A better understanding of cytokine signaling pathways involving transcription factors of the STAT family in T cells responding to allostimulation will improve interpretation of immune-regulatory functions after organ transplantation. For this purpose, we established a novel method for monitoring phosphoprotein responses in alloreactive T cells. CFSE-labeled PBMCs from healthy human volunteers were cultured in completely allogeneic combinations and then fixed and permeabilized for phosphor (p)-specific STAT1, 3, 4, 5, and 6 intracellular staining. Cells were also stained with anti-CD4 and anti-CD8 mAbs in combination with various immunosuppressants. The sequential halving of CFSE-fluorescence can be used to track cell division in populations of proliferating cells, which allows intracellular profiling of STAT phosphorylation in proliferating cells of each T-cell subset by multicolor FCM analysis. In both CD4+ and CD8+ T cells proliferating in response to allostimulation, p-STAT1, STAT3, and STAT5 levels were significantly upregulated; however, p-STAT4 and STAT6 levels remained low. These results reflect the cytokine profiles of culture supernatants of allogeneic MLR, i.e., IFN-α, IL-6, and IL-2 levels increased but IL-4 levels remained undetectable. Addition of anti-IL-2Rα mAb reduced the proliferation of alloreactive CD8+ T cells but did not influence the proliferation of CD4+ T cells. In the proliferating CD4+ T cells, p-STAT5 level was specifically reduced but p-STAT1, STAT3, and STAT6 were unaffected, resulting in no appearance of foxp3+ Treg cells. Addition of anti-IL-6R mAb did not influence the proliferation of alloreactive CD4+ T cells. In this case, p-STAT3 level was specifically reduced but p-STAT1 and STAT5 levels were unaffected in alloreactive CD4+ T cells, resulting in the expansion of Treg cells in the MLR products. Clinical doses of CNIs markedly inhabited the T cell proliferation, and completely inhibited phosphorylation of all STAT in alloreactive CD4+ T cells. Whereas clinical doses of MPA and mTOR inhibitors showed less inhibitory effects on the T cell proliferation, however, significantly inhibited p-STAT1 and STAT3 levels while p-STAT5 level was unaffected in alloreactive CD4+, resulting in the expansion of Treg cells. The combined anti-IL-6R mAb and MPA/mTOR significantly induced p-STAT 5 level in alloreactive CD4+ T cells without elevation of p-STAT1 and STAT3 levels. Hence, such combined immunesuppresants may be a novel paradigm of inducing/expanding Treg cells after organ transplantation.

CITATION INFORMATION: Tanaka Y, Ohdan H. Impact of Immunosuppressants on Inducing Regulatory T Cells in Human Allo-Immune Responses. Am J Transplant. 2016;16 (suppl 3).

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