Likewise blockade of PD-1 signaling reverses tumor-induced T-cell exhaustion and enhances the functions of CD8+ T cells [37, 38] In the present work, PDL-1 and PDL-2 do not seem to be involved in the regulation of CD4+ T cells as we could not observe an effect on responding CD4+ T cells after neutralizing of these ligands on DX5+CD4+-modulated DCs (data not shown). Nonetheless, their high expression levels on DCs after modulation with DX5+CD4+ supernatants, combined with the phenomena described above, also point this website to the multiple pathways implemented DX5+CD4+ T cells to steer the outcome of T-cell responses. These pathways do not only involve

the modulation of cytokine secretion but also the expression of molecules known to affect T-cell responses. Whether the action check details of DX5+CD4+ T cells on DC function and phenotype is responsible for the effects observed in disease models is not known. Nonetheless, our findings are in line with data obtained in vivo indicating a preferential reduction of Th-1-associated IgG2a against collagen type II in CIA model following adoptive transfer of DX5+CD4+ T cells [19]. Likewise, some of our findings resemble the findings observed in studies focusing on FoxP3+ CD25+CD4+ Treg-cell-mediated immune regulation. Like DX5+CD4+ T cells, CD25+CD4+

Treg cells can exert their suppressive effect directly and indirectly via suppressing T-cell responses and altering the phenotype and the function of DCs, respectively. In addition, human Treg cells inhibit the maturation and antigen presentation of monocyte-derived DCs to become poor APCs [7, 10, 13-17]. Together our results point to the plethora of pathways

affected by DX5+CD4+ T cells that could be involved Nintedanib order in the control of autoimmune responses. Understanding of these pathways might be instrumental to further define potential immune modulating strategies with the aim to counteract autoimmune diseases. D011.10 (OVA-specific TCR Tg) mice were used for the generation of bone marrow DCs and for the isolation of CD4+ T cells and C57BL/6 mice were used for the generation of DX5+CD4+ T cells and DX5−CD4+ T cells. D011.10 mice were housed and bred in the animal facility of the Leiden University Medical Center. C57BL/6 mice were purchased from Charles River. Experiments were performed in accordance with our institutional guidelines on animal use in research. Splenocytes were isolated from spleens of mice that were injected three times (7, 5, and 3 days before purification of DX5+CD4+ T cells) intraperitoneally with 1 × 106 immature DCs in PBS. RBCs were lysed and CD4+ T cells were purified by positive selection using Dynal CD4 positive isolation kit (Invitrogen). Afterwards, DX5+ and DX5− cells were isolated from CD4+ T cells derived from the same mice using CD49 (DX5) MicroBeads (Miltenyi Biotec). The purity was 80–90%. DX5+CD4+ and DX5−CD4+ T cells were isolated from the same mice.