Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.

Mouse cells were stained at 4°C for 60 min with the antibodies.

For flow cytometry experiments, samples were acquired on a 5-laser BD LSRFortessa (BD Biosciences) or a 5-laser Cytek Aurora. For cell sorting experiments, memory T cells from the spleen (CD8α+Vα2+CD45.1+) were sorted according to the gating strategy described in Figure 1I using a 5-laser BD FACSAria III (BD Bioscience) or a 4-laser Beckman Coulter Cytoflex SRT (>95% purity). Data was analysed on Flowjo v10 (Treestar) or OMIQ (https://www.omiq.ai/).