Cutting Edge: B Cell-Intrinsic T-bet Expression Is Required To Control Chronic Viral Infection

Abstract

The role of Ab and B cells in preventing infection is established. In contrast, the role of B cell responses in containing chronic infections remains poorly understood. IgG2a (IgG1 in humans) can prevent acute infections, and T-bet promotes IgG2a isotype switching. However, whether IgG2a and B cell-expressed T-bet influence the host-pathogen balance during persisting infections is unclear. We demonstrate that B cell-specific loss of T-bet prevents control of persisting viral infection. T-bet in B cells controlled IgG2a production, as well as mucosal localization, proliferation, glycosylation, and a broad transcriptional program. T-bet controlled a broad antiviral program in addition to IgG2a because T-bet in B cells was important, even in the presence of virus-specific IgG2a. Our data support a model in which T-bet is a universal controller of antiviral immunity across multiple immune lineages.

Fig. 1. B cell expressed T-bet and antiviral IgG2a required for control of cl13 infection

(A) (A) Flow cytometry of T-bet and IgM in splenic CD4⁻CD8⁻CD19⁺B220⁺IgD⁻CD138⁻ B cells after Arm infection. Number of T-bet⁺ B cells per spleen (bottom). (B) B220⁺CD19⁺IgD⁻GL7⁻CD38⁺T-bet⁺ B cells in WT, cHet, and cKO at d5 p.i. (spleen), quantified to the right. (C) Viral titers in the blood of WT, cHet, and cKO mice (left), and in kidney at d64 p.i. (Middle). The frequency of mice in each group with detectable serum virus over time after cl13 infection (Right). Log-rank score by survival statistics (score <0.05 indicates a significant difference in time to clearance from the serum) (D) LCMV-specific serum IgG2a (i.e. IgG2c; called IgG2a throughout) in infected WT, cHet, cKO, and uninfected mice. (E) LCMV-specific serum IgM at d7 p.i. (F) LCMV-specific IgG1, IgG2a, IgG2b, IgA, and IgG3 in the serum of infected mice at d64 p.i. (left). Total serum IgG in serum of cl13 infected mice at d64 p.i.(right) (G) IgG2a^−/− mice and WT littermate controls infected with cl13 and viral titers and IgG2a measured at d120 p.i.. (H) cHet or cKO mice were infected with cl13 and cKO mice were treated i.p. at d22, 27 and 32 p.i. with 200 μl of serum from WT mice at d120 p.i. with cl13. Treated cKO, untreated cKO, and untreated cHet mice analyzed for viral titers at d35 p.i. in the serum and kidneys. n=4-7 mice per experiment, each experiment repeated at least 2 times. ** p<0.01, *** p<0.001

Fig. 2. B cell responses to acutely resolved LCMV Arm infection are largely normal in cHet and cKO mice

(A) Memory B cells from the spleens at d30 p.i. with Arm were analyzed for expression of IgG2a and IgG1. (B) Serum LCMV-specific IgG2a and IgM were quantified over time after Arm infection. * p<0.05, n=2-6 mice/group.

Fig. 3. GC and memory B cells populations in T-bet deficient B cells after cl13 infection

(A) GC B cells (CD4⁻CD8⁻NK1.1⁻B220⁺CD19⁺IgD⁻GL7⁺CD38⁺) were analyzed in spleens of cl13 infected mice at d18 p.i. (B) GC (GL7⁺CD38⁻) and memory B cells (GL7⁻CD38⁺) were examined at d64 p.i. (C) Memory B cells at d64 p.i. were analyzed for expression of IgG2a and IgG1. n=3-5 mice per experiment, each experiment repeated at least 2 times. * p<0.05, ** p<0.01

Fig. 4. T-bet controls a broad antiviral gene expression program including genes involved in migration, proliferation, and antibody glycosylation

CD3⁻NK1.1⁻CD19⁺B220⁺ were sorted as IgD⁺CD38⁺, IgD⁻IgM⁻T-bet⁺, and IgD⁻IgM⁻T-bet⁻ B cells from cl13 infected T-bet-GFP reporter mice at d10 p.i. (A) Selected genes differentially expressed between T-bet⁺ and T-bet⁻ B cells, row normalized. (B) Genes significantly upregulated in T-bet⁺ B cells were compared to a microarray of T-bet^+/+ vs T-bet^−/− CD8⁺ T cells by GSEA. (C) Selected genes were confirmed by flow cytometry (top), with the MFI of each selected target quantified (bottom). (D) CD3⁻CD4⁻CD8⁻CD11c⁻NK1.1⁻Ter119⁻CD45.2⁺B220⁺CD19⁺IgD⁻CD138⁻GL7⁻CD38⁺ memory B cells were analyzed in the spleens, Peyer’s patches, LP, and IEL of infected mice at d10 p.i. (top). Memory B cells were analyzed for expression of T-bet and IgM, with the frequency of T-bet⁺IgM⁻ cells quantified (bottom). (E) IgD⁻ memory B cells were quantified in WT and cKO mice in the spleen, peyer’s patches, LP, and IEL (top). The same cells were examined for the frequency of IgM⁻T-bet⁺ cells (bottom). n=2-5 mice per experiment, each experiment repeated at least 2 times. * p<0.05, *** p<0.001