Background: Tumor infiltrating lymphocytes (TILs) assessed via light microscopy are prognostic and predictive in the early stage and advanced triple negative and HER2-amplified breast cancer (BC). Higher TILs can also identify patients more likely to benefit from anti-PD-1 therapy. In this study we interrogated T cell subsets that comprise high TILs to determine if distinct subpopulations are key mediators of anti-tumor immunity. Methods: We characterised TILs with a focus on CD3+ T cells in 129 primary and metastatic BC samples using flow cytometry, bulk RNASeq on flow sorted T cell populations, multiplex immunohistochemistry and microdroplet based single cell 3' mRNA sequencing on the 10X Genomics Chromium platform. Cell type specific gene expression signatures were determined from differential expression between putative T cell subpopulations. These signatures were investigated in clinical cohorts, including trial cohorts treated with pembrolizumab. Results: High TIL Infiltrates consisted primarily of CD3+ T cells, with both CD8 and CD4 populations. Unsupervised clustering of single cell sequencing identified 9 CD8 and CD4 subpopulations with distinct gene expression profiles. In addition to Tregs and CD8 effector memory (TEM) T cells, we found a CD8+ tissue resident memory (TRM) population expressing greater levels of T-cell checkpoints and cytotoxic markers compared to effector memory cells. In 2 primary tumours and 1 liver metastasis, bulk RNASeq of flow sorted TEM and TRM corroborated the single cell mRNASeq results. T cell receptor profiling (TCR) in the 3 samples found non-overlapping repertoires in the 2 primary tumours, but overlap in one metastatic lesion, suggesting divergent developmental origins in the breast, but the potential for nascent TRM differentiation in a metastatic niche. Clustering of these TCRs suggested differing antigen specificities between TRM and non-TRM CD8 T cells. Using Metabric data, the CD8 TRM gene expression signature was prognostic for disease free survival (DFS) in primary TNBCs (n=329, log-rank p=0.003), and was able to further stratify cases with high and low CD8A expression for DFS (log-rank p = 0.03). The CD8 TRM signature was enriched in baseline tumour samples of responders (n = 9) compared with non-responders (n=36) in 45 patients with metastatic melanoma treated with T cell checkpoint blockade (p < 0.0001). Additional single cell sequencing data with TCR sequencing will be combined with these initial results, and an independent data set of single cell mRNASeq and TCR Seq on CD3+ BC TILs will be used to confirm our findings. Cell type specific signatures will be explored in additional clinical cohorts including KEYNOTE-086, and presented at the meeting. Conclusion: Using single cell profiling of the immune microenvironment in BC we demonstrate that high TIL BCs contain multiple T cell subpopulations with different functional and prognostic significance. Our approach identified a CD8 TRM population with a distinct gene expression profile and strong expression of key immune checkpoints likely representing the presence of true tumor specific immunity. This population may be a key target of immune checkpoint blockade. Citation Format: Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, Salgado R, Teo ZL, Dushyanthen S, Byrne A, Luen SJ, Fox SB, Speed TP, Mackay LK, Neeson PJ, Loi S. Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-03.