Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4 + T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.gene regulation | tolerance | autoimmunity

Although mechanisms leading to brain-specific inflammation and T cell activation have been widely investigated, regulatory mechanisms of local innate immune cells in the brain are only poorly understood. In this study, to our knowledge we show for the first time that MHC class II+CD40dimCD86dimIL-10+ microglia are potent inducers of Ag-specific CD4+Foxp3+ regulatory T cells (Tregs) in vitro. Microglia differentially regulated MHC class II expression, costimulatory molecules, and IL-10 depending on the amount of IFN-γ challenge and Ag dose, promoting either effector T cell or Treg induction. Microglia-induced Tregs were functionally active in vitro by inhibiting Ag-specific proliferation of effector T cells, and in vivo by attenuating experimental autoimmune encephalomyelitis disease course after adoptive transfer. These results indicate that MHC class II+CD40dimCD86dimIL-10+ microglia have regulatory properties potentially influencing local immune responses in the CNS.

CD44 is a prominent activation marker which distinguishes memory and effector T cells from their naïve counterparts. It also plays a role in early T cell signaling events as it is bound to the lymphocyte-specific protein kinase and thereby enhances T cell receptor signalling. Here, we investigated whether IFN-γ and IL-17 producing T helper cells differ in their CD44 expression and their dependence of CD44 for differentiation. Stimulation of CD4+ T cells with allogeneic dendritic cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive IL-17 producing T helper cells were mainly CD44+++ as compared to IFN-γ+ T helper cells, which were CD44++. This effect was enhanced under polarizing conditions. T helper 17 polarization led to a shift towards the CD44+++ population, whereas T helper 1 polarization diminished this population. Furthermore, blocking CD44 decreased IL-17 secretion, while IFN-γ was barely affected. Titration experiments revealed that low T cell receptor and CD28 stimulation supported T helper 17 rather than T helper 1 development. Under these conditions CD44 could act as a co-stimulatory molecule and replace CD28. Indeed, rested CD44+++CD4+ T cells contained already more total and especially phosphorylated zeta-chain-associated protein kinase 70 as compared to CD44++ cells. Our results support the notion, that CD44 enhances T cell receptor signaling strength by delivering lymphocyte-specific protein kinase, which is required for induction of IL-17 producing T helper cells.

Primary T cell activation and effector cell differentiation is required for rejection of allogeneic grafts in na€ ıve recipients. It has become evident, that mitochondria play an important role for T cell activation. Expression of several mitochondrial proteins such as TCAIM (T cell activation inhibitor, mitochondrial) is down-regulated upon T cell receptor triggering. Here we report that TCAIM inhibited spontaneous development of memory and effector T cells. CD4 þ T cells from Tcaim knock-in (KI) mice showed reduced activation, cytokine secretion and proliferation in vitro. Tcaim KI T cells tolerated allogeneic skin grafts upon transfer into Rag-1 KO mice. CD4 þ and CD8 þ T cells from these mice did not infiltrate skin grafts and kept a na€ ıve or central memory phenotype, respectively. They were unable to acquire effector phenotype and functions. TCAIM altered T cell activation-induced mitochondrial distribution and reduced mitochondrial reactive oxygen species (mROS) production. Thus, TCAIM controls T cell activation and promotes tolerance induction probably by regulating TCR-mediated mitochondrial distribution and mROS production.

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The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long-term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti-CD4 antibody (aCD4). Here, we investigated whether adding TGF-β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg-cell generation and function. Murine CD4 + T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF-β+RA or aCD4+Rapa. Addition of TGF-β+RA or Rapa resulted in an increase of CD25 + Foxp3 + -expressing T cells. Expression of CD40L and production of IFN-γ and IL-17 was abolished in aCD4+TGF-β+RA aTreg cells. Additionally, aCD4+TGF-β +RA aTreg cells showed the highest level of Helios and Neuropilin-1 co-expression. Although CD25 + Foxp3 + cells from all culture conditions displayed complete demethylation of the Treg-specific demethylated region, aCD4+TGF-β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF-β+RA aTreg cells suppressed effector T-cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF-β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells. Keywords: Alloreactive regulatory T cells · Effector T cells · Nondepleting anti-CD4 monoclonal antibody · Retinoic acid · TGF-βAdditional supporting information may be found in the online version of this article at the publisher's web-site In recent years, adoptive transfer of Treg cells has gained major attention as an alternative or complementary therapy to conventional immunosuppressive treatments with the ultimate aim of reducing the side effects of conventional drugs [12,13]. Since only 5-10% of the circulating CD4 + cells in an organism are Foxp3 + Treg cells, their potential use for cell therapy seems to be limited and the peripheral population would require expansion [14]. Isolated CD4 + CD25 + cells frequently undergo expansion in the presence of aCD3/ aCD28 Ab and IL-2. Allo-specific expanded Treg cells seem to be more potent in suppressing chronic rejection, graft versus host disease (GvHD) and autoimmune diseases than polyclonal Treg cells. For example it was shown that antigen-specific expanded Treg (alloreactive Treg (aTreg)) cells could suppress experimental autoimmune diabetes more effectively than polyclonally expanded Treg cells [15].We have shown previously that in vitro culture of total murine CD4 + or CD25 − CD4 + cells in the presence of alloantigen and a nondepleting anti-CD4 antibody results in the enrichment of CD25 + CD62L + Foxp3 + T cells effective in controlling graft survival in vivo in an alloantigen-specific manner [16]. Although the in vitro enriched aTreg cells were effective in vivo, the protocol still has some limitations. To obtain almost pure Treg-c...

Cell therapy by tolerogenic dendritic cells has become an attractive therapeutic option in transplantation. We could recently show that high expression of TCAIM, formerly known as TOAG-1, in dendritic cells (DCs) is associated with stable tolerance following transplantation. Here, we investigated transcriptional control of TCAIM in murine bone marrow derived DCs (BMDCs) and human MUTZ-3 derived DCs (M3DCs). We have cloned the human TCAIM promoter to identify regulatory elements by site directed mutagenesis. In addition, we performed luciferase reporter as well as ChIP assays. Furthermore, BMDCs or M3DCs were pre-incubated with cAMP elevating drugs prior to LPS-mediated maturation. Phenotype and tolerogenic potential of DCs were investigated by qPCR, flow cytometry, CBA and MLR. Site-directed mutagenesis, reporter assays and ChIP defined TCAIM core promoter containing three CREB responsive elements. Co-transfections revealed positive regulation of promoter activity by NFATc1 and C/EBPβ. Furthermore, pre-incubation of M3DCs or BMDCs with cAMP elevating drugs leading to CREB activation resulted in high TCAIM expression, inhibited DC maturation, diminished pro-inflammatory cytokine release and induced high frequency of CD4+CD25+FoxP3+ Tregs in DC-T cell co-cultures. These results indicate that transcriptional activation of TCAIM is cAMP/CREB dependent and further amplified by NFATc1 and C/EBPβ, resulting in induction of tolerogenic potential in human M3DCs and murine BMDCs.

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