CD11b expression divides human B1 cells into two functionally distinct subsets. (A–D) Sort-purified populations of naive B cells (CD20⁺CD27⁻CD43⁻), CD11b⁻ B1 cells (CD20⁺CD27⁺CD43⁺CD11b⁻), and CD11b⁺ B1 cells (CD20⁺CD27⁺CD43⁺CD11b⁺) were obtained from adult peripheral blood and umbilical cord blood samples, and sort-purified memory (mem) B cells (CD20⁺CD27⁺CD43⁻) were obtained from adult blood. (A and B) B cells were cultured at 10⁶ cells per ml for 5 d, after which supernatants were evaluated for secreted IgM by ELISA. Mean values are shown along with lines indicating SEM for six adult blood samples (A) and four cord blood samples (B) for each population. (C and D) B cells were irradiated and co-cultured 1:2 with negatively selected allogeneic CD4⁺ T cells for 5 d, after which proliferation was measured by incorporation of tritiated thymidine during the last 8 h of triplicate cultures. Mean cpm values are shown, along with lines indicating SEM for four adult blood samples (C) and three cord blood samples (D) for each population. (E and F) Mononuclear cells from adult peripheral blood samples were immunofluorescently stained for CD20, CD27, CD43, CD11b, and CD86 and were then evaluated by flow cytometric analysis. (E) Levels of CD86 and CD11b expressed by gated CD20⁺CD27⁺CD43⁺ B1 cells in a representative adult peripheral blood sample are shown. Representative results from 1 of 15 comparable experiments are shown. (F) Mean values for the proportion of CD86⁺ cells among naive B cells (CD20⁺CD27⁻CD43⁻), memory (mem) B cells (CD20⁺CD27⁺CD43⁻), CD11b⁻ (11b⁻) B1 cells (CD20⁺CD27⁺CD43⁺CD11b⁻), and CD11b⁺ (11b⁺) B1 cells (CD20⁺CD27⁺CD43⁺CD11b⁺) are shown with lines indicating SEM for 15 adult blood samples. (G) RNA was prepared from sort-purified populations of naive B cells (CD20⁺CD27⁻CD43⁻), memory (mem) B cells (CD20⁺CD27⁺CD43⁻), and 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 of CD86 transcripts is shown for each population in the form of a heat map. (H) Sort-purified and irradiated CD20⁺CD27⁺CD43⁺CD11b⁺ B1 cells were co-cultured with allogeneic CD4⁺ T cells, as in C, with (CD86 NA) or without (normal) anti-CD86 neutralizing antibody. For anti-CD86–treated cultures, B1 cells were exposed to antibody for 1 h before addition of T cells. Mean cpm values are shown along with lines indicating SEM for three adult peripheral blood samples analyzed in sextuplicate.