The production of alloantibodies can undoubtedly contribute to the mechanisms that lead to injury and destruction of an allograft by binding to the graft and causing tissue damage through multiple mechanisms, including perturbing the function of graft endothelial cells, the fixation of complement, and enhancing the activity of effector leukocytes by forming a bridge between the target tissue and the effector cell through a mechanism known as antibody-dependent cytotoxicity (ADCC). In particular, antibody deposition and complement fixation may contribute to many of the changes associated with acute and chronic rejection (1).
Diagrammatic representations of rejection pathways often show the B cell and the antibody producing plasma cells as one of the effector pathways in the rejection cascade. However, it is important to remember that B cells can also act in other roles, including as antigen presenting cells, and therefore have the capacity to amplify the immune response to the allograft.
Acute Rejection Can Occur in the Absence of Alloantibody and B cells
To try and address the question of whether B cell tolerance is essential, one could ask if there is any evidence to suggest that B cells and/or the production of alloantibody is essential for acute rejection? Early in the investigations carried out to determine the contribution of the different components of the immune system to the rejection process, it was shown that the transfer of serum containing alloantibodies was not sufficient on its own to trigger acute rejection of an allograft (2). These data were interpreted as indicating that cellular immunity, as we now know the T cells, was more important in triggering acute rejection, since the transfer of immune cells rather than just serum from sensitized animals was able to trigger acute graft rejection. As the tools available to dissect the immune response have become more sophisticated, these data were confirmed and extended by studies showing that allografts are rejected by mice deficient in antibody production, as well as by mice that have no B cell deficiency.
In contrast to the lack of evidence that B cells and alloantibody were essential, data accumulated showing that T cells were absolutely required for graft rejection. These include studies showing that allografts are not rejected in the absence of T cells (3) and that grafts are rejected by immunodeficient animals reconstituted with T cells alone (4–6). Indeed, data from my own laboratory using T cell receptor transgenic mice as a source of T cells with a defined alloantigen specificity has confirmed these findings and shown that less than 1000 alloantigen specific T cells purified from T cell receptor transgenic mice with a single specificity for donor alloantigen can trigger graft rejection in the absence of B cells (7).
Targeting T Cells is Sufficient to Prevent Acute Rejection in Naive Recipients
In young, naive hosts that have not been previously exposed to donor alloantigens and who do not have high frequencies of leukocytes that can cross react with alloantigen or preformed alloantibodies, targeting T cells by eliminating them or inhibiting their function is sufficient to prevent acute allograft rejection. There are many studies, both experimental and clinical, showing that agents such as immunosuppressive drugs and polyclonal or monoclonal antibodies that inhibit T cell function or deplete T cells in vivo are very effective at preventing acute graft rejection (8). Moreover, in the context of tolerance, many experimental studies have shown that inducing tolerance in alloantigen reactive T cells alone is sufficient to achieve long-term graft survival and operational tolerance to donor alloantigens, including work from my own group (9). In addition, dissecting the mechanisms responsible for operation tolerance to alloantigens in naive recipients shows that deletion and/or suppression/regulation of donor reactive T cells are sufficient and dominant for both inducing and maintaining tolerance in vivo (10).
Alloantibodies Can Participate In or Trigger Allograft Rejection
While there is no evidence that alloantibodies alone are able to trigger acute allograft rejection, there are many data showing that alloantibodies can contribute to the rejection process. The clearest demonstration is hyperacute rejection, which occurs within hours after transplantation of vascularized organs into recipients with preformed donor-specific antibodies. In this special setting, activation of cellular immune mechanisms is not necessary, as the binding of the preformed alloantibody and the effector mechanisms it triggers is sufficient. This type of early humoral allograft rejection was first described in the context of clinical kidney transplantation in the late 1960s (11).
Preformed alloantibodies can develop in patients that have rejected a previous transplant, after transfusions, and in multiparous women. The preformed antidonor antibodies are usually specific for allogeneic major histocompatibility complex (MHC) antigens, and bind to donor endothelial cells thereby disrupting endothelial cell homeostasis and activating the complement cascade. Complement-mediated destruction of the endothelial cell layer exposes the subcellular matrix to the blood stream, initiating platelet aggregation and thrombus formation. In general, as a result of rigorous screening before transplantation, hyperacute rejection is avoided in clinical transplantation.
Preformed antibodies reactive with blood group antigens can also trigger hyperacute allograft rejection when there is an ABO blood group incompatibility between the donor and the recipient. However, there are also examples in the literature (such as pediatric cardiac transplantation) where ABO incompatibility does not result in rejection (12). ABO-incompatible heart transplantation during infancy has been shown to result in development of B cell tolerance to donor blood group A and B antigens. Tolerance in this setting has been found to occur by elimination of donor-reactive B lymphocytes and may be dependent upon persistence of some degree of antigen expression. These findings suggest that intentional exposure to nonself blood group A and B antigens may prolong the window of opportunity for ABO-incompatible transplantation (13).
Interestingly, preformed antibodies that can react to alloantigens have also been observed in about 1% of the general population who have not been specifically sensitized as above. These antibodies are likely to arise through cross-reactivity with other antigens to which the individual has been previously exposed, including viral antigens. Whether these cross-reactive antibodies can contribute to graft rejection remains unclear.
In many situations, alloantibodies will be produced by the host after transplantation. However, the appearance of alloantibodies does not necessarily lead to graft rejection. In some situations, the alloantibody produced can contribute to the rejection process.
In clinical transplantation, a correlation between the presence of alloantibody and both acute and delayed graft loss has been reported. The hallmark of this type of rejection is the deposition of one of the components of the complement system, C4d, in the graft (14,15). Indeed, early detection of C4d deposition has been linked to a poorer graft outcome. In other specialized settings, alloantibody may play a more dominant role. For example, in an experimental study where donor and recipient rats were mismatched for a single MHC class I molecule, transfer of anti-class-I antibody was shown to trigger rejection (16,17). However, it remains a matter of debate whether alloantibody production after transplantation is the cause of graft loss or simply a witness to an earlier cellular immune response that is really responsible for the damage.
Alloantibodies Can Contribute to Allograft Survival
Interestingly, there is also evidence that alloantibody production after transplantation can contribute to graft survival. Thus, not only are there data showing that B cells and/or alloantibodies are not required for rejection, there are also a large number of studies demonstrating that in certain circumstances, alloantibodies can actually enhance allograft survival (18). As a result, such antibodies were called enhancing antibodies when they were first described. There are also other situations where antibodies can have a positive impact on graft survival later in the response to the graft. Although preformed alloantibodies and naturally-occurring xenoreactive antibodies can undoubtedly cause hyperacute rejection (see above), there are also data showing that these same antibodies can be responsible for graft accommodation (19); a process where a graft becomes refractory to further damage after the initial response.
The mechanisms responsible for graft prolongation by enhancing antibodies, and for accommodation, are not fully characterized. Allografts transplanted into recipients treated with enhancing antibodies have been shown to be more rapidly infiltrated by T cells and to induce expression of donor MHC antigens, suggesting that an accelerated response to donor alloantigens occurs in treated recipients (20–22). However, precisely how this influences the rejection response remains unclear. The specificity of enhancing antibodies is also not known, although it has been suggested that part of the repertoire may be anti-idiotypic (23).
B Cells and Tolerance
Small resting B cells can induce tolerance to defined antigens in vivo. The most successful demonstration of this phenomenon was the induction of tolerance to the male specific antigen H-Y following the infusion of small resting male B cells into syngeneic female recipients (24). The tolerant state induced was robust, as male skin grafts transplanted onto females treated with the resting B cells intravenously were accepted. The infusion of small resting B cells was shown to switch off the response of naive T cells but not of memory T cells.
When the ability of small resting B cells to induce unresponsiveness to multiple minor and major histocompatibility antigens was investigated, we found that while effective in switching off responses to minor antigens, infusion of small resting B cells before transplantation did not impact the response to major antigens (25). Indeed, we were able to show that the small resting B cells were activated after infusion into MHC disparate donors and recipients and that additional measures, such as blockade of the CD40-CD154 pathway, were required to reveal their tolerogenic potential in this setting (26).
Mechanisms of B cell Tolerance
B cell tolerance to self antigens is achieved by two mechanisms, deletion and functional inactivation of self reactive B cells. The precise mechanism selected depends on the form of the self antigen to which tolerance is induced. Data from experimental studies shows that B cell tolerance to soluble antigens is induced by functional silencing or B cell anergy (27), whereas B cell tolerance to membrane-bound antigens is induced by deletion (28). These same mechanisms can most likely be harnessed for inducing tolerance to alloantigens, xenoantigens, and blood group antigens (13,29,30). Indeed, there is evidence from studies in mixed chimerism models that alloantigen specific B cells as well as T cells are deleted from the repertoire in stable mixed chimeras (29). Clearly, if B cell tolerance could be induced and maintained, this would enable alloantibody production to be prevented throughout the posttransplant course and ensure that the potentially harmful effects of alloantibody were completely avoided.
Situations Where B cell Tolerance Would Be Desirable or Advantageous for Transplantation Tolerance
The conclusion from reviewing the literature in this area is that the requirement for B cell tolerance is context dependent, with quality, quantity, specificity, functional properties, and the time at which alloantibodies appear all contributors to the overall picture that determines whether B cell tolerance is essential. In truly naive recipients that have no reactivity to donor alloantigens before transplantation, either as a result of the presence of T cells and B cells expressing antigen receptors specific for alloantigens or T cells and B cells that can cross-react with alloantigens, B cell tolerance does not appear to be essential. However, in marked contrast, in primed recipients in whom preformed antibodies are present or where there are large numbers of primed T cells that can promote alloantibody production very rapidly after transplantation, strategies to target and tolerize B cells (thereby preventing antibody mediated damage to the graft) would be very advantageous. In this setting, selective targeting of B cells that produce antidonor antibodies would be most beneficial to ensure that antibodies could still be produced to other antigens as required to maintain protective immunity particularly against pathogens. Recipients who are truly naive may be very rare in the setting of adult transplantation. Individuals exposed to the environment and who have experienced a series of infections throughout their lives may already have some antibodies that can cross react with alloantigens as mentioned earlier. In these situations, effective strategies for inducing B cell tolerance may also be beneficial, enabling long-term graft function to be improved.
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