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

Abstract

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.

Figure 1

Gr-1⁺CD11b⁺F4/80⁺ cells from RMA-S tumor-bearing mice express RAE-1 and suppress T cell proliferation. (A) Peripheral blood leukocytes (PBLs) of naive and tumor-bearing mice isolated on day 21 after tumor cell inoculation were stained with mAbs specific for Gr-1 and CD11b in combination with mAbs directed against F4/80 and CD124 (IL-4Rα). Plots are gated on Gr-1⁺CD11b⁺ cells. Histograms were further gated on Gr-1⁺CD11b⁺F4/80⁻ (R1) or Gr-1⁺CD11b⁺F4/80⁺ (R2) cells to analyze the expression of CD124 (solid lines) compared with the respective isotype-matched control Ig (gray filled histograms). Flow cytometric analysis of PBLs of one representative of 5 animals per group is shown. (B) PBLs from tumor-bearing mice were gated on Gr-1⁺CD11b⁺F4/80⁻ (R1) or Gr-1⁺CD11b⁺F4/80⁺ (R2) as described above and analyzed for the expression of Ly6G. Gated cells are also depicted in a forward and side scatter plot. (C) PBLs from naive and tumor-bearing mice (day 21) were stained with mAbs specific for Gr-1 and F4/80 in combination with a mAb directed against panRAE-1 (solid lines) or the respective isotype-matched Ig controls (gray filled histograms). Histograms are gated on Gr-1⁺F4/80⁻ (R1) or Gr-1⁺F4/80⁺ (R2). For R1 and R2, percentages among all PBLs are indicated in the plots. (A-C) One representative experiment of at least 3 independent experiments is shown.

Figure 2

Gr-1⁺CD11b⁺F4/80⁺ cells from RMA-S tumor-bearing mice suppress T-cell proliferation. Sorted Gr-1⁺CD11b⁺F4/80⁺ (A) or Gr-1⁺CD11b⁺F4/80⁻ (B) cells isolated from tumor-bearing mice on day 21 after tumor cell inoculation were cocultured with splenocytes (spl) from naive mice for 72 hours with ConA at the indicated ratios. Proliferation was assessed by ³H-thymidine incorporation assay. Data show mean plus or minus standard deviation (SD) of triplicate cultures. Asterisks indicate statistical significance (P < .01) determined by Student t test. Representative data selected from 3 independent experiments are shown.

Figure 3

Gr-1⁺CD11b⁺F4/80⁺ cells from RMA-S tumor-bearing mice do not affect NK cell proliferation and cytotoxic activity. (A) NK cells isolated from naive mice were cultured with 400 U/mL IL-2 for 48 hours in the absence or presence of Gr-1⁺CD11b⁺F4/80⁺ cells from tumor-bearing mice at the indicated ratios. Proliferation was determined by ³H-thymidine incorporation assay. (B) Purified Gr-1⁺CD11b⁺F4/80⁺ or Gr-1⁺CD11b⁺F4/80⁻ cells from tumor-bearing mice were cultured with NK cells from naive mice for 4 hours and used as effector cells against YAC-1 targets at the indicated ratios. Specific killing was determined by a standard 4-hour ⁵¹Cr-release assay. Data represent mean plus or minus SD of triplicate cultures. (A,B) One representative experiment of 2 independent experiments is shown.

Figure 4

NK cells cocultured with Gr-1⁺CD11b⁺F4/80⁺ cells produce large amounts of IFN-γ. (A-D) Purified Gr-1⁺CD11b⁺F4/80⁺ or Gr-1⁺CD11b⁺F4/80⁻ cells from tumor-bearing mice were cocultured with NK cells at the indicated ratios. Supernatants were harvested after 24 hours and IFN-γ amounts were determined by ELISA. (A) NK cells from naive mice were cultured alone or in the presence of Gr-1⁺CD11b⁺F4/80⁺ cells (left panel) or Gr-1⁺CD11b⁺F4/80⁻ (right panel) in the absence of IL-12. (B) NK cells were cultured in IL-12 in the absence or in the presence of Gr-1⁺CD11b⁺F4/80⁺ (left panel) or Gr-1⁺CD11b⁺F4/80⁻ (right panel) cells at the indicated ratios. (C) Tumor-infiltrating Gr-1⁺CD11b⁺F4/80⁺ cells were purified and cultured with NK cells in the presence of IL-12. (D) NK cells isolated from C57BL/6 wt or IFN-γ^−/− mice were cultured in IL-12 in the absence of presence of Gr-1⁺CD11b⁺F4/80⁺ cells. Data depict mean plus or minus SD of triplicate cultures and represent one of 2 (A,C,D) or one of 5 (B) independent experiments. Asterisks indicate statistical significance (P < .01) determined by Student t test. ND indicates not detectable.

Figure 5

Induction of IFN-γ production is NO independent and is partially mediated by NKG2D. (A) NK cells purified from naive mice were cultured in IL-12 in the absence or presence of Gr-1⁺CD11b⁺F4/80⁺ cells isolated from RMA-S tumor bearing mice at the indicated ratios. As indicated, L-NMMA, D-NMMA, or medium only were added. Supernatants were harvested after 24 hours and IFN-γ production was determined by ELISA. (B) Gr-1⁺CD11b⁺F4/80⁺ cells were purified from RMA-S tumor-bearing mice. Gr-1⁺CD11b⁺F4/80⁺ cells were cultured alone or cocultured with splenocytes (spl.) from naive C57BL/6 mice with ConA in the absence or presence of the NO inhibitor L-NMMA or its inactive isoform D-NMMA at the indicated ratios for 72 hours. Proliferation was determined by ³H-thymidine incorporation assay. (C) NK cells were cultured in IL-12 in the absence or presence of Gr-1⁺CD11b⁺F4/80⁺ cells from tumor-bearing mice at a 1:1 ratio using a standard plate or a transwell system. IFN-γ production was determined by ELISA. (D) Gr-1⁺CD11b⁺F4/80⁺ cells were sorted from PBL of tumor-bearing mice. Fab fragments of the anti-NKG2D mAb or of an isotype-matched control mAb were added to cocultures of NK cells and Gr-1⁺CD11b⁺F4/80⁺ cells. Data are shown as mean plus or minus SD of triplicates. One representative experiment of 3 experiments is shown. *P < .05. ND indicates not detectable.

Figure 6

Neutralization of IFN-γ, and depletion of NK1.1⁺ or Gr-1⁺ cells in vivo leads to enhanced tumor growth. C57BL/6 mice (n = 10) were treated with PBS (), isotype-matched control mAb (□), a neutralizing anti–IFN-γ mAb (A, ■), a depleting anti-NK1.1 mAb (B, ■), or an anti–Gr-1 mAb (C and D, ▴) and were inoculated subcutaneously with either RMA-S (A,B,C) or RMA lymphoma cells (D). (A-D) Tumor growth was monitored and is presented as mean plus or minus SD. Asterisks indicate statistical significance of P < .01, determined by a one-way analysis of variance (ANOVA) test. One representative experiment of at least 2 independent experiments is shown.

Figure 7

Gr-1⁺CD11b⁺F4/80⁺ cells are eliminated by NK cells in vitro and in vivo. (A) IL-2–activated NK cells were cultured with Gr-1⁺ cells isolated from PBLs of tumor-bearing mice at the indicated effector-target ratios for 12 hours. Percentages of Gr-1⁺CD11b⁺F4/80⁺ cells among Gr-1⁺ were determined by flow cytometry. Data are presented as mean plus or minus SD of triplicate cultures. One representative experiment of 3 independent experiments is shown. (B) NK cells were isolated from naive C57BL/6 mice. NK cells (4-6 × 10⁶) were injected intravenously into C57BL/6-Ly5.1 tumor-bearing mice (n = 9) at day 18 or 19 after tumor cell inoculation. Percentages of Gr-1⁺CD11b⁺F4/80⁺ and Gr-1⁺CD11b⁺F4/80⁻ cells among total PBLs were determined by flow cytometry 4 hours before and 5 hours after transfer. Injected NK cells were excluded by gating on Ly5.2-negative cells. In control experiments, mice were mock-treated as described in “Methods.” *P < .05. NS indicates not statistically significant.