A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens
Vishal J. Sindhava ... Ann Marshak-Rothstein, Michael P. Cancro
Vishal J. Sindhava ... Ann Marshak-Rothstein, Michael P. Cancro
Published May 1, 2017
Citation Information: J Clin Invest. 2017;127(5):1651-1663. doi:10.1172/JCI89931.
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Categories: Research Article Immunology

A TLR9-dependent checkpoint governs B cell responses to DNA-containing antigens

Abstract

Mature B cell pools retain a substantial proportion of polyreactive and self-reactive clonotypes, suggesting that activation checkpoints exist to reduce the initiation of autoreactive B cell responses. Here, we have described a relationship among the B cell receptor (BCR), TLR9, and cytokine signals that regulate B cell responses to DNA-containing antigens. In both mouse and human B cells, BCR ligands that deliver a TLR9 agonist induce an initial proliferative burst that is followed by apoptotic death. The latter mechanism involves p38-dependent G1 cell-cycle arrest and subsequent intrinsic mitochondrial apoptosis and is shared by all preimmune murine B cell subsets and CD27– human B cells. Survival or costimulatory signals rescue B cells from this fate, but the outcome varies depending on the signals involved. B lymphocyte stimulator (BLyS) engenders survival and antibody secretion, whereas CD40 costimulation with IL-21 or IFN-γ promotes a T-bet+ B cell phenotype. Finally, in vivo immunization studies revealed that when protein antigens are conjugated with DNA, the humoral immune response is blunted and acquires features associated with T-bet+ B cell differentiation. We propose that this mechanism integrating BCR, TLR9, and cytokine signals provides a peripheral checkpoint for DNA-containing antigens that, if circumvented by survival and differentiative cues, yields B cells with the autoimmune-associated T-bet+ phenotype.

Authors

Vishal J. Sindhava, Michael A. Oropallo, Krishna Moody, Martin Naradikian, Lauren E. Higdon, Lin Zhou, Arpita Myles, Nathaniel Green, Kerstin Nündel, William Stohl, Amanda M. Schmidt, Wei Cao, Stephanie Dorta-Estremera, Taku Kambayashi, Ann Marshak-Rothstein, Michael P. Cancro

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Addition of BLyS prevents AM14 and WT B cells from undergoing proliferat...

Figure 1

Addition of BLyS prevents AM14 and WT B cells from undergoing proliferation-associated cell death following stimulation with BCR-delivered TLR9 ligands.

Representative FACS analysis at 60 hours (A) and percentage of live divided cells at 48, 60, and 72 hours (C) in AM14 CD23+ splenocytes cultured with the indicated stimuli in the presence or absence of BLyS. Dead cells were stained with TO-PRO-3, while CFSE dilution indicates proliferation. (B and D) Representative FACS analysis at 60 hours (B) and percentage of live divided cells at 48, 60, and 72 hours (D) in C57BL/6 CD23+ splenocytes cultured with the indicated stimuli in the presence or absence of BLyS. Diagram in B (inset) depicts the structure of STIC9. (E and F) Representative FACS analysis at 60 hours (E) and percentage of live divided cells 60 hours after STIC9 stimulation (F) in C57BL/6 and Tlr9–/– CD23+ B cells. (G) Immunoblot analysis of p-SYK in protein extracts isolated from CD23+ C57BL/6 splenocytes cultured with the indicated stimuli. Fold-change differences in expression are shown compared with unstimulated cells. Values in parentheses indicate the molecular weight. (H) FACS analysis of B cells from Nur77-GFP reporter mice at 5 hours, cultured with the indicated stimuli, with or without TLR9 inhibitor (Inh18) as described previously (84). Gray-filled area represents no stimulation; black line represents F(ab′)2 fragments of anti-IgM; gray line represents ODN 1826; blue line represents F(ab′)2 fragments of anti-IgM plus ODN 1826; and red line represents STIC9. Max, maximum. (I) Representative FACS plots show the proliferation and survival of C57BL/6 CD23+ splenocytes cultured for 60 hours with SA-linked biotinylated (Bio) ODN 1826 and biotinylated F(ab)2, in the presence or absence of BLyS. Data represent a minimum of 3 independent experiments with 3 mice each. Error bars indicate the mean ± SEM. *P < 0.05 and ***P < 0.001, by 2-tailed Student’s t test. “–” signifies unstimulated cells.