PROJECT SUMMARY/ABSTRACT One of the most intriguing and understudied aspects of male reproductive physiology is the ability of the epididymis to prevent the development of immune responses against autoantigenic spermatozoa, while initiating very efficient immune responses against pathogens. The long-term goal of this application is to develop new strategic therapies for common disorders such as male infertility and epididymitis, and to identify novel targets for male-contraception. The overall objective is to elucidate how epididymal epithelial clear cells (CCs) communicate with immunocytes to contribute to the “immune privileged” environment that is optimal for sperm maturation and storage. We have recently showed a completely novel role for CCs in immune activation and sperm tolerance. Importantly, these cells establish intimate contact with region-specific heterogeneous subsets of mononuclear phagocytes (MPs) in the epididymis. Our central hypothesis is that CCs are strategically positioned to work in a concerted manner with immune cells to survey the epididymis barrier and regulate the balance between inflammation and immune tolerance in the post-testicular environment. The rationale is based on our preliminary data showing that CCs and MPs communicate in the steady state epididymis and also in the presence of infection or injury, and that both cell types express different tolerogenic mediators allowing the modulation of sperm tolerance. Therefore, these cells play a central role in the epididymal mucosa. In the first specific aim, we will determine CC – MP communication networks that initiate immune activation during epididymitis. In this aim, two mouse models of epididymitis will be used: injection of bacterial antigens into the cauda lumen, and epididymal-testicular torsion that induces sterile inflammation. In the second specific aim, we will evaluate CC - MP crosstalk after ablation of sperm tolerance in the epididymis, induced by depletion of regulatory T cells using diphtheria toxin-based transgenic mice. The research proposed in this application will use an innovative multidisciplinary approach that employs powerful immunology techniques rarely used to study reproductive physiology, 3D confocal microscopy, transcriptomics analysis, flow cytometry, cell sorting and bioinformatic analysis, as well as the use of mouse models to describe how specialized epithelial cells and immunocytes communicate in the epididymis. Notably, we will provide an in- depth characterization of three specific epididymal cell types: CCs and two subsets of MPs. Our proposed research is significant because it is expected to provide new insights into major immunoregulatory mechanisms by which epididymal CCs together with MPs protect spermatozoa against harmful antigens and autoimmunity. Ultimately, such knowledge will fill important gaps related to male reproductive biology by addressing crucial concepts of mucosal immunology and cell– cell interactions, all of wh...