Sex- and species-specific contribution of CD99 to T cell costimulation during multiple sclerosis

Background Differences in immune responses between women and men are leading to a strong sex bias in the incidence of autoimmune diseases that predominantly affect women, such as multiple sclerosis (MS). MS manifests in more than twice as many women, making sex one of the most important risk factor. However, it is incompletely understood which genes contribute to sex differences in autoimmune incidence. To address that, we conducted a gene expression analysis in female and male human spleen and identified the transmembrane protein CD99 as one of the most significantly differentially expressed genes with marked increase in men. CD99 has been reported to participate in immune cell transmigration and T cell regulation, but sex-specific implications have not been comprehensively investigated. Methods In this study, we conducted a gene expression analysis in female and male human spleen using the Genotype-Tissue Expression (GTEx) project dataset to identify differentially expressed genes between women and men. After successful validation on protein level of human immune cell subsets, we assessed hormonal regulation of CD99 as well as its implication on T cell regulation in primary human T cells and Jurkat T cells. In addition, we performed in vivo assays in wildtype mice and in Cd99-deficient mice to further analyze functional consequences of differential CD99 expression. Results Here, we found higher CD99 gene expression in male human spleens compared to females and confirmed this expression difference on protein level on the surface of T cells and pDCs. Androgens are likely dispensable as the cause shown by in vitro assays and ex vivo analysis of trans men samples. In cerebrospinal fluid, CD99 was higher on T cells compared to blood. Of note, male MS patients had lower CD99 levels on CD4+ T cells in the CSF, unlike controls. By contrast, both sexes had similar CD99 expression in mice and Cd99-deficient mice showed equal susceptibility to experimental autoimmune encephalomyelitis compared to wildtypes. Functionally, CD99 increased upon human T cell activation and inhibited T cell proliferation after blockade. Accordingly, CD99-deficient Jurkat T cells showed decreased cell proliferation and cluster formation, rescued by CD99 reintroduction. Conclusions Our results demonstrate that CD99 is sex-specifically regulated in healthy individuals and MS patients and that it is involved in T cell costimulation in humans but not in mice. CD99 could potentially contribute to MS incidence and susceptibility in a sex-specific manner. Supplementary Information The online version contains supplementary material available at 10.1186/s13293-024-00618-y.


Figure S2 .
Figure S2.Identification of immune cell subsets in PBMC cohort.(A-C) Representative gating strategy for identification of (A) T cells, (B) dendritic cells as well as (C) B cells, NK cells and monocytes.

Figure S3 .
Figure S3.Testosterone impact on CD99 expression of human T cells.(A) CD99 surface expression on T cells of anti-CD3 and anti-CD28 stimulated cryopreserved PBMCs (n = 3 males) treated with different concentrations of testosterone or dihydrotestosterone.Expression values are normalized to the individual vehicle control for every time point.(B) CD99 surface expression on T cells of unstimulated cryopreserved PBMCs (n = 3 males) treated with different concentrations of testosterone or dihydrotestosterone.Expression values are normalized to the individual vehicle control for every time point.(C) Total testosterone and bioavailable testosterone serum levels of trans men (n = 5).The male serum reference range is indicated by the grey shade (total testosterone: 1.93-7.4ng/mL, bioavailable testosterone: 1.4-4.3μg/l).Each color represents the same individual donor.Data are shown as mean ± SEM.Statistics: (A, B) two-way ANOVA with Tukey post-hoc; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure S4 .
Figure S4.Identification of immune cell subsets in trans men cohort.(A-C) Representative gating strategy for identification of (A) T cells, (B) dendritic cells, B cells, NK cells and monocytes.

Figure S7 .
Figure S7.CD99 dynamics in human T cells upon stimulation.Cryopreserved PBMCs from healthy controls (n = 3 females) were treated with anti-CD3 and anti-CD28 mAbs and cultured for 72 hours.(A) Raw CD99 expression values of memory and naïve T cells was analyzed by flow cytometry.(B) Raw CD69 expression values of memory and naïve T cells as a marker for T cell activation analyzed by flow cytometry.Data are shown as mean ± SEM.Statistics: (A) two-way ANOVA with Dunnett post-hoc; (B) two-way ANOVA with Tukey post-hoc; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure S8 .
Figure S8.Sex-specific analysis of anti-CD99 treatment on cell proliferation in human T cells.Anti-CD3 and anti-CD28 stimulated cryopreserved T cells from 27 healthy controls (n = 12 women and n = 15 men) were treated with anti-CD99 mAb (clone HCD99) or the respective isotype control antibody and proliferation was tracked by cluster formation in the IncuCyte® for 7 days.Data are shown as mean ± SEM.Statistics: Wilcoxon matched-pairs signed rank test; *P < 0.05; **P < 0.01; ***P < 0.001.

Table S2 .
Characteristics of MS patients and non-neuroinflammatory disease patients included in CD99 surface expression analysis of blood and CSF samples.

Table S3 .
Characteristics of healthy individuals included in CD99 surface expression analysis of PBMCs in trans men cohort.

Table S4 .
Antibodies used for flow cytometry.

Table S5 .
Primers, oligonucleotides and respective restriction sites used for the generation of CD99 vectors.