However, the inhibitory effect was found in the SN of R-DC-induced Treg (Fig. 2A). Both purified CD4+ and CD8+ peripheral blood T cells were cocultured with R-DC and each of their SNs contained this suppressive factor (Fig. 2B and C), because the SN again showed a strong T-cell inhibitory capacity. This factor was not released by naïve T cells, isolated from human CB, as the SN of naïve T cells cocultured with R-DC was not inhibitory (Fig. 2D). Additionally, naïve T cells cocultured with GSI-IX R-DC did not show a reduced proliferation (Supporting Information Fig. 1A and B). This contrasts strongly to the finding that in

the coculture of peripheral blood T cells and R-DC, T-cell proliferation is impaired 12. Thus, R-DC-mediated inhibition is specific for CD4+ and CD8+ effector T cells, but not for naïve T cells. Inducible Treg can develop from mature T-cell populations under certain conditions, e.g. upon stimulation with tolerogenic DC. They act via release of soluble factors such as IL-10, a well established inhibitory molecule. In order to Neratinib elucidate if the inhibitory effect was mediated through IL-10 or other factors, we added the SN of our R-DC-induced Treg to an MLR and investigated whether the inhibitory effect was reversible with neutralizing Ab to IL-10, TGF-β, or IFN-α 11, 19.

The levels of the respective factors in the T-cell/R-DC SN were determined previously 12. The inhibitory quality of the SN of R-DC-induced old Treg was not reversible with mAb against IL-10, IFN-α, and TGF-β (Fig. 3A). The inhibitory effect of IL-10, TGF-β or IFN-α on a T-cell/DC coculture and the reversibility of this effect with neutralizing Ab is depicted in Supporting Information Fig. 2. Furthermore, size fractionation of the T-cell/R-DC SN revealed that the inhibitory factor is found in the >50 kDa fraction and not in the <50 kDa molecular weight range (Fig. 3B). The observation that the inhibitory factor is expected to be >50 kDa leads us to investigate IL-35, a heterodimeric cytokine consisting of EBI3 and the p35 subunit of IL-12 with inhibitory

function and a molecular size of 78 kDa 5. We found that T cells cultured with R-DC showed elevated levels of EBI3 and p35 mRNA, but no changes in the p28 levels, which forms IL-27 together with EBI3 (Fig. 4A). Furthermore, intracellular stainings showed that EBI3 was also upregulated at the protein level in peripheral blood T cells, stimulated with R-DC in comparison to T cells cocultured with DC (Fig. 4B, left column). In naïve T cells stimulated with R-DC we did not observe an upregulation of EBI3 (Fig. 4B, right column). P35 is constitutively expressed in DC or R-DC stimulated peripheral blood T cells or naïve T cells (Fig. 4B). This is in accordance to previous findings, which show that p35 is constitutively expressed in various types of human T cells 6.