Tumor-infiltrating monocytic myeloid-derived suppressor cells mediate CCR5-dependent recruitment of regulatory T cells favoring tumor growth.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of myeloid cells in cancer patients and tumor-bearing mice that potently inhibits T cell responses. During tumor progression, MDSCs accumulate in several organs, including the tumor tissue. So far, tumor-infiltrating MDSC subpopulations remain poorly explored. In this study, we performed global gene expression profiling of mouse tumor-infiltrating granulocytic and monocytic (MO-MDSC) subsets compared with MDSCs from peripheral blood. RMA-S lymphoma-infiltrating MO-MDSCs not only produced high levels of NO and arginase-1, but also greatly increased levels of chemokines comprising the CCR5 ligands CCL3, CCL4, and CCL5. MO-MDSCs isolated from B16 melanoma and from skin tumor-bearing ret transgenic mice also expressed high levels of CCL3, CCL4, and CCL5. Expression of CCR5 was preferentially detected on regulatory T cells (Tregs). Accordingly, tumor-infiltrating MO-MDSCs directly attracted high numbers of Tregs via CCR5 in vitro. Intratumoral injection of CCL4 or CCL5 increased tumor-infiltrating Tregs, and deficiency of CCR5 led to their profound decrease. Moreover, in CCR5-deficient mice, RMA-S and B16 tumor growth was delayed emphasizing the importance of CCR5 in the control of antitumor immune responses. Overall, our data demonstrate that chemokines secreted by tumor-infiltrating MO-MDSCs recruit high numbers of Tregs revealing a novel suppressive role of MDSCs with potential clinical implications for the development of cancer immunotherapies.

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway.

The triggering receptor expressed on myeloid cells 1 (TREM-1) has been implicated in the production of proinflammatory cytokines and chemokines during bacterial infection and sepsis. For downstream signal transduction, TREM-1 is coupled to the ITAM-containing adaptor DAP12. Here, we demonstrate that Bruton tyrosine kinase (Btk), a member of the Tec kinases, becomes phosphorylated upon TREM-1 triggering. In U937-derived cell lines, in which expression of Btk was diminished by shRNA-mediated knockdown, phosphorylation of Erk1/2 and PLCγ1 and Ca²âº mobilization were reduced after TREM-1 stimulation. Importantly, TREM-1-induced production of the pro-inflammatory cytokines, TNF-α and IL-8, and up-regulation of activation/differentiation cell surface markers were impaired in Btk knockdown cells. Similar results were obtained upon TREM-1 stimulation of BMDCs of Btk(-/-) mice. The analysis of cells containing Btk mutants revealed that intact membrane localization and a functional kinase domain were required for TREM-1-mediated signaling. Finally, after TREM-1 engagement, TNF-α production by PBMCs was reduced in the majority of patients suffering from X-linked agammaglobulinemia (XLA), a rare hereditary disease caused by mutations in the BTK gene. In conclusion, our data identify Btk as a positive regulator in the ITAM-mediated TREM-1/DAP12 pathway and suggest its implication in inflammatory processes.

Mononuclear myeloid-derived "suppressor" cells express RAE-1 and activate natural killer cells.

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice and potently suppress T-cell activation. In this study, we investigated whether MDSCs regu-late natural killer (NK)-cell function. We discovered that mononuclear Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from RMA-S tumor-bearing mice do not suppress, but activate NK cells to produce high amounts of IFN-gamma. Gr-1(+)CD11b(+)F4/80(+) MDSCs isolated from tumor-bearing mice, but not myeloid cells from naive mice, expressed the ligand for the activating receptor NKG2D, RAE-1. NK-cell activation by MDSCs depended partially on the interaction of NKG2D on NK cells with RAE-1 on MDSCs. NK cells eliminated Gr-1(+)CD11b(+)F4/80(+) MDSCs in vitro and upon adoptive transfer in vivo. Finally, depletion of Gr-1(+) cells that comprise MDSCs confirmed their protective role against the NK-sensitive RMA-S lymphoma in vivo. Our study reveals that MDSCs do not suppress all aspects of antitumor immune responses and defines a novel, unexpected activating role of MDSCs on NK cells. Thus, our results have great impact on the design of immune therapies against cancer aiming at the manipulation of MDSCs.

Metalloprotease-mediated tumor cell shedding of B7-H6, the ligand of the natural killer cell-activating receptor NKp30.

Natural killer (NK) cells are potent immune effector cells capable of mediating antitumor responses. Thus, during immunoediting, tumor cell populations evolve strategies to escape NK-cell-mediated recognition. In this study, we report a novel mechanism of immune escape involving tumor cell shedding of B7-H6, a ligand for the activating receptor NKp30 that mediates NK-cell binding and NK-cell-mediated killing. Tumor cells from different cancer entities released B7-H6 by ectodomain shedding mediated by the cell surface proteases "a disintegrin and metalloproteases" (ADAM)-10 and ADAM-17, as demonstrated through the use of pharmacologic inhibitors or siRNA-mediated gene attenuation. Inhibiting this proteolytic shedding process increased the levels of B7-H6 expressed on the surface of tumor cells, enhancing NKp30-mediated activation of NK cells. Notably, we documented elevated levels of soluble B7-H6 levels in blood sera obtained from a subset of patients with malignant melanoma, compared with healthy control individuals, along with evidence of elevated B7-H6 expression in melanoma specimens in situ. Taken together, our results illustrated a novel mechanism of immune escape in which tumor cells impede NK-mediated recognition by metalloprotease-mediated shedding of B7-H6. One implication of our findings is that therapeutic inhibition of specific metalloproteases may help support NK-cell-based cancer therapy.