Peptides derived from alpha-helices of allogeneic class I major histocompatibility complex antigens are potent inducers of CD4+ and CD8+ T cell and B cell responses after cardiac allograft rejection.

We studied the rejection of cardiac allografts in a rat strain combination (PVG.R8 to PVG.1U) disparate for a single class I MHC antigen (RT1.Aa) to test the extent by which this molecule is recognized as peptides in association with recipient MHC molecules during graft rejection and the contribution of this recognition process to the rejection reaction. Three synthetic peptides that correspond to the portions of alpha-helices of the alpha 1 (P1, P2) and alpha 2 (P3) domains of the donor RT1.Aa molecule were used in this study. Splenocytes from heart allograft recipients at rejection responded in a proliferation assay to all 3 peptides and in a cytotoxic assay to peptides P1 and P2. The peptide-mediated proliferation and cytolytic reactions were blocked by antibodies against CD4/class II MHC and CD8 molecules. Serum from graft recipients at rejection contained significant titers of antibodies to peptides. Presensitization of graft recipients with the peptides resulted in a marked increase in peptide-mediated T cell and antibody responses. Although all 3 peptides were effective in eliciting active immune responses, the P3-mediated response was minimal when compared with those mediated by P1 and P2. Recipients presensitized with the peptides rejected their grafts in 5 days compared with 6 days for unsensitized animals. Recipients presensitized with donor-irradiated splenocytes and aortic endothelial cells, on the other hand, rejected their grafts in 1 and 3 days, respectively, which suggests that immunization with the whole RT1.Aa molecule is required to stimulate accelerated rejection of the graft. This rejection was associated with high titers of donor cell-specific antibodies that exhibited moderate cross-reactivity with the peptides. Our results clearly demonstrate that (1) the donor RT1.Aa molecule is recognized as peptides in the context of recipient class I and class II MHC molecules during the rejection of heart allografts, and (2) peptides derived from this molecule are highly immunogenic in that they contain epitopes recognized by CD4+ and CD8+ T cells and alloantibodies. Immune responses elicited by these peptides, however, did not significantly affect the rate of rejection. These results suggest that acute rejection of allografts may be mediated primarily by the direct recognition of intact MHC molecules.

Shirwan H ，Leamer M ，Wang HK ，Makowka L ，Cramer DV ... -  《TRANSPLANTATION》

Indirect T cell allorecognition and alloantibody-mediated rejection of MHC class I-disparate heart grafts.

Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RTIu recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the alpha1 and alpha2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.

Pettigrew GJ ，Lovegrove E ，Bradley JA ，Maclean J ，Bolton EM ... -  《JOURNAL OF IMMUNOLOGY》

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4(+) T cells.

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A(u), were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20-100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from nai;ve animals. The proliferative response was primarily mediated by CD4(+) T cells recognizing the RT1.A(a) molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4(+) T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.

Shirwan H ，Mhoyan A ，Yolcu ES ，Que X ，Ibrahim S ... -  《TRANSPLANT IMMUNOLOGY》

Complement contributes to the rejection of complete and class I major histocompatibility complex--incompatible cardiac allografts.

We have demonstrated previously that the terminal complement component C6 contributes to the acute rejection of ACI cardiac allografts by PVG recipients. ACI rats differ from PVG rats at major and minor histocompatibility antigens and ACI cardiac allografts stimulate vigorous alloantibody responses in PVG rats. We have now bred the C6 deficiency onto four PVG congenic rat strains to determine the effects of C6 on cardiac allograft survival across individual donor-recipient major histocompatibility complex (MHC) disparities. Hearts from C6-deficient PVG.1A (RT1a) donors were transplanted heterotopically to fully MHC-incompatible C6-sufficient and C6-deficient PVG.1L (RT1(1)) recipients, as well as from C6-deficient PVG.R8 (RT1.AaBu) donors to MHC class I incompatible C6-sufficient and C6-deficient PVG. 1U (RT1.AuBu) recipients. Hearts from PVG.1A (C6-) female donors were rejected acutely (7 to 9 days; n = 5) by fully MHC disparate female PVG.1L (C6+) recipients, but they survived significantly longer in female PVG.1L (C6-) recipients (13 to >50 days; n = 6). Slightly better survival resulted in male PVG.1L (C6-) heart transplant recipients of male PVG.1A (C6-) hearts (19 to >50 days [n = 5] vs 6 to 9 days for C6+ male PVG.1L recipients [n = 10]). The C6 deficiency had an even greater effect in PVG.1U recipients of class I MHC disparate PVG.R8 hearts (>50 day survival in C6- PVG.1U recipients [n = 5] vs 6 to 7 days in C6+ recipients [n = 8]). The cardiac allografts elicited similarly vigorous immunoglobulin M and G alloantibody responses in the C6- and C6+ recipients as measured by flow cytometry. At the time of acute rejection, the hearts in the C6+ recipients demonstrated extensive vascular endothelial destruction. In contrast, rejection of hearts by C6- recipients was characterized by endothelialitis, but there was little destruction of the endothelium and limited proliferation of smooth muscle cells in the intima. These results demonstrate that the terminal complement component C6 can contribute to the rejection of class I or complete MHC-incompatible hearts in rats that have been characterized as "high" alloantibody responders.

Qian Z ，Jakobs FM ，Pfaff-Amesse T ，Sanfilippo F ，Baldwin WM 3rd ... -  《JOURNAL OF HEART AND LUNG TRANSPLANTATION》

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: a role for alloantibody.

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8+ T cells and the mechanism whereby CD4+ T cells are able to independently mediate rejection. In this study of rejection of RT1Aa class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1u recipients, we show that elimination of CD8+ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4+ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1u athymic nude rats reconstituted with CD8+ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4+ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4+ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4+ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4+ T cell-dependent alloantibody plays a decisive role in effecting rejection.

Morton AL ，Bell EB ，Bolton EM ，Marshall HE ，Roadknight C ，McDonagh M ，Bradley JA ... -  《EUROPEAN JOURNAL OF IMMUNOLOGY》