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Knit directory: 5_gd_Tcell/1_analysis/

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Kerrie L. Foyle1,4, Hon Yeung Chan1,4, Ha M. Tran1, Jimmy Breen1, John E. Schjenken1,2,3, and Sarah A. Robertson1,5,*

Author affiliations:

1 The Robinson Research Institute and Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia.

2 Hunter Medical Research Institute, Infertility and Reproduction Research Program, New Lambton Heights, NSW 2305, Australia.

3 School of Environmental and Life Sciences, Discipline of Biological Sciences, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia

4 These authors contributed equally

5 Lead contact

* Corresponding author: Sarah A. Robertson, The Robinson Research Institute, Adelaide Medical School, University of Adelaide, Adelaide, SA 5005, Australia.

Phone: +61883134094 E-mail:

Conflict of Interest Statement: The authors have declared that no conflict of interest exists

Abstract

Seminal fluid elicits an immune response in the uterine mucosa after mating that impacts embryo implantation and pregnancy, but the underlying molecular and cellular events are unclear. In this study, we report RNA sequencing to analyse the uterine response to seminal fluid after mating. Females exposed to seminal fluid of intact males exhibited gene expression changes on D3.5 post-coitum (pc) just prior to embryo implantation, compared to females mated with males surgically rendered seminal plasma deficient. Functional enrichment analysis revealed genes related to T cell activation amongst those with the largest fold-changes. Using flow cytometry we then showed profound changes in uterine T cell abundance and phenotype regulated by seminal fluid contact. While CD4+ and CD8+ T cells were elevated by seminal fluid, the most conspicuous change was in CD4-CD8- T cells expressing γδ T cell receptors (TCR). Mating with intact males caused a 8.3-fold increase in γδ T cell abundance compared to estrous virgin females, and a 22.4-fold increase in the proportion of γδ T cells expressing proliferation marker Ki67. Vγ6+ cells were the most abundant subpopulation in the uterus, followed by Vγ4+ and Vγ1+ T cells, and all three were similarly expanded after mating. Seminal plasma was critical for γδ T cell accumulation and activation in the endometrium, and similar changes occurred in uterine-draining lymph nodes but not spleen. These findings identify γδ T cells as prominent in the immune response to seminal fluid and imply key roles in uterine immune regulation and reproductive success.