THE ROLE OF CTLA4 NEGATIVE SIGNALING PATHWAY IN THE INDUCTION OF ACQUIRED THYMIC TOLERANCE IN VIVO

Abstract

664 Injection of antigen into the thymus of adult animals induces a state of antigen-specific systemic tolerance in several experimental autoimmune and transplantation models, but the exact mechanisms of acquired thymic tolerance have yet to be fully elucidated. In order to study the mechanisms of acquired thymic tolerance we developed a mouse model of intathymic injection of antigen in normal and OVA TCR transgenic mice. In normal BALB/c mice we have shown that intrathymic injection of OVA induces Th1 cell unresponsiveness and prevents peripheral expansion of antigen-specific CD4+ T cells after immunization with OVA in vivo. Activated CD4+ T cells appear to traffic to the thymus where they get anergized and ultimately deleted. Injection of OVA into the thymus of OVA TCR transgenic mice results in early deletion by apoptosis of double positive thymocytes followed by deletion of some but not all single positive thymocytes, and prolonged anergy of surviving thymocytes and peripheral T lymphocytes (J. Immunol., in press). Recent data from Abul Abbas indicate that negative signaling by CTLA4 is important in the induction of T cell anergy in a model of peripheral tolerance in vivo. In this study we investigated the role of CTLA4 engagement in the induction of acquired thymic tolerance in vivo. The same model of intrathymic injection of OVA in BALB/c mice was used. Peripheral immunization of unmanipulated control mice with OVA induces a high proliferative response as well as IL-2 and IFN-γ secretion by peripheral lymphocytes with OVA stimulation in vitro. Intrathymic injection of OVA 24-48 hours prior to peripheral immunization with OVA leads to a state of antigen-specific tolerance as shown by a decreased proliferative response to OVA as well as a significant decrease in antigen-specific IL-2 and IFN-γ production by lymphocytes in response to OVA in vitro, as compared to control mice and to mice injected intrathymically with control antigen. In order to investigate the role of CTLA in the induction as well as maintenance of acquired thymic tolerance, different groups of mice were treated with 100µg/day i.p. of a blocking anti-CTLA4 mAb on days -3, -2, -1 from immunization (group 1), on the day of immunization (group 2), 3 days post-immunization (group 3), or 8 days post-immunization (group 4). Animals were tolerized by intrathymic injection of OVA 24-48 hours before immunization. CTLA4 blockade abrogated thymic tolerance only when anti-CTLA4 was administered on day 3 post-immunization, as determined by reversal of T cell unresponsiveness in vitro. We conclude that the CTLA4 negative signaling pathway is important in the induction but not maintenance phase of acquired thymic tolerance. The observation that CTLA4 blockade around the time of thymic injection of antigen (i.e. before immunization) fails to abrogate induction of acquired thymic tolerance is consistent with the upregulation patterns of CTLA4 on activated T cells, but more interestingly, it supports our hypothesis that T cells need to be activated (in this case by immunization) and then traffic to the thymus where they become anergized and ultimately deleted.