Deep profiling deconstructs features associated with memory CD8+ T cell tissue residence

Tissue resident memory CD8+ T cells (Trm) control infections and cancer and are defined by their lack of recirculation. Because migration is difficult to assess, residence is usually inferred by putative residence-defining phenotypic and gene signature proxies. We assessed the validity and universality of residence proxies by integrating mouse parabiosis, multi-organ sampling, intravascular staining, acute and chronic infection models, dirty mice, and single-cell multi-omics. We report that memory T cells integrate a constellation of inputs— location, stimulation history, antigen persistence, and environment— resulting in myriad differentiation states. Thus, current Trm-defining methodologies have implicit limitations, and a universal residence-specific signature may not exist. However, we define genes and phenotypes that more robustly correlate with tissue residence across the broad range of conditions that we tested. This study reveals broad adaptability of T cells to diverse stimulatory and environmental inputs and provides practical recommendations for evaluating Trm cells. Overall design: Memory P14 CD8+ T cell populations specific to lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) were created for RNA-seq and transcriptome analyses. First, 5 x 10^4 naïve CD45.1+ P14 CD8+ T cells were adoptively transferred into naïve C57BL/6J mice followed by i.p infection with 2x10^5 PFU LCMV Arm. At >30 days p.i., i.v. antibody labeling was performed with anti-CD8a 3 minutes prior to euthanizing the mice and removal of tissues. We then created single cell suspensions from the excised tissues. Cells were stained with anti-CD8b, anti-CD45.1, anti-CD44, anti-CD69, anti-CD62L, and Ghost Dye Red 780 (Tonbo Biosciences) flow antibodies. Subsequently, LCMV Arm specific Tcm (CD8b+CD45.1+CD69-CD62L+), Tem (CD8b+CD45.1+CD69-CD62L-), and Trm (CD8b+CD45.1+CD69+CD62L-) populations were sorted from cell suspensions of pooled secondary lymphoid organs (SLO) with a FACSAria II (BD Biosciences). Extravascular Trm (identified as i.v. antibody negative CD8b+CD45.1+CD69+CD62L- P14 cells) were sorted from single-cell suspensions originating from the small intestine (SI) epithelial and lamina propria compartments, as well as the female reproductive tract (FRT). Extravascular LCMV Arm specific CD69+CD62L- P14 cells in the SI and FRT tissues were presumed to be Trm based on prior parabiosis studies. Bystander activated (CD8+CD45.1+CD69+) P14 cells were sorted from pooled SLO of LCMV Arm immune mice 48 hours after i.v. VSV-NJ infection. Recently stimulated (CD8+CD45.1+CD69+) P14 cells were sorted from pooled SLO cell suspensions from mice 4 days after LCMV Arm infection. Endogenous CD69+ H-2Db/gp33 tetramer+CD8+ T cells were sorted from pooled SLO of mice 50 days after LCMV Cl13 infection in CD4 T cell depleted mice. Naïve (CD44 low) P14 CD8+ T cells were sorted from pooled SLO cell suspensions from CD45.1+/Thy1.2+ P14 transgenic mice. After sorting to obtain cell populations of interest, RNA isolation, integrity assessment, and sequencing was performed. Sequencing libraries were prepared with the Clontech SMARTer Stranded Total RNA-seq Kit v2 (Pico Input Mammalian Kit). 50 base pair single-end sequences were then generated from the with the HiSeq 2500 Illumina.