A) Suppression assay using various ratios of MDSCs from tumor-bearing WT and Lilrb3^−/− bone marrow (left) and spleen (Right). Co-culture with splenocytes from naïve OT-II transgenic mice, stimulated with OT-II peptide. Proliferation count per minute: CPM ×1,000, Bars, and SD from triplicate wells. N=7, p = 0.8788 B) WT and Lilrb3^−/− MDSCs from bone marrow and spleen were co-cultured with OT-II splenocytes at a 1:4 ratio for 5 days, followed by staining with CD4, CD25, and Foxp3 antibodies. The CD4⁺ cell population was gated to show Treg cell percentages. Data presented are from one of seven reproducible experiments. C) Lilrb3^−/− splenic MDSCs exhibit an M1 phenotype. Splenic MDSCs from WT or Lilrb3^−/− tumor-bearing mice were cultured in culture media in the absence of any stimulation for 24 hrs. Arginase activity, and IL-10, NO, and TNFα production in the culture supernatant were measured. D) Purified splenic MDSCs were stained for scavenger receptor B (CD36), mannose receptor (CD206), IL-4R, Tie2, PDL1, CD80, CD86, MHC class II, and CC7. Expression was evaluated in the Gr-1⁺CD115⁺ cell population. Gray line: isotype control, Blue line: wild type MDSC, Red line: Lilrb3^−/− MDSC. Data was from one of three reproducible experiments. E) Real time PCR for CCR7 and CCR2 expression with GAPDH internal control (*, P < 0.05; ***, P < 0.001).

OVA-LLC-bearing MaFIA mice (CD45.2) were left untreated or depleted of CD115⁺ cells followed by adoptive transfer of CD45.1 OT-II T cells and reconstitution with WT or Lilrb3^−/− MDSC. A) Tumor (OVA)–specific CD4⁺CD25⁺Foxp3⁺ Treg cells in the tumor and spleen. TILs were isolated and stained with antibodies against CD45.1, CD4, CD25, and Foxp3 or isotype controls followed by analysis with flow cytometry. Contour plots were gated on the CD45.1⁺CD4⁺ population. One of three reproducible experiments (n = 6–7 per group) is presented. B) Percentage of tumor (OVA)– specific Treg cells in the tumor (fourth versus third column) C) Tumor weight. Tumors were resected from tumor-bearing mice and weighed (fourth versus third column,). Data were combined from three reproducible experiments (n = 6). D) Proliferative response of tumor (OT-II)–specific T cells from recipient MaFIA mice. CD45.1⁺ OT-II T cells were re-isolated from the spleen of recipient mice and stimulated with OVA peptide (1 μg/mL) in the presence of irradiated naïve splenocytes for 3 days. Cells were pulsed with [³H]-thymidine for the last 8 hours of culture (fourth versus third column, **P = 0.003). E) Tumor vascularity assessed by immunofluorescent CD31 (red) staining with DAPI (blue) background in OVA-LLC tumors from mice that received injections of WT or Lilrb3^–/– MDSCs.

A and C) Western blots were performed using MDSCs stimulated with IFN-γ (50 ng/ml), IL-10 (50 ng/ml), IL-13 (50 ng/ml), or LPS (100 ng/ml) for 10 and 30 min. Mean gray value was measured by ImageJ 1.43, relative activation is calculated by dividing phospho-protein by total protein or β-actin (* p<0.05, ** p<0.01). B) Purified MDSCs were treated with IFN-γ (50 ng/ml) or IL-10 (50 ng/ml) for 10 min, followed by intracellular phosphorylation staining of p-STAT1 and p-STAT-3. Thin line: isotype control, Gray line: WT MDSC, Black line: Lilrb3^−/− MDSC. MFI were calculated (second versus first column, * p = 0.030, fourth versus third column, ** p = 0.005), Data shown is from one of five reproducible experiments.

A) Real-time quantitative PCR results for PIRA and PIR-B. Relative copy numbers were calculated in relation to β-actin. Black column: WT MDSCs, white column: Lilrb3^−/− MDSCs. Data shown is from one of two reproducible experiments. B) Splenocytes from naïve or tumor-bearing mice were stained with Gr-1, CD115, and PIR-A and -B antibodies, which stains both PIR-A and PIR-B. Expressions of PIR-A and -B were compared by gating on the Gr-1⁺CD115⁺ MDSC population. Thin line: isotype control, Gray line: wild type MDSC, Black line: Lilrb3^−/− MDSC. Data shown is from one of four reproducible experiments. C) MDSCs from WT, Lilrb3^−/−, and β2m deficient tumor-bearing mice or WT MDSC treated with NSC 87877 (SHP inhibitor) were cultured in the presence or absence of IL-13 (50 ng/ml) for 24 hours. INOS and ARG1 activity, and TNFα and IL-10 production in supernatant were measured by colorimetric assay and ELISA, respectively (*P < 0.05, **P < 0.01). D) MDSCs from SHP-1 mutated me^v/me^v tumor bearing mice had an M1 phenotype while FcRγ-deficient MDSCs had an M2 phenotype. Gr-1⁺CD115⁺ MDSCs were purified from WT, Lilrb3^−/−, me^v/me^v, and FcRγ-deficient tumor bearing mice. ARG1 activity, NO production, and IL-10 and TNF-α secretion were measured by colorimetric assay and ELISA, respectively.

A) CFSE-labeled CD4⁺CD25⁺ Treg cells from OT-II transgenic mice were co-cultured with MDSCs from tumor-bearing mice, stimulated with 0.5 μg/ml OT-II peptide for 3 days, and stained with Foxp3 and CD4 antibodies. Proliferation percentages are shown in the dot plot (upper panel) and histograms (lower panel). B) MDSC-mediated Treg cell activation in the presence of various inhibitors and anti-IL-10. Data shown are from representative of 6 reproducible experiments.

A) LLC tumor was implanted subcutaneously in WT or Lilrb3^−/− mice (n=6) and tumor size was measured every 6 days for 30 days. *, P < 0.05; **, P < 0.01. B) Real time quantitative PCR was performed on Gr-1⁺CD115⁺ MDSCs isolated from TILs. Relative copy number was calculated based on β-actin. Black column: WT TIL MDSCs, white column: Lilrb3^−/− TIL MDSCs. Data presented is from one of four reproducible experiments, **, P < 0.01. C) TILs from WT or Lilrb3^−/− tumor tissue were stained for Gr-1, CD115, CD36, CD206, Tie2, and IL-4R. Expressions of these proteins were compared via histogram. Grey line: isotype control, Blue line: WT MDSC, Red line: Lilrb3^−/− MDSC. Data shown is from one of four reproducible experiments. D) CD11b (green), CD206 (yellow), CCR7 (red), and DAPI (blue), or F4/80 (green), ARG1 (yellow), iNOS (red), and DAPI (blue) immunofluorescent staining of LLC tumor tissues from WT or Lilrb3^−/− mice. CD11b, CD206, CCR7 were co-localized with DAPI separately or merged, 4 specimens per group per staining combination were examined. E) Tumor cytotoxicity mediated by monocytic MDSCs. Tumor killing activities were measured as percentage of tumor cell death. Data shown are from one of four reproducible experiments. F) Effect of adoptive cell transfer of MDSC on subcutaneous LLC tumor growth. Student's t-test was performed by comparing MDSC transfer group with MDSC adoptive transfer group (*P < 0.05, **P < 0.01). G) The effect of MDSC adoptive transfer on lung metastasis. Upper panel: Long-term survival rate of treated mice. Lower panel: Lung weight. The survival rate of mice receiving Lilrb3^−/− MDSCs is significantly higher that that of mice injected with WT MDSC (Log-rank test, P = 0.0036). On day 25, representative mice were sacrificed and lung weights were measured. Lungs from mice receiving Lilrb3^−/− MDSCs weighed significantly less than those from mice receiving PBS or WT MDSC (**P < 0.01).