Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: The concurrent increases of MDSCs and B cells in breast cancer mice and surface molecules of splenic B cells and MDSCs in tumor or normal mice. Wide-type BALB/c mice were injected subcutaneously with 1 × 106 4T1 murine breast cancer cells, and were sacrificed at day 21 after tumor inoculation. (A) The brief descriptions of tumor-bearing status were shown in the left and middle panel. The overall splenic cells were analyzed from tumor-bearing and tumor-free mice (right panel). (B) Paraffin-embedded spleen tissues were collected from tumor-bearing mice to detect CD19+B cells (left panel) and CD11b+ MDSCs (right panel) by IHC staining (original magnification × 200 and × 400). (C) The percentage of B cells was detected using anti-CD19 Ab from tumor and normal splenic cells. (D) Splenic MDSCs were stained for CD11b and Gr-1, and detected by FC (right panel). The percentages are indicated in the representative dot plots (left two panels). (E) Isolated splenic MDSCs were assessed for their ability to suppress CD3+ T-cell proliferation at a 1:1, 3:1 or 5:1 of Gr-1+CD11b+cell: T-cell ratio. (F) FC analysis of immune checkpoint molecules including PD-1, PD-L1, CTLA-4, Tim-3, CD200R and CEACAM showing their expression frequencies in B cells. (G) Expression of the co-stimulatory molecules and activation markers including CD80, CD86, MHC II, CCR6 and CD62L on the surface of splenic CD19+ B cells from 4T1 tumor-bearing and control mice. (H) Immune checkpoint molecules on MDSCs were measured by FC from normal and tumor-bearing mice. The results are shown as the mean±SEM from five independent experiments with 8 mice per group per experiment. The t test or One-way ANOVA analysis were used for statistical analysis.* = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant. FC, flow cytometry; MDSC, Myeloid-derived suppressor cells; IHC, immunohistochemistry.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Injection, Immunohistochemistry, Staining, Isolation, Expressing, Activation Assay, Flow Cytometry, Derivative Assay

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: MDSCs regulate B-cell function through a contact-dependent mechanism. Purified splenic B cells were cultured alone or with MDSCs in the presence or absence of IL-4 (200 ng/ml) plus anti-CD40 antibody (10μg/ml) (A) The proliferation of CD19+ B cells was assessed by FC using BrdU labeling method. (B) CD19+B cell apoptosis was detected by FC using an Apoptosis Detection Kit. (C) The expression of immune checkpoint molecules (upper panel) and co-stimulatory or activation markers (bottom panel) on the surface of B cells were analyzed by FC. (D) The antibody subtypes (IgA, IgG, and IgM) and cytokine (TNF-α, TGFβ1) concentrations were determined from the culture supernatants after 48 h with specific ELISA Kit. ELASA data were collected from 5 independent experiments. (E) IL-10 and (F) IFN-γ production in the co-cultured groups were both detected by ELISA (left panels) and FC analysis (right panels). Data represent the mean±SEM of 5 independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant, as determined with One-way ANOVA analysis. FC, flow cytometry; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; MDSCs, Myeloid-derived suppressor cells; IL, interleukin; IFN, interferon.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Cell Function Assay, Purification, Cell Culture, Labeling, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: A typical PD-1−PD-L1+CD19+ B-cell subtype exert immuno-regulatory properties. (A) The left two panels show a representative dot plots of PD-1−PD-L1+ B cells gated on CD19+ cells from normal or tumor-bearing spleen. The right panel depicted values from 8 independent experiments. (B) Representative FC analysis showing the percentage of PD-1−PD-L1+CD19+ B cells at 48 h in the presence or absence of MDSCs in the culture system (B1). B2 shows summarized data from 5 independent experiments. (C) The percentage of PD-1−PD-L1+CD19+ B cells in BrdU-labeled B cells at 48 h with or without MDSCs from 8 independent experiments. (D) The percentage of CD5+ and CD5+CD1dhi B cells in tumor-bearing or tumor-free mice (D1) and (D2) in the MDSC-co-cultured system was evaluated by FC. (E) The intra-cellular IL-10 level of B cells was analyzed by FC in 4T1 or normal mice (E1) and (E2) in the MDSC-co-cultured group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant as determined with the t test. MDSC, myeloid-derived suppressor cells; FC, flow cytometry; IL, interleukin.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Labeling, Cell Culture, Derivative Assay, Flow Cytometry

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: PD-1−PD-L1+CD19+ B cells display immuno-regulatory properties on activated T cells. PD-1−PD-L1+CD19+ B cells were isolated from the splenic cells of tumor-bearing mice. T cells were cultured for 2 days with or without B cells for the subsequent analyses. (A) Regulation of CD3+ T-cell proliferation by different B cell subsets is shown in relative to T cells cultured in the absence of B cells from 8 independent experiments. (B) The apoptosis of CD3+ T cell was detected by FC at 48 h following incubation with splenic CD19+ cells, PD-1−PD-L1+CD19+cells or CD5+B cells from tumor-bearing mice. (C) The percentage of Tregs (CD4+CD25+CD125low) with or without B cell incubation was evaluated by FC analysis. (D) Cytokine concentrations (IL-10, IFN-γ, TGFβ1, and IL-2) were determined by FC to analyze T-cell intra-cellular production. Data were collected from 5 independent experiments. Data represent the mean ± SEM of 5 independent experiments. * = P < 0.05; ** = P < 0.01; *** = P < 0.001, ns = not significant as determined with t test or One-way ANOVA. BrdU, bromodeoxyuridine; MDSCs, myeloid-derived suppressor cells; Tregs, regulatory T cells; FC, flow cytometry; TNF, tumor necrosis factor; TGF, transforming growth factor; IL, interleukin; IFN, interferon.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Isolation, Cell Culture, Incubation, Derivative Assay, Flow Cytometry

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: The distribution of different B cell subsets in BM, peripheral blood, spleen and TDLNs. (A) PD-1−PD-L1+CD19+ B cells was expanded in the BM, peripheral blood, spleen, LNs and primary tumor tissue in tumor-bearing or normal mice . Representative dot plots of FC in LNs were shown. (B) The percentage of Gr-1+CD11b+MDSCs and representative dot plots in tumor-bearing or tumor-free mice from 5 independent experiments with triplicate samples was shown. (C) Normal splenic B cells were cultured with 4T1 cells or 4T1-supernatant, and the percentage of PD-1−PD-L1+CD19+ B cells was detected by FC. (D) The percentage of CD5+CD19+ B cells (E) CD5+CD1dhi CD19+B cells and (F) intra-cellular staining was with PE-anti-IL-10 antibody to detect IL-10+ B cells determined by FC. Data show mean±SEM of 7 independent experiments. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant as determined by t test. BM, bone marrow; LN, lymph node; TDLN, tumor-draining lymph node; MDSC, Myeloid-derived suppressor cells.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Cell Culture, Staining, Derivative Assay

Journal: Oncoimmunology

Article Title: A novel MDSC-induced PD-1 − PD-L1 + B-cell subset in breast tumor microenvironment possesses immuno-suppressive properties

doi: 10.1080/2162402X.2017.1413520

Figure Lengend Snippet: PD-1−PD-L1+CD19+ Bregs were expanded in the peripheral blood of breast cancer patients. The percentages of indicated cell types were determined in 35 breast cancer patients and 20 healthy female controls. The results are shown as the mean±SEM. of the percentage of (A) CD19+ B cells, and (B) CD45+CD33+CD13+CD14−CD15− MDSCs in the periphery blood. (C) PD-1−PD-L1+ Bregs were expanded in the peripheral blood of breast cancer patients when compared with healthy controls. Representative dot plots were shown in the left panel. (D) A scatter plot for the percentage of PD-1−PD-L1+CD19+ B cells in two groups. (E) The correlation of the PD-1−PD-L1+ CD19+ cells with tumor stages. (F) The histopathological characteristics and other basic information of the patients involved. The analysis was determined by unpaired t test. * = P <0.05, *** = P < 0.001, ns = not significant. Bregs, regulatory B cells; MDSC, Myeloid-derived suppressor cells.

Article Snippet: The tissue sections were stained using anti-mouse antibody to CD19 (Abbiotec) for B cells and CD11b (Abcam) for MDSCs analysis.

Techniques: Derivative Assay

Journal: Cancer Cell

Article Title: Determinants of anti-PD-1 response and resistance in clear cell renal cell carcinoma

doi: 10.1016/j.ccell.2021.10.001

Figure Lengend Snippet:

Article Snippet: Rabbit anti-human monoclonal anti-CD19 [SP291] , Spring Bioscience , Cat#M5914; RRID N/A.

Techniques: Staining, Sequencing, Next-Generation Sequencing, Derivative Assay, Methylation, Software