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. 2024 Feb 9;15:1229. doi: 10.1038/s41467-024-45201-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — 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.