Phenotyping on the front lines

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Identified about a decade ago, tissue-resident memory T cells (TRM) provide antigen-specific protection from pathogens and viruses in peripheral tissues. Characterized by their residency in tissues and distinct inability to recirculate, TRM 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), TRM 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 TRM 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 TRM is thought to be a fruitful strategy for enhancing current immunotherapy approaches and vaccination efficacy. In this article, we will review common TRM markers and address the development of TRM 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 TRM(Table 1, below).

Key cell-surface markers for TRM

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 (TCM; CD45RA CCR7+ CD62L+), and effector memory (TEM; CD45RA CCR7 CD62L) T cells, as well as terminally differentiated effector memory cells re-expressing CD45RA (TEMRA; CD45RA+ CCR7 CD62L) [8]. TEM 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, TCM survey secondary lymphoid organs for cognate antigen, while naïve cells are typically localized to the blood, spleen, and lymph nodes [8].

CD69: Human TRM 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 TRM. Although CD69 is a marker of early T cell activation, most TRM 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 TRM in most tissues, and it functions as a critical antagonist of S1PR1 (CD363) activity [11]. The CD69+ TRM 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 TRM, 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 TRM 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+ TRM in mucosal sites and skin [7,8,9]. TRM 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 TRM 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 TRM [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 TRM biology. Its expression, however, does not distinguish the TRM subset from other CD8+ T cell populations, and it is mainly used as a marker of previous T cell activation, as it also labels TCM and TEM [11]. The role of CD44 in TRM may include regulation of cell–cell interactions, cell adhesion, migration, or lymphocyte activation.

TRM surface marker signature

The Farber lab at the Columbia University Medical Center has reported a core TRM surface marker signature consistent across tissues and diverse human donors and expressed in both CD4+ and CD8+ T cells [7]. In their approach, TRM are identified as CD69+, while TEM are CD69. When compared to CD69 TEM, CD69+ TRM 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+ TRM show downregulated expression of S1PR1, CD62L, CX3CR1, and KLF2 relative to CD69 TEM, a phenotype consistent with the retention of TRM within tissues [7,9].

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

The study of transcriptional regulation in TRM is ongoing. Perhaps as a function of their diversified tissue distribution with myriad metabolic niches, TRM 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 TRM tissue residency. TRM 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 TRM [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 TRM may be limited) [5,9]. Current research is focused on identifying the components of the general and tissue-specific signaling pathways that regulate TRM.

Flow cytometry panels for TRM

Taking these data into consideration, flow cytometry panels for TRM 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 TRM panels [9]. TRM 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].

Figure 1. Example of a flow cytometry panel for TRM. C57BL/6 mouse lymph node cells were stained with Invitrogen eBioscience CD3 eFluor 450, CD44 PE–eFluor 610, CD25 PE, CD62L Super Bright 780, CD4 FITC, CD8 PerCP–eFluor 710, and CD69 APC monoclonal antibodies. Viability was determined using Invitrogen eBioscience Fixable Viability Dye eFluor 506. For analysis, viable CD3+ T cells (C) were gated from singlet-gated (A) mouse lymph node cells (B). These CD3+ T cells were further gated by CD44+ and CD25 expression (memory and activation/Treg phenotype markers, respectively) (D), and then into CD4+ and CD8+ subsets (E). CD69 and CD62L expression identifies TRM cells (CD69+ CD62L), TCM cells (CD69 CD62L+), and TEM cells (CD69 CD62L) in the CD4+ population (F) and the CD8+ population (G). For Research Use Only. Not for use in diagnostic procedures. Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company.

Antibody selection tools for your flow cytometry panels

TRM 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 TRM. 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.

Marker typeMarkerLocationNotesClone (selected* Cat. No.)
Mouse
Core markerCD69SurfaceFound on most TRM; also expressed by activated cellsH1.2F3 (12-0691-82)
General phenotype (not tissue-specific)CD45SurfaceGeneral lymphocyte marker30-F11 (12-0451-82)
CD3SurfaceGeneral T cell marker145-2C11 (12-0031-82); 17A2 (12-0032-82); eBio500A2 (12-0033-82)
CD4SurfaceCD4+ T cell subset markerGK1.5 (12-0041-82); RM4-5 (12-0042-82); RM4-4 (12-0043-82)
CD8SurfaceCD8+ T cell subset marker53-6.7 (12-0081-82)
CD279 (PD-1)SurfaceHighly expressed on TRMJ43 (12-9985-82); RMP1-30 (12-9981-82)
CD101SurfaceSubsets of TRMMoushi101 (12-1011-82)
CD11aSurfaceSubunit of LFA-1M17/4 (12-0111-82)
CD44SurfaceExpression gradient: naïve < effector < memory T cellsIM7 (12-0441-82)
CD127 (IL-7R)SurfaceSubsets of TRMA7R34 (12-1271-82)
KLRG1SurfaceNegative or low expression on TRM2F1 (12-5893-82)
CCR7 (CD197)SurfaceNegative on TRM; expressed by TCM4B12 (12-1971-82)
CD62L (L-selectin)SurfaceNegative on TRM; expressed by TCMMEL-14 (12-0621-82)
CD45.1
CD45.2
SurfaceMay be used for parabiosis studiesA20 (12-0453-82)
104 (12-0454-82)
Blimp-1Nuclear 5E7 (12-9850-82)
Mucosal and barrier sitesCD103 (integrin αE)SurfaceSubsets of TRM2E7 (12-1031-82)
Skin and liverCD186 (CXCR6)SurfaceSubsets of TRMDANID2 (12-9186-82)
SkinCD183 (CXCR3)Surface CXCR3-173 (12-1831-82)
LiverLFA-1Surface M17/4 (12-0111-82); M18/2 (12-0181-82)
Human
Core markerCD69SurfaceFound on most TRM; also expressed by activated cellsFN50 (12-0699-42)
General phenotype (not tissue-specific)CD45SurfaceGeneral lymphocyte markerHI30 (12-0459-42); 2D1 (12-9459-42)
CD3SurfaceGeneral T cell markerUCHT1 (12-0038-42); SK7 (12-0036-42); OKT3 (12-0037-42); HIT3a (12-0039-42)
CD4SurfaceCD4+ T cell subset markerRPA-T4 (12-0049-42); OKT4 (12-0048-42); SK3 (12-0047-42)
CD8SurfaceCD8+ T cell subset markerRPA-T8 (12-0088-80); OKT8 (12-0086-42); HIT8a (12-0089-42); SK1 (12-0087-42)
CD45RASurfaceNaïve T cell marker; negative on TRMHI100 (12-0458-42)
CD45ROSurfaceMemory T cell markerUCHL1 (12-0457-42)
CD279 (PD-1)SurfaceHighly expressed on TRMMIH4 (12-9969-42); eBioJ105 (14-2799-80)
CD127 (IL-7R)SurfaceSubsets of TRMeBioRDR5 (12-1278-42)
NOTCH1IntracellularPossible TRM metabolic markermN1A (12-5785-82)
CRTAM (CD355)SurfaceUpregulated on some TRM subsets (adhesion molecule)Cr24.1 (12-3559-42)
S1PR1 (CD363)SurfaceDownregulated on TRMSW4GYPP (50-3639-42)
CX3CR1SurfaceDownregulated on TRM vs. circulating TRM2A9-1 (12-6099-42)
CCR7 (CD197)SurfaceNegative on TRM; expressed by TCM3D12 (12-1979-42)
CD62L (L-selectin)SurfaceNegative on TRM; expressed by TCMDreg-56 (12-0629-42)
CD101SurfaceSubsets of TRMBB27 (14-1019-82)
Mucosal and barrier sitesCD103 (integrin αE)SurfaceSubsets of TRMB-Ly7 (12-1038-42)
Lung and skinCD49a (integrin α1)Surface TS2/7 (46-9490-42)
LungCD183 (CXCR3)Surface CEW33D (12-1839-42)
SkinCD194 (CCR4)
CLA
Surface D8SEE (12-1949-42)
HECA-452 (50-9857-82)
Lymph nodeCD184 (CXCR4)
CD185 (CXCR5)
Surface 12G5 (12-9999-42)
MU5UBEE (12-9185-42)
LiverLFA-1Surface HI111 (11-0119-42); 6.7 (12-0189-42); R3.3 (BMS103FI)
*Antibodies are available as several different conjugates and in multiple packagings; go to thermofisher.com/antibodies to see a complete listing.

仅供科研使用,不可用于诊断目的。