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: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

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: 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: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

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: 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: Cell lines 4T1 mammary cells are of BALB/C origin and were acquired from the American Type Culture Collection (ATCC).

Techniques: Cell Culture, Staining, Derivative Assay