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Abstract 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.

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. Plain Language Summary: The immune system protects us from bacterial and viral infections and impacts the outcome of many diseases. Thus, understanding immunological processes is crucial to unravel pathogenic mechanisms and to develop new therapeutic treatment options. Sex is a biological variable affecting immunity and it is known that females and males differ in their immunological responses. Women mount stronger immune responses leading to more rapid control of infections and greater vaccine efficacy compared to men. However, this enhanced immune responsiveness is accompanied by female preponderance and susceptibility to autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis (MS). MS sex ratio varies around 2:1 to 3:1 with a steadily increasing incidence in female MS patients making sex one of the top risk factors for developing MS. However, the underlying biological mechanisms including sex hormones as well as genetic and epigenetic factors and their complex interplay remain largely unknown. Here, we discovered the gene and its encoded protein CD99 to be differentially expressed between women and men with men showing increased expression on many immune cell subsets including T cells. Since T cells are key contributors to MS pathogenesis, we examined the role of CD99 on T cells of healthy individuals and MS patients. We were able to identify CD99-mediated T cell regulation, which might contribute to sex differences in MS susceptibility and incidence indicating the importance to include sex as a biological variable. Of note, these differences were not reproduced in mice showing the necessity of functional research in humans. Highlights: Profiling of differential gene expression between sexes in the human immune system to delineate differential regulations that might impact immune responses. CD99 is genetically but not hormonally regulated on transcript and surface protein level with higher expression in men in healthy individuals as well as MS patients. Confirmation of CD99 as a critical co-stimulatory molecule on T cells, thus having an impact on T cell activation, proliferation and cluster formation. CD99 expression is elevated on T cells in the CSF compared to the blood. In male MS patients CD99 expression on CD4+ T cells from the CSF was lower in comparison to healthy men. Species difference in CD99 expression and its functional role with lack of differential CD99 expression in male and female mice as well as Cd99 deficiency not altering experimental autoimmune encephalomyelitis. [ABSTRACT FROM AUTHOR]