1. Volume 34, Issue 3, Pages 385-395 (March 2011)
Paired Immunoglobin-like Receptor-B Regulates the Suppressive Function and Fate of Myeloid-Derived Suppressor Cells 
Ge Ma, Ping-Ying Pan, Samuel Eisenstein, Celia M. Divino, Clifford A. Lowell, Toshiyuki Takai, Shu-Hsia Chen 
Immunity 
Volume 34, Issue 3, Pages (March 2011)
DOI: /j.immuni
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2. Immunity 2011 34, 385-395DOI: (10.1016/j.immuni.2011.02.004)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 M2 Phenotype Is Essential for Treg Cell Activation by MDSCs
(A) CFSE-labeled CD4+CD25+ Treg cells from OT-II transgenic mice were cocultured 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 six reproducible experiments.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Lilrb3−/− Splenic MDSCs but Not Bone Marrow-Derived MDSCs Exhibit an M1 Phenotype
(A) Suppression assay with various ratios of MDSCs from tumor-bearing WT and Lilrb3−/− bone marrow (left) and spleen (Right). Coculture with splenocytes from naive OT-II transgenic mice, stimulated with OT-II peptide, is shown. Proliferation count per minute: CPM × Bars and SD from triplicate wells are shown (∗p < 0.05).
(B) WT and Lilrb3−/− MDSCs from bone marrow and spleen were cocultured with OT-II splenocytes at a 1:4 ratio for 5 days and subsequently stained 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 hr. Arginase activity and IL-10, NO, and TNF-α 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. Colored lines correspond to the following: gray line, isotype control; blue line, wild-type MDSC; and 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 < 0.01; ∗∗∗p < 0.001).
Immunity  , DOI: ( /j.immuni )
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5. Figure 3
Lilrb3−/− Tumor Environment Retards Tumor Growth and Gives Rise to M1-Polarized MDSCs
(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. The black column showsWT TIL MDSCs, and the white column shows 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. Colored lines and their corresponding items are as follows: gray line, isotype control; blue line, WT MDSC; and red line, Lilrb3−/− MDSC. Data shown re 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, and CCR7 were colocalized with DAPI separately or merged, and four 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 comparison of the MDSC transfer group with without MDSC adoptive transfer control group (∗p < 0.05, ∗∗p < 0.01).
(G) The effect of MDSC adoptive transfer on lung metastasis. The left panel shows the long-term survival rate of treated mice. The right panel shows the lung weight. The survival rate of mice receiving Lilrb3−/− MDSCs is significantly higher than that of mice injected with WT MDSC (log-rank test, p = ). 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).
Immunity  , DOI: ( /j.immuni )
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6. Figure 4
PIR-B Is Essential for MDSC-Mediated Treg Cell Expansion, Immune Suppression, and Tumor Promotion
OVA-LLC-bearing MaFIA mice (CD45.2) were left untreated or depleted of CD115+ cells; this process was followed by adoptive transfer of CD45.1 OT-II T cells and reconstitution with WT or Lilrb3−/− MDSCs.
(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 and then analyzed 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 reisolated from the spleen of recipient mice and stimulated with OVA peptide (1 μg/mL) in the presence of irradiated naive splenocytes for 3 days. Cells were pulsed with [3H]-thymidine for the last 8 hr 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.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
PIR-B Regulates M1 versus M2 Polarization in MDSCs through Jak/STAT and TLR Pathways
(A, C, and D) Western blots were performed with 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, and 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, and this was followed by intracellular phosphorylation staining of p-STAT1 and p-STAT-3. The thin line refers to isotype control, the gray line refers to WT MDSC, and the black line refers to Lilrb3−/− MDSC. MFI were calculated (second versus first column, ∗p = 0.030, fourth versus third column, ∗∗p = 0.005), Data shown are from one of five reproducible experiments.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
Upregulation of PIR-A in Lilrb3−/− MDSCs, and Regulation of MDSC Polarization by PIR-B Blockade
(A) Real-time quantitative PCR results for PIR-A and PIR-B. Relative copy numbers were calculated in relation to β-actin. The black column shows WT MDSCs, and the white column shows Lilrb3−/− MDSCs. Data shown is from one of two reproducible experiments.
(B) Splenocytes from naive 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. The thin line refers to isotype control, the gray line refers to wild-type MDSC, and the black line refers to Lilrb3−/− MDSC. Data shown are from one of four reproducible experiments.
(C) MDSCs from WT, Lilrb3−/−, and β2m-deficient tumor-bearing mice or WT MDSC treated with NSC (SHP inhibitor) were cultured in the presence or absence of IL-13 or IFN-γ (50 ng/ml) for 24 hr. 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 mev/mev tumor-bearing mice had an M1 phenotype, whereas FcRγ-deficient MDSCs had an M2 phenotype. Gr-1+CD115+ MDSCs were purified from WT, Lilrb3−/−, mev/mev, 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.
Immunity  , DOI: ( /j.immuni )
Copyright © 2011 Elsevier Inc. Terms and Conditions