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.