CD300a is expressed on human B cells, modulates BCR-mediated signaling, and its expression is down-regulated in HIV infection by Rodolfo Silva, Susan Moir, Lela Kardava, Karen Debell, Venkateswara R. Simhadri, Sara Ferrando-Martínez, Manuel Leal, José Peña, John E. Coligan, and Francisco Borrego Blood Volume 117(22):5870-5880 June 2, 2011 ©2011 by American Society of Hematology

Flow cytometric analysis of CD300a expression on human peripheral blood and tonsil B cells. Flow cytometric analysis of CD300a expression on human peripheral blood and tonsil B cells. (A) Freshly isolated PBMCs were labeled with anti-CD19 and anti-CD300a mAb. The lymphocyte gate was determined according to the forward and side scatter parameters. The expression of CD300a was assessed on B cells (CD19+). Empty histogram represents binding of anti-CD300a mAb; and gray histogram, the binding of isotype control Ig. (B) Freshly isolated PBMCs were labeled with anti-CD19, anti-IgD, anti-CD27, and anti-CD300a mAb. The expression of CD300a for a representative donor was measured for the 4 subsets within the CD19+ gate (B cells) defined by the expression of IgD and CD27. The number in the histograms indicates the percentage of CD300a+ cells. (C) Single-cell suspensions from tonsils were labeled with anti-CD19, anti-IgD, anti-CD38, and anti-CD300a mAb. The expression of CD300a for a representative donor was measured in the 5 subsets of the CD19+ gate (B cells) defined by the expression of IgD and CD38. The number in the histograms indicates the percentage of CD300a+ cells. Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology

Regulation of the expression of CD300a on human B cells. Regulation of the expression of CD300a on human B cells. (A) Purified naive B cells were activated and cultured according to the protocol described in “Cell cultures.” The expression of CD27, CD300a, and CD305 was assessed at day 0 and day 7 of the cultures. Results are representative of 3 independent experiments. (B) Memory CD27+ B cells were purified and sorted according to the protocol described in “Cell cultures” and “Flow cytometric analyses” and stimulated with CpG 2006 for 5 days. Dot plots of IgD and CD27 expression at day 0 (before sorting) and day 5, and a histogram of CD38 expression for a representative culture is also shown (upper panels). Histogram of CD300a expression in the CD27+ and CD27++ cells and graphic representation of the average relative MFI ± SEM of CD300a expression (n = 4) (lower panels). (C) Purified B cells were stimulated with anti-IgG in the presence of IL-4 or TGF-β1 for 4 days according to the protocol described in “Cell cultures.” Flow cytometric analyses were performed to assess CD300a expression by the CD27+ cells. Histograms from a representative donor are shown (left panel) along with the graphic representation of the average relative MFI plus or minus SEM of CD300a expression (n = 4; right panel). Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology

CD300a down-regulates BCR-mediated activation signals. CD300a down-regulates BCR-mediated activation signals. (A) Purified human B cells (upper panel) or DT40 cells expressing CD300a (lower panel) were loaded with Fluo-4 and Fura-Red. Then, cells were acquired in a flow cytometer and stimulated with anti-BCR plus anti-CD300a mAb (gray line) or anti-BCR plus isotype control (black line), according to the protocol described in “Calcium mobilization assays.” Intracellular Ca2+ concentration was measured by the ratio of Fluo-4/Fura-Red as a function of time. Results are representative of 2 (human B cells) and 4 (DT40 cells) independent experiments. (B) DT40 cells expressing CD300a were transiently transfected with a NFAT luciferase reporter plasmid and stimulated with medium, antichicken BCR plus isotype control, or antichicken BCR plus anti-CD300a mAb as indicated. The measured luciferase activity was normalized to the activity obtained for the treatment with phorbol myristate acetate plus ionomycin. The data presented are the mean plus or minus SEM for 6 separate experiments. (C) Purified human B cells were transfected with nontarget or CD300a siRNA. Then, cells were stimulated with anti-Ig (IgG/A/M, IgM, or IgG) and CpG, and the proliferation was measured after 4 days of culture as described in “Cell cultures.” A representative donor is shown. Numbers in the upper left quadrant indicate the percentage of CD22+ cells that have divided one or more times (upper panel). Graphic representation of proliferating cells treated with CD300a siRNA normalized to the nontarget siRNA-treated cells (lower panel, left side) and the CD300a mRNA relative levels measured 4 days after transfection with siRNA (n = 3) are shown (lower panel, right side). Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology

Decreased frequency of CD300a+ B cells in HIV-infected patients. Decreased frequency of CD300a+ B cells in HIV-infected patients. PBMCs from healthy donors (HD), HIV-aviremic (HIV-AVIR) patients, and HIV-viremic (HIV-VIR) patients were labeled with anti-CD10, anti-CD19, anti-CD20, anti-CD21, anti-CD27, and anti-CD300a mAb. The lymphocyte gate was determined according to the forward and side scatter parameters. (A) The percentage of CD300a+ cells in the CD19+ gate (B cells) was determined. Each symbol represents a different donor. (B) The percentage of CD300a+ cells among CD21+ B cells (left panel) and CD21− B cells (right panel) was determined. Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology

Decreased CD300a expression on circulating mature B cells from HIV-infected patients. Decreased CD300a expression on circulating mature B cells from HIV-infected patients. For the healthy donors (HD), HIV-aviremic (HIV-AVIR) patients, and HIV-viremic (HIV-VIR) patients reported in Figure 5, CD300a expression was determined for each of the following mature (CD19+CD10−) circulating B-cell subsets: naive, resting memory, activated memory, atypical memory (exhausted), and plasmablasts. (Top panel) Dot plots and histograms from a representative person within the HD, HIV-AVIR, and HIV-VIR groups. (Bottom panel) The MFI for CD300a for each of the 5 B-cell subsets. Each symbol represents a different donor. Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology

Correlation of CD300a expression on B cells with HIV plasma viremia and CD4+ T-cell counts in HIV-infected patients. Correlation of CD300a expression on B cells with HIV plasma viremia and CD4+ T-cell counts in HIV-infected patients. (A) The correlation between HIV plasma viremia and CD300a expression on the mature (CD19+CD10−) circulating B-cell subsets from the HIV (combined aviremic and viremic) patients. (B) The correlation between CD4+ T-cell counts and CD300a expression on the mature (CD19+CD10−) circulating B-cell subsets from the HIV-aviremic and HIV-viremic patients. Rodolfo Silva et al. Blood 2011;117:5870-5880 ©2011 by American Society of Hematology