Fig 2: TLR7 and TLR8 evade X chromosome inactivation in the monocytes of men with Klinefelter syndrome. A RNA FISH analysis of CD14+ monocytes from men with KS (47,XXY). Confocal microscopy planes of cell nuclei hybridized with fluorescent probes for transcripts arising from TLR8 (red), TLR7 (green), and the Xa marker genes (pink). Nuclei are counterstained with DAPI (blue). The arrow heads indicate TLR8 (top) or TLR7 (bottom) transcript foci occurring either on both X chromosomes (right) or on the Xi only (left). B, C Quantification of XCI escape for TLR7 and TLR8. The forest plots show the percentage of XCI escape and its 95% CI in KS men (n = 5). The meta-analytical group mean and 95% CI is denoted by the red diamond

Fig 3: TLR7 and TLR8 are transcriptionally non-independent in CD4+ T cells. Quantitative analysis of RNA FISH experiments on stimulated CD4+ T cells from women and XY men. Cell counts from RNA FISH experiments were processed as in Fig. 4. The forest plots display the statistic of interest for each donor and its 95% CI (dots and whiskers), together with the meta-analytical group means (diamonds) and their 95% CIs (whiskers and shaded areas). In A–C, T cells were scored regardless of the chromosome of origin of the TLR7 and TLR8 transcripts (Any X). In D–F, only cells positive for the Xa probe (Xa+) were counted, and the data are restricted to TLR7 and TLR8 signals observed on those Xa+ male or female chromosomes. A, D Percentage of T cells positive for both TLR7 and TLR8 transcripts. B, E Analysis for the obs/exp ratio of the observed number of double-positive cells to the number of cells in this category expected under the hypothesis of independent transcription of TLR7 and TLR8. C, F Analysis for Yule’s Q coefficient of association in 2 × 2 tables. The p-values in A, D test the differences between group means; in B, C, E, and F, each p-value tests the divergence of a group summary value relative to the critical value, obs/exp = 1 or Q = 0; the two-tailed p-values were derived from the group CDs in the meta-analytical summarization. Women and euploid men display deviations of opposite signs from Q = 0, signifying transcriptional non-independence of TLR7 and TLR8

Fig 4: SAMPs activate the TLR8/MyD88/NF-κB axis in moDCs.(A) HEK293 cells stably transfected with human TLR7, TLR8, or luciferase alone (-) were stimulated with SCV2-RNA for 24 hours. NF-κB activation was evaluated as luciferase activity. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus (-) by 1-way ANOVA with Dunnett’s post hoc test. (B–E) moDCs were stimulated with SCV2-RNA as indicated (B) or pretreated with CU-CPT9a (1 μM) and then stimulated with SCV2-RNA or LPS for 1 hour (E). Nuclear extracts were blotted against NF-κB p65 and lamin B. One representative donor and densitometry of 3 donors are shown. *P < 0.05 versus untreated by 1-way ANOVA with Dunnett’s post hoc test; #P < 0.05 versus “SCV2-RNA” by paired Student’s t test. (C, left panel) moDCs were transfected with indicated siRNAs and target gene expression evaluated by qPCR. Results depict percentage of target gene expression (mean ± SEM n = 4). (C, right panel) moDCs transfected with indicated siRNAs were stimulated with SCV2-RNA for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production (n = 4); *P < 0.05 versus “ctr siRNA” by 1-way ANOVA with Dunnett’s post hoc test. (D) moDCs were transfected with indicated siRNAs and the expression of TLR8 was evaluated by qPCR (left y axis, white bars). IL-6 production upon SCV2-RNA stimulation was evaluated by ELISA (right y axis, gray bars). Data (percentage of expression/production) represent the mean ± SEM (n = 3); *P < 0.05 versus respective “ctr” by paired Student’s t test. (F) moDCs were pretreated with CU-CPT9a, then stimulated as indicated for 24 hours and IL-6 production evaluated by ELISA. Data are expressed as percentage of production for each individual stimulation (n = 3); *P < 0.05 versus respective “0” by 1-way ANOVA with Dunnett’s post hoc test.

Fig 5: TLR7 and TLR8 are responsible for primary DC activation by SAMPs.cDCs (A) and pDCs (B) were pretreated with increasing concentration of CU-CPT9a and then stimulated with SCV2-RNA (5 μg/mL) for 24 hours. Secreted TNF-α, IL-6, and IFN-α were quantified by ELISA. Data are expressed as percentage of production (n = 3); *P < 0.05 versus “0” by 1-way ANOVA with Dunnett’s post hoc test. (C) pDCs were pretreated (0 hours) with SCV2-RNA (5 μg/mL) or R848 (1 μg/mL) or left untreated for 24 hours, washed, and restimulated with R848 for an additional 24 hours. Secreted IFN-α and TNF-α were quantified by ELISA. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus “(-)” by 1-way ANOVA with Dunnett’s post hoc test. (D) cDCs were pretreated for 1 hour with chloroquine (CQ, 10 μM) and then stimulated with SCV2-RNA (5 μg/mL) for 24 hours. Secreted IL-6 (white bars) and TNF-α (gray bars) were evaluated by ELISA. Data are expressed as percentage of production (n = 3); *P < 0.05 versus respective “(-) SCV2-RNA” by paired Student’s t test. (E) pDCs were pretreated for 1 hour with CQ (10 μM) and then stimulated with SCV2-RNA (5 μg/mL) for 24 hours. Secreted IFN-α (white bars) and TNF-α (gray bars) were quantified by ELISA. Data are expressed as percentage of production (n = 3); *P < 0.05 versus respective “(-) SCV2-RNA” by paired Student’s t test.