Umbilical cord blood CD20⁺CD27⁺CD43⁺ B cells spontaneously secrete IgM and efficiently stimulate T cells. (A) Umbilical cord blood mononuclear cells were stained with immunofluorescent antibodies and evaluated by flow cytometry. The contour plot displays expression of CD27 and CD43 by gated CD20⁺ cells. Results shown represent 1 of 13 separate cord blood samples. (B) Sort-purified CD20⁺CD27⁻CD43⁻ (27⁻43⁻) and CD20⁺CD27⁺CD43⁺ (27⁺43⁺) cord blood B cells were plated at 1 × 10⁴ cells per well, incubated for 3 h at 37°C, and analyzed for IgM secretion by ELISPOT. Images shown are representative of three separate experiments on three different cord blood samples each done in triplicate. (C) Enumeration of ELISPOT results displayed as mean values for triplicate wells, with error bars indicating the SEM. Each bar graph indicates an individual experiment on a separate cord blood sample. (D) Sort-purified CD20⁺CD27⁻CD43⁻ and CD20⁺CD27⁺CD43⁺ cord blood B cells were cultured for 5 d, after which supernatants were evaluated for secreted IgM by ELISA. Each bar graph indicates an individual experiment on a separate cord blood sample. (E) Sort purified and irradiated CD20⁺CD27⁻CD43⁻ (27⁻43⁻) and CD20⁺CD27⁺CD43⁺ (27⁺43⁺) cord blood B cells were evaluated for the ability to drive allogeneic T cell proliferation as measured by tritiated thymidine incorporation for 8 h at the end of 5 d co-cultures. Data shown are representative of three separate experiments on three different cord blood samples each done in triplicate. Mean cpm values are displayed with error bars indicating SEM.

Umbilical cord blood CD20⁺CD27⁺CD43⁺ B cells exhibit tonic intracellular signaling. (A) Cord blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, and 12 min and then fixed, permeabilized and stained for surface antigens and intracellular phosphorylated PLC-γ2 with specific immunofluorescent antibodies. Because isotype control antibody staining by “fluorescence minus one” did not vary between the two cell populations being studied, only a single control tracing is shown. Results for one of three comparable experiments are shown. (B) Mean values of mean fluorescence intensity (MFI) are shown (with error bars indicating the SEM) for intracellular phosphorylated PLC-γ2 staining at various time points from three separate umbilical cord blood samples. (C) Cord blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, or 12 min, and then fixed, permeabilized, and stained for surface antigens and intracellular phosphorylated Syk with specific immunofluorescent antibodies. Results for one of three comparable experiments are shown. (D) Mean values of MFI are shown (with error bars indicating the SEM) for intracellular phosphorylated Syk staining at various time points from three separate umbilical cord blood samples.

Adult peripheral blood CD20⁺CD27⁺CD43⁺ B cells spontaneously secrete IgM and efficiently stimulate T cells. (A) Adult peripheral blood mononuclear cells were stained with immunofluorescent antibodies and evaluated by flow cytometry. The contour plot displays expression of CD27 and CD43 by gated CD20⁺ cells. Results shown represent 1 of 25 separate peripheral blood samples. (B) Sort-purified CD20⁺CD27⁻CD43⁻ (27⁻43⁻), CD20⁺CD27⁺CD43⁻ (27⁺43⁻), and CD20⁺CD27⁺CD43⁺ (27⁺43⁺) adult peripheral blood B cells were plated at 1 × 10⁴ cells per well, incubated for 3 h at 37°C, and analyzed for IgM secretion by ELISPOT. Images shown are representative of three separate experiments on three different adult blood samples each done in triplicate. (C) Enumeration of ELISPOT results displayed as mean values for triplicate wells, with error bars indicating SEM. Each bar graph indicates an individual experiment on a separate adult blood sample. (D) Sort-purified CD20⁺CD27⁺CD43⁺, CD20⁺CD27⁺CD43⁻, and CD20⁺CD27⁻CD43⁻ adult blood B cells were cultured for 5 d, after which supernatants were evaluated for secreted IgM by ELISA. Each bar graph indicates an individual experiment on a separate adult blood sample. (E) Sort-purified and irradiated CD20⁺CD27⁻CD43⁻ (27⁻43⁻), CD20⁺CD27⁺CD43⁻ (27⁺43⁻), and CD20⁺CD27⁺CD43⁺ (27⁺43⁺) adult peripheral blood B cells were evaluated for the ability drive allogeneic T cell proliferation as measured by tritiated thymidine incorporation for 8 h at the end of 5-d cultures. Data shown are representative of three separate experiments on three different blood samples each done in triplicate. Mean cpm values are displayed with error bars indicating SEM.

Adult peripheral blood CD20⁺CD27⁺CD43⁺ B cells exhibit tonic intracellular signaling. (A) Adult blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, or 12 min, and then fixed, permeabilized, and stained for surface antigens and intracellular phosphorylated PLC-γ2 with specific immunofluorescent antibodies. Because isotype control antibody staining by “fluorescence minus one” did not vary between the three cell populations under study, only a single control tracing is shown. Results for one of three comparable experiments are shown. (B) Mean values of MFI are shown (with error bars indicating the SEM) for intracellular phosphorylated PLC-γ2 staining at various time points from three separate adult blood samples. (C) Adult blood mononuclear cells were unexposed (0) or were exposed to pervanadate for 4, 8, or 12 min, and then fixed, permeabilized, and stained for surface antigens and intracellular phosphorylated Syk with specific immunofluorescent antibodies. Results for one of three comparable experiments are shown. (D) Mean values of MFI are shown (with error bars indicating the SEM) for intracellular phosphorylated Syk staining at various time points from three separate adult peripheral blood samples.

CD20⁺CD27⁺CD43⁺ B1 cells display two typical B1 cell specificities. Adult peripheral blood mononuclear cells were immunofluorescently stained with specific antibodies and with either PC-BSA-fluorescein (A and B) or biotinylated DWEYS tetramer plus PE-streptavidin (C and D), as described in the Materials and methods. Representative results are shown in A and C for three different B cell populations (CD20⁺CD27⁺CD43⁺ B1 cells, CD20⁺CD27⁺CD43⁻ memory B cells, and CD20⁺CD27⁻CD43⁻ naive B cells). (top) Gated CD20⁺CD43⁻ cells; (bottom) gated CD20⁺CD27⁺CD43⁺ cells. Aggregate results are shown in B and D, with the proportion of B cells that bound antigen displayed on the Y axis as mean ± SEM, n = 4.

CD20⁺CD5⁺ B cells are not the same as CD20⁺CD27⁺CD43⁺ B1 cells. Adult peripheral blood mononuclear cells were immunofluorescently stained with specific antibodies. The proportion of CD20⁺CD27⁺CD43⁺ B1 cells that express CD5 (75 ± 2.5%; n = 46) is shown in A; the proportions of CD20⁺CD5⁺ B cells that phenotype as CD20⁺CD27⁺CD43⁺ (34 ± 3.0%; n = 46), CD20⁺CD27⁺CD43⁻ (4.8 ± 0.41%), and CD20⁺CD27⁻CD43⁻ (61 ± 3.1%) are shown in B. Representative flow cytometry data are shown in Fig. S2.

CD20⁺CD27⁺CD43⁺ B1 cells decline with age in normal individuals. (A) Cord blood (n = 6) and peripheral blood (n = 43) samples were stained with immunofluorescent antibodies and evaluated by flow cytometry. CD20⁺CD27⁺CD43⁺ B cells are expressed as a percentage of the total number of CD20⁺CD27⁺ cells at the ages indicated. The line through the datapoints represents a cubic regression curve fit (R-squared = 0.070494). (B) Mean values for the proportion of CD43⁺ B cells among CD20⁺CD27⁺ B cells are shown for the indicated age intervals with error bars indicating the SEM.

Expression of CD69 and CD70 differentiates B1 cells from activated memory and naive B cells. (A) CD20⁺CD27⁺CD43⁻ memory and CD20⁺CD27⁻CD43⁻ naive B cells were sort-purified from adult peripheral blood and were cultured with SAC (0.005%) plus IL-2 (5 ng/ml) for 4 d, after which surface expression of CD43, CD69, and CD70 was determined by immunofluorescent staining and flow cytometry. Unstimulated CD20⁺CD27⁺CD43⁺ B1 cells were immunofluorescently stained and analyzed for CD69 and CD70 at the same time. CD69 and CD70 expression is displayed for CD43⁺ B cells. Results are shown for one of three separate samples. Fluorescence minus one isotype control staining is shown in solid gray for each population. (B and C) Memory and naive B cells from adult blood were cultured for 4 d with SAC plus IL-2, PMA at 100 ng/ml plus ionomycin at 400 ng/ml (P + I), or anti-CD40 at 10 µg/ml plus anti-IgM at 7 µg/ml (αCD40 plus αIgM), and immunofluorescently stained, after which surface expression of CD43, CD69, and CD70 was determined. Unstimulated CD20⁺CD27⁺CD43⁺ B1 cells were immunofluorescently stained and analyzed at the same time. The proportion of CD43⁺ B cells that stained positively for CD69 (B) and for CD70 (C) is shown. Mean values for three separate adult blood samples for each population are shown along with error bars indicating SEM.

Human B1 cells are not contained within the CD20⁻ B cell population. (A) Umbilical cord blood and adult peripheral blood mononuclear cells were stained with immunofluorescent antibodies and evaluated by flow cytometry. Expression of CD20 and CD19 is displayed. Representative results for one of three cord blood and one of three adult blood samples are shown. (B) CD19⁺CD20⁻ B cells were sort-purified from adult blood and plated at 800 cells per well, incubated for 1 h at 37°C, and analyzed for IgM secretion by ELISPOT. The image shown is representative of three separate experiments on three different adult blood samples. (C) Adult blood CD19⁺CD20⁻ B cells (thin black lines) and CD20⁺CD27⁺CD43⁺ B1 cells (thick black lines) were treated with the phosphatase inhibitor pervanadate. At the indicated times, B cells were immunofluorescently stained and phosphorylation of PLC-γ2 (C) and Syk (D) was analyzed as described in the legend to Fig. 4. Results for one of three comparable experiments are shown.