CHEMOKINE REGULATION OF NKT CELLS, AND ACAID

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Chemokines are associated with a variety of inflammatory and autoimmune diseases for their role in recruitment of cells to the sites of immune induction and inflammation. However, few studies have explored a role for chemokines in peripheral tolerance induction and no studies are reported for specific chemokines for NKT cells. This research plan will explore the role of specific chemokines in negative regulation of an immune inflammatory response and specifically in the recruitment of NKT cells to the site of tolerance induction. Anterior Chamber Associated Immune Deviation (ACAID) is associated with the selective negative regulation of delayed type hypersensitivity (DTH) after the introduction of antigen into the eye. An accumulation of splenic NKT cells is absolutely required for the development of ACAID. Preliminary data show that MIP-2 is associated with NKT cell recruitment. We will use molecular, cellular and morphological techniques to identify and define the role of the chemokines in the tolerance inducing process. Aim One will define the chemokine profile by RNase Protection Assay and multi-color flow cytometry. The second aim will use classic chemotaxis assays to define the migratory response of NKT cells to ACAID associated chemokines, along with molecular techniques to confirm the expression of chemokine receptors on these cells. The third aim will explore the mechanisms of chemokine-NKT cell interaction during ACAID induction by reconstituting NKT cell knockout mice with NKT cells from chemokine receptor knockout mice. A local adoptive transfer assay will measure if regulator T cells were generated. Finally, the fourth aim will explore the hypothesis that the cells recruited to the spleen for the induction of ACAID must necessarily form interactive cell clusters. After identifying such clusters and cells within, we will modulate chemokines and potential adhesion molecules and evaluate the outcome by quality and quantity of clusters. NKT cell defects and/or deficiency is associated with a variety of autoimmune disorders in both mice and humans (lupus, type 1 diabetes, systemic scleroderma). Thus, the mechanisms of NKT cell mediated induction of T regulatory cells might function in the maintenance of self tolerance in a variety of organs and tissues in addition to immune privileged sites such as the eye. The novel postulates about chemokines, innate cells and peripheral tolerance presented in this proposal, represent a heretofore totally unexplored area of research.