Rapamycin, unlike cyclosporine A, enhances suppressive functions of in vitro -induced CD4 + CD25 + Tregs

Background. A growing body of data shows that CD4 + CD25 + regulatory T cells (Tregs) can induce transplantation tolerance by suppressing immune responses to allograft antigens. However, both the generation and the suppressive capacity of CD4 + CD25 + Tregs can be substantially affected by different immunosuppressive drugs used in clinical transplantation. The goal of this study was to compare the effects of cyclosporine A and rapamycin on the induction and suppressive functions of human CD4 + CD25 + Tregs in vitro . Methods. CD4 + CD25 + Tregs were induced in two-way mixed lymphocyte reaction (MLR) in the presence of rapamycin (Treg-Rapa) or cyclosporine A (Treg-CsA). GITR mAbs and ant-PE-FOXP3 staining sets. Suppressive capacity of induced Tregs was evaluated by their capability to inhibit anti-CD3 Ab-triggered proliferation of peripheral blood mononuclear cells (PBMCs), as measured by flow cytometry. The concentration of TGF- β 1 in culture supernatants was measured by enzyme-linked immunosorbent assay. Results. Although both rapamycin and cyclosporine A suppressed the induction of CD4 + CD25 + Tregs during MLRs, this effect was significantly more pronounced in cells cultured with cyclosporine. On the other hand, only rapamycin significantly decreased the percentage of CD4 + CD25 + Tregs which expressed GITR, a negative regulator of Treg ’ s suppressive capacity. Importantly, Treg-Rapa, unlike Treg-CsA, displayed significant suppressive activity and were capable of inhibiting the proliferation of anti-CD3 Ab-activated PBMCs. This activity was likely mediated by TGF- β 1. Conclusions. Rapamycin, unlike cyclosporine A, does not inhibit the function of CD4 + CD25 + Tregs. This implies that rapamycin could contribute to the development of transplantation tolerance by promoting the induction of functional CD4 + CD25 + Tregs. Moreover, our results suggest that rapamycin could be combined with functional Tregs. Abstract Background. Blockade of CD40 – CD40 ligand (CD154) interactions protects against renal injury in adriamycin nephropathy (AN) in immunocompetent mice. To investigate whether this protection relied on adaptive or cognate im-munity, we tested the effect of CD40 – CD154 blockade in severe combined immunodeficient (SCID) mice. Methods. SCID mice were divided into three groups: normal, AN + hamster IgG (ADR+IgG group) and AN + anti-CD154 antibody (MR1) (ADR+MR1 group). AN was induced by tail vein injection of 5.2 mg/kg of adriamycin (ADR). Hamster IgG (control Ab) or MR1 was adminis-tered intraperitoneally on days 5, 7, 9 and 11 after ADR injection. Histological and functional data were collected 4 weeks after ADR injection. In vitro experiments tested the effect of soluble and cell-bound CD154 co-cultured with CD40-expressing cells [macrophages, mesangial cells and renal tubular epithelial cells (RTEC)]. Results. All experimental animals developed nephropathy. Compared to the ADR+IgG group, ADR+MR1 animals had significantly less histological injury (glomerulosclero-sis and tubular atrophy) and functional injury (creatinine clearance). Kidneys of ADR+MR1 animals had significantly less macrophage infiltration than those of ADR +IgG animals. Interestingly, expression of CD40 and CD41 (a platelet-specific marker) was significantly less in ADR+MR1 animals compared to ADR+IgG animals. In vitro , CD154 blockade significantly attenuated upregulation of CCL2 gene expression by RTEC stimulated by activated macrophage-conditioned medium. In contrast, platelet-induced upregulation of macrophage and mesangial cell proinflammatory cytokine gene expression were not CD154-dependent. Conclusion. CD40 – CD154 blockade has a significant innate renoprotective effect in ADR nephrosis. This is poten-tially due to inhibition of macrophage-derived soluble CD154.