Jiye Li

Regulatory Cells Potentiate the Efficacy of IL-4 Gene Transfer by Up-Regulating Th2-Dependent Expression of Protective Molecules in the Infectious Tolerance Pathway in Transplant Recipients

We have previously shown that the tolerant state in allograft recipients can be maintained and perpetuated by an “infectious” T cell-dependent regulatory mechanism. Hence, 1) treatment of LEW rats with RIB-5/2, a CD4 nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts; 2) donor-specific tolerance can be then transferred by spleen cells into new cohorts of test allograft recipients; and 3) putative regulatory CD4+ Th2-like cells are instrumental in this tolerance model. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the interactions between intragraft adenovirus-IL-4 gene transfer and systemic infusion of regulatory cells from tolerant hosts. Unlike individual treatment regimens, adjunctive therapy with adenovirus-IL-4 and suboptimal doses of regulatory spleen cells was strongly synergistic and extended donor-type test cardiac allograft survival to about 2 mo. RT-PCR-based expression of intragraft mRNA coding for IL-2 and IFN-γ remained depressed, whereas that of IL-4 and IL-10 reciprocally increased selectively in the combined treatment group, data supported by ELISA studies. In parallel, only adjunctive treatment triggered intragraft induction of molecules with anti-oxidant (HO-1) and anti-apoptotic (Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32) functions, both in the early and late posttransplant phases. Hence, systemic infusion of regulatory cells potentiates the effects of local adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway in transplant recipients.

Evidence of T cell clonality in the infectious tolerance pathway: implications toward identification of regulatory T cells.

We have shown that a rare population of regulatory CD4+ T cells plays a key role in the acquisition of infectious tolerance in rat sensitized recipients of cardiac allografts pretreated with nondepleting anti-CD4 mAb. This study was designed to analyze the TCR V&bgr; expression patterns in this transplantation model. First, we used V&bgr;-specific RT-PCR to show that there was no differential usage of TCR V&bgr; genes by T cells mediating rejection or tolerance. Indeed, graft-infiltrating lymphocytes expressed most of the 22 known rat TCR V&bgr; genes in both recipient groups, suggesting unrestricted TCR V&bgr; repertoire in alloreactive T cells. Then, we applied CDR3 spectrotyping of TCR &bgr;-chain to assess the clonality of T cells at different anatomic sites. CDR3 size restriction, indicative of the presence of T cell clones, was observed in graft-infiltrating lymphocytes but not in draining lymph nodes or spleen of tolerant hosts. Consisent with the clonal expansion, T cells in tolerated grafts exhibited the memory phenotype at a much higher percentage as compared with peripheral lymphoid organs. Moreover, in tolerated graft-infiltrating lymphocytes, the CD3 size restriction occurred in limited V&bgr; gene families, with V&bgr;8.1 and V&bgr;18 most frequently detected. Hence, T cells at the graft site of tolerant recipients contain T cell clones expressing selective V&bgr; genes. This phenotypic characteristics of the tolerogenic GILs may potentially be used as a novel marker to identify operational regulatory T cells in organ allograft recipients.

Selectin-Mediated Interactions Regulate Cytokine Networks and Macrophage Heme Oxygenase-1 Induction in Cardiac Allograft Recipients

Host sensitization to major histocompatibility complex (MHC) antigens is among the most critical of problems facing heart transplantation. Selectins are postulated to mediate the early adhesive events in the recruitment of leukocytes at the allograft site. We investigated the significance of selectin–P-selectin glycoprotein ligand-1 (PSGL-1)–mediated in vivo interactions in the immune cascade leading to rejection of cardiac allografts in skin presensitized rats. Infusion of a soluble recombinant form of PSGL-1 (rPSGL-Ig) during skin graft–mediated sensitization prevented Day 1.0 ± 0.1 “accelerated” rejection in sensitized rat recipients, and prolonged cardiac allograft survival to Day 3.8 ± 1.0 (p < 0.001). This therapy significantly depressed serum IgM levels and decreased intragraft expression of Th1 type cytokines (IL-2 and IFN-γ) as well as of IL-1β and MCP-1, as compared with controls, without affecting the initial number of infiltrating mononuclear cells (MNC). A profound decrease in graft-infiltrating MNC was recorded at 24 hours in rPSGL-Ig–treated rats. The expression of heme oxygenase-1 (HO-1), an inducible heat shock protein 32 that protects against oxidative cell/tissue injury, was found in approximately 14-fold higher levels in the rPSGL-Ig–treated recipients as compared with controls. The HO-1 overexpression in rPSGL-Ig–treated hosts, primarily by infiltrating macrophages, was accompanied by virtual absence of myocardial infarcts and decreased frequency of TUNEL + cells at the graft site. Moreover, down-regulation of HO-1 expression by zinc protoporphyrin, an HO-1 antagonist, decreased expression of antiapoptotic Bag-1 molecule in recipients conditioned with rPSGL-Ig. Thus, the blockade of selectin–PSGL-1 interactions depresses intracardiac allograft expression of Th1 type cytokines, and might inhibit the differentiation of Th1 type cells. In addition, it up-regulates HO-1 expression and protects against myocardial infarction and apoptosis. Hence, this study reports on a previously unrecognized role of selectin–PSGL-1–mediated interactions after in vivo alloantigenic challenge.