T-bet+ B cells are activated by and control endogenous retroviruses through TLR-dependent mechanisms

Abstract

Endogenous retroviruses (ERVs) are an integral part of the mammalian genome. The role of immune control of ERVs in general is poorly defined as is their function as anti-cancer immune targets or drivers of autoimmune disease. Here, we generate mouse-strains where Moloney-Murine Leukemia Virus tagged with GFP (ERV-GFP) infected the mouse germline. This enables us to analyze the role of genetic, epigenetic and cell intrinsic restriction factors in ERV activation and control. We identify an autoreactive B cell response against the neo-self/ERV antigen GFP as a key mechanism of ERV control. Hallmarks of this response are spontaneous ERV-GFP⁺ germinal center formation, elevated serum IFN-γ levels and a dependency on Age-associated B cells (ABCs) a subclass of T-bet⁺ memory B cells. Impairment of IgM B cell receptor-signal in nucleic-acid sensing TLR-deficient mice contributes to defective ERV control. Although ERVs are a part of the genome they break immune tolerance, induce immune surveillance against ERV-derived self-antigens and shape the host immune response.

Fig. 1. Generation and expression of ERV-GFP in the mouse strain EGT-315.

a Schematic genome and virus structure of ERV-GFP Mo-MuLV. Magnification: env-GFP fusion protein (green). b Detection of ERV-GFP by PCR in genomic DNA of embryonic stem (ES) cells and mouse embryonic fibroblasts (MEFs). c Expression of ERV-GFP RNA transcripts analyzed by Q-PCR in MEFs and ES cells. Single aliquots of uninfected (wt) and ERV-GFP infected ES cells and MEFs were tested. Duplicate measurements ES V6.5 ERV-GFP mean = 0.039 (technical replication), SD = 0,004 and MEF ERV-GFP, mean = 1.1, SD = 0.18 (technical replication). Statistics of technical replication with ordinary one-way ANOVA, ***p = 0.0003. d ERV-GFP protein detection by flow cytometry. Anti-CD90.2 staining was used to differentiate ES cells from MEF feeder cells in co-culture. e Microphotograph of ERV-GFP infected ES-cell colony and MEFs. ERV-GFP (green; feeder cells) and nuclear counterstaining with DAPI (gray; all cells). f Flow cytometry for ERV-GFP expression in blood from 3-week old founder mouse EGT-315 backcrossed to C57BL/6 background and negative controls (C57BL/6 and EGT-314 non-transgenic founder, upper row). Example of in vitro infection assay of WEHI-231 cells by plasma of EGT-315 Tlr7^−/− mice (genotype: Tlr3^+/-Tlr7^−/−Tlr9^+/-) (5 positive of n = 6) and EGT-315 wild type Tlr7^−/− littermates (n = 3) and C57BL/6 (n = 3) (lower row). g Representative flow cytometry of neonatal ERV-GFP activation in spleen, on day 1 and day 6 and 7 in EGT-315 C57BL/6 (n = 0/2 on day 1 and 3/3 on day 6) and EGT-315-Tlr7^−/− mice (genotype: Tlr3^+/- Tlr7^−/− Tlr9^+/-) (n = 0/2 and 4/6 on day 7). h Flow cytometry of cell type-specific expression of ERV-GFP in cells of spleen and bone marrow of F1-4 EGT-315 B6 mice (black bars, n = 5) as well as EGT-315-Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (white bars, n = 5). Green indicates the percentage of ERV-GFP⁺ cells relative to 100% of the cell type. EGT-315 B6 Spleen: B cells (mean = 7.6, SD = 4.3), T cells (mean = 9.2, SD = 7.5), Granulocytes (mean = 27, SD = 14.1), DCs (mean = 9, SD = 4.2). EGT-315 B6 bone marrow: B cells (mean = 3.2, SD = 2.4), T cells (mean = 8.9, SD = 9.3), Granulocytes (mean = 2.8, SD = 2.5), DCs (mean = 2.8, SD = 0.042). EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− spleen: B cells (mean = 17.7, SD = 5.7), T cells (mean = 15, SD = 6.3), Granulocytes (mean = 54, SD = 21.2), DCs (mean = 34, SD = 16.8). EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− bone marrow: B cells (mean = 28.5, SD = 13.6), T cells (mean = 25, SD = 0.31), Granulocytes (mean= 34.6, SD = 4.7), DCs (mean = 26.6, SD = 11.3). i Confocal microphotograph from small intestine of 2.5-month old mice F1-4; helper T cells (CD4), B cells (B220) and nuclei (DAPI). GFP signal indicates ERV-GFP expression. Intestines of 2.5-month old mice representative for C57BL/6 mice (n = 4), EGT-315 B6 mice (n = 4), and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (n = 6). Scale bar 20 µm. Right pannel, summary of ERV-GFP positive crypts (green) compared to negative crypts (black) from generations F1-4 EGT-315 B6 mice (n = 4), and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (n = 5). EGT-315 B6 31,7% (83 GFP⁺ crypts of 262) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice 54,3% (139 GFP⁺ crypts of 256). Source data are provided as a Source Data file.

Fig. 2. ERV-GFP expression in vivo is influenced by modifier genes from the 129/Sv genetic background but not by epigenetic DNA methylation and suppressed by human cell intrinsic restriction factor Apobec3G (hA3).

a Reintroduction of 129/Sv genes by backcross of EGT-315 B6 (F9-F11) (mean = 0.79, SD = 2.23, n = 40) with 129/Sv increases ERV-GFP expression in EGT-315 B6 x 129/Sv (F1) mice (mean = 8.91, SD = 14.0, n = 32). C57BL/6 (mean = 0, SD = 0, n = 9) and (129/Sv x C57BL/6) F1 wt littermates (mean = 0.063, SD = 0.122, n = 9). Red bars show mean. Flowcytometry of blood leukocytes at 3 weeks of age. ***p = 0.0008. Ordinary one-way ANOVA Tukey´s multiple comparsion test. b Epigenetic DNA methylation status of a 549 bp DNA fragment of ERV-GFP in GFP positive (+) vs GFP negative (-) cells isolated from spleen cells of EGT-315 mice. DNA was digested with methylation sensitive HpaII (recognition site CCGG) and normalized with Plcg2 specific PCR w/o HpaII recognition site. ES-cells are ERV-GFP containing but GFP-expression negative and ERV-GFP infected WEHI-231 cells strongly express GFP. From individual mice sorted GFP⁺ cells (mean = 4.91, SD = 4.32, n = 9), sorted GFP^- cells (mean = 3.3, SD = 1.9, n = 15), WEHI-231 GFP⁺ (mean = 0.37, SD = 0.12, n = 4), ES cells GFP^- (mean = 26.9, SD = 8.3, n = 4). Sorted GFP⁺ cells vs Sorted GFP^- cells ^nsP = 0.752. Sorted GFP⁺ cells vs ES cells GFP^- ****P < 0.0001. Sorted GFP^- cells vs ES cells GFP^- ****P < 0.0001. WEHI-231 GFP⁺ vs ES cells GFP^-****P < 0.0001. Ordinary one-way ANOVA Tukey´s multiple comparsion test. Summary of 3 experiments. c Q-PCR of RNA expression of the GFP and Env region (549 bp) of ERV-GFP transcribed in spleen of EGT-315 mice (left panel). Sequence analysis of RNA expression analysis by RT-PCR-transcripts of EGT-315 mice with and without human Apobec3 (hA3Tg). Each dot depicts the mean number of mutations of 4–8 clones sequenced per individual mouse of the different genotypes (right panel). EGT-315 B6 (green bar, mean = 1, SD = 1.9, n = 4), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 0.06, SD = 0.09, n = 5), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− hA3 (purple bar, mean = 7.3, SD = 6.0, n = 5). Statistics of mutations EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− versus EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− hA3 *p = 0.028 by Ordinary one-way ANOVA Tukey´s multiple comparsion test. Source data are provided as a Source Data file.

Fig. 3. Properties of the env-GFP membrane autoantigen that lead to TLR-dependent anti-GFP IgG response.

a Comparison of anti-GFP IgG antibody response measured by GFP-specific ELISA for EGT-315 B6 (green dots, n = 9) vs different GFP reporter mice, Nkx6.1 Venus (n = 9), CAT-GFP (n = 7), Foxo-1-GFP (n = 6), mb1-GFP (n = 6), Blimp1-GFP (n = 6), IL4/GFP (n = 8) and EGT-315 Tlr3^-/-Tlr7^-/-Tlr9^-/--hA3 transgenic mice (n = 3). b Stratification of ERV-GFP expression and anti-GFP IgG production. Upper panel: flow cytometry of ERV-GFP expression as % positive peripheral blood cells per mouse. EGT-315 B6 mice (green, total n = 30) were subdivided into three groups: high percentage of ERV-GFP positive cells (green dots, n = 5), low (green squares, n = 11) and negative (green triangles, n = 14). EGT-315 Tlr7^-/- (genotype: Tlr3^+/-Tlr7^−/−Tlr9^+/-) (blue triangles, n = 6) and C57BL/6 (black rhombuses, n = 6). Lower panel: corresponding anti-GFP IgG titers measured by GFP-specific ELISA. Statistics by two-tailed unpaired t-test with Welch´s correction, upper pannel GFP: EGT-315 B6 ERV-GFP^high vs ERV-GFP^low ***P < 0,0001; EGT-315 B6 ERV-GFP^high vs. ERV-GFP^negative ****P < 0,0001. lower pannel anti-GFP IgG: EGT-315 B6 ERV-GFP^high vs ERV-GFP^low **P = 0,0013; EGT-315 B6 ERV-GFP^high vs ERV-GFP^negative ***P = 0,0004. c Western blot of antibody production of EGT-315 mice against GFP protein blotted on stripes. Stripes were incubated with plasma from 2 to 4-month old mice. Visualization was done by anti-IgG-HRP detection. Representative for EGT-315 Tlr7^−/− mice (genotype: Tlr3^+/-Tlr7^−/−Tlr9^+/-) (n = 4) EGT-315 Tlr7^+/- mouse (n = 1), EGT-315 C57BL/6 mice (n = 10 positive from 11), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 4) and C57BL/6 mice (n = 5). d Env-GFP is expressed on the surface of cells. Flow cytometry of inhibitory anti-GFP mAb binding to ERV-GFP expressing cells. Biotinylated anti-GFP monoclonal Ab clone 174 was used to stain NIH-3T3 ERV-GFP. Anti-GFP mAb174 and Streptavidin-Alexa Fluor 633 (blue), only Streptavidin Alexa Fluor 633 (red) (n = 3). e Schematic genome structure of the recombinant Mo-MuLV used to generate EGT-315 (pMOV-GFP) and the EZGT-332/3 (pZAPM-GFP) mouse strains. f Comparison of anti-GFP IgG response of individual mice. Genotype: C57BL/6 (mean = 1.2, SD = 1.3, n = 18), Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 0.5, SD = 0.5, n = 19), EGT-315 B6 (mean = 452.4, SD = 672.9, n = 37), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 0.48, SD = 0.48, n = 27), EZGT B6 (mean = 19, SD = 58.6, n = 29), EZGT Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 0.68, SD = 0.58, n = 16); EGT-315 B6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001; EGT-315 B6 vs EZGT B6 ****P < 0.0001. Ordinary one-way ANOVA Tukey´s multiple comparsion test. Summary of 3 experiments. Source data are provided as a Source Data file.

Fig. 4. The GFP-specific IgG response engages spontaneous germinal centers containing ERV-GFP immune complexes.

a GFP virus containing immune complexes on FDC network in B cell follicles and germinal centers of EGT-315 mice examined by confocal microphotography. Representative of spleens from EGT-315 B6 (n = 6) and EGT-315 Tlr3^-/-Tlr7^-/-Tlr9^-/- (n = 4) mice (scale bar 20 µm). Two left panels, ERV-GFP (green) and DAPI (blue). Right panels, spontaneous GCs stained with PNA-Alexa Fluor 647 (Peanut Agglutinin, red). Nuclei were stained with DAPI (blue). ERV-GFP (green). White triangels point to weak ERV-GFP signal in follicles. b ERV-GFP deposition in B cell follicles of EGT-315 B6 mice. Left, confocal microphotography representative of spleens stained with PNA-Alexa Fluor 647 (Peanut Agglutinin, red) and CD19-PE (blue). ERV-GFP is shown in green. Middle, microphotography of ERV-GFP deposition in standard immunflourescence (IF) in green. Right, quantification of ERV-GFP (green) vs ERV-GFP negative (black) B cell follicles of individual mice: EGT-315 B6 young (positive 7.5%; 15 + /186-; n = 5; mean age = 1m19d), EGT-315 B6 old (2%; 6 + /297-; n = 12; mean age = 5 m 24d), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (positive 0%; 0 + /348-; n = 10; mean age = 4m5d) and C57BL/6 and Tlr3^−/−Tlr7^−/−Tlr9^−/− (positive 0%; 0 + /151-; n = 4; mean age = 8m14d). c Identification of IgD^-GL7⁺ GC B cells in spleen. Left panel, exemplary flow cytometry of anti-IgD vs GL7 staining (Blue gate, IgD^-GL7⁺). Right  panel, statistical analysis of GL7⁺ B cells of C57BL/6 (mean = 111, SD = 117, n = 14) Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 59, SD = 33, n = 12), EGT-315 B6 (mean = 363, SD = 232, n = 16), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 62, SD = 52, n = 15). C57BL/6 vs. EGT-315 B6 ****P < 0.0001; EGT-315 B6 vs. EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. Ordinary one-way ANOVA Tukey’s multiple comparisons test. Summary of 8 experiments. d Q-PCR mRNA expression analysis of purified splenic B cells from individual mice. Values are x-fold expression relative to houskeeping gene actin. ERV-GFP (pMOV-GFP, left) C57BL/6 (black bar, mean = 0, SD = 0, n = 3), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 0, SD = 0, n = 2), EGT-315 B6 (green bar, mean = 1.7, SD = 2.3, n = 7), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 122.7, SD = 84.6, n = 3), Summary of 7 experiments; **P = 0.0096; AID (middle) C57BL/6 (black bar, mean = 0.41, SD = 0.17, n = 3), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 0.13, SD = 0.1, n = 3), EGT-315 B6 (green bar, mean= 1.36, SD = 0.96, n = 15), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 0.09, SD = 0.04, n = 8), Summary of 8 experiments; **P = 0.0019. T-bet (right) C57BL/6 (black bar, mean = 0.41, SD = 0.06, n = 3), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 0.48, SD = 0.34, n = 3), EGT-315 B6 (green bar, mean = 0.84, SD = 0.34, n = 15), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 0.46, SD = 0.25, n = 10), Summary of 8 experiments, **P = 0.0074; For all Q-PCR statistics we used two-tailed unpaired t-tests. Source data are provided as a Source Data file.

Fig. 5. ERV-GFP recognition results in upregulation of IFN-γ, IgG2c, increases T-bet⁺ ABCs and is dependent on T-bet.

a T-bet positive B cells identified by intracellular flow cytometry. Left, examples of dot plot analysis of the four genotypes, red line demarcates T-bet negative vs positive cells. Lower example, cells of a leukemic EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mouse. Right, summary of % T-bet⁺ cells of individual mice: C57BL/6 (mean = 1.07, SD = 0.463, n = 3), Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 0.29, SD = 0.15, n = 3), EGT-315 B6 (mean = 1.75, SD = 0.189, n = 8), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 0.52, SD = 0.19, n = 7). EGT-315 B6 vs. EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. Ordinary one way ANOVA multiple comparisons test. Summary of 5 experiments. b ABCs in EGT-315 B6 mice. Gating strategy of CD19⁺ splenic B cells from C57BL/6 (2.3, n = 1), EGT-315 B6 (mean = 35.97, SD = 19.1, n = 3), EGT-315 TLR3^−/−TLR7^−/−TLR9^−/− (mean = 4.0, SD = 0.33, n = 2) and EGT-315 T-bet^−/− mice (mean = 0.67, SD = 0.15, n = 2). DN defines CD23^- CD21^- B cells. Box in DN panel defines both CD11c⁺ CD11b⁺ and CD11c⁺ CD11b^- B cells analyzed for T-bet expression. Right panel, summary of mice tested for % T-bet positive CD11c⁺ cells. c Overlay of histogram for GL7 of DN (red) and T-bet⁺ CD11c⁺ ABC population (blue) of EGT-315 B6 mice (n = 3). GL7 positive B cells are present in the DN B cell population but not in the ABC population in spleen of individual EGT-315 B6 mice. DN (mean = 9.48 SD = 0.627, n = 3), T-bet⁺ CD11c⁺ ABC ⁽mean = 1.14 SD = 0.752, n = 3), ***P = 0.0002. Unpaired Two-tailed t-test with Welch´s correction. d Serum IFN-γ levels in pg/ml measured by cytometric bead array for mouse inflammatory cytokines in individual sera of C57BL/6 (black bar, mean = 337, SD = 40, n = 10), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 338, SD = 26, n = 14), EGT-315 B6 (green bar^, mean = 392, SD = 34, n = 16), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 275, SD = 37, n = 16). For statistics a Two-tailed Mann–Whitney U-test was applied. EGT-315 B6 vs. C57BL/6 **P = 0.0029, EGT-315 B6 vs. EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. Experiment is a summary of two experiments. e Serum IgG2c levels measured by IgG2c-specific ELISA in aribitrary units (AU) in C57BL/6 (mean = 683, SD = 918, n = 13), Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 83, SD = 62, n = 14), EGT-315 B6 (mean = 1442, SD = 1088^, n = 21). EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 121, SD = 120, n = 23) mice. C57BL/6 vs EGT-315 B6 *P = 0.0186; Tlr3^−/−Tlr7^−/−Tlr9^−/− vs EGT-315 B6 ****P < 0.0001; EGT-315 B6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. Ordinary one-way ANOVA Tukey´s multiple comparisons test. Summary of 3 experiments. f ERV-GFP expression and anti-GFP antibody response in EGT-315 T-bet^−/− mice. Flow cytometry of generation matched (F11-F12) EGT-315 B6 mice (mean = 0, SD = 0, n = 9) EGT-315 T-bet^−/− (mean = 10.5, SD = 14.1, n = 26) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (mean = 2.9, SD = 5.2, n = 17). % GFP positive cells in PBMC of 3-week old mice (left panel). EGT-315 B6 vs EGT-315 T-bet^−/− *P = 0.0327. 10 out of 26 mice (38.5%) of the EGT-315 T-bet^−/− mice display very high ERV-GFP expression (>10%). Red bars are mean values. Statistics with Ordinary one-way ANOVA Tukey´s multiple comparisons test. Summary of 3 experiments. Anti-GFP IgG (middle) EGT-315 B6 mice (mean = 504, SD = 773, n = 9) EGT-315 T-bet^−/− (mean = 10.2, SD = 11.3, n = 14) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (mean = 4.5, SD = 2.5, n = 8). EGT-315 B6 vs EGT-315 T-bet^−/− *P = 0.0242; EGT-315 B6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− *P = 0.0485; EGT-315 T-bet^−/− vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^{-/- ns}P = 0.9995; Statistics with Tukey´s multiple comparisons test. Anti-GFP IgG2c (right panel) response of 2–3 month old mice measured by ELISA. EGT-315 B6 mice (mean = 1374, SD = 2873, n = 9) EGT-315 T-bet^−/− (mean = 9.3, SD = 24.3, n = 14) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (mean = 1, SD = 3, n = 8). All ^nsP-values > 0.1. Summary of 2 experiments. g Confocal microphotograph from small intestine. GFP signal indicates ERV-GFP expression. Intestines of 2.5-month old mice representative for C57BL/6, EGT-315 B6 and EGT-315 T-bet^−/− mice. Right, summary of ERV-GFP positive crypts (green) compared to negative crypts (black) EGT-315 B6 mice (2.2% GFP⁺, total crypts analyzed 792 of n = 21), EGT-315 T-bet^−/− (12.8% GFP⁺, total crypts analyzed 601 of n = 9) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (37.2% GFP⁺, total crypts analyzed 487 of n = 16). Summary of 4 experiments. h EGT-315 T-bet^−/− mice succumb to T-ALL. Summary of mice analysed: EGT-315 B6 (3/34, 8.8%). EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (5/13, 38%). EGT-315 T-bet^−/− (6/11, 55%). The mice examined were 7 month or older. Each dot represents a single mouse. *T-ALL cells from an EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mouse show increased T-bet expression. **Two out of six lymphomas of EGT-315 T-bet^−/− mice were ERV-GFP positive (green dots). Source data are provided as a Source Data file.

Fig. 6. ERV-GFP enhances natural ERV (MuLV of C57BL/6) expression, anti-MuLV antibody production, anti-DNA IgG production and immune complex deposition in kidneys.

a MuLV specific mRNA expression analyzed by Q-PCR of purified splenic B cells from individual mice. Actin was used to normalize expression. ERV (MuLV): C57BL/6 (black bar, mean = 0.04, SD = 0.03, n = 4), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 1216, SD = 800, n = 4), EGT-315 B6 (green bar, mean = 0.89, SD = 0.23, n = 9), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 7202, SD = 6050, n = 6), Summary of 6 experiments; **P = 0.0055 by unpaired Two-tailed t test. b Anti-MuLV IgG production is increased in EGT-315 B6 mice. MuLV-specific ELISA of plasma from C57BL/6 (mean = 252.7, SD = 315.4, n = 19), Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 11.9, SD = 7.3, n = 8), EGT-315 B6 (mean = 2711, SD = 1936, n = 16 including 5 pools purple dots), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 9.1, SD = 6.7, n = 15 including 5 pools), red lines depict mean value. Summary of 3 experiments; C57BL/6 vs EGT-315 B6 ****P < 0.0001. Statistics with Ordinary one-way ANOVA Tukey´s multiple comparisons test. c Anti-DNA IgG production is increased in EGT-315 B6 mice. DNA (ss and ds) specific ELISA of plasma from C57BL/6 (black bar, mean = 2.1, SD = 0.86, n = 6), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, mean = 0.67, SD = 0.24, n = 6), EGT-315 B6 (green bar, mean = 5.1, SD = 2.9, n = 6), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, mean = 0.9, SD = 0.37, n = 6). **P = 0.0055. Statistics with Ordinary one-way ANOVA Tukey´s multiple comparisons test. d Confocal microphotography of immune complex deposition containing IgG and ERV-GFP in kidney glomeruli representative of EGT-315 B6 (n = 7) but not in C57BL/6 (n = 4) and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− mice (n = 2). Anti-IgG Alexa Fluor 633, DAPI (nucleus) and ERV-GFP are shown. Scale bar 20 µm. Source data are provided as a Source Data file.

Fig. 7. Presence of ERV-GFP and absence of nucleic-acid Tlr-sensing results in systemic IgE overproduction.

a Serum IgE levels measured by IgE-specific ELISA from individual mice: C57BL/6 (mean = 0.43, SD = 0.79, n = 7), Tlr3^−/−Tlr7^−/−Tlr9^−/−(mean = 0.11, SD = 0.33, n = 9), EGT-315 B6 (mean = 0.06, SD = 0.24, n = 18) EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 5.5, SD = 8.0, n = 17). Red lines depict mean value. Tlr3^−/−Tlr7^−/−Tlr9^−/− vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− *P = 0.035. EGT-315 B6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− **P = 0.0061. Ordinary one-way ANOVA Tukey´s multiple comparisons test. Summary of 3 experiments. b Basophil-bound IgE levels measured by flow cytometry of spleen cells from individual mice: C57BL/6 (mean = 373, SD = 252; n = 14), Tlr3^−/−Tlr7^−/−Tlr9^−/−(mean = 196, SD = 131, n = 11), EGT-315 B6 (mean = 128, SD = 73, n = 15), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 939, SD = 190, n = 16). Left exemplary flow cytometry panel, and right panel statistical evaluation of MFI of the IgE⁺CD23^- basophil population (red circle). C57BL/6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. EGT-315 B6 vs EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ****P < 0.0001. Statistics with Ordinary one-way ANOVA Tukey´s multiple comparisons test. Summary of 12 experiments. Source data are provided as a Source Data file.

Fig. 8. Combined deficiency of Tlr3 Tlr7 Tlr9 in B cells impairs IgM-BCR induced B cell survival/proliferation but upregulates CD23 expression induced by Tlr4 in vitro.

a Flow cytometry plots of IgM vs IgD staining of splenic B cells representative of C57BL/6 (n = 7), Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 7), EGT-315 B6 (n = 6), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 6). Squares: mature B cells (left), transitional B cells (right upper), immature B cells (right lower). Summary of 9 experiments. b Follicular (gate upper middle) and marginal zone (gate lower right) B cell distribution in Tlr deficient mice. Exemplary flowcytometry of spleen cells from C57BL/6 (n = 3), Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 2), EGT-315 B6 (n = 6), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 5) using the B cell markers CD23 and CD21. Summary of 3 experiments. c GFP-specific IgM measured by GFP specific ELISA from individual mice: C57BL/6 (mean = 1160, SD = 476, n = 4), Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 392, SD = 229, n = 6), EGT-315 B6 (mean = 3249, SD = 2037, n = 29), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (mean = 312, SD = 171, n = 8). Red lines depict mean value. EGT-315 B6 vs. EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− ***P = 0.0004. Ordinary one-way ANOVA Tukey’s multiple comparisons test. d Survival/proliferation of purified B cells isolated from individual mice 72 h after in vitro stimulation (no stimulus, LPS = Tlr4 ligand, anti-IgM = BCR ligand, R848 = Tlr7 ligand, 1668 PTO = Tlr9 ligand) measured by flow cytometry using propidium iodide to distinguish live vs dead cells. C57BL/6 (black bar, for anti-IgM: mean = 15.1, SD = 5.9, n = 9), Tlr3^−/−Tlr7^−/−Tlr9^−/− (red bar, for anti-IgM: mean = 3.2, SD = 2.0, n = 9), EGT-315 B6 (green bar, for anti-IgM: mean = 18.6, SD = 6.3, n = 8), EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− (blue bar, for anti-IgM: mean = 4.0, SD = 3.0, n = 7). Summary of 12 experiments, 2 way ANOVA, Sidak´s multiple comparisons test ****P < 0.0001. e Role of Btk in survival/proliferation of purified B cells 72 h after in vitro stimulation. Anti-IgM was used with and without the addition of the Btk-inhibitor Ibrutinib. Cells from individual mice were tested. C57BL/6 and EGT-315 B6 combined (green bar, for anti-IgM: mean = 11.9, SD = 4.3, n = 6), Tlr3^−/−Tlr7^−/−Tlr9^−/− and EGT-315 Tlr3^−/−Tlr7^−/−Tlr9^−/− B6 combined (blue bar, for anti-IgM: mean = 1.7, SD = 1.1, n = 6). Summary of 5 experiments, 2-way ANOVA, Sidak´s multiple comparisons test, ****P < 0.0001. f Tlr3^−/−Tlr7^−/−Tlr9^−/− B cells induced by LPS (Tlr4-ligand) upregulate CD23. Upper panels, LPS-mediated in vitro induction of CD23 is higher in Tlr3^−/−Tlr7^−/−Tlr9^−/− B cells compared to wild type B cells. Flowcytometry examples of stimulated B cells from C57BL/6 and Tlr3^−/−Tlr7^−/−Tlr9^−/− mice for 72 h with medium alone (red), LPS (10 μg/ml, blue) and LPS + IL-4 (LPS 10 μg/ml+ IL-4 250 U/ml, green). Right panels, Summary of MFI of CD23 from three independent experiments. Mann-Whitney test Two-tailed pairwise comparison ***P = 0.0007 and **P = 0.0031. B6 (n = 10), Tlr3^−/−Tlr7^−/−Tlr9^−/− (n = 7). Source data are provided as a Source Data file.