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Accumulating evidence indicates that, in absence of CD8+ T-cell activation, CD4+ T-cell-mediated allograft rejection is associated with a dominant Th2-cell response and eosinophil infiltrates. In this study, we analyzed the mechanisms by which CD8+ T cells regulate alloreactive CD4+ T-cell priming and differentiation into interleukin 4 (IL-4)-producing cells. We showed that interferon gamma (IFN-gamma) production by CD8+ T cells was dispensable for the inhibition of Th2-cell development, as well as tissue eosinophilia and type 2 cytokine production in the rejected grafts. Since we noticed that CD8+ T cells not only suppressed Th2 differentiation, but also down-modulated the overall priming of alloreactive CD4+ T cells, we evaluated whether CD8+ T cells act by limiting the accumulation of donor-derived dendritic cells (DCs) in lymph nodes. We found that indeed, alloreactive CD8+ T cells rapidly eliminated allogeneic DCs from T-cell areas of draining lymph nodes, through a perforin-dependent mechanism. Thus, our data demonstrate that cytotoxic T lymphocyte (CTL)-mediated clearance of allogeneic DCs is a negative feedback mechanism that limits the duration of alloantigen presentation in draining lymph nodes, thereby modulating the amplitude and polarization of the primary alloreactive CD4+ T-cell responses.

Although much progress has been made in understanding the role of NK cells in bone marrow transplantation, little is known about their function in CD4 T cell-mediated allograft rejection. We have previously shown that in the absence of CD8 T lymphocyte priming, the in vivo default development pathway of alloreactive CD4 T cells was strongly biased toward Th2 phenotype acquisition. In this study, we investigate the impact of NK cells on the activation and differentiation of alloreactive CD4 T cells in various donor/recipient combinations. Our data demonstrate that defective inhibition of host NK cells by donor APCs including dendritic cells (DCs) results in diminished allospecific Th cell responses associated with the development of effector Th cells producing IFN-gamma rather than type 2 cytokines. Turning host NK cells off was sufficient to restore strong alloreactive CD4 T cell priming and Th2 cell development. Similar results were obtained by analyzing the effect of NK cell activation on CD4 T cell responses to skin allografts. However, despite the dramatic effect of NK cells on alloreactive Th1/Th2 cell development, the kinetics of skin graft rejection were not affected. Thus, Th2 differentiation is a major pathway of alloreactive CD4 T cell development during solid organ transplant rejection, as long as host NK and CD8 T cells are not activated. We propose the hypothesis that MHC class I-driven interactions between donor DCs and host NK cells or CD8 T cells might result in DC-carried signals controlling the dynamics of alloreactive CD4 T cell priming and polarization.

The mechanisms that influence the polarization of CD4 T cells specific for allogeneic MHC class II molecules in vivo are still poorly understood. We have examined the pathway of alloreactive CD4 T cell differentiation in a situation in which only CD4 T cells could be activated in vivo. In this report we show that priming of adult mice with allogeneic APC, in the absence of MHC class I-T cell interactions, induces a strong expansion of type 2 cytokine-producing allohelper T cells. These alloantigen-specific CD4 T cells directly recognize native allogeneic MHC class II molecules on APC and secrete, in addition to the prototypic Th2 cytokines IL-4, IL-5, and IL-10, large amounts of TGF-beta. The default Th2-phenotype acquisition is not genetically controlled and occurred both in BALB/c and C57BL/6 mice. CD8 T cells are the principal cell type that controls CD4 T cell differentiation in vivo. Furthermore, we demonstrate that strong Th2 priming can be induced not only with allogeneic splenocytes but also with a low number of bone marrow-derived dendritic cells. Finally, using a passive transfer system, we provide direct evidence that CD8 T cell expansion in situ promotes alloreactive Th1 cell development principally by preventing their default development to the Th2 pathway in a mechanism that is largely IFN-gamma independent. Therefore, this work demonstrates that type 2 cytokine production represents a dominant pathway of alloreactive CD4 T cell differentiation in adult mice, a phenomenon that was initially thought to occur only during the neonatal period.