Much research has been devoted to improving our understanding of the molecular mechanisms by which vascularized xenografts are rejected. Only on the basis of such knowledge can we devise therapeutic interventions capable of modifying, on the one hand, the recipient's immune response, and, on the other, the antigenicity of grafts from animal donors, using genetic engineering. The first barrier to xenotransplantation between species such as the pig and primates is hyperacute rejection (HAR*), a process involving xenoreactive natural antibodies (XNA) and the complement-dependent activation of endothelial cell (EC). After HAR comes acute vascular rejection (AVR) or delayed xenograft rejection (DXR), a multifactorial process in which mechanisms incriminated include the action of induced xenoreactive antibodies (Xabs), EC, and platelet activation, leading to a prothrombotic and pro-inflammatory environment and antibody-dependent cell-mediated cytotoxicity. In the preclinical pig-to-baboon model, AVR associated with an increase in Xabs now seems to be the major obstacle to successful xenografting, being remarkably resistant to conventional immunosuppressants (1). Anti-Gal antibodies, the principal XNA and Xabs in this model, are produced in both T cell dependent and T cell independent processes and are therefore partially resistant to conventional T cell-specific immunosuppressant agents. In the concordant hamster-to-Lewis rat model, in which XNAs are not abundant enough to induce HAR, anti-hamster Xabs (mostly of the IgM isotype) are rapidly induced and AVR occurs within 3 days after grafting. This is not a delayed form of HAR, because it is not primarily driven by complement activation; inactivating complement with cobra venom factor (CVF) fails to prolong graft survival. Also, T cell activation is not primarily involved; rejected hamster hearts are infiltrated with only a few T cells and cyclosporin (CsA) has no pronounced effect on graft survival. Nevertheless, the combination of CVF and CsA for 10 days makes prolonged survival possible in the presence of CsA alone. The survival of xenografts in this model with CsA alone after initial complement inactivation may be explained by several findings. First, parallel to survival during CsA treatment, anti-apoptotic and anti-oxidant genes are expressed in graft endothelium (2). Second, there is a reduction in the antihamster serum response, which has been associated with a loss in the ability to produce Tindependent xenoantibodies (3). Third, CsA directly suppresses EC expression of adhesion molecules and chemoattractants (B. Charreau et al., submitted). Finally, it may be that in this model the initial complement inactivation is necessary for ECs to adapt without being damaged and for ECs to express protecting genes, whereas CsA inhibits the serum response as well as the activation of T cells and ECs. Pertinent to this idea are our recent observations that T cells and antihamster xenoantibodies are rapidly induced after the withdrawal of CsA (4). Without complement inactivation, long-term survival with CsA alone can also be achieved by using a treatment that suppresses the synthesis of T cell-dependent and T cell-independent Xabs. A 2-to-4 week treatment with leflunomide and CsA makes extended survival possible using CsA alone. Survival in this case was based on accommodation (involving a reduction in the expression of adhesion molecules by ECs) and on a hamster-specific, T cell-independent B cell tolerance (3, 5, 6). The CsA in these experiments could be replaced by FK506 (7). These data provide increasing evidence that modifying the antibody response to xenografts, in addition to conventional immunosuppressants, can result in long-term survival, at least in the hamster-to-rat model. Combining CsA with other antiproliferative drugs (e.g., as deoxyspergualin, which reduces B cell secretion of Xabs) also brings about long-term xenograft survival (8). Suppressing xenoantibodies, therefore, seems to be functionally equivalent to suppressing complement activation, in that both allow CsA to prevent T cell activation and reduce the serum response. To find out whether the same is true in pig-to-primate xenotransplantation will require experiments aimed at depressing the CsA-independent xenoantibody response in primates, too. But the high doses of antiproliferative drugs necessary to achieve a significant effect on Xabs in primates are likely to prove toxic. Attempts have been made to reduce the secretion of XNA without severe side effects by using anti-immunoglobulin (Ig) antibodies to target membrane IgM and IgD molecules on B cells. B cells can be separated into phenotypically distinct subpopulations. Immature B cells are IgMlowIgDlow CD45Rlow. Mature B cells include B-1 cells (IgMhigh IgDlow CD45Rlow), mainly found in the peritoneal cavity, and B-2 cells (IgMlow IgDhigh and CD45Rhigh), originating from the bone marrow and found in the spleen and lymph nodes. There are also B cells from the marginal zone of the spleen, which are IgMhigh IgDlow and CD45Rhigh. A recent report from Yang et al. (9) shows that XNAs, at least in the mouse, originate from different B cell compartments, including both B1 and B2 cells. We, therefore, know that for efficient XNA depletion to be achieved, all B cell compartments will need to be targeted. Targeting IgM with anti-IgM antibodies in the rat inhibits B cell differentiation in the bone marrow and causes down-regulation of IgM and IgG XNA (10, 11). This treatment, combined with CsA for the first few weeks after xenotransplantation, also makes long-term survival of cardiac concordant xenografts possible with CsA alone (Soares et al., personal communication) and prolongs the survival of pig-to-baboon kidney xenografts (Gianello, personal communication). In this issue of Transplantation, Saxton et al. confirm that an initial reduction in B cell activity in the rat prolongs the survival of hamster xenografts and makes extended survival possible with CsA alone. Treating Lewis rats with cOX6 (anti-RT1-B class II) monoclonal antibody reduces the B cell (CD45RA-positive) count, the formation of anti-hamster Xabs, and the deposition of IgM and C3 on heart xenografts. Whether inhibiting B cells with antibodies (anti-Ig or anti-class II) will result in T cell-independent B cell tolerance like that caused by leflunomide remains to be determined. The mechanisms by which anti-class II antibodies bring about their effects on B cells are not fully understood. In mice, a signal mediated through class II molecules results in the proliferation of preactivated B cells (12) and in the death of resting B cells (13). Administering anti I-A in mice results in the disappearance and reappearance of B cells (14). In RT1-B rats, the administration of cOX6 clearly induces at least a partial depletion, despite the non-complement-fixing nature of the OX6 IgG1 isotype. Anti-class II treatment, in addition to suppressing B cell activity directly, probably also blocks the presentation of hamster xenoantigens by Lewis RT1-B class II antigens on Lewis APCs. Alternatively, it may modify the priming of MHC-restricted recipient T helper cells so that they become capable of actively suppressing allograft rejection (as suggested by Smith et al. (15)). OX6 is known to completely block a mixed lymphocyte reaction between RT1-B and RT1-D lymphocytes. In the hamster-to-Lewis rat xenotransplantation model, the mechanism by which cOX6 exerts its effect in the early phase of rejection is unlikely to be inhibition of a T cell mediated effector response, because there are very few T cells infiltrating the xenograft at the time of rejection (3 days). There is a possibility that a blocking effect exerted by cOX6 on recipient APCs, in addition to the depletion effect illustrated, contributes to the modulation of B cell priming by T helper cells in a way that renders them incapable of producing anti-hamster antibodies. A putative direct negative signaling to B cells through class II molecules, independent of any presentation, could also explain the suppression of a T cell-independent antibody response by which AVR is known to be induced. The data gathered so far in the hamster-to-rat xenotransplantation model have important implications for pig-to-primate trials. They point out the importance of CsA-resistant induction of Xabs in AVR and suggest that if the B cells responsible for the secretion of those Xabs can be inhibited (e.g., by the use of anti-B cell bioreagents), no further CsA-resistant immune mechanism or molecular incompatibility remains to relay Xabs in a process of accelerated rejection.
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Bernard Vanhove (1999) studied this question.