Journal: Oncoimmunology

Article Title: Reciprocal influence of B cells and tumor macro and microenvironments in the Apc Min/+ model of colorectal cancer

doi: 10.1080/2162402X.2017.1336593

Figure Lengend Snippet: B lymphocytes are expanded in tumor-draining LNs of ApcMin/+ mice. (A) Representative pictures of hematoxylin/eosin-stained intestine sections of 18-weeks old Wt and ApcMin/+ mice (left) and histological scoring of intestinal adenomas (right). Each symbol of the scatter plot depicts individual mice (n = 12) among the Wt and ApcMin/+ groups. (B, D) Percentages of viable B cells were analyzed among leukocytes isolated from the spleen, peritoneum, inguinal and mesenteric LNs. In all conditions, cells were treated with LPIM for 5 h before staining. A flow cytometric analysis based on the dual staining with an anti-CD19 mAb and the green Live/Dead probe was performed. (B) B cell frequencies were compared in 18-weeks old Wt and ApcMin/+ mice. Dot plots for one representative experiment are shown. The CD19 vs. Live/Dead plot allows to identify viable B cells (gated) and excludes both non-B cells, which are negative for CD19 staining, and dead cells, that are positively stained with the Live/Dead probe. (C) B cell frequencies were evaluated in 10- and 18-weeks old animals. Each symbol depicts individual mice among the Wt and ApcMin/+ groups (at least n = 5) while horizontal lines indicate the mean value ± SEM. (D) B cell frequencies were evaluated in the CT-26, ApcMin/+ and AOM/DSS models of CRC. Each symbol depicts individual mice among the control and tumor-bearing group (at least n = 3). In the CT-26 model, inguinal LNs were analyzed as draining (triangle symbol) and non-draining (square symbol) the tumor site. Horizontal lines indicate the mean value ± SEM. (E) Cells were isolated from mesenteric LNs of 18-weeks old Wt and ApcMin/+ mice and directly underwent the staining procedure for Ki-67 detection. Percentages of Ki-67+ cells were calculated among viable CD19+ cells. A representative dot plot and bar graphs reporting the mean values (+ SEM) of 3 independent experiments are shown. (F) Relative expression of cxcl12, cxcl13, ccl19, ccl21 and ccl20, normalized to the housekeeping gene g3pdh, was analyzed in the total cellular population isolated from the mesenteric LNs of Wt and ApcMin/+ mice (n = 4). For each chemokine, the Wt sample with the lowest expression was used as control and set to one. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

Article Snippet: Cell preparation and B/MDSC co-culture conditions Mouse CD19 MicroBeads (Miltenyi) were used for the positive selection of B lymphocytes from total peritoneal cells.

Techniques: Staining, Isolation, Control, Expressing

Journal: Oncoimmunology

Article Title: Reciprocal influence of B cells and tumor macro and microenvironments in the Apc Min/+ model of colorectal cancer

doi: 10.1080/2162402X.2017.1336593

Figure Lengend Snippet: Tumor progression leads to discrete alterations of IL-10-competent B cell homeostasis in the ApcMin/+ model of CRC. Percentages of IL-10-competent B cells were analyzed among leukocytes isolated from the spleen, peritoneum, inguinal and mesenteric LNs. In all conditions, cells were treated with LPIM for 5 h before staining with the green Live/Dead probe and the anti-mouse CD19 and anti-mouse IL-10 mAbs. (A) Dot plots from one representative experiment show frequencies of IL-10+ cells among total viable B cells within the indicated gates in 18-weeks old Wt and ApcMin/+ animals. Dead cells were excluded from the analysis using the CD19 vs. Live/Dead dot plot (data not shown). (B) The IL-10-competent B cell frequencies were evaluated in 10- and 18-weeks old animals. Each symbol depicts individual mice among the Wt and ApcMin/+ groups (at least n = 5) while horizontal lines indicate the mean value ± SEM. (C) The IL-10-competent B cell frequencies were evaluated in the CT-26, ApcMin/+ and AOM/DSS models of CRC. Each symbol depicts individual mice among the control and tumor-bearing group (at least n = 3). In the CT-26 model, inguinal LNs were analyzed as draining (triangle symbol) and non-draining (square symbol) the tumor site. Horizontal lines indicate the mean value ± SEM. *p < 0.05; **p < 0.01; ns: not significant.

Article Snippet: Cell preparation and B/MDSC co-culture conditions Mouse CD19 MicroBeads (Miltenyi) were used for the positive selection of B lymphocytes from total peritoneal cells.

Techniques: Isolation, Staining, Control

Journal: Oncoimmunology

Article Title: Reciprocal influence of B cells and tumor macro and microenvironments in the Apc Min/+ model of colorectal cancer

doi: 10.1080/2162402X.2017.1336593

Figure Lengend Snippet: Wt and ApcMin/+ splenic B cells respond similarly to activating stimuli. (A) B cells were purified from the spleen of Wt and ApcMin/+ mice, CFSE labeled and cultured either alone (NST) or in the presence of anti-CD40 mAb (a-CD40), LPS or CpG for 72 h. CFSE profiles of B cell proliferation in the different conditions are shown for one representative experiment while bar graphs indicate mean (+ SEM) of the B cell proliferation index from 5 independent experiments. (B) The frequencies of B cells competent to express cytoplasmic IL-10 following a 5 h stimulation with LPIM were analyzed in purified Wt and ApcMin/+ B cells cultured for 48 h with anti-CD40 mAb. The condition in which cells received only monensin during the last 5 h of culture was used to correctly discriminate IL-10+ from IL-10− B cells. Dot plots for one representative experiment are shown together with the frequencies of IL-10+ cells among total CD19+ lymphocytes. Bar graphs indicate mean (+ SEM) percentages of CD19+IL-10+ cells from 6 independent experiments. (C) B cells were cultured either alone (NST) or in the presence of a-CD40 mAb, LPS or CpG for 48 h. Cell supernatants were collected and IL-10 and LAP-1 levels detected by ELISA. Bar graphs indicate mean (+ SEM) concentrations from 6 independent experiments and comparison were performed against the NST condition. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

Article Snippet: Cell preparation and B/MDSC co-culture conditions Mouse CD19 MicroBeads (Miltenyi) were used for the positive selection of B lymphocytes from total peritoneal cells.

Techniques: Purification, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Oncoimmunology

Article Title: Reciprocal influence of B cells and tumor macro and microenvironments in the Apc Min/+ model of colorectal cancer

doi: 10.1080/2162402X.2017.1336593

Figure Lengend Snippet: B cells with a CD21low/− phenotype are enriched in the spleen of tumor-bearing ApcMin/+ mice. Splenocytes were isolated from 18-weeks old Wt and ApcMin/+ mice, stained with anti-CD21, -CD23 and -CD19 (A, C) or with anti-CD1d, -CD5, and -CD19 (B) mAbs and analyzed by flow cytometry. (A, B) Histogram and dot plots show the analysis of one representative mouse per condition while scatter plots report the mean ( ± SEM) percentages of CD19+ cells with the phenotype of newly formed (NF), CD21low, transitional 2-marginal zone precursor (T2-MZP), marginal zone (MZ) and follicular (FO) cells (A) or of CD1dhi, CD1d+CD5−, CD1d+CD5low and CD1d+CD5+ cells (B). Each symbol depicts individual mice among the Wt and ApcMin/+ groups (n = 6 in A; n = 8 in B). (C) The percentages of CD19+ cells with a NF, CD21low, T2-MZP, MZ and FO phenotype were analyzed in 10- and 18-weeks old Wt and ApcMin/+ mice (n = 6). *p < 0.05; **p < 0.01; ns: not significant.

Article Snippet: Cell preparation and B/MDSC co-culture conditions Mouse CD19 MicroBeads (Miltenyi) were used for the positive selection of B lymphocytes from total peritoneal cells.

Techniques: Isolation, Staining, Flow Cytometry

Journal: Oncoimmunology

Article Title: Reciprocal influence of B cells and tumor macro and microenvironments in the Apc Min/+ model of colorectal cancer

doi: 10.1080/2162402X.2017.1336593

Figure Lengend Snippet: Tumor progression induces a strong IgA response in ApcMin/+ mice. (A) Representative analysis of CD24, CD93, CD45R and CD138 expression on CD21low, FO and MZ B cells from the spleen of 18-weeks old ApcMin/+ mice. Single-color histograms in which cell percentages (y-axis) are plotted against log fluorescence intensity of the specific Ag are the result of a 4-color staining. Filled and empty histograms indicate isotype-matched controls and specific Abs, respectively. For each of the 4 phenotypic markers in analysis, a plot overlaying the expression patterns among the CD21low, FO and MZ populations is shown on the right. (B) Representative dot plots showing the CD19+CD45R+ and CD19+CD45Rlow/− subsets in Wt and ApcMin/+ mice. Non-B cells were excluded from the analysis. Bar graphs indicate mean (+ SEM) percentages of CD19+CD45R+ and CD19+CD45Rlow/− cells among total B lymphocytes from 4 independent experiments. (C) Representative dot plots showing the CD138neg, CD138int and CD138high subsets in Wt and ApcMin/+ mice. Percentages of CD138high cells are also indicated. Non-B cells were excluded from the analysis. Bar graphs indicate mean (+ SEM) percentages of CD138neg, CD138int and CD138hi cells among total B lymphocytes from 6 independent experiments. (D) B cells were cultured either alone (NST) or in the presence of a-CD40 mAb, LPS or CpG for 48 h. Cell supernatants were collected and IgM, IgG and IgA levels detected by ELISA. Bar graphs indicate mean (+ SEM) concentrations from 4 to 6 independent experiments. (E) Total IgM, IgG and IgA were measured by ELISA in the serum of 18-weeks old Wt and ApcMin/+ mice (n = 8). (F) B cells purified from the peritoneum and spleen of 18-weeks old Wt and ApcMin/+ mice were stained for CD45R and IgA and analyzed by flow cytometry. Dot plots are shown together with the percentages of IgA+ cells and are representative of n = 2 (peritoneum) and n = 4 (spleen) experiments. Non-B cells were excluded from the analysis. The scatter plot reported on the right is the result of the analysis of IgA+ cells in the spleen of 10- and 18-weeks old Wt and ApcMin/+ mice. (G) Wt and ApcMin/+ splenocytes were stained for CD19 and CXCR4, CXCR5, CCR6, CCR7, CCR9 or CCR10. The scatter plot reports the mean fluorescence intensity (MFI) of each chemokine receptor. Each symbol depicts individual mice among the Wt and ApcMin/+ groups (at least n = 4) while horizontal lines indicate the mean value ± SEM. For CCR10, the dot plots of one representative experiment are shown with indicated the percentages of CD19+CCR10+ cells. *p < 0.05; **p < 0.01; ***p < 0.001; ns: not significant.

Article Snippet: Cell preparation and B/MDSC co-culture conditions Mouse CD19 MicroBeads (Miltenyi) were used for the positive selection of B lymphocytes from total peritoneal cells.

Techniques: Expressing, Fluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Purification, Flow Cytometry