Mechanism of acquired thymic tolerance induced by a single major histocompatibility complex class I peptide with the dominant epitope: Differential analysis of regulatory cytokines in the lymphoid and intragraft compartments

Background. We have recently shown that intrathymic injection of a combinat ion of immunogenic WAG-derived or Wistar-Furth (WF) (RT1.A(u)) major histoc ompatibility complex class I peptides induces acquired systemic tolerance t o cardiac and islet allografts in the WF-to-ACI rat combination and therefo re hypothesized that identification of the class I peptide dominance may pl ay an important role in the induction of antigen (Ag)-specific tolerance. T his study defined the peptide with the dominant epitope among the seven syn thetic RT1.4(u) peptides and analyzed the immunoregulatory cytokines within the lymphoid and intragraft compartments associated with acquired thymic t olerance. Methods. ACI recipients were pretreated with intrathymic (IT) injection of 300 mu g of the individual seven RT1.A(u) peptides 7 days before WF or Lewi s cardiac transplantation. Cytokine profile of mixed lymphocyte reaction su pernatants of T cells obtained from the thymus, mesenteric lymph nodes, spl een, peripheral blood leukocytes, and graft infiltrating cells after donor (WF) or third-party (Lewis) Ag stimulation were measured by enzyme-linked i mmunosorbent assay, whereas cytokine gene expression was determined by reve rse transcription-polymerase chain reaction. Results. Only IT injection of peptide 5 (93-109) among the seven RT1.A(u) p eptides induced donor-specific tolerance to cardiac allografts in the WF-to -ACI rat combination, In addition, intravenous injection of peptide 5 did n ot prolong WF graft survival in ACI recipients. Analysis of cytokine produc tion by the tolerant recipients showed significant Ag-specific reduction in the production of interleukin (IL)-2 and interferon-gamma (IFN-gamma) in t he thymus, mesenteric lymph nodes, spleen, and peripheral blood leukocytes, which was not associated with a concomitant Ag-specific increase in IL-4 a nd IL-10 production, Measurement of cytokine mRNA expression confirmed unde tectable IL-2 and significant reduction of IFN-gamma, which was associated with high levels of IL-4 and IL-10 expression in all lymphoid tissues. Alth ough the long-term functioning grafts showed high levels of IL-4 and IL-10 production and mRNA expression associated with no detectable IL-2 expressio n, grafts rejected at 20 days by recipients pretreated with the nondominant peptide 4 (residues 60-82) expressed high levels of IL-2 and low levels of IL-10 without any detectable IL-4. Of interest is our finding that both re jected and accepted grafts consistently expressed lFN-gamma. Conclusions. We have defined the short segment of RT1.A(u) peptide with the dominant antigenic epitope that induces acquired thymic tolerance by the p referential deletion/anergy of allospecific Th1 cells in the thymus and the prevention of expansion of Ag-specific T cells in the peripheral lymphoid tissues. Although there was a predominance of Th2 cytokines in the tolerize d grafts and Th1 cytokines in rejected grafts, IFN-gamma was expressed in b oth allografts. Thus, it appears that acquired thymic tolerance is associat ed with Th1 deletion/anergy rather than activation of Th2 cells.