To assess whether MO-MDSCs sensitize T cells to Fas-mediated apoptosis, the Fas agonistic antibody Jo2 or control antibody were added to the cocultures. Fas ligation massively induces CD8+ T-cell death in the presence of MO-MDSCs at 42 h, but not in any other condition, in agreement with the Fas expression data (Fig. 6B). These findings clearly illustrate that splenic MO-MDSCs further augment the activation-induced upregulation

of Fas and sensitize CD8+ T cells to Fas-mediated apoptosis. Finally, we analyzed to which extent splenic MDSC subsets affect the cytotoxic activity of CD8+ T cells. One of the major pathways utilized Daporinad in vitro by CTLs to eliminate target

cells is via granzyme B exocytosis [8]. Following 3 days of OVA stimulation, PMN-MDSCs had no effect on the presence of granzyme B in the remaining viable OT-1 T cells, while MO-MDSCs significantly reduced its expression in those cells (Fig. 7A), suggesting that MO-MDSC-treated CD8+ T cells have a diminished killing capacity. Therefore, viable CD8+ T cells were purified from OVA-stimulated cocultures and their cytotoxic activity was assessed against EG7-OVA and control EL-4 cells. In agreement with the granzyme B data, only MO-MDSCs were able to strongly reduce antigen-specific cytotoxicity (Fig. 7B). When MO-MDSCs were only added during the 4 h effector phase, Bumetanide neither the effect on CTL cytotoxicity could be recorded (Supporting Information signaling pathway Fig. 13A), nor were the MO-MDSCs from EG7-OVA tumor bearers killed by the OVA-specific CTLs (Supporting Information Fig. 13B). These data show that, although both splenic MDSC subsets diminish the number of CTLs due to their antiproliferative effect, only MO-MDSCs

also actively impede the formation of mature CTLs, but cannot obstruct the cytotoxic activity of existing mature CTLs. CD8+ T-cell activation and differentiation is a tightly regulated process, involving massive alterations in surface marker expression, cytokine secretion, and proliferative, migratory, and cytotoxic potential. Evidence exists that these features can be regulated independently from each other [3, 4], for example, upon interaction with immunoregulatory cells such as Treg cells [9]. MO- and granulocytic (PMN-) MDSCs both interfere with CD8+ T-cell proliferation [11, 12], but their effects on other features of early CD8+ T-cell activation are largely unknown. Here, we show that splenic MDSC subsets differentially modulate multiple aspects of CD8+ T-cell activation, encompassing both inhibitory and stimulatory effects, resulting in a distinct functional outcome (for overview: Supporting Information Table 1).