CD45RB Glycosylation and Ig Isotype Define Maturation of Functionally Distinct B Cell Subsets in Human Peripheral Blood

Abstract

Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.

Keywords: B cell subset heterogeneity; CD27; CD45RB glycosylation; memory B cells; naive B cells.

Figure 1

B cells within subsets separated based on CD45RB glycosylation and CD27 expression feature differential Ig isotype usage. (A) Representative biaxial CD45RB/CD27 plot gated from singlet viable CD19+CD38^lo cells ( Figure S1A ). (B) Percent of B cells in each quadrant, as in (A), for seven biological replicates (healthy adults) each composed of two technical replicates over three independent experiments. (C) I_g-isotype usage per quadrant. ND, not determined. Black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p < 0.05, **p < 0.01.

Figure 2

CD45RB glycosylation marks antigen-primed B cells independent of CD27 expression. (A) Representative gating strategy for cell-sorting B cell subsets from healthy donor peripheral blood CD19+ pre-isolated B cells by flow cytometry. Gated from singlet viable CD19+ cells ( Figure S1B ). Table shows the classification of subsets. (B) Frequency of subsets within CD19+ cells, for seven biological replicates each composed of two technical replicates over three independent experiments. (C) Mean number of mutations per Ig gene (healthy adults, n = 3) and (D) Cumulative frequency distribution of mutations in the Ig repertoire in the different B cell subsets. Different colored lines represent different donors. (E) mean V_H-CDR3 length from cell-sorted B cell subsets (healthy adults, n = 3). Bars and black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

CD45RB glycosylation, combined with Ig isotype, marks functionally distinct B cell subsets. B cell subsets were cultured with TD-stimulation of CD40L-expressing 3T3s in the presence of IL-21 (50ng/ml) for 10 days (six biological replicates each consisting of two technical replicates over two independent experiments). (A) Representative biaxial CD27/CD38 FACS plot after 10 days of TD culture. The formation of (B) MBCs (CD27+CD38-) (C) IgG+ B cells, and (D) ASCs (CD27+CD38+) was measured using flow cytometry. (E) Cumulative IgM secretion measured in culture supernatants using ELISA at day 10 (n = 6). B cell subsets were cultured with TI-stimulation of CpG ODN (0.1μM) and anti-IgM-F(ab’)₂ (1μg/ml) for 7 days (four biological replicates each consisting of two technical replicates). The formation of (F) MBCs and (G) ASCs was measured using flow cytometry. Black lines depict mean values. Statistical differences were determined using a Friedman analysis of variance and Dunn’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.