Isolation and Characterization of Human Tissue Resident Memory T cells

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Abstract The majority of immune cells in the human body exist within the tissues rather than in the circulation. Nevertheless, most of our knowledge of the human immune system is biased towards the characterization and understanding of circulating immune cell populations because the latter are readily sampled, whereas cells in tissues are difficult to obtain and/or are limited to single sites of disease. Tissue‐resident cells differ from circulating cells due to tissue‐specific niche adaptations that influence their phenotype and function. For instance, T cells in tissues, resident memory (TRM cells), exhibit tissue‐specific properties that allow optimal protection from infection due to an acquired ability to coordinate rapid and efficacious pathogen clearance. Thus, to fully understand T‐cell responses in various pathological conditions one must focus on the properties of TRM cells and how they have been shaped by their environment. Moreover, one must sample and analyze T cells from multiple tissues, optimally from the same individual, to determine how infectious, cancer, or autoimmune challenge is affecting homeostatic function. Our longstanding collaboration with the organ procurement organization, LiveOnNY, provides unique access to multiple lymphoid, mucosal, and peripheral tissues from organ donors where consent for research use has been obtained. These samples have enabled characterization of human tissue resident memory T cells and other immune cell types across a variety of tissues. Concomitant with this endeavor, we developed and refined a series of methodologies critical for extracting immune cells from tissue for the purpose of phenotypic and mechanistic interrogation. Here, we describe our optimized protocols for processing select human tissues and the requisite coordination and considerations for their maximal yield and tissue quality. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of immune cells from blood‐rich sites [spleen (SPL), blood (BLD), bone marrow (BOM)] Basic Protocol 2: Isolation of immune cells from lymph nodes, tonsils, and thymus [iliac lymph nodes (ILN), lung lymph nodes (LLN), mesenteric lymph nodes (MLN), colonic lymph nodes (CLN), hepatic lymph nodes (HLN), tonsils (TON), thymus (THY)] Basic Protocol 3: Isolation of immune cells from the lungs [lung (LNG), bronchioalveolar lavage (BAL)] Basic Protocol 4: Isolation of immune cells from the intestines [jejunum epithelial layer (JEL), jejunum lamina propria (JLP), colon epithelial layer (CEL), colon lamina propria (CLP)] Basic Protocol 5: Isolation of immune cells from the liver (LVR) Basic Protocol 6: Immune cell staining for flow cytometry

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