Human CD11b+ B1 cells are not monocytes: A reply to 'Gene profiling of CD11b+ and CD11b- B1 cell subsets reveals potential cell sorting artifacts'

Griffin et al., respond: In January 2011, we reported the unique CD20+CD27+CD43+ phenotype of human B1 cells (Griffin et al., 2011a). In December 2011, we reported that the human B1 cell pool consists of functionally distinct CD11b− and CD11b+ populations, with the former particularly proficient at antibody secretion and the latter particularly efficient in stimulating T cells (Griffin and Rothstein, 2011). We also noted that the frequency of CD11b+ B1 cells is increased in the circulation of patients with lupus. As part of this work, we included microarray data that pointed to transcriptional differences between CD11b− and CD11b+ B1 cells. We have read with great interest the letter by Reynaud and Weill, who have mined the microarray data we deposited in the National Center for Biotechnology Information (NCBI) repository and drawn inferences, incorrectly as it turns out, regarding the nature and origin of CD11b− and CD11b+ B1 cells. Reynaud and Weill speculated that CD11b− B1 cells represent B cell–T cell doublets because this population, in aggregate, expressed genes characteristic of T cells along with genes characteristic of B cells. The potential issue of B cell–T cell doublets, raised previously by these same authors (Descatoire et al., 2011), is a topic we have already addressed by demonstrating that our sort-purified B1 cells exist as singlets (Griffin and Rothstein, 2011; Griffin et al., 2011b). At the same time, we identified procedures that can be used to circumvent the potential problem of doublet events (Griffin et al., 2011b). In our published work, we reported that B1 cells sort-purified without CD3− gating contained an estimated 1–3% CD3+ events (Griffin and Rothstein, 2011; Griffin et al., 2011b). We have now sort-purified CD11b− B1 cells again, exactly as was done to generate our published microarray results, and have analyzed three independent postsort samples for CD3 expression, along with purified T cells (Fig. 1 A). We again find a very small proportion of CD3+ events (1.9 ± 0.66%; mean ± SEM) in the CD11b− B1 cell population. This very low level of CD3 positivity is likely attributable to a minor degree of T cell contamination that could account for the appearance of very low levels of some characteristically T cell transcripts among CD11b− B1 cells. In keeping with this, flow cytometric analysis of unsorted CD19-enriched PBMCs demonstrates a small degree of CD2, CD7, CD8, and CD3 staining among CD11b− B1 cells that is eliminated by CD3 exclusion and subsequent analysis of CD3−CD20+CD27+CD43+CD11b− B1 cells (Fig. 1, B and C). Importantly, when CD3 exclusion is combined with sort purification, expression of T cell genes such as CD7, CD8, and granzyme A is essentially eliminated (to