A small CD11b(+) human B1 cell subpopulation stimulates T cells and is expanded in lupus - PubMed (original) (raw)

Figure 1.

CD11b expression divides human B1 cells into two phenotypically distinct subsets. (A) Adult peripheral blood mononuclear cells were immunofluorescently stained for CD20, CD27, CD43, and CD11b, and were then evaluated by flow cytometric analysis. Two gating strategies are shown using nuclear staining with Hoechst 33342 (Hoechst) and FSC-H by FSC-A doublet gating to separate CD20+CD27+CD43+ B1 cells into CD11b+ and CD11b− populations for a representative adult blood sample with isotype control displayed for CD11b. (B) Adult peripheral blood and umbilical cord blood mononuclear cells were stained for IgD, CD14, CD11c, and CD5 in addition to CD20, CD27, CD43, and CD11b, and then evaluated by flow cytometric analysis. Expression of IgD, CD14, CD11c, and CD5 by naive (CD20+CD27−CD43−) and memory (CD20+CD27+CD43−) B cells and by CD11b− and CD11b+ B1 cells (CD20+CD27+CD43+) is shown for representative adult and cord blood samples (one of three each) with isotype control in solid gray. (C) RNA was prepared from sort-purified populations of naive (CD20+CD27−CD43−) and memory (CD20+CD27+CD43−) B cells, and from CD11b− and CD11b+ B1 cells (CD20+CD27+CD43+), obtained from three normal adult individuals for each B cell population and analyzed for gene expression by microarray. Expression levels for CD11b, CD14, and CD11c transcripts are shown in the form of a heat map. (D) Adult peripheral blood mononuclear cells were stained with fluorescent antibodies that recognize CD19, CD43, and CD11b and sorted into populations containing naive/memory B cells, CD11b+ B1 cells, and CD11b− B1 cells. Sort-purified B cells were then stained with a fluorescent antibody targeting CD14, fixed, adhered to poly-lysine–coated slides, and examined for immunofluorescence sequentially at three wavelengths by confocal microscopy. Representative results from one of three comparable experiments are shown. (E) Adult peripheral blood mononuclear cells were stained for CD20, CD27, CD43, CD11b, and CD80 and were then evaluated by flow cytometric analysis. CD80 expression by naive (CD20+CD27−CD43−) and memory (CD20+CD27+CD43−) B cells, and by CD11b+ and CD11b− B1 cells (CD20+CD27+CD43+), is shown as colored lines, along with isotype control (solid gray). Representative results from one of three comparable experiments are shown. (F) Adult peripheral blood mononuclear cells were stained for CD20, CD27, CD43, CD11b, and CD71 and were then evaluated by flow cytometric analysis. CD71 expression by naive (CD20+CD27−CD43−) and memory (CD20+CD27+CD43−) B cells, and by CD11b+ and CD11b− B1 cells (CD20+CD27+CD43+) and CD11b+CD20−CD71+ cells, is shown as colored lines, along with isotype control (solid gray). Representative results from one of three comparable experiments are shown.