Effects of two classes of immunosuppressive agents, mTOR inhibitors vs. calcineurin inhibitors, on the generation and function of human alloreactive T helper cells (Th1, Th17 and Treg)
La Monica, Giovanna (2011) Effects of two classes of immunosuppressive agents, mTOR inhibitors vs. calcineurin inhibitors, on the generation and function of human alloreactive T helper cells (Th1, Th17 and Treg). [Tesi di dottorato] (Inedito)
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Successful transplantation requires the prevention of allograft rejection and complete reduction of the effects on the immune system by any immunosuppressive agent. The commonly immunosuppressive drugs used in clinical such as calcineurin inhibitor (tacrolimus, TAC) and mTOR inhibitor (sirolimus, SRL) affect naïve T cell differentiation and memory T cell expansion; however, the biological and biochemical changes induced by these drugs on the generation and expansion of different subpopulations of T helper cells are not fully elucidated yet. CD4 positive (CD4+) T cells are known to orchestrate and regulate adaptive immune responses and play an important role in allograft rejection or tolerance. CD4+ T cells, upon activation and expansion, develop into different effector T helper cell subsets and produce distinct cytokine profiles and mediate separate effector functions. From a functional perspective, CD4+ T cells can be classified into effector T helper cells (Th1, Th2, Th17, Tfh) and regulatory T cells (Tregs). In the transplant setting, prevailing evidence shows that both effector Th1, Th17 cells and cytokines IFN-g, IL-17 are involved in the process of allograft rejection whereas Treg and Th2 cells favor long-term graft survival. The effects of Tacrolimus and Sirolimus on these subsets were studied because a balance between graft-destructive effector T cells and graft-protective regulatory T cells toward dominance of Tregs may promote clinical transplant tolerance. Therapies targeting inhibition of pathogenic effector T cells, promotion of Treg cells and directing against the mediators of intragraft inflammation may have profound effects on the rejection process and induce long-term graft acceptance. In this study, alloreactive CD4+ T cells in a MLR culture (Mixed-Lymphocyte reaction) with responder naïve T cells and allogeneic APCs (antigen presenting cells), were generated. Alloreactive CD4+ T cells were enriched and restimulated with autologous APCs plus anti-CD3 stimulation in the absence or the presence of TAC or SRL or their combination. Although both TAC and SRL inhibit alloreactive T helper cell proliferation and various cytokine productions, the intensity and kinetics for TCR-induced T helper subpopulations are markedly differently affected between the two drugs. TAC at 2-5ng/ml significantly inhibited over 90% of the productions of IFN-g and IL-17 from the supernatants of bulk cell cultures, and the percentages of IFN-g (Th1) and IL-17-secreting cells (Th17), whereas SRL at high concentration (20ng/ml) had moderate inhibition on IFN-g and IL-17 productions (30% and 60% respectively). When IL-2 was added to the culture, TAC still exerted similar inhibition while SRL completely lost its inhibition on IFN-g expression. In contrast, in the presence of IL-2, FOXP3 expressing cells (Tregs) were markedly increased in SRL treatment compared to the controls (Averagely 2-fold increase), whereas TAC treatment was not changed and did not show a decreased trend. When the two drugs were used in combination, we found that TAC at 2ng/ml with SRL at 2-5ng/ml achieved the maximal effect in inhibiting the productions of IFN-g and IL-17 while maintaining a high level of FOXP3 expression. SRL treatment did not affect the plasticity or reprogramming of Tregs, but significantly decreased FOXP3+IFN-g and FOXP3+IL-17+ populations when used in combination with very low dose of TAC. When an inflammatory setting was mimicking by adding proinflammatory cytokines (IL-1, IL-6, TNF-α) to the cell culture, there was a significant decrease of the generation of SRL-derived FOXP3+Treg cells. SRL-derived Tregs expressed normal Treg surface markers, were anergic to allostimulations, and functionally suppressed the proliferation of allogeneic effector T cells, and Th1 and Th17 alloimmune responses. Without affecting FOXO3/FOXP3 interaction, SRL markedly decreased DNMT1 expression. DNMT1 is a FOXP3 promoter demethylation and it may account for its long-term induction. Furthermore, it is a prerequisite for stable FOXP3 expression and suppressive phenotype of Tregs. These findings can help to guide the clinical use of immunosuppressive drugs to promote Treg expansion and to control Th1 and Th17 alloimmunity.
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