MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses - PubMed (original) (raw)

. 2014 Dec 19;346(6216):1486-92.

doi: 10.1126/science.346.6216.1486.

Zeping Hu 2, Xiaolei Shi 2, Xiaohong Li 1, Xiaoming Zhan 1, Xiao-Dong Li 3, Jianhui Wang 3, Jin Huk Choi 1, Kuan-wen Wang 1, Tiana Purrington 1, Miao Tang 1, Maggy Fina 1, Ralph J DeBerardinis 2, Eva Marie Y Moresco 1, Gabriel Pedersen 4, Gerald M McInerney 4, Gunilla B Karlsson Hedestam 4, Zhijian J Chen 3, Bruce Beutler 5

Affiliations

MAVS, cGAS, and endogenous retroviruses in T-independent B cell responses

Ming Zeng et al. Science. 2014.

Retraction in

Abstract

Multivalent molecules with repetitive structures including bacterial capsular polysaccharides and viral capsids elicit antibody responses through B cell receptor (BCR) crosslinking in the absence of T cell help. We report that immunization with these T cell-independent type 2 (TI-2) antigens causes up-regulation of endogenous retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a mitochondrial antiviral signaling protein (MAVS)-dependent RNA sensing pathway or reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a second, sustained wave of signaling that promotes specific immunoglobulin M production. Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings suggest that ERV and two innate sensing pathways that detect them are integral components of the TI-2 B cell signaling apparatus.

Copyright © 2014, American Association for the Advancement of Science.

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Figures

Figure 1

Figure 1. Cytosolic DNA and RNA sensing pathways are essential for induction of the TI-2 antibody response

(A) Serum NP-specific IgM on day 4.5 post-immunization with NP-Ficoll. (B) Serum NP-specific IgM on day 4.5 post-immunization with NP-LPS. (C) Serum βgal-specific IgG on day 14.5 post-immunization with rSFV-encoded βgal. (D) Serum NP-specific IgM on day 4.5 post-immunization of Rag2−/− mice adoptively transferred 1 day prior to immunization with splenic and peritoneal B cells from donor mice of the indicated genotypes. Data points represent individual mice. P values were determined by one-way ANOVA and post hoc Tukey test; in B and C, no significant difference was found between any mutant genotype and C57BL/6J. Results are representative of 2–3 independent experiments.

Figure 2

Figure 2. cGAMP is elevated within antigen-specific B cells following TI-2 immunization and is sufficient to drive B cell activation in vitro and in vivo

(A) CD86, MHC class II, and CD25 expression, and BrdU incorporation by GFP+ or GFP− splenic CD19+ B cells 36 hours after transfection with a GFP expression plasmid. N = 3 C57BL/6J mice, 3 Stinggt/gt mice. (B) cGAMP level measured by liquid chromatogryph-tandem mass spectrometry (LC-MS/MS) in 2×105 NP-specific or non-NP-specific splenic CD19+ B cells from C57BL/6J mice on day 4.5 post-immunization with NP-Ficoll (N = 3) or in naïve mice (N = 3). Upper panels, chromatograms of cGAMP. Lower panel, cGAMP abundance normalized to cell number for each sample. (C) Time course of cGAMP levels in NP-specific or non-NP-specific B cells from C57BL/6J mice (N = 3) immunized with NP-Ficoll. (D and E) CD86 expression (D) or the percentage of BrdU+ splenic B cells (E) after treatment with cGAMP or vehicle for 2 days in vitro. N = 3 C57BL/6J mice, 3 Stinggt/gt mice. (F) Serum NP-specific IgM on day 5 post-immunization with NP-Ficoll plus cGAMP or NP-Ficoll plus vehicle. (G) HLA-DR or CD69 expression by human B cells isolated from healthy donor peripheral blood after treatment with cGAMP or vehicle for 2 days in vitro. MFI, mean fluorescence intensity. Data points represent individual mice or humans (B, F, G). P values were determined by one-way (A–B, D–F) or two-way ANOVA and post hoc Tukey test (C) or Student’s t test (G). Results are representative of 2–3 independent experiments.

Figure 3

Figure 3. TI-2 antigen immunization induces expression of ERV mRNA and cDNA that are detected by cytosolic sensors in antigen-specific B cells

Splenic NP-specific or non-NP-specific CD19+ B cells were collected from C57BL/6J mice (A, C and D) or IghB1-8+ transgenic mice (B) 4.5 days post-immunization with NP-Ficoll (N = 3 per experiment). (A) Transcript levels of the indicated ERVs measured by RT-qPCR of mRNA isolated from NP-specific or non-NP-specific B cells. Data were normalized to GAPDH mRNA levels in the same cells. Due to copy number differences the magnitude of upregulation of different ERVs cannot be directly compared in this experiment. (B) IghB1-8+ transgenic mice express a recombined variable region derived from an NP-binding antibody in place of the endogenous 3′ Igh-D element (DQ52) and the Igh-J elements. RT-qPCR of the indicated ERV mRNAs immunoprecipitated with RIG-I from NP-specific or non-NP-specific B cells. Data were normalized to the level of GAPDH mRNA bound to RIG-I in the same samples, which represents a non-specific interaction equivalent in NP-specific and non-NP-specific cells as shown in fig. S7E. (C) qPCR of ERV DNA in the cytoplasmic fraction of NP-specific or non-NP-specific B cells. Data were normalized to GAPDH intronic DNA levels in the same cells. Note that endogenous eMLV and MMTV were amplified with primers targeting spliced cDNAs; these species likely represent a minority of the cytoplasmic eMLV and MMTV cDNAs and thus may not precisely reflect total eMLV and MMTV cDNA levels. (D) RT activity in the indicated B cells from C57BL/6J mice. (E and F) Splenic CD19+ B cells from mice of the indicated genotypes were treated with NP-Ficoll plus RT inhibitors (AZT, NVP, ddI) or NP-Ficoll plus vehicle for 2 days, and CD86 (E) or GL7 expression (F) was measured in NP-specific B cells. N = 3 mice for each genotype. MFI, mean fluorescence intensity. (G) Serum NP-specific IgM on day 4.5 post-NP-Ficoll immunization of mice pretreated for 3 days with RT inhibitors (AZT, NVP) or vehicle. RT inhibitor treatment continued after immunization until measurement of serum IgM. (H) Serum NP-specific IgM on day 4.5 post-immunization with NP-Ficoll. Data points represent individual mice (D, G, H). P values were determined by Student’s t test (A–C) or one-way ANOVA and post hoc Tukey test (D–H). n.d., not detected. Results are representative of 2–3 independent experiments.

Figure 4

Figure 4. cGAMP elevation and ERV transcription are induced by BCR signaling

(A–C) Mouse splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hr. (A) CD86 and MHCII expression. (B) Transcript levels of the indicated ERVs measured by RT-qPCR of isolated mRNA. Data were normalized to GAPDH mRNA levels in the same cells. N = 3 mice per genotype. (C) Chromatograms of cGAMP in C57BL/6J B cells measured by LC-MS/MS. N = 3 mice. (D) Human B cells from healthy donor peripheral blood (N = 3 individuals) were cultured in vitro and stimulated with anti-IgM, anti-IgM + Ibrutinib, or vehicle for 48 hr. Transcript levels of the indicated human ERVs measured by RT-qPCR of isolated mRNA. (E) Serum NP-specific IgM on day 4.5 post-immunization with NP-Ficoll. (F) Transcript levels of the indicated ERVs measured by RT-qPCR of mRNA isolated from NP-specific or non-NP-specific B cells. Data were normalized to GAPDH mRNA levels in the same cells. N = 3 mice per genotype. (G) Activation marker expression by human B cells treated as in D, or with 0.06 μM cGAMP for 48 hr. MFI, mean fluorescence intensity. Data points represent individual mice or humans (A, E, G). P values were determined by Student’s t test (B, D–F) or one-way ANOVA and post hoc Tukey test (A, G). Results are representative of 2 independent experiments.

Figure 5

Figure 5. NF-κB is required for ERV induction and is activated by BCR and MAVS signaling

(A, B) Splenic NP-specific or non-NP-specific CD19+ B cells were collected from mice 4.5 days post-immunization with NP-Ficoll. (A) Cytokine expression in the indicated B cells from C57BL/6J mice. (B) Transcript levels of the indicated ERVs measured by RT-qPCR of mRNA isolated from NP-specific or non-NP-specific B cells. N = 3 mice. No significant difference was found between NP+ and NP− cells for any ERV tested. (C–D) Splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hr. (C) Levels of phospho-p65 (left) and phospho-p105 (right). N = 3 mice. (D) Levels of p65 in the nuclear fraction of cells. (E) Levels of phospho-p105 in splenic NP-specific and non-NP-specific CD19+ B cells or naïve B cells on day 6 post-NP-Ficoll immunization. N = 3 mice per genotype. MFI, mean fluorescence intensity. Data points represent individual mice (A, D). P values were determined by Student’s t test (B) or one-way ANOVA and post hoc Tukey test (A, C–E). Results are representative of 2 independent experiments.

Figure 6

Figure 6. TI-2 antibody responses to Streptococcus pneumoniae PS1 and PS3 and to the commercial vaccine Pneumovax 23 require cytosolic DNA and RNA sensing pathways

(A) Serum PS1- and PS3-specific IgM on day 4.5 post-immunization with S. pneumoniae PS1 and PS3. (B and C) Serum PPV-23-specific IgM (B) and IgG (C) on day 5.5 post-immunization with Pneumovax 23. Data points represent individual mice. P values were determined by one-way ANOVA and post hoc Tukey test (A) or Student’s t test (B and C). Results are representative of 2–3 independent experiments.

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