Flow Cytometry Antibodies for Identifying Tissue-Resident Memory T Cells

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Identified about a decade ago, tissue-resident memory T cells (T_RM) provide antigen-specific protection from pathogens and viruses in peripheral tissues. Characterized by their residency in tissues and distinct inability to recirculate, T_RM have been identified in lung, skin, liver, brain, intestinal, and mucosal tissues [1-4]. While initial studies focused on their persistence at sites of previous infection and their long-lived role in combating reinfection (through immediate effector function and accelerated recruitment of circulating immune cells), T_RM have also been reported to accumulate in the tumor microenvironment, including those of epithelial (ovarian, pancreatic, colorectal, and lung) and nonepithelial (malignant glioma and melanoma) origin [5].

The efficacy of immune checkpoint inhibitors (anti–PD-1, anti– PD-L1, and others) in cancer treatment has led researchers to postulate that T_RM are key players in the tumor microenvironment, capable of initiating and maintaining antitumor responses due to their high levels of expression of inhibitory receptors. Potential mechanisms of action include rapid and local antigen-specific proliferative responses, with expression of effector molecules to promote inflammation and immune cell recruitment and differentiation (IFNγ, TNFα, IL-2, IL-17) and to direct target-cell lysis (perforin, granzyme B) [6,7]. Therefore, increased activation of T_RM is thought to be a fruitful strategy for enhancing current immunotherapy approaches and vaccination efficacy. In this article, we will review common T_RM markers and address the development of T_RM flow cytometry panels. Thermo Fisher Scientific offers a wide variety of Invitrogen primary antibodies and antibody conjugates for flow cytometry that recognize positive and negative markers for T_RM(Table 1, below).

The peripheral blood T cell population contains a variety of subsets that can be identified by expression of CD45RA, CCR7 (CD197), and CD62L (L-selectin). These subsets include naïve (CD45RA⁺ CCR7⁺ CD62L⁺), central memory (T_CM; CD45RA^– CCR7⁺ CD62L⁺), and effector memory (T_EM; CD45RA^– CCR7^– CD62L^–) T cells, as well as terminally differentiated effector memory cells re-expressing CD45RA (T_EMRA; CD45RA⁺ CCR7^– CD62L^–) [8]. T_EM play an important role in immune surveillance and are a major subset of both CD4⁺ and CD8⁺ T cell populations found in peripheral tissues [7,9]. In contrast, T_CM survey secondary lymphoid organs for cognate antigen, while naïve cells are typically localized to the blood, spleen, and lymph nodes [8].

CD69: Human T_RM are typically associated with the expression of the surface markers CD69, CD49a (integrin α1), and CD103 (integrin αE); these markers, along with CD44, are useful for studying mouse T_RM. Although CD69 is a marker of early T cell activation, most T_RM express CD69 under steady-state conditions, without expression of other activation markers such as CD25, CD38, and HLA-DR [10]. CD69 is therefore an important distinguishing cell-surface marker constitutively expressed on T_RM in most tissues, and it functions as a critical antagonist of S1PR1 (CD363) activity [11]. The CD69⁺ T_RM population is phenotypically and transcriptionally distinct from recirculating CD69^– memory T cells in both tissues and blood, each having a defined gene expression signature that includes molecules associated with adhesion, migration, and regulation [6,7].

CD49a: CD49a (integrin α1) is also an important marker expressed in some T_RM, as it acts with CD29 (integrin β1) to form the heterodimeric molecule VLA-1, which can bind collagen and laminin and promotes tissue residency [5,12].

CD103: CD103 (integrin αE) expression in T_RM is variable [7]. CD103 is upregulated after exposure to TGF-β, and it complexes with integrin β7 on the T cell surface to allow adherence through binding to CD324 (E-cadherin) on epithelial cells [11]. CD103 expression is restricted to CD8⁺ T_RM in mucosal sites and skin [7,8,9]. T_RM that exist outside of epithelial tissues generally lack CD103 expression, although they may express other adhesion molecules such as LFA-1, which is a heterodimeric integrin composed of CD11a (LFA-1α) and CD18 (LFA-1β). In mice, LFA-1 is present on CD103^– liver-resident T_RM and is thought to allow binding of CD54 (ICAM-1) on liver sinusoidal endothelial cells. It is not yet known if this marker is expressed on human liver-resident T_RM [13].

CD44: CD44 is a C-lectin–containing glycoprotein that is expressed on leukocytes and other cell types and serves as a receptor for hyaluronic acid (HUA), which is an extracellular matrix component produced by vascular endothelial cells and other immune cells [11]. CD44 also binds to other matrix proteins like fibronectin, laminin, and collagen [11]. In mice, CD44 is considered a core marker with a functional role in T_RM biology. Its expression, however, does not distinguish the T_RM subset from other CD8⁺ T cell populations, and it is mainly used as a marker of previous T cell activation, as it also labels T_CM and T_EM [11]. The role of CD44 in T_RM may include regulation of cell–cell interactions, cell adhesion, migration, or lymphocyte activation.

The Farber lab at the Columbia University Medical Center has reported a core T_RM surface marker signature consistent across tissues and diverse human donors and expressed in both CD4⁺ and CD8⁺ T cells [7]. In their approach, T_RM are identified as CD69⁺, while T_EM are CD69^–. When compared to CD69^– T_EM, CD69⁺ T_RM show upregulated expression of CD103 (integrin αE), CD49a (integrin α1), CRTAM (CD355), CD186 (CXCR6), CD279 (PD-1), MKP3 (DUSP6), and IL-10. Notably, expression of CD186 (CXCR6) allows for migration into peripheral tissues. Furthermore, CD69⁺ T_RM show downregulated expression of S1PR1, CD62L, CX3CR1, and KLF2 relative to CD69^– T_EM, a phenotype consistent with the retention of T_RM within tissues [7,9].

Other markers of interest expressed specifically in CD8⁺ CD69⁺ T_RM are ICOS and IRF4 [7]. CD101 is also upregulated on CD8⁺ T_RM, as compared with CD4⁺ T_RM in the lung and spleen, and may be involved in inhibition of T cell activation and proliferation [7,9]. CD28 and CD127 (IL-7R) may be useful for delineating cell activation and homeostasis [4]. T_RM development and maintenance are regulated by CD122 (IL-15R) and CD127 (IL-7R), and the latter receptor is expressed on a majority of CD69⁺ T_RM [6]. Other homing and retention markers may be utilized to distinguish T_RM in specific compartments, such as CLA (CD162, PSGL-1), CCR8, CCR10, FABP4, and FABP5 on skin-resident T_RM, and CCR9 and integrin α4β7 (LPAM-1) on intestine-resident T_RM [5].

The study of transcriptional regulation in T_RM is ongoing. Perhaps as a function of their diversified tissue distribution with myriad metabolic niches, T_RM are currently thought to rely on combinations of transcription factors, and a unifying lineage-restricted master regulator has not yet been identified [11]. That said, several transcription factors have been identified as important for T_RM tissue residency. T_RM are reported to downregulate the T-box transcription factors EOMES (negative) and T-bet (low) following TGF-β signaling [5,6,14]. RUNX3 enhances granzyme B and CD103 expression, whereas NOTCH1 appears to regulate metabolism in T_RM [6]. Other transcription factors may contribute to tissue residency programs, such as BLIMP-1 and HOBIT, which can repress CCR7, S1PR1, KLF2, and TCF-1 expression (although some reports suggest the role of HOBIT in T_RM may be limited) [5,9]. Current research is focused on identifying the components of the general and tissue-specific signaling pathways that regulate T_RM.

Taking these data into consideration, flow cytometry panels for T_RM may include a core set of markers such as CD45, CD69, CD3, CD4, CD8, CD279 (PD-1), CD103 (integrin αE), CD186 (CXCR6), CD19 (negative), CD45RA (negative), CCR7 (negative), and CD62L (negative) (Figure 1). Further clarity would be provided by inclusion of CX3CR1 and S1PR1 markers in the panels [6]. CD44 is useful in mouse T_RM panels [9]. T_RM may also upregulate CD28 and CD127 (IL-7R), although this upregulation appears to depend on tissue localization [7,8]. Further memory phenotypes may be identified using KLRG1 and CD27. Tissue-specific homing markers, such as LFA-1, CCR8, CCR10, CLA, FABP4, FABP5, CCR9, and integrin α4β7 (LPAM-1) may also be of interest [6].

T_RM are an important and still-emerging subset in oncology, potentially other disease research, and vaccine design. Further studies are needed to illuminate their development and roles in disease protection and progression. Table 1 lists selected Invitrogen flow cytometry antibodies and antibody conjugates for the study of T_RM. Search our complete portfolio of primary and secondary antibodies.

Table 1. Invitrogen flow cytometry antibodies and selected antibody conjugates for the study of tissue-resident memory T cells.

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