Immune responses in trans men during gender-affirming hormone therapy become more like those of cisgender men

A recent study in Nature examined the effects of gender-affirming hormonal treatment (GAHT) on the immune system in trans gendermen.

Background

Men and women differ in their severity levels for infectious and inflammatory disorders due to differences in their immune systems. For example, men are more likely to die from coronavirus disease 2019 (COVID-19), while women are at a higher risk of post-acute COVID-19 sequelae and elicit stronger reactions to vaccines. Studies also report more frequent adverse effects among women.

Immunological sex differences are due to genes, hormones, and behavior. However, their significance and impact on disease risk remains unclear. Transgender people’s hormone levels change after gender-affirming testosterone treatment (GAHT). It is crucial to understand the effects of GAHT on immune responses in transgender individuals to elucidate the mechanisms underlying the differences in disease severity and immune reactions among men and women.

About the study

The present study investigated the immunomodulatory effects of gonadal steroids in humans.

Between 2016 and 2023, the study included 23 trans males registered at Swedish transgender medical centers. At 18 to 37 years of age, they received 1,000 mg of Nebido testosterone therapy after being assigned female at birth. Five participants received 750 mg doses due to low body mass index (BMI) values. The study participants had normal sex hormone levels and no prior testosterone therapy. They provided blood samples at baseline, three months, and one year after testosterone therapy. The study excluded individuals with autoimmune disorders, immunodeficiencies, or ongoing infections.

Liquid chromatography and tandem mass spectrometry measured sex hormone and steroid hormone levels in serum. They used bulk ribonucleic acid sequencing (RNA-seq) and single-cell RNA-seq to study gene expression changes. NicheNet analysis predicted downstream effects on additional immune cell groups. The single-cell assay for transposase-accessible chromatin with high-throughput sequencing (sc-ATAC-seq) measured changes in chromatin accessibility at various binding sites. The binding sites included those for Janus kinase-signal transducer and activator of transcription (JAK-STAT), activator protein-1 (AP-1), nuclear factor kappa B (NFκB), and mitogen-activated protein kinase (MAPK).

The researchers used flow cytometry to evaluate the functional effects of GAHT on lymphocytes. They profiled immune cells using mass cytometry and spectral flow cytometry to measure androgen receptor (AR) and estrogen receptor (ESR) expression, peripheral blood mononuclear cell (PBMC) stimulation, and intracellular labeling. The Olink test generated data on plasma proteins. Enzyme-linked immunosorbent assays (ELISA) measured interferon subtypes in the plasma and PBMC supernatants.

To determine the effects of high testosterone and low estrogen on immune cells, researchers collected blood samples from 11 cisgender females. They then pretreated them with testosterone and fulvestrant to simulate the loss of ESR-mediated signals. Researchers examined sc-mRNA-seq datasets of male and female participants to investigate whether the differences in immune responses may explain the varied responses in cisgender individuals. They used mixed-effects models and principal component analysis (PCA).

Results

Testosterone is essential for modifying the balance between tumor necrosis factor (TNF) and type I interferon (IFN-I). TNF is pro-inflammatory, whereas IFN-I exerts antiviral effects. Attenuating IFN-I responses in plasmacytoid monocytes and dendritic cells while potentiating monocyte responses achieves increased TNF, interleukin-6 (IL-6), and IL-15 production, as well as downstream stimulation of NF-κB-mediated genes and enhancement of IFN-γ responses. This occurs naturally among natural killer (NK) cells. Gender-diverse replies in public databases support these findings among trans men.

Bioavailable testosterone levels increased in blood, but oestradiol and progesterone concentrations declined in three months. Testosterone treatment led to immune cell changes, but overall white blood cell counts remained steady. Plasmacytoid dendritic cells (pDCs) showed changes in their phenotype. CD81 levels rose after three months. Three months after testosterone treatment, interferon-stimulated exonuclease gene 20 (ISG20), poly(ADP-ribose) polymerase family member 14 (PAPR14), myxovirus resistance protein 1 (MX1), and SP110 were less activated.

The plasmacytoid dendritic cells downregulated the suppressor of cytokine signaling (SOCS) regulators. SOCS3 inhibited IFN-I responses during influenza infection. Testosterone treatment reduced IFN-α responses in monocytes. However, it increased TNF responses to R848 stimulation. Testosterone treatment increased SLAM Family Member 7 (SLAMF7) in T cells and monocytes. GAHT and DHT pretreatment activated NFκB pathways for 20 hours, but Fulvestrant did not.

Professor Petter Brodin, Garfield Weston Chair and Professor of Paediatric immunology at Imperial College London, who led the work while based at the Karolinska Institutet, said: “These findings have implications for us all. For the first time, we have been able to identify which parts of a person’s immune system are directly regulated by sex hormones rather than genetic sex differences. This could have significant impact not only on our understanding of how different diseases affect males and females differently, but also to develop new treatments which could help in everything from immune diseases to cancer.” 

Conclusion

The study found that testosterone levels rise during gender-affirming hormone therapy and oestradiol levels decrease. This affects the cross-regulation between IFN-I and TNF. This increases serum levels of receptor activators of nuclear factor kappa beta ligand (RANKL) and colony-stimulating factor 1 (CSF1). It also improves chromatin accessibility at NFκB transcription factor binding sites. TNF responses increase in an androgen receptor-dependent manner.

This indicates that the cross-regulation between IFN-I and TNF occurs at the system level, and sex hormones regulate it. DHT increased NFκB responses to lipopolysaccharide but inhibited IFN-I, SOCS1, and SOCS3. Studies with longer follow-ups are required to evaluate the long-term impacts of hormonal treatment on immunity and disease risk.