Summary
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.
Graphical abstract
Flow cytometry
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/).
Figure 1. (A) InfinityFlow analysis pipeline. Naive P14 Thy1.1+ T cells were primed using LCMV, isolated from various tissues 60 dpi, barcoded, and stained with a backbone antibody panel. Cells were stained with >200 PE-conjugated antibodies (LEGENDScreen), acquired by flow cytometry and analyzed using InfinityFlow.
qPCR
RNA was extracted from sorted samples using RNeasy Plus Micro Kit (QIAGEN) according to the manufacturer’s instructions. mRNA was converted into cDNA using High Capacity cDNA Reverse Transcription Kit, and genes of interest were preamplified using TaqMan PreAmp Master Mix (ThermoFisher Scientific). Gene expression was analysed by real-time PCR using the StepOnePlus Real-Time PCR System (ThermoFisher Scientific), TaqMan Fast Advanced Master Mix, and the following TaqMan probes (all from Thermo Fisher Scientific): Hprt Mm00446968_m1, Sirt1 Mm01168521_m1, Tbp Mm00446973_m1. Cycle-threshold values were determined for genes individually, and gene expression was normalized according to the 2-dCt method to the housekeeping gene Hprt or Tbp.
Beckman Coulter Life Sciences
CytoFLEX SRT Benchtop Cell Sorter
Optical Filters: 450/45, 525/40 (2), 585/42, 610/20 (3), 660/10, 675/30, 690/50, 710/50, 712/25, 780/60 (3)
Lasers: 405 nm, 488 nm, 561 nm, 638 nm
Nozzle Diameter(s): 100 µm
Sorting Performance: With a target population of 5% and a threshold rate of <10,000 events per second, >99% purity and >80% of Poisson's expected yield
Collection Vessels: 12x75 mm tubes, 15 mL conical tubes, 96-well plates, 96-deep-well plates, 6-, 24-, 48-, 96- and 384-well plates, slides
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