TCRβ expression in CD4−CD8− double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4+CD8+ double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCRβ+ DN thymocytes and found that there are at least three functionally distinct subsets of TCRβ+ DN thymocytes: TCRβ+ DN3E, TCRβ+ DN3L, and TCRβ+ DN4. Survival and proliferation of TCRβ+ DN3E were independent of Gads, but survival and proliferation of TCRβ+ DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCRβ+ DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCRβ+ DN3L cells. Bcl-2 expression was not dependent on Gads in TCRβ+ DN4 cells, but proliferation of TCRβ+ DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads−/− DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCRβ-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.

Ag recognition by OVA-reactive OT-II (I-Ab restricted) and DO11.10 (I-Ad restricted) TCR-Tg CD4+ T cells after heterotopic transplantation of OVA transgene-expressing tracheal grafts was examined as a model of minor histocompatibility Ag (mHAg)-induced chronic allograft rejection. In response to airway allotransplantation with grafts expressing the OVA transgene, these TCR-Tg CD4+ T cells expressed the activation markers CD69 and CD44, demonstrated evidence of blastogenesis, underwent multiple rounds of cell division leading to their clonal expansion in the draining lymph node, and proceeded to differentiate to a effector/memory T cell phenotype based on a reduction in the expression of CD45RB. These mHAg-specific TCR-Tg CD4+ T cells responded equally well to fully MHC-mismatched tracheas and to class II-deficient allografts, demonstrating that donor mHAg recognition by recipient CD4+ T cells does not rely on Ag presentation by donor-derived APC. The activation of mHAg-specific TCR-Tg CD4+ T cells after their adoptive transfer into recipient mice given MHC-matched, but mHAg-disparate, airway allografts was associated with their movement into the allograft and the near uniform destruction of the transplanted airway tissue secondary to the development of obliterative airways disease. These results demonstrate that an activation of mHAg-reactive CD4+ T cells in the draining lymph node by recipient APC that indirectly express graft mHAg-derived peptide/class II MHC complexes precedes responder T cell proliferation and differentiation, and leads to the eventual migration of these alloreactive T cells to the transplanted airway tissue and the promotion of chronic graft rejection.

To investigate the role of TCR signaling in the exit of CD4+ T cells from cell cycle, we took advantage of a low frequency TEa T cell adoptive transfer technique as well as the Y-Ae mAb to interrupt Ag/MHC recognition before the completion of clonal expansion. Termination of TCR signaling after 36 h of Ag exposure caused an immediate reduction in cell size and deceleration of G1—>SG2M phase cell cycle progression. As a consequence, clonal expansion in the absence of durable TCR signaling decreased by two-thirds. Thus, CD4+ T cells scan for the presence Ag throughout their clonal expansion response, and continuously adjust their rate of cell growth and G1—>S phase transition to match their intensity of TCR signaling.

Human T cells responding against transplanted allogeneic lung tissue have been implicated in late graft failure secondary to obliterative bronchiolitis. This obliterative airways disease (OAD) also develops in heterotopic murine tracheal allografts in association with graft infiltration by both CD8+ and CD4+ T cells. To date, there has been little evidence to suggest that directly alloreactive CD8+ T cells either promote chronic rejection or lead to the development of OAD following airway allotransplantation. Using Ld-specific TCR-Tg 2C CD8+ T cells adoptively transferred into wild-type B6 (H-2b) mice and the transplantation of BALB/c (H-2d) tracheal allografts, we now show that the direct recognition of donor-specific class I MHC molecules by host CD8+ T cells leads to their activation, clonal expansion within the graft, and differentiation to an effector phenotype with the capacity to induce airway fibrosis. In addition, these experiments demonstrate that ongoing direct alloantigen recognition within the transplanted airway tissue is necessary for the recruitment and retention of these directly alloreactive CD8+ T cells. Thus, these experiments are the first to definitively show a role for directly alloreactive CD8+ T cells in the chronic rejection that leads to OAD.