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 COMPARTMENTS1

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Abstract
We have recently shown that intrathymic injection of a combination of immunogenic WAG-derived or Wistar-Furth (WF) (RT1.Au) major histocompatibility complex class I peptides induces acquired systemic tolerance to cardiac and islet allografts in the WF-to-ACI rat combination and therefore hypothesized that identification of the class I peptide dominance may play an important role in the induction of antigen (Ag)-specific tolerance. This study defined the peptide with the dominant epitope among the seven synthetic RT1.Au peptides and analyzed the immunoregulatory cytokines within the lymphoid and intragraft compartments associated with acquired thymic tolerance. ACI recipients were pretreated with intrathymic (IT) injection of 300 μg of the individual seven RT1.Au peptides 7 days before WF or Lewis cardiac transplantation. Cytokine profile of mixed lymphocyte reaction supernatants of T cells obtained from the thymus, mesenteric lymph nodes, spleen, peripheral blood leukocytes, and graft infiltrating cells after donor (WF) or third-party (Lewis) Ag stimulation were measured by enzyme-linked immunosorbent assay, whereas cytokine gene expression was determined by reverse transcription-polymerase chain reaction. Only IT injection of peptide 5 (93-109) among the seven RT1.Au peptides induced donor-specific tolerance to cardiac allografts in the WF-to-ACI rat combination. In addition, intravenous injection of peptide 5 did not prolong WF graft survival in ACI recipients. Analysis of cytokine production by the tolerant recipients showed significant Ag-specific reduction in the production of interleukin (IL)-2 and interferon-γ (IFN-γ) in the thymus, mesenteric lymph nodes, spleen, and peripheral blood leukocytes, which was not associated with a concomitant Ag-specific increase in IL-4 and IL-10 production. Measurement of cytokine mRNA expression confirmed undetectable IL-2 and significant reduction of IFN-γ, which was associated with high levels of IL-4 and IL-10 expression in all lymphoid tissues. Although the long-term functioning grafts showed high levels of IL-4 and IL-10 production and mRNA expression associated with no detectable IL-2 expression, 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 rejected and accepted grafts consistently expressed IFN-γ. We have defined the short segment of RT1.Au peptide with the dominant antigenic epitope that induces acquired thymic tolerance by the preferential 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 tolerized grafts and Th1 cytokines in rejected grafts, IFN-γ was expressed in both allografts. Thus, it appears that acquired thymic tolerance is associated with Th1 deletion/anergy rather than activation of Th2 cells.