are not known. Our preliminary investigations indicate that, in addition to indirect modulation via APCs, MSCs exert a direct immunomodulatory effect on alloreactive T cells. We define 3 specific aims to answer the following questions: Aim 1: What are the mechanisms by which MSCs inhibit generation of alloreactive Th1 cells? Aim 2: What are the mechanisms by which MSCs inhibit effector function of alloreactive Th1 cells? And finally Aim 3: How do MSCs promote the generation of tolerance- inducing Tregs? Our preliminary data implicate specific soluble and surface-bound immunoregulatory molecules. Based on these data, we propose 3 hypotheses: (1) MSCs negatively regulate early T cell activation via the surface-bound molecule ALCAM, resulting in decreased generation of Th1 cells; (2) MSC- secreted IL11 suppresses Th1 function by antagonizing IFNγ and Tbet expression; and (3) MSCs skew the differentiation of naïve T cells toward Foxp3+ Tregs via CD80/CTLA-4 interaction. Principal objective of this project is to define the molecular mechanism by which MSCs interact with T cells to regulate alloimmunity. In this proposed supplement, we plan to apply our understanding of the immunomodulatory and cytoprotective functions of MSCs to promote the survival of ocular cells following nitrogen mustard exposure. In the parent grant, we investigate how MSCs suppress ocular inflammation in a model of adaptive immunity. Here, we will investigate whether MSCs or their secreted immunoregulatory and growth factors provide similar protective functions following vesicant-induced injury.