Research Article

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

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Science  19 Dec 2014:
Vol. 346, Issue 6216, pp. 1486-1492
DOI: 10.1126/science.346.6216.1486

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  1. Fig. 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 after immunization with NP-Ficoll. (B) Serum NP-specific IgM on day 4.5 after immunization with NP-LPS. (C) Serum β-gal–specific IgG on day 14.5 after immunization with rSFV-encoded β-gal. (D) Serum NP-specific IgM on day 4.5 after immunization of Rag2−/− mice adoptively transferred 1 day before 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 analysis of variance (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 two or three independent experiments.

  2. Fig. 2 cGAMP is elevated within antigen-specific B cells after 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(GFP, green fluorescent protein) splenic CD19+ B cells, 36 hours after transfection with a GFP expression plasmid. N = 3 C57BL/6J mice and 3 Stinggt/gt mice. (B) cGAMP level measured by liquid chromatography–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 after 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 and 3 Stinggt/gt mice. (F) Serum NP-specific IgM on day 5 after 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 and treated with cGAMP or vehicle for 2 days in vitro. MFI, mean fluorescence intensity. Data points represent individual mice or humans [(B), (F), and (G)]. P values were determined by one-way [(A), (B), and (D) to (F)] or two-way ANOVA and post hoc Tukey test (C) or Student’s t test (G). Results are representative of two or three independent experiments.

  3. Fig. 3 TI-2 antigen immunization induces expression of ERV mRNA and cDNA that is 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 after immunization with NP-Ficoll (N = 3 mice per experiment). (A) Transcript levels of the indicated ERVs measured by reverse transcription quantitative PCR (RT-qPCR) of mRNA isolated from NP-specific or non–NP-specific B cells. Data were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels in the same cells. Because of copy number differences, the magnitude of up-regulation 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 nonspecific 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. Endogenous ecotropic murine leukemia virus (eMLV) and mouse mammary tumor virus (MMTV) were amplified with primers targeting spliced cDNAs; these species probably 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, and 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. (G) Serum NP-specific IgM on day 4.5 after NP-Ficoll immunization of mice pretreated for 3 days with RT inhibitors (AZT and NVP) or vehicle. RT inhibitor treatment continued after immunization until measurement of serum IgM. (H) Serum NP-specific IgM on day 4.5 after immunization with NP-Ficoll. Data points represent individual mice [(D), (G), and (H)]. P values were determined by Student’s t test [(A) to (C)] or one-way ANOVA and post hoc Tukey test [(D) to (H)]. n.d., not detected. Results are representative of two or three independent experiments.

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

    (A to C) Mouse splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hours. (A) CD86 and MHC class II 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 hours. Transcript levels of the indicated human ERVs measured by RT-qPCR of isolated mRNA. (E) Serum NP-specific IgM on day 4.5 after 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 hours. Data points represent individual mice or humans [(A), (E), and (G)]. P values were determined by Student’s t test[ (B) and (D) to (F)] or one-way ANOVA and post hoc Tukey test [(A) and (G)]. Results are representative of two independent experiments.

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

    (A and B) Splenic NP-specific or non–NP-specific CD19+ B cells were collected from mice 4.5 days after 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 and D) Splenic B cells were cultured in vitro and stimulated with anti-IgM or vehicle for 22 hours. (C) Levels of phospho-p65 (left) and phospho-p105 (right) are shown. 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 after NP-Ficoll immunization. N = 3 mice per genotype. Data points represent individual mice [(A) and (D)]. P values were determined by Student’s t test (B) or one-way ANOVA and post hoc Tukey test [(A) and (C) to (E)]. Results are representative of two independent experiments.

  6. Fig. 6 TI-2 antibody responses to S. 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 after immunization with S. pneumoniae PS1 and PS3. (B and C) Serum PPV-23–specific IgM (B) and IgG (C) on day 5.5 after 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 two or three independent experiments.

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