The primary requirment for CD4 T cells in acute cardiac rejection is well known (4
). Traditionally, CD4 T cells are generally thought to mediate cellular destruction indirectly via CD4 T cell “help” for cytotxic CD8 T cells (29
) and B cells and/or or via bystander inflammatory cytokine destruction with TNF-α or IFN-γ (30
). However, in addition to playing a helper role in initiating rejection, CD4 T cells also can be sufficent to act as effectors of acute rejection via direct recognition of donor MHC class II molecules (7
). Thus, while ‘indirect’ (host APC-dependent) recognition has increasingly been appreciated as a major pathway of allograft recognition, direct engagment of donor MHC antigens can be sufficient for acute rjection. While CD4 T cells can clearly reject cardiac allografts, the specific effector mechanism(s) utilized by these cells to mediate rejection was unclear. Importantly, largely in vitro studies indicate that CD4 T cells can mediate direct cellular cytotoxicity either via Fas/FasL (13
) or via perforin-dependent (20
) mechanisms. However, thus far relatively little in vivo evidence has shown a role for direct CD4 T cell toxicity in host defense (15
). This study set out to determine whether Fas/FasL interactions and/or perforin played a role in the specific setting of CD4 T cell mediated cardiac allograft rejection.
Results demonstrate that CD4 T cell-mediated cardiac rejection indeed requires the alternative use of either allograft Fas expression or effector CD4 T cell perforin. It is noteworthy that while perforin appeared to play little role in CD4 T cell cytotoxicity in vitro, there was a clear requirement for perforin-dependent reactivity in vivo. Because the sensitivity to cytolytic mediators is likely dictated by the target cell used, such results highlight the importance of examining the role of candidate effector pathways in vivo. In any case, findings strongly suggest that direct cardiac allograft rejection by CD4 T cell-mediated cardiac rejection is largely dependent on cytolytic activity. Interstingly, these results are quite similar to our previous studies of CD8 T cell-mediated islet allograft rejection (27
). In that report, we found that perforin and Fas also were alternative but necessary mediators of acute rejection. It is intriguing to consider that direct CD4 or CD8 T cell mediated rejection may not greatly differ in their respective mechanisms of rejection. We would propose that the mechanism of rejection (e.g. cytolytic versus other cellular or humoral pathways) may be dictated by the nature of allograft recogntion (e.g. ‘direct’ versus ‘indirect’) rather than by the specific T cell subset involved. So, while CD4 T cells can certainly contribute to both acute and chronic allograft rejection in a variety of ways, when the role of the CD4 T cell is constrained to that of a primary, direct effector cell, the array of effector molecules employed may also be somwhat restricted.
A caveat to these findings is that defined immune receptors/pathways can have influences on immune reactivity beyond a defined effector role, potentially leading to ambiguous results or experimental artifacts. For exammple, while Fas-deficient (lpr
) cardiac allografts were acutely rejected by wild-type CD4 T cells, we found that there was a moderate delay in rejection by FasL (CD95L) deficient T cells. However, it is important to note that FasL clearly has been shown to play an agonist role in T cell activation and function, presumably independent of the death-inducing property of this molecule (33
). Other studies have demonstrated a pro-costimulatory effect of Fas signaling in that Fas or FADD-deficient T cells cells have markedly diminished proliferative capacity (35
). Thus, it is possible that some delay in acute rejection by gld
T cells is due to an impaired intrinsic reactivity of these cells rather than the loss of FasL as an effector molecule. We found that gld
CD4 T cells were almost completely non-responsive to allogeneic APCs in vitro (not shown), consistent with this agonist role for reverse signaling through FasL in T lymphocytes (33
). The finding that Fas-deficient (lpr
) allografts do not
have prolonged survival would be consistent with this interpretation of the results. Thus, experimental results must be viewed considering the potential multiple properties of immnoreceptors/effector molecules in immune responses.
The histologic evaluation of cardiac allografts in this study mirrored our in vivo
adoptive transfer experimental findings. More specifically, whereby individual deficiency in either donor Fas or CD4 T cell perforin demonstrated acute cellular rejection, the simultaneous removal of donor Fas and CD4 T cell perforin only demonstrated infiltration by CD4 T cells without evidence of significant cardiomyocyte damage. This histologic appearance strongly suggests that activated CD4 T cells are able to home to the graft (intact Signal 1, Signal 2 and MHC-restricted targeting), but once there are incapable of acutely rejecting the graft (loss of effector CD4 T cell function). It is interesting to note that while Fas-deficient allografts are not acutely rejected by PFPKO CD4 T cells, they do develop evidence of vasculopathy (chronic rejection). As MHC Class II expression is intact on both the donor allograft and the host tissues, both the direct and indirect pathways of antigen presentation are operational. As previous work in our laboratory has shown that MHC Class II deficient (B6 C2D) donor allografts are not acutely rejected by naïve CD4 T cells in immune deficient CB.17 SCID hosts but do develop evidence of vasculopathy (7
), the current results again implicate an indirect
CD4 T cell in the pathogenesis of chronic rejection (vasculopathy). This result is not completely surprising as other studies have also implicated the indirect CD4 T cell in the pathogenesis of chronic cardiac allograft rejection (38
). Importantly, this finding demonstrates the utility of this model for the future study of the molecular pathogenesis of vasculopathy initiated by indirect CD4 T cells.
In conclusion, results of these studies demonstrate that acute CD4 T cell-mediated cardiac allograft rejection requires donor Fas and CD4T cell perforin expression in an obligate and parallel fashion. Additionally, the simultaneous loss of donor Fas expression and effector CD4 T cell perforin expression abrogates rejection by impairing the effector mechanism of the CD4 T cell and not by inhibiting recognition (Signal 1) or costimulation (Signal 2). To our knowledge, this is the first demonstration of a requirement for a directly cytotoxic CD4 T cell in an in vivo model of transplantation. Consequently, potential therapies to abrogate clinical acute cardiac rejection should take into consideration the potential need for inhibiting the required molecular machinery for direct CD4 CTL function. Additionally, these results highlight the redundancy of the immune system and underscore the probability that therapies will likely require more than the inhibition or targeting of individual molecules or pathways to be effective. Finally, the loss of donor Fas expression and CD4 T cell perforin expression completely abrogated acute rejection but did not impair the development of chronic rejection (vasculopathy). This finding implicates the indirect CD4 T cell in the pathogenesis of vasculopathy and demonstrates that the development of vasculopathy initiated by CD4 T cells occurs independently of allograft Fas and CD4 T cell perforin expression.