Mononuclear myeloid-derived “suppressor” cells express RAE-1 and activate natural killer cells by Norman Nausch, Ioanna E. Galani, Eva Schlecker, and Adelheid Cerwenka Blood Volume 112(10):4080-4089 November 15, 2008 ©2008 by American Society of Hematology

Gr-1+CD11b+F4/80+ cells from RMA-S tumor-bearing mice express RAE-1 and suppress T cell proliferation. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

Gr-1+CD11b+F4/80+ cells from RMA-S tumor-bearing mice suppress T-cell proliferation. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

Gr-1+CD11b+F4/80+ cells from RMA-S tumor-bearing mice do not affect NK cell proliferation and cytotoxic activity. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

NK cells cocultured with Gr-1+CD11b+F4/80+ cells produce large amounts of IFN-γ. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

Induction of IFN-γ production is NO independent and is partially mediated by NKG2D. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

Neutralization of IFN-γ, and depletion of NK1 Neutralization of IFN-γ, and depletion of NK1.1+ or Gr-1+ cells in vivo leads to enhanced tumor growth. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology

Gr-1+CD11b+F4/80+ cells are eliminated by NK cells in vitro and in vivo. 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. Norman Nausch et al. Blood 2008;112:4080-4089 ©2008 by American Society of Hematology