4B); however, NK cells from 4T1/IL-1β-tumor-bearing mice expressed 5–10 times less CD27 protein than NK cells from the other mice (Fig. 4B). Moreover, the tumor-bearing mice contained less CD11b+ NK cells in the bone marrow (Fig. 4A (right) and B) indicating a block in the differentiation of NK cells in these mice. In contrast to the BM, the total number of splenic NK cells was five-fold

increased in both groups of tumor-bearing mice (Fig. 4A). More importantly, CD11b+ and KLRG-1+ cells were absent from 4T1/IL-1β-tumor-bearing mice, while splenic NK cells from 4T1-tumor-bearing mice expressed CD11b and KLRG1 at levels and frequencies comparable to naïve mice (Fig. 4B and C). Further analyses showed a rapid down-modulation of NKG2D but not NKp46 expression by NK cells after injection of 4T1- and 4T1/IL-1β-tumor cells. The reduced expression of NKG2D occurred earlier and was more pronounced Z-VAD-FMK clinical trial in 4T1/IL-1β- than in 4T1-tumor bearing mice (Fig. 5A and data not shown). To explore whether the MDSC subsets were involved in the reduction of NKG2D expression by NK cells, we sorted Ly6Clow MDSC and Ly6Cneg MDSC from the spleens of 4T1- or 4T1/IL-1β-tumor-bearing mice,

respectively, and co-cultured them for 24 h with selleck kinase inhibitor splenocytes from naïve Rag2−/− mice in the presence of IL-2. We observed a stronger reduction of NKG2D expression by Rag2−/− NK cells when co-cultured with Ly6Cneg MDSC as compared with Ly6Clow MDSC (Fig. 5B, top). Furthermore, transwell experiments revealed GNAT2 that NKG2D downregulation was cell–cell contact dependent (Fig. 5B, middle). We obtained similar results in vivo after adoptive transfer of purified Ly6Cneg MDSC and Ly6Clow MDSC, respectively,

into naïve Rag2−/− mice. NK cells from Rag2−/− mice given Ly6Cneg MDSC displayed reduced expression of NKG2D 2 days after transfer, while NKG2D levels remained unchanged on NK cells from mice transplanted with Ly6Clow MDSC (Fig. 5B, bottom). Together, these results indicated that MDSC subsets induce the downregulation of NKG2D on the cell surface of NK cells and that Ly6Cneg MDSC were more potent in this process in vitro and in vivo. We next addressed whether the down modulation of NKG2D expression was associated with functional impairment of NK cells in vivo. We adoptively transferred enriched MDSC isolated from BM and spleen of 4T1- or 4T1/IL-1β-tumor-bearing mice, respectively, intravenously into naïve BALB/c mice and challenged them 2–3 days later with luciferase-expressing YAC-1 target cells (Luc-YAC-1). As few as 7–8 h thereafter, NK cell activity was significantly lower in mice that had received MDSC from the BM and spleen of 4T1/IL-1β-tumor-bearing mice as compared to those having received MDSC from 4T1-tumor-bearing mice or Gr-1+CD11b+ cells from naive mice (Fig. 5C). There was no clearance of Luc-YAC-1 cells in NK-deficient Rag2−/−IL-2Rβ−/− mice within the 8-h period confirming NK cells as the effectors (Supporting Information Fig. 5).