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. 2022 Dec 14;13:1082154. doi: 10.3389/fimmu.2022.1082154

Figure 1.

Figure 1

Differential pO2 controls human B cell differentiation into CD27+ and antibody secreting cell compartments. (A) Schematic overview of B cell in vitro culture system. 250 resting human naive B cells (CD19+CD27-IgD+) were stimulated using a human CD40L-expressing feeder cell layer (subtype ‘High’ (12),); recombinant human IL-4 (25ng/ml) and IL-21 (50ng/ml); and cultured at 5% pCO2 and 21, 3, or 1% pO2 for a maximum of 11 days. (B) Representative biaxial CD27/CD38 FACS plots after 5, 7, and 11 days of culture at 21, 3, or 1% pO2. (C) Quantification of the percentages of CD27 and CD38 subpopulations within total CD19+ B cells over time (n = 9). (D) CD138 expression within the CD27+CD38+ antibody secreting cell population (n = 6). (E) Cumulative secretion of IgM and IgG measured in culture supernatants after 11 days (n = 9). (F) gMFI of AID expression over time (n = 3). (G) Percentage of IgG+ cells within CD19+ B cells, combined surface and intracellular staining (n = 8). Bars represent means of biological replicates each composed of 2 technical replicates in (D, F) 2, (E, G) 3 or (C) 4 independent experiments. Statistical differences were determined using (C, F) mixed-effects analysis using Tukey’s test for multiple comparisons (D, E, G) repeated measures one-way ANOVA using Tukey’s test for multiple comparisons. *p < 0.05, ***p < 0.001, ****p < 0.0001.