Investigating sex differences in T regulatory cells from cisgender and transgender healthy individuals and patients with autoimmune inflammatory disease: a cross-sectional study

Abstract

Background: Sexual dimorphisms, which vary depending on age group and pubertal status, have been described across both the innate and adaptive immune system. We explored the influence of sex hormones on immune phenotype in the context of adolescent health and autoimmunity.

Methods: In this cross-sectional study, healthy, post-pubertal cisgender individuals (aged 16-25 years); healthy, pre-pubertal cisgender individuals (aged 6-11 years); transgender individuals (aged 18-19 years) undergoing gender-affirming treatment (testosterone in individuals assigned female sex at birth and oestradiol in individuals assigned male sex at birth); and post-pubertal cisgender individuals (aged 14-25 years) with juvenile-onset systemic lupus erythematosus (SLE) age-matched to cisgender individuals without juvenile-onset SLE were eligible for inclusion. Frequencies of 28 immune-cell subsets (including different T cell, B cell, and monocyte subsets) from each participant were measured in peripheral blood mononuclear cells by flow cytometry and analysed by balanced random forest machine learning. RNA-sequencing was used to compare sex and gender differences in regulatory T (Treg) cell phenotype between participants with juvenile-onset SLE, age-matched cis-gender participants without the disease, and age matched transgender individuals on gender-affirming sex hormone treatment. Differentially expressed genes were analysed by cluster and pathway analysis. Suppression assays assessed the anti-inflammatory function of Treg cells in vitro.

Findings: Between Sept 5, 2012, and Nov 6, 2019, peripheral blood was collected from 39 individuals in the post-pubertal group (17 [44%] cisgender men, mean age 18·76 years [SD 2·66]; 22 [56%] cisgender women, mean age 18·59 years [2·81]), 14 children in the cisgender pre-pubertal group (seven [50%] cisgender boys, mean age 8·90 [1·66]; seven [50%] cisgender girls, mean age 8·40 [1·58]), ten people in the transgender group (five [50%] transgender men, mean age 18·20 years [0·47]; five [50%] transgender women, mean age 18·70 years [0·55]), and 35 people in the juvenile-onset SLE group (12 [34%] cisgender men, mean age 18·58 years [2·35]; 23 [66%] cisgender women, mean age 19·48 [3·08]). Statistically significantly elevated frequencies of Treg cells were one of the top immune-cell features differentiating young post-pubertal cisgender men from similarly aged cisgender women (p=0·0097). Treg cells from young cisgender men had a statistically significantly increased suppressive capacity in vitro compared with those from cisgender women and a distinct transcriptomic signature significantly enriched for genes in the PI3K-AKT signalling pathway. Gender-affirming sex hormones in transgender men and transgender women induced multiple statistically significant changes in the Treg-cell transcriptome, many of which enriched functional pathways that overlapped with those altered between cisgender men and cisgender women, highlighting a hormonal influence on Treg-cell function by gender. Finally, sex differences in Treg-cell frequency were absent and suppressive capacity was reversed in patients with juvenile-onset SLE, but sex differences in Treg-cell transcriptional signatures were significantly more pronounced in patients with juvenile-onset SLE compared with individuals without juvenile-onset SLE, suggesting that sex hormone signalling could be dysregulated in autoimmunity.

Interpretation: Sex-chromosomes and hormones might drive changes in Treg-cell frequency and function. Young post-pubertal men have a more anti-inflammatory Treg-cell profile, which could explain inflammatory disease susceptibilities, and inform sex-tailored therapeutic strategies.

Funding: Versus Arthritis, UK National Institute for Health Research University College London Hospital Biomedical Research Centre, Lupus UK, and The Rosetrees Trust.

Figure 1

Comparison of immune-cell subsets in young-post pubertal cisgender men and women Comparison of 28 immune-cell subsets in 17 young, post-pubertal cisgender men versus 22 young, post-pubertal cisgender women using the balanced random forest model approach. (A) out of bag error rate of the balanced random forest was 0·1795 (82·05% accuracy). (B) Receiver operator characteristic curve analysis used to validate the model, providing an area under the curve of 0·8476 (84·76% accuracy; 82·61% sensitivity; and 81·25% specificity), with 10-fold cross-validation classification accuracy of 77·40%. (C) The top ten variables contributing to the balanced random forest: higher mean decrease in Gini score represents a higher importance of the variable. (D) Heatmap of p values comparing T-cell subset immunophenotyping in cisgender men versus cisgender women. Cumulative cell frequency (E) and absolute counts (F) Tresp-cell (CD4⁺CD25^–CD127⁺) and Treg-cell (CD4⁺CD25⁺CD127^–) frequencies; data are mean (SE). (G) Treg-cell mediated suppression of activated Tresp cells in four cisgender men and four cisgender women detected using cell trace violet and flow cytometry following 72 h activation using soluble anti-CD3 and anti-CD28 in the presence of monocytes. Treg-cell to Tresp-cell ratios of 1:1 1:2, 1:4, and 0:1 were assessed (appendix 1 p 5); each leftward peak represents a round of proliferation. (H) Suppressive capacity of Treg cells at varying Treg-cell to Tresp-cell ratios in cisgender men compared with cisgender women, calculated using the fold change of proportion of Tresp-cell proliferation with Treg cells (1:1, 1:2, and 1:4) compared with Tresp cells without Treg cells (0:1); data are mean (SE). CM=central memory. EM=effector memory. EMRA=effector memory re-expressing CD45RA. Treg=regulatory T. Tresp=responder T. *Significant p value following 10% false discovery rate adjustment for multiple comparisons.

Figure 2

Comparison of Treg-cell transcriptomic profiles between young post-pubertal cisgender men and women FACS-sorted Treg cells (CD4⁺CD25⁺CD127^–) from five young, post-pubertal cisgender men and five young, post-pubertal cisgender women were analysed by RNA-sequencing and whole genome expression was compared by sex. (A) Volcano plot displaying log₂ fold changes and log₁₀ p values of DEGs between cisgender men and women, where coloured points represent statistically significant DEGs below p value threshold of 0·01). (B) Hierarchical clustering heatmap (Clustvis, Pearson's) of normalised gene counts of statistically significantly altered DEGs between cisgender men and women. (C) Pathway analysis plot displaying the –log₁₀ p values of enriched DEG pathway ontology terms of Treg cells between cisgender men and women using the 82 statistically significant DEGs. (D) Pathway analysis plot displaying the p value and enrichment ratios of enriched DEG pathway ontology terms of Treg cells between cisgender men and women using the extended gene list, incorporating both original seed genes and the genes from respective protein–protein interaction networks; the size of the points are relative to the number of genes contributing to that pathway. Box and whisker plots displaying Treg-cell gene expression by normalised counts of genes associated with the PI3K–AKT signalling (E), altered translation initiation pathways (F), and FOXP3 expression (G); data are mean (SE). DEG=differentially expressed gene. ER=oestrogen receptor alpha. Treg=regulatory T. Tresp=responder T.

Figure 3

Assessment of Treg-cell functional gene expression and frequency in cisgender and transgender individuals Violin plots displaying circulating Treg-cell (CD4⁺CD25⁺CD127^–) frequencies between seven pre-pubertal cisgender boys and seven cisgender girls, assessed by t test (A), and 17 post-pubertal cisgender men, 22 cisgender women, five transgender men, and five transgender women, assessed by one-way ANOVA (B); measured by flow cytometry; data are mean (SE). FACS-sorted Treg cells (CD4⁺CD25⁺CD127^–) from five young, post-pubertal cisgender men and five cisgender women and five transgender men and five transgender women were analysed by RNA-sequencing and whole genome expression was compared by gender. Volcano plots displaying log₂ fold changes and log₁₀ p values of DEGs between transgender men and cisgender women (C) and transgender women and cisgender men (D): the red and blue points represent statistically significantly DEGs below p value threshold (p<0·01). Cluster significance –log₁₀ p values of enriched DEG pathway ontology terms of Treg cells between transgender men and cisgender women (E). Hierarchical clustering heatmap (Clustvis, Pearson's) of normalised gene counts of DEGs found in the functional genetic pathway ontology terms in for transgender men versus cisgender women (F, G). Cluster significance –log₁₀ p values of enriched DEG pathway ontology terms of Treg cells between transgender women versus cisgender men (H). Hierarchical clustering heatmap (Clustvis, Pearson's) of normalised gene counts of DEGs found in the functional genetic pathway ontology terms in for transgender women versus cisgender men (I, J). Box and whisker plots of Treg-cell gene expression by normalised counts of genes associated with the (K) cytokine signalling, and (L) cell growth and activation pathways; data are mean (SE). ANOVA=analysis of variance. DEGs=differentially expressed genes. FACS=fluorescence-activated cell sorting. Treg=regulatory T. Tresp=responder T.

Figure 4

Sex and gender specific transcriptomic and functional changes in Treg cells Cohort details and gender-specific terminology descriptions are reported in appendix 1 (p 1). FACS-sorted Treg cells (CD4⁺CD25⁺CD127^–) from five young, post pubertal cisgender men, five cisgender women, five transgender men, and five transgender women were analysed by RNA-sequencing and whole genome expression was compared by gender. (A) Overlap of DEGs (p<0·01) from Treg cells between different group comparisons: cisgender men versus cisgender women, transgender men versus cisgender women, and transgender women versus cisgender men. (B) Treg-cell gene expression by normalised counts of genes that overlapped in all gender comparisons; data were assessed by one-way ANOVA and are mean (SE). (C) Sparse partial least squares discriminant analysis plot clustering each gender group using normalised gene counts of the overlapping genes (n=24); individual distribution points and confidence ellipses (ovals) are plotted for each gender group. (D) Treg-cell gene expression by normalised counts of the top ranked loaded genes for component 1 (appendix 1 p 11); data were assessed by one-way ANOVA and are mean (SE). Treg-cell mediated suppression of activated Tresp cells. Treg cells, Tresp cells, and monocytes were isolated from four young, post-pubertal cisgender men, four cisgender women, three transgender men, and four transgender women. (E) Proliferation of Tresp cells at varying Treg-cell to Tresp-cell ratios in individuals of different genders. (F) Suppressive capacity of Treg cells at a Treg-cell to Tresp-cell ratio of 1:1 between different gender groups, calculated using the fold change of the percentage of Tresp-cell proliferation with Treg cells (1:1) compared with Tresp-cell proliferation without Treg cells (0:1); data were assessed with one-way ANOVA and are mean (SE; appendix 1 p 5). DEGs=differentially expressed genes. FACS=f luorescence-activated cell sorting. ANOVA=analysis of variance. Treg=regulatory T. Tresp=responder T. Unstim=Unstimulated.

Figure 5

Sex differences in Treg cells in patients with juvenile-onset SLE (A) Cumulative cell frequency flow cytometry data of Treg cells (CD4⁺CD25⁺CD127^–) comparing 17 young post-pubertal healthy cisgender men, 12 cisgender men with juvenile-onset SLE, 22 young post-pubertal healthy cisgender women, and 23 cisgender women with juvenile-onset SLE; data were assessed by t test and are mean (SE). (B) Treg cell-mediated suppression of activated Tresp cells in five cisgender men and five cisgender women with juvenile-onset SLE, and four cisgender men and four cisgender women without juvenile-onset SLE; data were assessed with t test and are mean (SE). (C) Log₂ fold changes and log₁₀ p values of DEGs between five cisgender men and five cisgender women with juvenile-onset SLE, coloured points represent statistically significantly upregulated DEGs below p value threshold (p<0·01). Treg-cell gene expression by normalised counts of hormone (gender) (D) or sex chromosome specific genes (E) that were significantly associated with SLE by open target analysis (five per group); data were assessed with one-way ANOVA and are mean (SE); open bars are participants without juvenile-onset SLE and closed bars are participants with juvenile-onset SLE. (F) Overlap of DEGs (p<0·01) from Treg cells comparing patients with juvenile-onset SLE with healthy participants, stratified by sex (cisgender men and cisgender women). (G) Pathway analysis bar charts of enriched pathway ontology terms from sex-unique or overlapping DEGs. ANOVA=analysis of variance. SLE=systemic lupus erythematosus. DEGs=differentially expressed genes. Treg=regulatory T. Tresp=responder T. Unstim=unstimulated.