[Aim] Although the various potential benefits of everolimus over other immunosuppressants in organ transplantation have been described, the precise sensitivity of this drug to differentiation into various T-helper (Th) cell and B cell subsets in humans remains to be elucidated.
[Methods] We have recently established a method to quantify STAT family phosphorylation at the single cell level in alloreactive T cells. CFSE-labeled PBMCs were cultured in fully allogeneic combinations, and then fixed and stained for phosphor specific STAT1, STAT3, STAT4, STAT5, and STAT6, together with anti-CD4/CD8 mAbs, followed by multiparameter FCM analyses. We have also established an in vitro B cell activation model that facilitates inducible differentiation into B-1a, B-1b, and B-2 cells. To differentiate B-0 cells into B-1a cells, resting PBMC B cells were treated with anti-IgM F(ab′)2, a TI-2 Ags analog. Further, for B-1b cell differentiation, resting B cells were treated with a combination of anti-IgM F(ab′)2 and LPS, a TLR4 agonist. For B-2 cell differentiation, resting B cells were activated with feeder cells of 3T3 fibroblasts transfected with CD40L and BAFF. The sensitivities to various drugs were tested using these T cell and B cell activation models.
[Results] In both CD4+ and CD8+ T cells proliferating in response to allostimulation, levels of pSTAT1, pSTAT3, and pSTAT5 were significantly upregulated, but levels of pSTAT4 and pSTAT6 remained low. Addition of either cyclosporine or tacrolimus markedly inhibited T cell proliferation and wholly reduced pSTAT1, pSTAT3, and pSTAT5 levels even in barely proliferating alloreactive CD4+ T cells. In contrast, addition of everolimus mildly inhibited T cell proliferation and selectively reduced levels of pSTAT1 and pSTAT3 but maintained pSTAT5 levels in the proliferating alloreactive CD4+ T cells, which were positive for Foxp3. Addition of either cyclosporine or tacrolimus in the B cell activation assay exclusively inhibited the differentiation into B-1a cells, but did not affect the differentiation into B-1b and B-2 cells. In contrast, everolimus had inhibitory effects on the differentiation into all B cell subsets.
[Conclusions] Everolimus specifically inhibited differentiation into Th-1 and Th-17 cells, but maintained differentiation into T-reg cells in response to allostimulation. Everolimus inhibited the differentiation into not only B-1 cells but also B-2 cells, which are thought to be precursors of anti-HLA antibodies.
To cite this abstract in AMA style:Tanaka Y, Yamashita M, Ohdan H. Comprehensive Analyses of Inhibitory Effects of the mTOR Inhibitor Everolimus on the Differentiation into Subsets of Human T-Helper Cells and B Cells [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/comprehensive-analyses-of-inhibitory-effects-of-the-mtor-inhibitor-everolimus-on-the-differentiation-into-subsets-of-human-t-helper-cells-and-b-cells/. Accessed December 1, 2023.
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