TLR7 escapes X chromosome inactivation in immune cells · Solange Grunenwald,2 Catherine...

SC I ENCE IMMUNOLOGY | R E S EARCH ART I C L E

AUTO IMMUNE D I S EASE

1Centre de Physiopathologie Toulouse-Purpan, Université de Toulouse, CNRS, INSERM,Université Paul Sabatier, Toulouse, France. 2Service d’Endocrinologie, MaladiesMétaboliques et Nutrition, Hôpital Larrey, Centre Hospitalier Universitaire (CHU)de Toulouse, Toulouse, France. 3Service Pédiatrie-Unité d’Endocrinologie de l’Hôpitaldes Enfants, CHUde Toulouse, Toulouse, France. 4Institut Cochin, INSERMU1016, CNRSUMR8104, Université Paris-Descartes, Paris, France.*Corresponding author. Email: [email protected]

Souyris et al., Sci. Immunol. 3, eaap8855 (2018) 26 January 2018

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TLR7 escapes X chromosome inactivation inimmune cellsMélanie Souyris,1 Claire Cenac,1 Pascal Azar,1 Danièle Daviaud,1 Astrid Canivet,1

Solange Grunenwald,2 Catherine Pienkowski,3 Julie Chaumeil,4

José E. Mejía,1 Jean-Charles Guéry1*

Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key path-ogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prev-alence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary Xchromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is en-coded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chro-mosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasma-cytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chro-mosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than themonoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations.Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27+ plasma cells. In ad-dition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immu-noglobulin G class switch during the TLR7-driven, T cell–dependent differentiation of naive B lymphocytes intoimmunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with addedresponsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing tobiallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and inmen with Klinefelter syndrome.

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INTRODUCTIONToll-like receptor 7 (TLR7), together with TLRs 3, 8, and 9, forms afamily of intracellular nucleic acid sensors maintained under strongpurifying selection, which attests to their essential role in host survivalto viral infection (1). TLR7 localizes to the endosomal compartment,where it binds microbial or self-derived single-stranded RNA ligands(2). In plasmacytoid dendritic cells (pDCs), TLR7 engagement elicitsstrong type I interferon (IFN) production and is critical to the induc-tion of antiviral immune responses (3). TLR7 is also an essentialcomponent of antibody-mediated immunosurveillance against the re-activation of endogenous retroviruses (4).

RNA-containing immune complexes associated with severe sys-temic lupus erythematosus (SLE) (5) are recognized by TLR7, anddisease severity in lupus-prone mice depends on the Tlr7 gene dose(6–8). TLR7 deficiency protects mice against lupus by decreasing theproduction of autoantibodies to ribonucleoproteins and mitigateslupus-associated kidney disease (9). In contrast, Tlr7 overexpressioninduces systemic autoimmunity even in a non–lupus-prone geneticbackground (6). B cell–intrinsic TLR7 signaling is essential to thedevelopment of spontaneous germinal centers, autoantibody produc-tion, and systemic inflammation (10, 11), which supports the notionthat RNA sensing through TLR7 represents a central mechanism ofchronic B cell activation in SLE.

Like other autoimmune disorders, SLE is markedly sex-biased,and female-to-male incidence peaks at 9:1 in adulthood (12). SLE

susceptibility correlates with the number of X chromosomes inthe karyotype and is thus increased in men with Klinefelter syn-drome (47,XXY; KS) (13), whereas a lower risk has been suggestedfor women affected with Turner syndrome (45,X) (12). These obser-vations support a gene-dose effect of X chromosome loci in SLE pre-disposition (13, 14).

The gene encoding TLR7maps to the short arm of the X chromo-some. In placental mammals, dosage compensation for X-linkedgene products between XX females and XY males is achieved bythe random silencing of one of the two X chromosomes during theearly development of female embryos (15, 16). This essential mech-anism, called X chromosome inactivation (XCI), was first thought tobe extremely stable in adult somatic cells, with only a minority ofgenes, notably those in the pseudoautosomal regions shared withthe Y chromosome, endowed with biallelic expression (17–19). How-ever, studies of global X-linked gene expression in rodent-humansomatic cell hybrids, using limited numbers of available exonic poly-morphisms, found that up to 30% of X-linked genes, in and outsidethe pseudoautosomal regions, exhibited some variable degree of es-cape from XCI (17–19). Recently, advances in high-throughput andsingle-cell technologies enabled a systematic RNA sequencing analy-sis of up to 29 tissues in hundreds of individuals, which confirmedthat about 23% of X-linked genes show incomplete XCI, with varia-bility between tissues and individuals (19, 20). Because TLR7 is a non-pseudoautosomal locus, we investigated whether this gene escapedfrom XCI in women and KS males. Here, we demonstrate that TLR7is transcribed on both X chromosomes in a large proportion of thepDCs, B cells, and monocytes from normal women and KS males.Focusing on B lymphocytes, we show that biallelism leads to increasedTLR7 gene products and imparts a selective advantage at key TLR-dependent development checkpoints of effector B cells.

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RESULTSTLR7 evades XCI in female primary immune cellsWe developed an assay to measure the relative abundance of TLR7transcripts derived from either X chromosome in female cells atsingle-cell resolution (Fig. 1A). The assay used the allele-specific KASP(KBiosciences) polymerase chain reaction (PCR) chemistry (21) to dis-criminate a diallelic (G or C) single-nucleotide polymorphism (SNP) ofTLR7 in the 3′ untranslated region of the mRNA (rs3853839; Fig. 1B).We were able to analyze, in this way, immune cells from women whocarried allele C on one X chromosome and allele G on the other. Wecalculated the relative proportions of the two alleles from the ratio ofthe respective endpoint fluorescence signals in the KASP PCRamplification; for this, we built a standard curve based on genomicDNA samples from men of G/0 and C/0 genotypes in different mixratios (Fig. 1, C and D). B lymphocytes, monocytes, and pDCs wereindividually sorted by the gating strategy in fig. S1A, and each cellwas classified as monoallelic or biallelic for TLR7 expression whenthe relative abundance of the minor allele (i.e., the less representedallele among total TLR7 transcripts in each cell) fell below or above


15%, respectively. This ex vivo approach categorized primary B lym-phocytes into the two monoallelic groups predicted by the paradigmof stochastic XCI (15, 16) plus a substantial stratum of biallelic cellswhere TLR7 escaped from XCI (Fig. 1E; green or blue, monoallelic;red, biallelic). pDCs split up in a similar way, as did monocytes (Fig.1, F and G). The accuracy of this allele-of-origin classification wasverified by sequencing the TLR7 complementary DNA (cDNA) am-plimers from random monoallelic and biallelic B cells (Fig. 1H), andwe confirmed single-cell resolution by assaying mixedmale B cells ofG/0 and C/0 genotypes (1:1), which generated only correct monoal-lelic calls (Fig. 1I). Last, similar biallelic cell frequencies were foundin a comparison of KASP results for SNP rs3853839 and for thealternative TLR7 exonic polymorphism rs179008 (A to T substi-tution near the 5′ terminus of exon 3; Fig. 1B), ruling out an SNP-dependent bias in the allele-specific PCR (fig. S1B). The allelicexpression pattern of TLR7 among B cells, monocytes, and pDCswas consistent across a cohort of eight heterozygous women (Fig. 1J):30% of cells were biallelic, on average, with some interindividual var-iability but similar monoallelic/biallelic ratios between same-donor

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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) andC/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) Representativeallele-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; eachdot 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 KASPmonoallelic (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/0genotypes 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.

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cell types. Among steady-state CD19+ B lymphocytes, biallelismlevels were similar between CD27− IgM+ IgD+ (naive) and CD27+

IgM− IgD− (memory) cells in a cohort of 13 heterozygous women(fig. S1C).

In addition, we observed simultaneous TLR7 transcription on bothX chromosomes using RNA fluorescence in situ hybridization (RNAFISH; Fig. 2) (22). A fraction of female CD27+ B cells, pDCs, andmonocytes exhibited two nuclear foci of nascent TLR7 RNAs at twosites of transcription, one of which colocalized with the inactive Xchromosome (Fig. 2 and fig. S2). The inactive X territory was detectedby using probes against the XIST (X-inactive specific transcript) non-coding transcripts that coat the chromosome to initiate XCI and formthe so-calledXIST cloud (23). Further hybridizations onB cells showedthe expected double signals from SLC25A6, a pseudoautosomal geneon the tip of Xp constitutively expressed from the active and the in-active X (17), paired with signals from TLR7 nascent transcripts onboth chromosomes (movie S1). These experiments confirmed that TLR7escapes from XCI in a proportion of immune cells.

Escape from X inactivation increases TLR7 gene productsTo measure the effect of escape from X inactivation on TLR7mRNAlevels, we quantified total TLR7 transcripts by real-time PCR in singleCD19+ CD27− IgD+ naive B cells from three women and determinedin parallel the relative abundance of allelic TLR7 transcripts in eachcell (Fig. 3A). The relative abundance of TLR7 transcripts from theminor allele in each cell ranged from ≈0% (apparent TLR7 mono-allelism) to nearly 50% (apparent balanced biallelism). Because genetranscription is stochastic and may occur in nonsynchronous burstson either allele, single-cell analysis may count stably and transientlymonoallelic cells together (24).We considered that cells where the mi-nor allele accounted for less than 10% ofTLR7 transcripts [as per (17)]represented the stratum with the highest likelihood of stable mono-allelism and defined a reciprocal biallelic stratum of cells where themi-nor allele accounted for 35 to 50% of total TLR7 transcripts (Fig. 3A).The two strata were sharply different as regards the proportions of


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low (below the first-quartile threshold) and highTLR7 expression cells(P < 10−4, two-sided Fisher’s exact test). Low-expression cells weredepleted in the biallelic stratum (Fig. 3B), and median TLR7 expres-sion increased 1.8-fold relative to the monoallelic stratum (1.27 ver-sus 0.70 times the population mean; Fig. 3B, white dots), consistentwith a shift from one to two contributing alleles in the burst-likestochastic transcription model (24).

In parallel toTLR7, we quantitated by single-cell real-time PCR thetranscripts from 15 well-expressed housekeeping and TLR signaling–related genes, but these were not significantly elevated inTLR7 bialleliccells (fig. S3A). We calculated the relative expression of TLR7 withreference to 12 autosomal and 3 X chromosome genes by the DCt

method on a per-cell basis. As expected, we observed a significantdifference between monoallelic and biallelic cells when TLR7 ex-pression was normalized to nine autosomal genes and to the threeX-linked genes (IL2RG, IRAK1, and TMSB4X; Fig. 3C and fig. S3, Bto E). This indicated that greater TLR7 expression in biallelic cellswas specific (i.e., did not mirror a hypothetical increase across theentire gene panel) and suggested that the inactive X chromosomewas not globally reactivated (although our sample of X-linked geneswas small). Principal components analysis of the real-time PCR dataset showed a groupwise difference between the monoallelic and bial-lelic cells in the first and second principal components (Fig. 3D). Wecould not correlate increased TLR7 transcripts in biallelic naive Bcells with any significant changes in other assayed genes. TMSB4Xlies only 85 kb from TLR7 and reportedly evades XCI (25); elevatedTLR7 transcripts over the TMSB4X internal control in biallelic cells(−0.534 DCt units on average) suggest that the escape from XCI ofTLR7 and TMSB4X is uncoupled despite the close physical linkage.Consistent with biallelic gene expression in a large cell subset,Westernblot analysis (using a newly validated monoclonal antibody to the Cterminus of human TLR7; see Materials and Methods and fig. S4)showed the full-length and proteolytically mature forms of TLR7 tobe increased 1.38- and 1.31-fold, respectively, in resting peripheralblood mononuclear cells (PBMCs) from women relative to the malecells (Fig. 4).

TLR7 escape from X inactivation enhances the B cellresponse to TLR7 engagementWe investigated the B cell response to TLR7 engagement attending tosex and to the allelic pattern of TLR7 expression. In the presence oftype I IFN, nucleic acid sensor (TLR7 or TLR9) engagement elicitsproliferation and immunoglobulin secretion by B cells (Fig. 5A)(26). We stimulated CD19+ B cells in vitro for 6 days with IFN-b,an up-regulator of TLR7 expression (27), and with either TLR7 orTLR9 ligands. The frequencies of proliferating plasma cell (PC)–committed (CD27hi) and naive (CD27−) B cells showed that theCD27hi PC lineage population (28) expand more vigorously in femalethan in male cells (Fig. 5B), but only when TLR7-stimulated. Onehypothesis was that this bias could arise from TLR7 biallelic expres-sion in women’s cells. TLR7 biallelism was enriched among CD27hi

PCs that proliferated in response to TLR7 engagement specifically(P = 0.016, Wilcoxon’s paired test; Fig. 5C). Proliferating CD27−

(naive) B cells trended in the same direction, but the difference wasnot conclusive. Biallelic CD27hi PC lineage B cells were counterselectedupon TLR9 engagement (Fig. 5C), consistent with the notion thatTLR7 and TLR9 compete for binding to UNC93B1, the chaperoneprotein that regulates the trafficking of both receptors to the endo-somal compartment (29, 30). This predicts that, at least under IFN-b

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Fig. 2. Direct visualization by RNA FISH of TLR7 escape from X inactivationin B cells from women. RNA FISH was performed on CD19+ CD27+ B cells from awoman donor after 2 hours of in vitro stimulation with IFN-b. The images showtwo confocal planes from a Z-stack after hybridization with fluorescent probes forTLR7 (red) and XIST (green) transcripts. Fluorescent TLR7-specific probes were gen-erated from a collection of PCR amplimers mapping to nonrepetitive regions andcovering 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 asenlarged images (right). Nuclei are counterstained with DAPI.

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stimulation, greater TLR7 expression in biallelic B cells should result inlower levels of functional TLR9, and the converse in monoallelic cells.

Activation of human naive B cells requires a combination of T cellhelp and TLR engagement (31, 32). Figure 6A illustrates this depen-dency using an in vitromodel (32, 33) that measured proliferation andimmunoglobulin class switch in response to T cell–dependent signalsand ligands for TLR7 (CL097) or, alternatively, TLR9 (class B CpGoligodeoxynucleotides).OnCD19+CD27− IgG−naive B cells from fivewomen, this model showed the frequency of TLR7 biallelic B cells tobe significantly enriched among immunoglobulin G (IgG)–switchedproliferative cells after TLR7 but not TLR9 engagement (Fig. 6B).Wefurther analyzed these data to quantify the effect of biallelism on classswitch. In each instance of B cell donor and TLR ligand used to pro-mote class switch, wewrote the biallelic andmonoallelic cell counts forthe IgG+ and IgG− populations as a 2 × 2 contingency table (table S6),by analogy with a case-control study of epidemiological association(34). We next pooled all the 2 × 2 tables for the same TLR ligand ina statistical meta-analysis to extract a summary odds ratio (OR). Thesummary OR measured the strength of the association between bialle-lism and IgGclass switch and is formally the estimated ratio of the oddsof class switch in biallelic cells to the odds of this outcome in mono-allelic cells (Fig. 6C). By this approach, we observed non-associationoverall between biallelism and IgG expression in response to the


TLR9 ligand (summary ORTLR9, ≈1; Fig. 6C). In contrast, uponTLR7 engagement, biallelic cells weremore likely to undergo IgG classswitch than monoallelic cells, with summary ORTLR7 = 2.4, 95% con-fidence interval (CI) [1.6 to 3.7] (Fig. 6C).

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Fig. 3. Increased TLR7 transcripts in biallelicnaive B cells. (A) Total TLR7 mRNA (raw Ctvalues in real-time PCR) plotted against the rel-ative abundance (RA) of the less expressed allelein each cell according to the allele-of-origin assay.Each dot denotes one cell; n = 134 cells pooledfrom three female donors. Q1, low-expressionrange as in (B). Highest-likelihood monoallelicand biallelic strata are color-coded as orange(RA < 10%) and blue (RA > 35%), respectively.(B) Violin plots for the same experiment, totalTLR7 expression normalized to the mean. Thefirst-quartile (Q1) threshold splits cells into loweror higher expression groups; two-sided Fisher’sexact test for proportions in the monoallelic andbiallelic strata. (C) Relative TLR7 transcript quan-titation in the monoallelic and biallelic strata.Each dot represents one cell’s DCt with referenceto TLR9 (difference of means, −0.567 Ct units); theTLR7-adjacent gene, TMSB4X (−0.534); MYD88encoding a TLR-signaling factor (−0.797); andhousekeeping gene B2M (−0.743); P values fromWelch’s t test (TLR9, TMSB4X, and MYD88) ortwo-sided Mann-Whitney test (B2M). (D) Principalcomponents analysis of the single-cell transcriptquantitation data set for 30 immunity, chromo-some X, and control genes, showing the individ-ual cells and group centroids for the monoallelicand biallelic strata.

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Fig. 4. Enhanced expression of TLR7 protein in PBMCs from women. TheTLR7 140-kDa (full-length; A) and 75-kDa (proteolytically mature; B) forms ofthe protein were quantitated separately and normalized to b-actin in three Westernblot experiments on nonstimulated PBMCs. Cells from 26 males and 26 femaleswere analyzed, and the measurements for female cells were normalized to the meanfor male cells in each experiment. The donors were all hemizygous or homozygousfor the major alleles of the exonic TLR7 SNPs, namely, allele A of rs179008 and allele Cof rs3853839. P values from Student’s t test.

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Because immunoglobulin class switch represented <10% of out-comes in thismodel (e.g., 4.45% for TLR7 and 7.03% for TLR9; Fig. 6A),the OR makes a good approximation to the relative risk (35). WithORTLR7 = 2.4, the propensity of biallelic cells to differentiate intoisotype-switched cells in the presence of T cell help and TLR7 agonistligands was thus more than twofold greater than that of monoalleliccells. In addition, ORTLR7 enabled the percentage of IgG

+ cells due tobiallelism to be estimated as the population attributable fraction(PAF) = 29% (34), 95% CI [14 to 45%] (assuming 30% biallelism asobserved earlier under IFN-b stimulation; Fig. 1J and Materials andMethods). This experiment confirmed that TLR7 escape from XCImay endow the B cell compartment with added responsiveness toTLR7 ligands and contribute a large proportion of immunoglobulinclass switch events.

The TLR7 allelic expression profile is unstable in EBV-transformed B cellsTLR7 escape fromXCI was reported recently in human Epstein-Barrvirus (EBV)–transformed lymphoblastoid B cells (36, 37). We inves-tigated whether monoallelic or biallelic expression was stable in theprogeny of lymphoblastoid cell clones. Similar to the primary B cellsof the female donor, EBV-transformed cells comprised three subsetswith monoallelic or biallelic TLR7 expression (fig. S5A). We character-


ized the TLR7 allele of origin in clonal subcultures derived by limitingdilution (fig. S5, B and C) and selected a predominantly monoallelicsubline, F2, for further subcloning. Follow-up at single-cell resolutionof three clonal sublines (F2-2G7 and F2-3E10 plus G9 derived fromthe initial lymphoblastoid population) evidenced a striking relaxationof monoallelic expression over time in culture, steadily convergingtoward biallelism in one-half of the cells (fig. S5, D and E).

TLR7 evades X inactivation in immune cells from KS malesThe carriage of a supernumerary X chromosome in KS multiplies therisk of developing SLE and other female-biased autoimmune pathol-ogies (13, 14).We examinedwhether higherTLR7 dosage arising fromXCI escape also operates in 47,XXY males. Similar to female singlecells, we observed biallelic TLR7 expression in 19.3 to 39% of primarypDCs, monocytes, and B lymphocytes from two KS males (Fig. 7, A toD) using either rs3853839 as above or, alternatively, rs179008 as aTLR7allelic marker. Improving the IFN-b stimulation step in the RNA FISHprotocol (see Materials and Methods) again showed biallelic TLR7expression at high frequencies (49 and 59%; Fig. 7, E and F) in CD19+

B cells from two other KS males. These results demonstrate that thefemale pattern of biallelic TLR7 expression also occurs in 47,XXYmalesand suggest TLR7 escape fromXCI as amechanism for the elevated riskof SLE in KS men.

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Fig. 5. Enhanced proliferative response of TLR7 biallelic B lymphocytes to TLR7 engagement. (A) Flow cytometric cell tracer [CellTrace Violet (CTV)] analysis ofCD19+ B cell proliferation upon treatment with IFN-b alone (IFN) or in combination with a TLR ligand, CL097 (TLR7) or CpG-B (TLR9). CD27 immunostaining discriminatedPC lineage committed (CD27) and naive (CD27−) B lymphocytes. NS, nonstimulated cells. (B) Sex bias in the effector proliferative response of CD27hi CTV low cells toCL097; ns, nonsignificant; two-sided Mann-Whitney test. Each dot represents one donor. (C) Relationship between the proliferative response to TLR engagement andTLR7 biallelism. Proliferating CTV low CD27hi and CTVlow CD27− cells were single cell–sorted, in parallel to nonproliferating CTVhigh B cells that were used as within-donorcontrols. The frequency of biallelic cells was determined in each of these populations. The deviation in percent biallelism among CTV low proliferating cells wascalculated with reference to nonproliferating CTVhigh cells; the value for cells stimulated through TLR7 (CL097) was then compared with the benchmark of same-donorcells stimulated through TLR9 (CpG-B); Wilcoxon’s paired test.

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DISCUSSIONXdisomy and polysomy are thought to contribute to the greater risk ofautoimmunity in women and also in KS men, who carry one or moresupernumerary X chromosomes (13, 14). We demonstrate here thatTLR7 gene products are increased in women owing to consistent es-cape fromXCI in a large proportion of B lymphocytes,monocytes, andpDCs. Previous studies have shown that TLR7 is required for the pro-duction of autoantibodies to ribonucleoproteins in SLE and thatTLR7-dependent B cell responses are TLR7 dose–sensitive (7–9, 38).In agreement with this, we observed enhanced TLR7-driven responsesof biallelic female B cells during key stages of effector B cell develop-ment. In addition, we found a significant TLR7 biallelism among im-mune cells of KS males. Together, our findings strongly support thehypothesis that TLR7 overexpression through biallelism is a candidatecontributor to the risk of SLE not only in women but also in 47,XXYmen (13, 14).

Our data show TLR7-expressing B cells as a mosaic population ofindividual cells expressing transcripts from either one or two alleles ata given time. The current model of stochastic burst–like gene expres-sion (24) predicts a similar pattern for autosomal genes expressed atlow-to-medium levels. In this model, transcription occurs not contin-uously but in pulses of RNA molecules from one allele or the other.The timing of these pulses is stochastic, and they alternatewith periodsof transcriptional inactivity. This model may explain that B cells withlow levels of TLR7 transcripts were almost absent among the biallelicpopulation (Fig. 3B), given that transcription fromone allele can occurduring the inactivity periods of the other if both are expressed. How-ever, as we note below, our functional experiments argue not for com-plete biallelism but for distinct monoallelic and biallelic cell subsetswith differential responsiveness to TLR7 ligands.


TLR engagement is necessary for immunoglobulin class switch inthe generation of antigen-specific antibody responses (31, 32). Weanalyzed in vitro the impact ofTLR7 biallelism on class switch throughthe response of naive B cells fromwomen to a TLR7 agonist and foundthat biallelic cells displaymore than twofold greater propensity to classswitch toward IgG than monoallelic cells. This is a conservative esti-mate of overall class switch propensity because we only analyzed forthe IgG isotype. We ascribed this effect to the higher dose of the pro-tein product ofTLR7 specifically, in light of the neutral outcome (sum-mary ORTLR9, ≈1) of TLR9 engagement. These results show that theobserved increase in TLR7 biallelism among class-switched IgG+ cellsdid not result fromTLR stimulation at large. The effects on the immu-noglobulin repertoire of overexpressing TLR7 in murine models oflupus have been addressed by several studies (38–40), which suggestthat increased TLR7 dose in women owing to biallelism could con-tribute to higher SLE risk by fostering a larger pool of PC lineage cellsproducing high-affinity IgG autoantibodies; these have been recog-nized as a factor in the pathogenesis of human SLE (41, 42).

TLR7 evaded XCI in all the female and KS male patients we havestudied thus far, with a wide range of biallelic cell frequencies but sim-ilar within-donor values in B lymphocytes, monocytes, and pDCs.Although this suggests intraindividual stability, variation in respectof physiological processes, age, genetics, and the environment remainsto be examined. Longitudinal investigation of biallelism during B celldifferentiation, activation, and proliferation is challenging becausesingle-cell longitudinal studies would require nondestructive sampleanalysis. In addition, our study does not directly demonstrate thatan increase in TLR7 expression drives autoimmunity in individualscarrying two X chromosomes. Because we are dealing with humanprimary cells, the participants of study were not directly amenable to

BA

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llelic

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%)

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Fig. 6. Association between TLR7 biallelic expression and immunoglobulin class switch. (A) Dependency of CD19+ CD27− naive B cells on combined T cell helpand TLR7 or TLR9 signals for proliferation and immunoglobulin class switch. Flow cytometry analysis using anti-IgG immunostaining and CTV. (B) Enrichment inbiallelism among class-switched CTV low IgG+ B cells versus CTV low IgG− cells when activated as in (A) through TLR7 or TLR9; observations paired by donor (n = 5women; 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.

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experimentation. Dissection of the mechanisms involved should beaided by cellular and animal models, although we note that XCI inmice appears to be more complete than in humans (19). To assessthe maintenance of XCI in the progeny of monoclonal B cells, we gen-erated EBV-transformed B cell clones, because escape from XCI hasbeen reported for TLR7 in lymphoblastoid cells (36, 37). However,marked degradation of monoallelism in these lines over time inculture suggested loose control of XCI as a consequence of EBV im-mortalization, which is known to cause large-scale epigenetic remo-deling of the host cells (43).

In conclusion, our study provides proof that TLR7 evades XCI inhumanB cells, pDCs, andmonocytes, endowingwomen andKSmaleswith subpopulations of immune cells where TLR7 expression isenhanced. In B lymphocytes, we found evidence for a causal relation-ship between biallelism and TLR7-dependent cell responsiveness.TLR7 function has been also well documented in pDCs (3) and otherspecialized cell types such as CD4+ T cells (44), monocytes (45), andneutrophils (46), implying a potential impact of sex-dependent TLR7dosage on multiple immune processes. This could be particularly rel-


evant to the TLR7-driven production of type I IFNs by pDCs, whichexhibits a strong female bias (47). Although the female sex hormone,estrogen, appears to be an important regulator of TLR responsivenessin these cells, an added nonredundant effect of X chromosome dosagehas also been suggested (48). The tissue distribution of biallelism andits longitudinal within-subject stability, variability between individ-uals, and functional consequences in different immune cell lineageswill be important topics of study in the context of the TLR7-dependentautoimmune and infectious pathologies of women and KS men.

MATERIALS AND METHODSDonors and ethical complianceThis study complied with the ethical principles of the Declaration ofHelsinki and with applicable French regulations. PBMCs of anony-mous, healthy blood donors at the Toulouse blood transfusion center(Etablissement Français du Sang) were from a biobank authorizedunder dossier number 2-15-36 by the competent ethics board (Comitéde Protection des Personnes Sud-Ouest et Outre-Mer II, Toulouse).

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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 representsone 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 andKS2; 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-b 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-b stimulation protocol boosting hybridization-detectable TLR7 transcripts (see Materials and Methods). Nuclei are counterstainedwith DAPI. (F) Proportions of monoallelic and biallelic B cells in two KS males (KS3 and KS4) as determined by RNA FISH.

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The study of KS donors was approved by this board under dossiernumber 1-16-28. Patients with 47,XXY karyotypes [aged 11 (KS3), 13(KS1), 16 (KS4), and 21 (KS2)] were recruited at the Toulouse Univer-sity Hospital, and informed consent was obtained in writing from thepatient or the patient’s parents or guardians as appropriate.

Genotyping and single-cell analysis of TLR7allelic expressionTyping for the frequent diallelic polymorphisms of TLR7, rs3853839(NM_016562.3:c.*881C>G) and rs179008 (NM_016562.3:c.32A>T),at the genomic and single-cell cDNA levels relied on custom KASPcompetitive allele-specific PCR assays (21) fromLGC. rs3853839 local-izes to the 3′ untranslated region in exon 3, 881 base pairs downstreamfrom the stop codon; rs179008 is a coding-sequence polymorphismnear the 5′ end of exon 3. The workflow for single-cell cDNA analysisis summarized in Fig. 1A. Immune cells from men or heterozygouswomen were sorted with a FACSAria II cell sorter (BD Biosciences)at one cell per well of a 96-well plate preloaded with a mediumcontaining 2% Triton X-100, RNaseOUT recombinant ribonucleaseinhibitor (1 U/ml; Thermo Fisher Scientific), 940 mM deoxynucleosidetriphosphates, and random hexamer primers (12.5 ng/ml). For RNAreverse transcription, single-cell lysates were supplemented with Max-ima H Minus reverse transcriptase (50 U per well) and the suppliedreaction buffer (Thermo Fisher Scientific). Before KASP typing, targetmolecules were PCR-amplified using TLR7 cDNA-specific primerpairs, and negative wells were screened out by quantitative real-timePCR with nested primers (table S1), using LightCycler 480 SYBRGreen I Master reagents from Roche. The quantitative real-time PCRand KASP fluorescence-based assays were performed in a LightCycler480 instrument (Roche). Relative allelic expression was calculated fromthe ratio of the 6-carboxyfluorescein (FAM) (allele C) and hexachloro-6-carboxyfluorescein (HEX) (allele G) fluorescence signals from the re-spective KASP probes; a four-parameter standard curve was generatedmonthly for each LightCycler 480 unit, using an R script based on packagedrc (Fig. 1, C andD) (49). BiallelicTLR7 expression in a cell was inferredwhen the relative expression values of TLR7 transcripts bearing thers3853839Gallelewere comprisedbetween 15 and 85%. Further informa-tionon the single-cell assay canbe found in the SupplementaryMaterials.

Immunostaining for flow cytometryHuman immune cellswere stainedwith the following fluorescentmouseantibody conjugates against specific surface markers: R-phycoerythrin(PE) anti-CD14 for monocytes; allophycocyanin (APC) anti-BDCA4(CD304), fluorescein isothiocyanate (FITC) anti-Lin (CD2/CD3/CD14/CD16/CD19/CD56/CD235a), and PE anti-CD123 for pDCs;and PE-Cy5 anti-CD19, APC anti-CD27, BV421 anti-IgA, BV510 anti-IgG, PE anti-IgM, and FITC anti-IgD for B cell subsets (table S2). Flowcytometry analysis was performed on a BD Biosciences LSR II orFACSAria II cell sorter, and the data were processed with the FlowJosoftware (FlowJo LLC).

RNA FISHRNA FISH was performed as described by Chaumeil and colleagues(22). Briefly, spreads of primary nonadherent cells on poly-L-lysine–coated coverslips were fixed for 10 min with 3% paraformaldehyde atroom temperature and permeabilized for 7 min in ice-cold cytoskeletalbuffer containing 0.5%TritonX-100 and 2mMvanadyl-ribonucleosidecomplex (New England Biolabs). The cells were dehydrated throughsuccessive ethanol baths, air-dried briefly, and incubated with the


labeled probes (described in the SupplementaryMaterials) overnightat 42°C. The coverslips were rinsed twice with 50% formamide in 2×SSC buffer and thrice with 2× SSC alone, and nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) in phosphate-buffered saline. The coverslips weremounted usingDako fluorescencemounting medium before microscopy on a Leica TCS SP8 or ZeissLSM 710 microscope using a 63× oil immersion objective. Image datawere processed with the Fiji software (50). Improved cell cultureconditions were applied to CD19+ B cells from KS patients, whichboosted hybridization-detectable TLR7 transcripts. The cells weregrown for 5 days in the presence of the imidazoquinoline CL097(250 ng/ml; InvivoGen) and human IFN-b (1 ng/ml; PeproTech),starved of IFN-b for 24 hours, and last, IFN-b–stimulated for 2 hoursimmediately before harvesting for RNA FISH.

High-throughput quantitative real-time PCRCells from specific immune compartments were flow-cytometricallyfractionated and then individually sorted using a C1 Single-Cell AutoPrep system (Fluidigm) at the Genomic and Transcriptomic (GeT) corelaboratory, Toulouse. Single-cell RNA was reverse-transcribed, and thecDNAs from genes of interest were amplified by PCR before quantita-tion by real-time PCR using the Fluidigm BioMark technology.

TLR activation assayCD19+ B cells were isolated to >93% purity by negative selection(Human B cell enrichment kit, STEMCELL Technologies), stainedwith a tracing dye (CellTrace Violet, Thermo Fisher Scientific), andseeded in 96-well U-bottom plates at 5 × 104 cells per well in RPMI1640 medium with 10% fetal bovine serum (FBS). The cells were thenstimulated with human IFN-b (1 ng/ml), class B CpG oligodeoxynu-cleotides (300 ng/ml; InvivoGen), or CL097 (250 ng/ml). After 6 days,the cells were labeled for CD19 and CD27 expression, and the prolifer-ative response was analyzed by flow cytometry.

T cell–dependent activation assayThe T cell– and TLR-dependent model of naive B cell activation wasimplemented as described by Ruprecht and Lanzavecchia (32). NaiveB lymphocytes were enriched by negative selection (EasySep human Bcell isolation kit, STEMCELL Technologies) on the PBMCs of healthydonors, and CD16− CD3− CD14−CD19+ CD27− IgG− IgA− cells weresorted on a FACSAria II cell sorter. CD4+ T cells were isolated fromthe same donor (CD4+ T cell isolation kit, human, Miltenyi Biotec)and irradiated at 20 grays. Naive B cells (1 × 104 per well) were loadedwith CellTrace Violet and cocultured with 2 × 104 irradiated T cells in96-well round bottom culture plates, using RPMI 1640 medium sup-plemented with 10% FBS; CD40L + enhancer (1 mg/ml; Enzo LifeSciences); interleukin-6 (IL-6), IL-10, and IL-12 (10 ng/ml each); F(ab′)2anti-human IgM-IgG-IgA (2.5 mg/ml); and the following superantigensat 0.5 ng/ml each: toxic shock syndrome toxin 1 and Staphylococcusaureus SeA, SeB, SeE, and SeC. TLR signaling was elicited by CL097(250 ng/ml; TLR7) or CpG-B (300 ng/ml; TLR9). After 7 days inculture, cells were stained for CD19, IgA, IgG, IgM, and IgD andsorted by differentiation status on a FACSAria II instrument.

EBV transformation of B cellsLymphoblastoid cell lines were generated by a standard protocol.Briefly, 106 PBMCs were incubated for 3 hours at 37°C with 1 ml ofEBV viral supernatant from B95-8 cells. The cells were washed, resus-pended in 1ml of RPMI 1640mediumwith 20% FBS in the presence of

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phytohemagglutinin (2 mg/ml), and grown in 1 well of a 24-well plateuntil the appearance of clumps of transformed B cells.

Statistical analysisPaired analysis of variance (ANOVA) and two-sided Welch’s t test,Mann-Whitney, Student’s t test, paired t test, and Wilcoxon’s pairedtest were performed using Prism 4 (GraphPad Software). All datasubjected to a parametric test satisfied the Shapiro-Wilk test of nor-mality at a = 0.05. The normality test and two-sided Fisher’s exact testwere performed with R. A summary OR and 95% CI were extractedfrom sets of 2 × 2 contingency tables by the exact meta-analysismethod of Liu and colleagues (51) implemented in R package gmeta.The PAF and its 95% CI were calculated from the summary OR usingthe following formula (34):

PAF ¼ PeðOR � 1ÞPeðOR � 1Þ þ 1

� 100 ð1Þ

where Pe is the prevalence of exposure to the risk factor. Here, Pe = 0.3,corresponding to 30% TLR7 biallelism in naive B cells (Fig. 1J).

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SUPPLEMENTARY MATERIALSimmunology.sciencemag.org/cgi/content/full/3/19/eaap8855/DC1Materials and MethodsFig. 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-celllevel.Fig. S3. Relationship between transcript quantitation and TLR7 monoallelic or biallelicexpression.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+ andIgG− B cells.Movie S1. RNA FISH visualization of TLR7 escape from X inactivation in a memory B lymphocyte.References (52–54)

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Acknowledgments: We thank M. Cremer for the gift of a XIST probe and L. Dupré for EBVinfectious supernatants. We also thank J. Plumas for the gift of the GEN2.2 and GENshTLR7cell lines. We are grateful to the staff of core laboratories for excellent technical support,especially A.-L. Iscache, A. Gendras, F. L’Faqihi, P.-E. Paulet, and R. Romieu-Mourez [Centre dePhysiopathologie Toulouse-Purpan (CPTP)]; F. Martins (GeT TQ); and E. Lhuillier (GeT Purpan).Funding: J.C. is supported by the Foundation for Cancer Research (ARC), France and theATIP-Avenir starting grant program (INSERM/CNRS/Plan cancer; France). Work at CPTP wassupported by grants from ARC, Arthritis Fondation Courtin, Fondation pour la RechercheMédicale (DEQ2000329169), Conseil Régional Midi-Pyrénées, and the French National Agencyfor Research on AIDS and Viral Hepatitis and by studentships to M.S. and P.A. from Fondationpour la Recherche Médicale (FDT20170437183) and SIDACTION, respectively. Authorcontributions:M.S., P.A., J.E.M., and J.-C.G. conceived and designed the laboratory investigation.J.-C.G., S.G., and C.P. conceived and designed the study of KS patients. M.S., P.A., J.E.M., andJ.-C.G. designed and performed the experiments with help from C.C., D.D., A.C., and J.C. M.S., C.C.,D.D., J.E.M., and J.-C.G. analyzed the data. M.S., J.E.M., and J.-C.G. wrote the manuscript withcontributions from co-authors. Competing interests: The authors declare that they have nocompeting interests. Data and materials availability: Plasmids and cell lines generated in thiswork can be provided upon request, subject to a material transfer agreement.

Submitted 6 September 2017Accepted 4 December 2017Published 26 January 201810.1126/sciimmunol.aap8855

Citation: M. Souyris, C. Cenac, P. Azar, D. Daviaud, A. Canivet, S. Grunenwald, C. Pienkowski,J. Chaumeil, J. E. Mejía, J.-C. Guéry, TLR7 escapes X chromosome inactivation in immune cells.Sci. Immunol. 3, eaap8855 (2018).

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escapes X chromosome inactivation in immune cellsTLR7

Julie Chaumeil, José E. Mejía and Jean-Charles GuéryMélanie Souyris, Claire Cenac, Pascal Azar, Danièle Daviaud, Astrid Canivet, Solange Grunenwald, Catherine Pienkowski,

DOI: 10.1126/sciimmunol.aap8855, eaap8855.3Sci. Immunol.

greater SLE risk in individuals with two X chromosomes.activated in SLE patients. On the basis of this, the authors propose that biallelic expression of TLR7 contributes toKlinefelter individuals. TLR7 binds single-stranded RNA and activates type I interferon signaling, a pathway that is also

) that is encoded from the X chromosome escapes X inactivation in B cells and myeloid cells in females andTLR7(. demonstrate that Toll-like receptor 7et alan important risk factor in SLE. Using sensitive quantification methods, Souyris

Klinefelter syndrome (47,XXY) also have increased incidence of SLE, suggesting that X chromosome dosage could be Nine of 10 individuals who develop systemic lupus erythematosus (SLE) are women. Furthermore, individuals with

The X chromosome link to lupus

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