Although without exact and consistent findings, various cell markers have been observed on TRM cells. We have summarized some of the key cell surface markers in the intestine based on recent studies.

CD103 and CD69 were defined as classic phenotypic markers on TRM cells (21) ( Figure 1 ). CD103 (αE integrin), a receptor for E-Cadherin, is predominantly expressed on intestinal CD8⁺ TRM cells, where it promotes the residency of TRM cells (22). It is considered to be a CD8⁺ TRM-residency-specific molecule (23), as most CD4⁺ T cells do not express CD103 (24). In normal barrier tissues, TGF-β signaling promotes CD103 expression, ensuring the long-term maintenance of CD103⁺ CD8⁺ TRM cells in nonlymphoid barrier tissues (25). During intestinal inflammation, the expression of CD103 can be influenced by cytokines such as interferon (IFN) -β and IL-12. These inflammatory mediators may modulate TGF-β-mediated CD103 expression, potentially inhibiting or altering its induction in different cellular contexts (26). Since E-cadherin is primarily expressed in the intestinal epithelium, it may have limited impact on the lamina propria where CD103⁺ cells locate (27).

CD69 serves as a marker of early T cell activation and is closely associated with T cell residency (28). During the development of TRM cells, TGF-β negatively regulates Forkhead Box Protein O1 (FoxOl) through the PI3K-Akt pathway, downregulating Kruppel-like factor 2 (KLF2) (29). KLF2 is a transcription factor critical for T cell trafficking, which directly downregulates sphingosine-1 phosphate receptor 1 (S1PR1) and indirectly upregulates CD69, preventing T cells egress from the intestine (30). The lack of S1PR1 is also a method to identify TRM cells. Compared with CD103, CD69 is a secondary marker, as CD69 is not an essential residency marker in the intestine of mice (31). However, in humans, CD69 is instrumental in distinguishing between TRM cells and circulating memory T cells (32).

Only 25% of TRM cells in the colon express both CD103 and CD69, TRM cells lacking CD103 or CD69 can also be found (33). Their generation depends on the location and whether they arise from local tissue infection (34), as these cells play a rapid response role in secondary infections (35). Compared to CD103⁺ TRM cells, intestinal CD103^- TRM cells have been demonstrated to possess greater effector functions, including the ability to produce higher levels of granzyme A (36). CD103^- TRM cells share similar transcriptional profiles with circulating T cells, rapidly producing IFN-γ, TNF-α, and IL-2 while retaining memory of infection within tissues after clearance (37, 38). The cytokine IFN-γ, largely regulated by signal transducer and activator of transcription 4 (STAT4), is a key driver of CD103^-TRM cell differentiation (39). Some CD8⁺ CD103^- TRM cells also express high levels of β2-integrin (40), while others express Killer Cell Lectin-like Receptor G1 (KLRG1), another receptor for E-Cadherin, which can compete with CD103 (41). Intestinal CD103^- TRM within the intraepithelial layer are derived from KLRG1⁺T cells (42). These KLRG1⁺ T cells exhibit enhanced survival and developmental plasticity, enabling the generation of CD103^- TRM (42).

Furthermore, CD49a, the α chain of integrin α1β1, is an immunomodulatory and adhesive molecule that interacts with type IV collagen in the lamina propria to establish tissue residency, promoting the accumulation of CD8⁺ TRM cells (43, 44) ( Figure 1 ). CD49a expression is upregulated in CD69⁺ TRM cells, contributing to the adhesion between T cells and intestinal epithelial cells (45). This is critical for the retention of TRM cells in the gut.

CD161, also called Killer Cell Lectin-Like Receptor C1 (KLRC1), is a C-type lectin-like receptor expressed by adult CD8⁺ T cells (46). It is a subtype of CD8⁺ T cells that produces significant amounts of cytotoxic meditators, including granzyme B and perforin. Additionally, these cells express high levels of the transcription factors Eomesodermin (EOMES) and T-box expressed in T cells (T-bet) (47), which predominantly regulate the expression of cytotoxicity mediators and cytokine production (48). Intermediate levels of CD161 are highly expressed in the colon. In inflamed tissues of IBD patients, CD161 is enriched in CD103⁺ TRM cells, and the majority of CD161 in the colon co-expresses CD69 (47). CD161^int CD8⁺T cells provide rapid immune protection against pathogens within the gut.

CD39 and CD73 are two regulatory markers expressed by TRM cells, involved in nucleotide-metabolizing that regulate immunity and inflammation (49). After the release of the inflammatory signal adenosine triphosphate (ATP), CD39 converts ATP to adenosine monophosphate (AMP), which is subsequently dephosphorylated by CD73 to produce adenosine (50). These enzymes facilitate the transition from ATP-driven, proinflammatory immune cell activity an adenosine-mediated, anti-inflammatory states. As these markers are typically expressed at high levels on regulatory T cells (51), reduced levels of CD39 with IBD may indicate a compromised regulatory function. Studies have shown that CD39 and CD73 are highly expressed on CD103⁺ CD8⁺ TRM cells, suggesting that these cells possess an immunosuppressive function (51). Human TRM cells expressing CD39 and CD73 may contribute to the maintenance of intestinal immune homeostasis.

Although CD69 and CD103 are considered the most prominent surface markers of TRM cells, they are not specific to TRM cells. Currently, the study of cell surface markers is not comprehensive, other potential markers of intestinal TRM cells include CD44 and CD101 (40). In particular, the specific TRM subsets in UC patients need to be further refined and studied.

Multiple metabolic pathways are involved in the generation and function of TRM cells. At present, a greater number of studies have been conducted on TRM cells in the small intestine than in the colon. Most of the findings were derived from experimental lymphocytic choriomeningitis virus (LCMV) mouse infection and clinical research in patients.

Purinergic Receptor P2X, Ligand Gated Ion Channel 7 (P2RX7) has recently been identified as a key signaling molecule associated with the formation of TRM cells. Given the abundant antigenic substances and microbiota in the colon, extracellular adenosine triphosphate (eATP) from the colon microbiota activates the P2RX7 (52), which enhances the sensitivity of TGF-β in CD8⁺ TRM cells (53). TGF-β has been proven to contribute to the development of TRM cell characteristics and the establishment of transcriptional networks (54). In the intestine, naive CD8⁺T cells are activated and differentiated in response to antigenic stimulation. TGF-β in the intestine environment accelerates the apoptosis of short-lived effector cells while promoting the rapid formation of memory precursor cells (55). This process occurs through the simultaneous downregulation of KLF2 and SIPR1, along with the upregulation of CD69 and CD103 (56) ( Figure 1 ). The signaling molecule Smad3 also contributes to CD103 regulation (57).

Additionally, P2RX7 is an ion channel that responds not only to eATP but also to Nicotinamide Adenine Dinucleotide (NAD⁺), playing a role in cell death via the ART2.2/P2RX7 pathway (58). Given its dual function, the role of P2RX7 in the development and function of TRM cells warrants further investigation. CD38 is an enzyme expressed on T cells that exerts regulatory effects on both the metabolism of NAD⁺ (59) and the development of TRM cells. CD38 serves as a marker distinguishing TRM cells from circulating memory T cells across various organs. Deletion of CD38 has been observed to impact TGF-β-dependent CD103⁺ TRM cell subpopulations in epithelial tissues (60).

Newly published research (61) has revealed that depletion of nutrient-dependent lysosomal signaling nodes activates the transcription factor EB (Tfeb), and deficiency in Follicular Lymphoma Overexpressed Gene (Flcn) enhances protective responses in intestinal TRM cells, with the Flcn-Tfeb axis mechanistically inhibiting retinoic acid-induced CCR9 expression to promote TGF-β-mediated lineage differentiation programs. Subsequently, the expression of CCR9 contributes to the differentiation of intestinal CD103⁺ TRM cells via retinoic acid signaling in mesenteric lymph nodes (62).

Although many new pathways are being discovered, studies on colonic TRM cells remain limited compared to those on small intestine TRM cells. Most of the mechanism-related studies have been conducted in mice and require further validation due to the heterogeneity of TRM cells.

The development, maintenance, migration, and function of intestinal TRM cells are regulated by a complex network of transcription factors. The transcriptional factors Blimp-1/Hobit and Eomes/T-bet play crucial but distinct roles in this process.

Blimp-1 and Hobit are transcription factors involved in TRM cell generation and regulation (63), encoded by Zfp683 and PRDM1 (64), respectively. They exhibit a high degree of overlap in their genomic binding sites, homologous sequences, and synergistic action that collectively drive T cell lineage differentiation (65). Approximately 30% of the genes related to tissue-resident functions in TRM cells are regulated by Blimp-1 and Hobit (64). Blimp-1 and Hobit suppress the expression of Klf2, S1pr1, T-cell transcription factor (Tcf), and C-C motif chemokine receptor 7 (Ccr7), thereby contributing to the residency of TRM cells in tissues. Ccr7 is a critical regulator of lymphocyte egress from peripheral tissues, which is usually absent in TRM cells (64). The core transcription factor Runx3 could induce chromatin accessibility and regulate Blimp-1 (66, 67). In CD8⁺T cells, Blimp-1 knockout alone is insufficient to induce granzyme B expression (68). However, Hobit is responsible for maintaining granzyme B expression, and in conjunction with Blimp-1, sustains the cytotoxic function of TRM cells (69). Following antigen rechallenge, TRM cells may experience a decrease in Hobit expression, leading to the formation of circulating memory cells (70). The development and maintenance of intestinal CD8⁺ TRM cells are impaired in mice with a double knockout Blimp-1 and Hobit, resulting in alleviated colitis (19). In a mouse model of colitis, the Hobit^- Blimp-1^- CD4⁺ TRM cells lead to reduced chemokines expression, impaired leukocyte recruitment, and decreased expression of proinflammatory molecules (71).

The T-box transcription factors Eomes and T-bet are involved in the complex regulatory mechanisms that control TRM cell differentiation and maintenance in the colon. Eomes is a key transcription factor to the differentiation and effector function of CD8⁺ T cells (72), inducing the production of proinflammatory substances such as IFN-γ, granzyme B, and perforin (73). Compared to the small intestine, the number and function of colonic TRM cells do not depend on EOMES (33). This phenomenon may be attributed to the distinct microenvironment present in the small intestine and colon. Ectopic expression of Eomes in a mouse model enhances the pathological properties of CD8⁺ T cells, supporting that Eomes promote inflammatory and pathological states in CD8⁺ TRM cells (73, 74). The coordinated downregulation of Eomes and T-bet promotes the generation of CD8⁺ CD103⁺ TRM cells and enhances their responsiveness to TGF-β signaling (75). A recent study (76) discovered that depletion of T-bet, a key molecule along with Hypermethylated in cancer 1 (Hic1) in regulating intestinal TRM cell differentiation, partially restored Hic1 expression and significantly rescued the formation of intestinal CD103⁺ TRM cells in the absence of TGF-β receptor signaling.