Multiomic and spatial immunophenotyping reveals a prominent Runx3⁺ resident T cell population in the healthy human breast

Abstract The role of Runt-domain related transcription factor 3 (Runx3) in the early development of T cells is well established, however recent work has implicated Runx3 as a driver of residency in barrier tissues such as the lung and intestinal epithelium, as well as in the tumor infiltrating lymphocytes of murine and human melanomas. We have identified a subpopulation of resident T cells in the healthy human breast exhibiting constitutive expression of Runx3. Employing single-cell RNA sequencing paired with concurrent sequencing of T cell receptors (TCRseq, 10× Genomics) and oligo-tagged antibodies (Totalseq, BioLegend) specific to canonical markers of memory and effector T cells, we compared the clonality and transcriptome of T lymphocytes isolated from reduction mammoplasty or tumor-patient-derived contralateral mastectomies to those obtained from matched patient peripheral blood. We subsequently utilized highly-multiplexed spatial immunophenotyping through Co-detection by indexing (CODEX, Akoya Biosciences) to infer the role of Runx3 +resident T cells in the healthy human breast based on informatic analysis of the associated cellular neighborhoods. Our analysis finds an abundant population of highly clonal CD8 +Runx3 +cytotoxic T lymphocytes consistently located in the milk-producing lobular and secretory ductal regions of the healthy adult breast, postulating roles in barrier immunity and tissue reconstruction. Furthermore, marked presence of these cells in the contralateral mastectomies of tumor-bearing patients suggests a protective antitumoral role in nonaffected or cleared tissue. Supported by NIH grant R01 (5R01CACA237376-03), T32 (T32-NS121727-01)