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We postulated that indirect recognition of MHC-derived peptides might modulate the alloresponse to donor antigen. In this study, we looked at the potential of two class I peptides derived from the alpha 1 and alpha 2 regions of the DA RT1Aa molecule. Lew responder rats were immunized by varying concentrations of two 25mer peptides covering residues 56-81 and 96-120. The injections were under the footpad and were repeated on day 14. The thickness of the footpads was measured to control delayed-type hypersensitivity (DTH). The animals were sacrificed on day 16 and the splenocytes were tested in mixed lymphocyte culture as responders against DA stimulator cells and CAP third-party splenocytes. In addition, the phenotype of the cells was measured using flow cytometry with antibodies against CD4, CD8, CD5, MHC class II, CD25, CD14 and CD19. Peptide 96-120 induced strong sensitization of the Lew recipient animals at concentrations of 200-500 micrograms (n = 4). The stimulation index was 2-3 times higher than that of untreated animals. Peptide 56-81 failed to induce sensitization at the concentrations used, but surprisingly induced a concentration-dependent immunosuppression that was highest at 400 micrograms (n = 4). In proliferation experiments responder Lew rats proliferated only to peptide 56-81 in vitro.

Chronic rejection of transplanted allografts is the major cause of graft loss after clinical solid organ transplantation. Recent data link the indirect presentation of allopeptides to chronic graft loss; thus, identification of immunodominant epitopes on major histocompatibility complex (MHC) antigens could significantly contribute to establishing novel ways for monitoring and managing chronic rejection. Here, we show that synthetic allo-MHC-derived peptides covering the polymorphic region 56 to120 of HLA-B7 modulate alloresponses. In particular, the 2 ␤-pleated sheet-derived peptides covering residues 91 to 105 and 96 to 120, respectively, but not sequences from the ␣1 helix, were presented by autologous peripheral blood lymphocytes to induce T-cell proliferation. In addition, the 2 ␤-pleated sheetderived peptides and the ␣1-derived peptide residues 60 to 75 abrogated lysis of HLA-B7 target cells by anti-HLA-B7 cytotoxic T lymphocytes (CTLs). Although most residues between 91 and 120 are normally not directly accessible to T cells, our results indicate that peptides derived from the lower surface of the peptidebinding groove of HLA-B7 are immunodominant in HLA-B7 alloresponses. To characterize the binding and stability of allopeptides to T cells, the 62-70 peptidederived from the 60-75 allopeptide that blocked cytotoxicity of anti-HLA-B7 CTL-was synthesized and coupled with fluorescein isothiocyanate. The peptide specifically labeled anti-B7 CTL, but not anti-HLA-A2 CTL as measured by flow cytometry. Peptide binding to CTL was specific at 4°C and remained stable for 12 hours, whereas it remained stable for less than 2 hours at 37°C. These studies allow the identification of HLA-B7 T-cell epitopes and reveal for the first time a novel, previously unrecognized application of synthetic HLA-derived allopeptides to visualize alloreactive T cells. IntroductionStructural studies show that T-cell receptor (TCR) contacts bind peptide and large stretches of ␣1 and ␣2 major histocompatibility complex (MHC) helices. 1,2 In general, endogenous peptides derived from allo-MHC are seen by T cells as foreign-eliciting alloresponses. 3 T cells may recognize peptide-induced conformational changes, and they may also recognize surface MHC structures in addition to or independent of bound peptide. 4 Furthermore, allospecific cytotoxic T lymphocytes (CTLs) that are peptidedependent and peptide-specific have been described in the literature. 5 In general, however, when donor and recipient MHCs are structurally similar, the endogenous peptide appears to be the dominant target of alloreactive T cells, 5-7 a phenomenon backed by clinical data in graft-versus-host disease. 8 Synthetic allopeptides have been used in many different models to modulate alloresponses and to study alloreactivity. [9][10][11] In other studies, class I-and class II-derived allopeptides were used to induce tolerance. 12-14 By studying a series of rat-derived synthetic allo-MHC peptides, immunodominant regions of allogeneic MHC molecules that modulate graft surviv...

A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.

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