CX3CR1 Signaling Drives Tertiary Lymphoid Structure Formation and Fertility Impairment Following Treg Depletion in the Epididymis 2251411

Abstract Introduction Using an autoimmune model induced by Regulatory T cell (Treg) depletion, we found B and T cells form tertiary lymphoid structures (TLSs) in the epididymis, producing autoantibodies and inflammation that impair male fertility. In this organ, mononuclear phagocytes (MPs) express CX3CR1, while epithelial cells produce CX3CL1. CX3CR1 drives TLS formation in other tissues. We studied whether CX3CR1 deficiency in CX3CR1EGFP/EGFP mice prevents MP-mediated B cell activation and TLS formation in the epididymal mucosa following Treg depletion. Methods CX3CR1EGFP/+ and CX3CR1EGFP/EGFP mice were bred with Foxp3-DTR mice. Treg depletion was induced by diphtheria toxin (DT) injections. Readouts were analyzed at 2 and 8 weeks using confocal microscopy, flow cytometry, ELISA, and fertility assessment. Results Following depletion, CX3CR1+ MPs in CX3CR1EGFP/+/Foxp3-DTR mice exhibited increased projections and phagocytosed antibody-opsonized sperm. We observed fewer B cells and TLSs in DT-treated CX3CR1EGFP/EGFP/Foxp3-DTR mice, indicating that CX3CR1 is critical for B cell recruitment and TLS formation. Anti-sperm antibodies persisted only in CX3CR1EGFP/+ mice at 8 weeks. CX3CR1 deficiency rescued the reduction in sperm count and fertility defects following Treg ablation by impairing MP phagocytosis. Conclusion CX3CR1 is essential for epididymal TLS formation. CX3CR1 represents a promising therapeutic target for mitigating immune-mediated reproductive dysfunction and chronic inflammation in male infertility disorders. Funding Source R01 HD104672 Topic Categories Basic Autoimmunity (BA)