Further studies also reported the existence of IgM– cells in CD27+CD43lo–int subpopulations, with one report noting that IgD– cells were more prevalent with increasing age [29, 31]. Further analysis of IgM+ cells within the CD27+CD43lo–int subpopulation showed there to be a proportion of IgMhi cells (data mTOR inhibitor not shown). As high expression of surface IgM is one of the discriminatory criteria for murine B1 cells [3], we re-ran our previous immunophenotyping analysis to distinguish between

IgMhigh and IgMlo CD20+CD27+CD43lo–int cells. We found a ninefold higher proportion of CD5+ cells within the IgMhigh subset compared to their IgMlow counterparts, which might indicate a closer phenotypic approximation to the ‘B1 cell’ population described previously [12] (data not shown). Sotrastaurin Nevertheless, discrepancies in the CD20+CD27+CD43+ cell immunophenotype we reported raised the need for a functional study which would match with our FACS results and reconfirm the functional B1 status of these putative B1 cells. The percentage and immunophenotype differences

found in the CD20+CD27+CD43lo–int cell subpopulation in CVID patients compared to healthy controls appeared not to be specific for this B cell subpopulation, but rather reflected a more general immune dysregulation in CVID. This could, potentially, be due to a lack of analysis using absolute counts of cells rather than percentages, which provides a much more accurate measure of difference [34]. We acknowledge this as a limitation of our study. A significantly increased percentage of CD21lo B cells within not the CD20+CD27+CD43lo–int subset in CVID patients compared to controls was observed. Although CD21lo B cells are known to have some innate-like features similar to murine B1 cells [14], our analysis showed that the proportion of CD21lo cells in the CD20+CD27+CD43lo–int was not

significantly different when compared with the proportion of CD21lo cells found in the CD20+CD27+CD43– cell subpopulation of the same patients. In addition, there was an observed lack of correlation with existing EUROclass classifications on CD21lo B cells; it is therefore likely that B1 cells and CD21lo innate-like B cells are not the same population. Further work investigating CVID and putative B1 B cells should focus on the functional aspects of B1 B cells, as any potential functional abnormalities have yet to be elucidated. In conclusion, our study showed that it is possible to use a rapid whole blood flow cytometric method to identify and analyse putative human B1 B cells. We demonstrated that CD20+CD27+CD43lo–int cells most probably represent a distinct subset within CD27+ B cells.