Epididymal tertiary lymphoid structure formation after Treg depletion sustains male subfertility 2733

Abstract Description Tregs are vital for maintaining immune tolerance in the testis and epididymis, where spermatozoa reside. Our previous studies showed that 2 weeks after Treg depletion, autoimmune orchitis, and epididymitis, characterized by anti-sperm antibody (ASA) production, reduced sperm count and motility, leading to subfertility. Here, we investigated the long-term effects of Treg depletion by injecting diphtheria toxin (DT, i.p.) in WT and Foxp3-DTR mice. Flow Cytometry showed that 8 weeks after Treg depletion, the myeloid cell infiltration had resolved, and T and B lymphocytes had increased in the testis and epididymis. ELISA detected elevated serum and tissue antibody levels against sperm and reproductive tract antigens. Confocal microscopy revealed organized T- and B-cell aggregates forming tertiary lymphoid structures (TLS). Soluble mediators associated with TLS formation and maintenance were upregulated in the Treg-depleted epididymis. Proteomic analysis revealed immune-related and fertility-associated sperm proteins targeted by serum and epididymal fluid autoantibodies. Sperm count and fertility were compromised, as evidenced by reduced litter size. Treg depletion induces a persistent proinflammatory response in the male reproductive tract, leading to TLS formation and chronic subfertility. These findings may aid in developing new treatments for male infertility and immuno-contraceptive strategies. Funding Sources NIH HD104672-01 MGH Physician/Scientist Development and Claflin Distinguished Scholar Award Lalor Foundation Fellowship Topic Categories Mucosal and Regional Immunology (MUC)