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Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.

We studied the effect of interferon-gamma (IFN-gamma) on the proliferation of lymphocytes from 10 B-type chronic lymphocytic leukemia (B-CLL) patients. In no instance did IFN-gamma induce a proliferative response whether used alone or in combination with anti-mu antibody (Ab). This was observed regardless of the responsiveness of a given patient's cells to interleukin 2 (IL 2) and to B cell growth factor (BCGF). In contrast IFN-gamma strongly and reproducibly synergized with IL 2 (but not with BCGF) to support B-CLL proliferation in five of the 10 patients. The effect of IFN-gamma was dose related and could be inhibited by an anti-IFN-gamma monoclonal Ab. A monoclonal Ab toward the IL 2 receptor molecule was also inhibitory. Preincubation with IFN-gamma potentiated the responsiveness of B-CLL to IL 2 in secondary cultures, showing that IFN-gamma exerts its effect before that of IL 2.

We studied the effect of recombinant interferon-alpha (IFN-alpha) on the proliferative response of normal human B cells and of lymphocytes from 10 patients with B cell-type chronic lymphocytic leukemia. We show that IFN-alpha, which has no effect on B cell proliferation when used alone, can synergize with anti-mu antibody and can support DNA synthesis by anti-mu activated B cells. This was observed with normal B cells and in 4 of 10 patients with B cell-type chronic lymphocytic leukemia. These four patients were all responders to B cell growth factor (BCGF), whereas the responsiveness to IFN-alpha was not correlated with that of IL-2. The positive functional relationship between IFN-alpha and BCGF in these patients is emphasized by the synergistic effect of these two factors in two of these patients, and the lack of inhibition of BCGF response by even supraoptimal concentrations of IFN-alpha. Among the subgroup of patients unable to respond to IFN-alpha, a synergy between IFN-alpha and IL-2 could be observed.

In the presence of anti-mu Ab, B-CLL lymphocytes are able to respond to cytokines such as IL2, BCGF and IFN-alpha. IL2 is most frequently an active factor. According to the B-CLL lymphocytes population, IL2 alone can induce growth or maturation of B cells. IFN-gamma and IL2 are inefficient on B-CLL lymphocyte proliferation, but they can profoundly modulate the response to IL2. IFN-gamma can synergize with IL2. In contrast IL4 profoundly and constantly inhibits the response to IL2.