Research ArticleAUTOIMMUNE DISEASE

TLR7 escapes X chromosome inactivation in immune cells

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Science Immunology  26 Jan 2018:
Vol. 3, Issue 19, eaap8855
DOI: 10.1126/sciimmunol.aap8855
  • Fig. 1 TLR7 escape from X inactivation in immune cells.

    (A) Workflow of the allele-of-origin analysis using a genetic marker observable in mature TLR7 transcripts. (B) Map of TLR7 and the useful exon 3 SNPs, rs179008 (A/T) and rs3853839 (C/G). (C) KASP assay of mixed genomic DNA samples from men genotyped G/0 (green) and C/0 (blue) for SNP rs3853839, showing the variation of fluorescence intensities according to the mix ratio. (D) Standard curve of the KASP assay. (E to G) Representative allele-of-origin profile of individual B cells (E), monocytes (F), and pDCs (G) from a woman of G/C heterozygous genotype, displaying a proportion of biallelic cells; each dot in the graphs represents one cell with monoallelic (blue or green) or biallelic (red) expression of TLR7. (H) Confirmation by Sanger sequencing (right) of the KASP monoallelic (green or blue) and biallelic (red) expression calls (left) in primary female B cells. (I) Single-cell resolution of the assay. Mixed male B cells of G/0 and C/0 genotypes correctly generate no biallelic events. (J) Frequency of single-cell biallelic TLR7 expression among pDCs (BDCA4+ CD123+), monocytes (CD14+), and B cells (CD19+) from eight heterozygous women (HD1 to HD8). The dotted line denotes the global average.

  • Fig. 2 Direct visualization by RNA FISH of TLR7 escape from X inactivation in B cells from women.

    RNA FISH was performed on CD19+ CD27+ B cells from a woman donor after 2 hours of in vitro stimulation with IFN-β. The images show two confocal planes from a Z-stack after hybridization with fluorescent probes for TLR7 (red) and XIST (green) transcripts. Fluorescent TLR7-specific probes were generated from a collection of PCR amplimers mapping to nonrepetitive regions and covering 7322 base pairs of the TLR7 gene. Arrows denote TLR7 transcripts (red) from the active [XIST RNA-negative (Xa)] and the inactive [XIST RNA-positive (Xi); green] X chromosome on two different confocal planes. Boxed areas are shown as enlarged images (right). Nuclei are counterstained with DAPI.

  • Fig. 3 Increased TLR7 transcripts in biallelic naive B cells.

    (A) Total TLR7 mRNA (raw Ct values in real-time PCR) plotted against the relative abundance (RA) of the less expressed allele in each cell according to the allele-of-origin assay. Each dot denotes one cell; n = 134 cells pooled from three female donors. Q1, low-expression range as in (B). Highest-likelihood monoallelic and biallelic strata are color-coded as orange (RA < 10%) and blue (RA > 35%), respectively. (B) Violin plots for the same experiment, total TLR7 expression normalized to the mean. The first-quartile (Q1) threshold splits cells into lower or higher expression groups; two-sided Fisher’s exact test for proportions in the monoallelic and biallelic strata. (C) Relative TLR7 transcript quantitation in the monoallelic and biallelic strata. Each dot represents one cell’s ΔCt with reference to TLR9 (difference of means, −0.567 Ct units); the TLR7-adjacent gene, TMSB4X (−0.534); MYD88 encoding a TLR-signaling factor (−0.797); and housekeeping gene B2M (−0.743); P values from Welch’s t test (TLR9, TMSB4X, and MYD88) or two-sided Mann-Whitney test (B2M). (D) Principal components analysis of the single-cell transcript quantitation data set for 30 immunity, chromosome X, and control genes, showing the individual cells and group centroids for the monoallelic and biallelic strata.

  • Fig. 4 Enhanced expression of TLR7 protein in PBMCs from women.

    The TLR7 140-kDa (full-length; A) and 75-kDa (proteolytically mature; B) forms of the protein were quantitated separately and normalized to β-actin in three Western blot experiments on nonstimulated PBMCs. Cells from 26 males and 26 females were analyzed, and the measurements for female cells were normalized to the mean for male cells in each experiment. The donors were all hemizygous or homozygous for the major alleles of the exonic TLR7 SNPs, namely, allele A of rs179008 and allele C of rs3853839. P values from Student’s t test.

  • Fig. 5 Enhanced proliferative response of TLR7 biallelic B lymphocytes to TLR7 engagement.

    (A) Flow cytometric cell tracer [CellTrace Violet (CTV)] analysis of CD19+ B cell proliferation upon treatment with IFN-β alone (IFN) or in combination with a TLR ligand, CL097 (TLR7) or CpG-B (TLR9). CD27 immunostaining discriminated PC lineage committed (CD27) and naive (CD27) B lymphocytes. NS, nonstimulated cells. (B) Sex bias in the effector proliferative response of CD27hi CTVlow cells to CL097; ns, nonsignificant; two-sided Mann-Whitney test. Each dot represents one donor. (C) Relationship between the proliferative response to TLR engagement and TLR7 biallelism. Proliferating CTVlow CD27hi and CTVlow CD27 cells were single cell–sorted, in parallel to nonproliferating CTVhigh B cells that were used as within-donor controls. The frequency of biallelic cells was determined in each of these populations. The deviation in percent biallelism among CTVlow proliferating cells was calculated with reference to nonproliferating CTVhigh cells; the value for cells stimulated through TLR7 (CL097) was then compared with the benchmark of same-donor cells stimulated through TLR9 (CpG-B); Wilcoxon’s paired test.

  • Fig. 6 Association between TLR7 biallelic expression and immunoglobulin class switch.

    (A) Dependency of CD19+ CD27 naive B cells on combined T cell help and TLR7 or TLR9 signals for proliferation and immunoglobulin class switch. Flow cytometry analysis using anti-IgG immunostaining and CTV. (B) Enrichment in biallelism among class-switched CTVlow IgG+ B cells versus CTVlow IgG cells when activated as in (A) through TLR7 or TLR9; observations paired by donor (n = 5 women; paired ANOVA). (C) Forest plots of the meta-analysis of the five-donor data in (B) for association between IgG+ cell prevalence and TLR7 biallelism. The meta-analysis P value tests the hypothesis that summary OR ≤ 1.

  • Fig. 7 TLR7 escape from X inactivation in KS males.

    Single-cell allele-of-origin assay of TLR7 transcripts in different immune cell populations; each dot represents one cell and is color-coded for allele assignment. The percentage of biallelic cells is indicated. (A and B) BDCA4+ CD123+ pDCs from two different KS patients (KS1 and KS2; heterozygous for SNPs rs179008 and rs3853839, respectively). (C) CD14+ monocytes. (D) CD19+ B cells. (E) RNA FISH visualization of TLR7 transcripts (red) in CD19+ B cells of a third KS male (KS3), after IFN-β stimulation and TLR7 engagement. Arrows denote TLR7 transcripts from the two X chromosomes (left) or a single X chromosome. These experiments benefited from an IFN-β stimulation protocol boosting hybridization-detectable TLR7 transcripts (see Materials and Methods). Nuclei are counterstained with DAPI. (F) Proportions of monoallelic and biallelic B cells in two KS males (KS3 and KS4) as determined by RNA FISH.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/19/eaap8855/DC1

    Materials and Methods

    Fig. S1. Determination of TLR7 monoallelic or biallelic expression at the single-cell level.

    Fig. S2. In situ visualization of monoallelic and biallelic TLR7 transcripts at the single-cell level.

    Fig. S3. Relationship between transcript quantitation and TLR7 monoallelic or biallelic expression.

    Fig. S4. Specific Western blot immunodetection of TLR7.

    Fig. S5. Unstable TLR7 allelic expression in EBV-transformed B cells.

    Table S1. PCR primer pairs used in the allele-of-origin assay.

    Table S2. Primary antibodies.

    Table S3. PCR primer pairs used in the preparation of the TLR7 RNA FISH probe.

    Table S4. PCR primer pairs used in the preparation of the XIST RNA FISH probe.

    Table S5. PCR primer pairs used in the preparation of the SLC25A6 RNA FISH probe.

    Table S6. 2 × 2 contingency tables of TLR7 biallelic and monoallelic cell counts in IgG+ and IgG B cells.

    Movie S1. RNA FISH visualization of TLR7 escape from X inactivation in a memory B lymphocyte.

    References (5254)

  • Supplementary Materials

    Supplementary Material for:

    TLR7 escapes X chromosome inactivation in immune cells

    Mélanie Souyris, Claire Cenac, Pascal Azar, Daniéle Daviaud, Astrid Canivet, Solange Grunenwald, Catherine Pienkowski, Julie Chaumeil, José E. Mejía, Jean-Charles Guéry*

    *Corresponding author. Email: jean-charles.guery{at}inserm.fr

    Published 26 January 2018, Sci. Immunol. 3, eaap8855 (2017)
    DOI: 10.1126/sciimmunol.aap8855

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Determination of TLR7 monoallelic or biallelic expression at the singlecell level.
    • Fig. S2. In situ visualization of monoallelic and biallelic TLR7 transcripts at the single-cell level.
    • Fig. S3. Relationship between transcript quantitation and TLR7 monoallelic or biallelic expression.
    • Fig. S4. Specific Western blot immunodetection of TLR7.
    • Fig. S5. Unstable TLR7 allelic expression in EBV-transformed B cells.
    • Table S1. PCR primer pairs used in the allele-of-origin assay.
    • Table S2. Primary antibodies.
    • Table S3. PCR primer pairs used in the preparation of the TLR7 RNA FISH probe.
    • Table S4. PCR primer pairs used in the preparation of the XIST RNA FISH probe.
    • Table S5. PCR primer pairs used in the preparation of the SLC25A6 RNA FISH probe.
    • Table S6. 2 ? 2 contingency tables of TLR7 biallelic and monoallelic cell counts in IgG+ and IgG B cells.
    • Legend for movie S1
    • References (52—54)

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). RNA FISH visualization of TLR7 escape from X inactivation in a memory B lymphocyte.

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