Asymmetric B cell division in the germinal center reaction

Abstract

Lifelong antibody responses to vaccination require reorganization of lymphoid tissue and dynamic intercellular communication called the germinal center reaction. B lymphocytes undergo cellular polarization during antigen stimulation, acquisition, and presentation, which are critical steps for initiating humoral immunity. Here, we show that germinal center B lymphocytes asymmetrically segregate the transcriptional regulator Bcl6, the receptor for interleukin-21, and the ancestral polarity protein atypical protein kinase C to one side of the plane of division, generating unequal inheritance of fate-altering molecules by daughter cells. Germinal center B lymphocytes from mice with a defect in leukocyte adhesion fail to divide asymmetrically. These results suggest that motile cells lacking constitutive attachment can receive provisional polarity cues from the microenvironment to generate daughter cell diversity and self-renewal.

Fig. 1

Asymmetric division of germinal center (GC) B cells. (A) Mitotic GC B cells from pooled spleens of 3 mice at d8 post-immunization stained for Bcl6, IL-21R, IRF4, or B220 (green), β-tubulin (red), and DNA (blue). Compared to tubulin, Bcl6 asymmetry 44%, n=16 mitoses, p<0.05; IL-21R asymmetry 43%, n=14, p<0.05; IRF4 asymmetry 11%, n=18, p>0.05; B220 asymmetry 11%, n=18, p>0.05. (B) Asymmetric mitoses of Bcl6 (filled) and tubulin (open) at d5, d8, d15 post-immunization, n=9, 16, 15, respectively, * = p<0.05. p values calculated with chi-squared tests. (C) Bcl6 and PKCζ co-segregate asymmetrically during mitosis at d8 post-immunization. Bcl6 and PKCζ were asymmetric in 44% and 38% of mitoses, respectively, and were 100% co-segregated when both were asymmetric, n=16 mitoses. (D) Isolated GC B cells undergoing cytokinesis maintain asymmetry. Bcl6, IL-21R, and PKCζ were asymmetric in 45%, 53%, and 61% of divisions, respectively, and had 100% co-segregation when both were asymmetric. Analysis of Bcl6 and IL-21R, n=13 cytokinetic cells; analysis of Bcl6 and PKCζ, n=13. All data are representative of 2 or more experiments.

Fig. 2

Polarity cues are required for asymmetric division of B cells. (A) Naive B cells simulated 36h with anti-IgM, with or without anti-CD40 to mimic a key contact-mediated signal from T_FH cells. Anti-CD40 was withheld, added solubly to media (“s”), or coated on cell culture plates (“p”). Left, representative microscopy of no added anti-CD40 (upper) and plate-bound anti-CD40 (lower). Right, quantification of mitoses exhibiting asymmetry of PKCζ in indicated conditions. Left-to-right, n=18, 20, 21 mitoses, respectively. (B) Upper row, B cells undergoing homeostatic proliferation in Rag1^−/− mice exhibit low incidence of asymmetric mitoses (11%, n=19 mitoses from pooled spleens of 3 recipients) compared to GC B cells from immunized mice (38%, n=16 mitoses); p<0.05. Lower rows, defective asymmetric division in adhesion-deficient mice. Left, GC B cells from 3 pooled spleens of d8 immunized wild-type (“WT”) and Icam1^−/− mice stained for indicated components. Right, quantification of GC B cell mitoses exhibiting asymmetry of Bcl6 and PKCζ from indicated genotypes. Left-to-right, n=33, 18, 16, 18 mitoses, respectively. NS = p>0.05, * = p<0.05, ** = p<0.01 by chi-squared test. Similar results obtained at d5. (C) Icam1^−/− mice have impaired antibody responses to immunization. Quantification of antibody secreting cells (ASCs) from immunized WT and Icam1^−/− mice d14 post-immunization, compared to adjuvant-only injected WT mice (“Control”). n=6 mice/group, NS = p>0.05; * = p<0.05 by unpaired student’s t-test. All data are representative of 2 or more experiments.