Germinal Center Hypoxia Potentiates Immunoglobulin Class Switch Recombination

Abstract

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O₂), hypoxic culture conditions (1% O₂) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4⁺ T cells. Reversal of GC hypoxia in vivo by breathing 60% O₂ during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1⁺ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.

Figure 1

The germinal center (GC) microenvironment is hypoxic and poorly vascularized. A) Tissue histology of hypoxic marker Hypoxyprobe of splenic GCs. Magnification is 20x. B) Flow cytometric analysis of splenic GCs for Hypoxyprobe staining. Red histogram is GC gate, grey histogram is non GC B cell gate. Gated on scatter, B220⁺ CD4^-. C) Quantification of GC B cells depicted in B. D) Tissue histology of splenic GCs of mice perfused with fluorescent tomato lectin to stain vasculature. E) Schematic of masking strategy for determining percent GC and follicle area within 40μm of lectin staining. F) Quantification of GC area that is within 40μm of perfused vessels. Each dot represents one GC or B cell follicle. Each bar represents an individual mouse. Bars placed at mean. Data is of splenic GCs 8 days following i.p. immunization with either NP-OVA/Alum or NP-CGG/Alum. Representative of at least two independent experiment. Four to ten mice per group.

Figure 2

Hypoxic culture conditions increase GL-7 Expression on B cells. A) Representative flow cytometry plots of purified resting B cells stimulated with αIgM and αCD40 in hypoxic or normoxic culture conditions. Gated on lymphocyte, singlet, live, B220⁺, CD4^-. B) Quantification of GL-7⁺ cells in A C) Relative expression of GL-7 on positive cells in A. Bars placed at geometric mean of quadruplicate wells from representative experiment. Representative of at least two independent experiments. *=p<0.01

Figure 3

Hypoxia accelerates Ig class switch recombination and plasma cell formation in vitro. A-B) Flow cytometric analysis and kinetics of B cell stimulated in normoxic or hypoxic incubators to undergo class switch recombination with αCD40 and IL-4. Gated on lymphocyte, live, singlet, CD138^-.C) RT-PCR analysis of looped-out circular IgG1 transcripts of B cells stimulated as in A. D, E) Representative flow cytometric plots and analysis and kinetics of plasma cell formation of cells stimulated as in A. Cells gated on lymphocyte, live, singlet. F,G) IgM and IgG1 antibody secreting cell (ASC) formation on day 4 of hypoxic or normoxic cultures stimulated as in A. *=p<0.05, **p<0.01. Representative of two to ten independent experiments. Samples were run in quadruplicate.

Figure 4

Respiratory hyperoxia suppresses the germinal center (GC) reaction during immunization. A) GC frequencies of mice 8 days following immunization breathing either normoxic (21%) or hyperoxic air (60%) from day 0 to 8 following immunization. Gated on lymphocyte, singlet, live, CD4^-, B220⁺. B) Flow cytometric plots and frequencies of T follicular helper (T_FH) cells of immunized mice 8 days following immunization and treated as in A. Gated on lymphocytes, singlet, live, B220^-, FoxP3^-, CD4⁺. C) Effect of breathing 60% O₂ on relative ICOS expression of T_FH. D) Representative IgM and IgG1 antibody secreting cell frequencies determined by ELISPOT. E) Effect of breathing 60% O₂ on plasma cell frequencies, gated on lymphocytes, singlet, live, B220^low. F) Effect of breathing 60% O₂ on serum antigen specific IgM. G) Effect of breathing 60% O₂ on serum IgG1. H-I) NP-binding GC IgM and IgG1 compartments and effect of breathing 60% O₂. Gated on lymphocytes, singlet, live, CD4^-, B220⁺, GL7⁺, CD38^-. J) Histological images of IgG1 within GCs on day 8 of mice treated as in A. Bars placed at geometric mean. Representative plots of two independent experiments where mice were immunized with either NP-OVA/Alum or NP-CGG/Alum and analyzed 8 days following immunization. Each dot represents one mouse. Representative of at least two independent experiments. Five to ten mice per group.