Double negative T cells decline in inflammatory reproductive dysfunction

Abstract

Abstract Double-negative T cells (DNTs; CD3 + CD4 − CD8 − ) have been implicated in immune regulation in autoimmune settings, but their relevance to reproductive tissue immune balance and inflammation-associated infertility remains unclear. Here, we define a population of TCRβ + NK1.1 − DNTs enriched in the mouse ovary and uterus and profiled its phenotype, tissue behavior, and function in inflammatory models associated with impaired fertility. In parallel, we performed RNA-seq on spleen- and thymus-derived NK1.1 − DNT and CD8 + T cell enriched population. Relative to CD8 + T cells, peripheral NK1.1 − DN T-cell-enriched populations displayed an activated, regulatory-like transcriptional profile with reduced Cd8a / Cd8b1 , Il7r , and cytotoxic effector markers, alongside increased expression of Pdcd1 , Lag3 , Tox , and Il10 . Ex vivo, FACS-sorted splenic DNTs produced low inflammatory cytokine output after CD3/CD28 stimulation and suppressed CD8 + T-cell proliferation primarily through contact-dependent mechanisms. In vivo, ovarian DNTs decreased following CD8-targeting depletion, and ovarian and uterine DNTs showed limited exchange in parabiosis. In chronic interferon-γ-driven inflammation (ARE −/− ) and zona pellucida 3-induced ovarian inflammation, reproductive tissues exhibited reduced DNT frequencies, a shift in the DNT: CD8 + T ratio favoring CD8 + T cells and increased activated CD8 + phenotypes. Finally, adoptive transfer of ex vivo FACS-sorted wild-type DNTs into ARE −/− females increased pregnancy frequency, supporting DNT-associated immunoregulation as a feature of inflammation-associated infertility.