VISUALIZATION OF ANTIGEN-SPECIFIC T-CELLS IN ACAID

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DESCRIPTION (Applicant's Description): The immune response in the eye has adapted to eliminate biological threats without significant nonspecific inflammation. Manipulation of this highly regulated immune response may lead to therapeutic strategies for prolonging transplant engraftment, including retinal cell transplantation, and mitigating autoimmune diseases. The injection of chicken ovalbumin (OVA) in the anterior chamber of the eye of mice induces a unique form of systemic tolerance called anterior chamber associated immune deviation or ACAID. ACAID is an excellent model for studying immune regulation. Upon a subsequent subcutaneous injection with an immunogenic form of OVA, these animals display reduced priming for delayed-type hypersensitivity responses. In addition, we have shown that cytolytic T-cell (CTL) responses are also reduced in ACAID. This suggests that CTL precursors are deleted or fail to expand in ACAID. Alternatively, CTL precursors are expanded but functionally inactivated. To determine the fate of CTL precursors, it is necessary to track these cells in vivo. However, antigen-specific CTL precursors in naive as well as immunized mice are below the level of detection by flow cytometry. To overcome this obstacle, T-cells, from transgenic mice, expressing a T-cell receptor specific to OVA peptide 257-264 complexed with H-2 Kb, will be transferred into naive syngeneic mice. This method increases the CTL precursor frequency while maintaining a normal physiological immune response. Donor T-cells will be enumerated in recipient mice following ACAID induction by flow cytometric analysis using OVA257-264/H-2Kb tetramers. The generation of ACAID includes active suppression. Splenic CD8+ T-cells have been shown to be the regulators of efferent suppression in ACAID. In addition, we have shown that gammadelta T-cells also contribute to efferent suppression. Using an in vitro assay of CTL suppression, we will determine if gammadelta T-cells are the efferent suppressor in ACAID or if gammadelta T-cells induce CD8+ alphabeta T-cells to become the efferent suppressor.