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 March 27, 2017
Citation Information: J Clin Invest. 2017;127(5):1651-1663. https://doi.org/10.1172/JCI89931.
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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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Figure 2

Cell death in response to STIC9 stimulation follows p38 MAPK–mediated cell-cycle arrest and mitochondrial apoptosis.

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Cell death in response to STIC9 stimulation follows p38 MAPK–mediated ce...
(A) Immunoblot analysis of caspase 9 and caspase 3 cleavage in protein extracts from CD23+ C57BL/6 splenocytes cultured for 60 hours with the indicated stimuli. Values in parentheses indicate the molecular weight. (B) Percentage of live divided CD23+ splenocytes from C57BL/6, RIP3–/–, and RIP3–/– caspase 8–/– mice following culture with the indicated stimuli. (C) Representative FACS plots of C57BL/6 and BCL-XL CD23+ splenocytes cultured for 60 hours with no stimulation or with STIC9 either loaded with CFSE and stained with TO-PRO-3 or stained with the mitochondrial stability–assessing dye JC-1. (D) Percentage of live divided C57BL/6 CD23+ splenocytes following stimulation with either anti-μ or STIC9 in the presence of various concentrations of the JNK inhibitor SP600125, the MEK1/2 inhibitor U0126, or the p38 inhibitor SB203580. Since vehicle and non-vehicle control groups showed no differences, the latter was used for controls in subsequent experiments. (E) Immunoblot analysis of caspase 9 cleavage as described in A. (F) Representative FACS plots assessing the mitochondrial stability of C57BL/6 CD23+ cells cultured for 60 hours with the indicated stimuli. (G) Percentage of live divided C57BL/6 CD23+ splenocytes following culture as in D with various p38 inhibitors. (H and I) FACS analysis measuring the cell-cycle status of C57BL/6 CD23+ splenocytes cultured for 48 hours with the indicated stimuli. (I) G0 and G1 phases were distinguished through Ki-67 staining. The gray area represents STIC9 alone; the dashed line represents STIC9 plus BLyS; and the solid black line represents STIC9 plus the p38 inhibitor SB203580. (J) Percentage of cells in the S/G2/M phase treated as in H. (J and K) FACS analysis measuring the cell-cycle status of BCL-XL–transgenic CD23+ splenocytes cultured for 48 hours. Error bars indicate the mean ± SEM; n ≥ 3 replicate analyses, and results are representative of 2 (B and E) or a minimum of 3 (A, C, D, and F–K) independent experiments. **P < 0.005 and ***P < 0.001, by 2-tailed Student’s t test. NS, not significant.