Mucosal-associated invariant T cells in digestive tract: Local guardians or destroyers?

Mucosa-associated invariant T cells (MAIT) are a class of innate-like T lymphocytes mainly presenting CD8 + phenotype with a semi-invariant αβ T-cell receptor, which specifically recognises MR1-presented biosynthetic derivatives of riboflavin synthesis produced by various types of microbiomes. As innate-like T lymphocytes, MAIT can be activated by a variety of cytokines, leading to immediate immune responses to infection and tumour cues. As an organ that communicates with the external environment, the digestive tract, especially the gastrointestinal tract, contains abundant microbial populations. Communication between MAIT and local microbiomes is important for the homeostasis of mucosal immunity. In addition, accumulating evidence suggests changes in the abundance and structure of the microbial community during inflammation and tumorigenesis plays a critical role in disease progress partly through their impact on MAIT development and function. Therefore, it is essential for the understanding of MAIT response and their interaction with microbiomes in the digestive tract. Here, we summarised MAIT characteristics in the digestive tract and its alteration facing inflammation and tumour, raising

Xingzhou Wang, Mengjie Liang and Peng Song contributed equally to this study. riboflavin metabolites produced by certain bacteria are recognised and processed by antigen-presenting cells (APCs), the MHC-I related molecule 1 (MR1) becomes activated and undergoes a conformational change [4]. This change allows MR1 to move from the endoplasmic reticulum to the cell membrane, where it can present the antigen to MAIT cells [4]. At the same time, the MR1 molecules expressed on the APCs are also highly evolutionary conserved, which makes them present only a limited number of antigens. Therefore, these characteristics make MAIT cells capable of rapidly responding to specific types of antigens owing to the broad innate recognition of the microbiomes without priming required for traditional adaptive T-cell responses. Upon recognition of antigens presented by MR1 and subsequent activation, MAIT cells secrete a variety of cytokines, including IFNγ, TNF-α and Granzyme B (GZMB), which can kill pathogens or infected cells [6,7].
In addition to the specific activation mechanism mediate by MR1 molecules, MAIT cells can also be activated by cytokines. MAIT cells express a variety of cytokine receptors, which can be activated by IL-7, IL-12, IL-15, IL-18, IL-23 and other cytokines [8][9][10][11][12][13]. IL-7 can directly induce MAIT cells to release cytolytic effector molecules [14]. In response to the bacteria or virus infection, APCs release IL-12, IL-15 and IL-18, which bind to the corresponding receptor on the surface of MAIT cells and activate their pro-inflammatory function [10,15]. IL-23, together with 5-OP-RU, has been found to be a vital regulator to control MAIT cell subpopulations into MAIT-17 cells and expansion in a T-cell receptor (TCR)dependent manner, which enhances control of Salmonella Typhimurium or Legionella longbeachae infection in mice [16]. However, in the case of Francisella tularensis infection, IL-23 and IL-12 together promote MAIT cell differentiation into MAIT-1 cells in a TCR-independent manner [17]. These results show that IL-23 can influence MAIT cell phenotype depending on the type of bacterial infection. Additionally, MAIT cells exhibit a CD103 + CD69 + resident memory phenotype, enabling them to be sensitive to cytokine changes in the local microenvironment and generate an immune response in a TCR-independent manner during the early stages of disease [18,19]. These characteristics make MAIT cells an important component of the mucosal immune barrier and act as an important player in the local immune response.
Gastrointestinal malignancies have long been important diseases threatening human health. In recent years, MAIT cells have been found to play an essential role in the host's resistance to microbial infection and in the occurrence and/or development of tumours. Moreover, recent studies have indicated that MAIT cells are not solely immune cells with cell-killing functions. Similar to traditional T cells, MAIT cells can differentiate into various phenotypes and release cytokines to regulate other immune cells. Therefore, the role of MAIT cells, which were previously thought to undertake the early antitumour immune response of tumours, in the occurrence and development of tumours is likely to be much more complicated than initially imagined, and there are still limitations in the cognition of their role in gastrointestinal malignancies. Our review summarises recent findings in the role and responsible mechanism of MAIT cells in gastrointestinal diseases and proposes some research directions worthy of attention.

Development of MAIT cells
MAIT cells initially develop in the thymus. Development of MAIT cells in the thymus is strongly dependent on the MR1-mediated mechanism, as riboflavin metabolites can travel through the body and arrive on thymus to activate MAIT cells [20]. Patients with MR1-mutation can lose MAIT cells in their bodies [21]. In humans, three developmental stages of MAIT cells can be defined based on the expression of CD27 and CD161 [22]: (1) After selective development of progenitor T cells to CD4 + CD8 + T cells in the thymus, MAIT cells develop to CD27 À CD161 À phenotype in the presence of MR1, which only exists in the thymus; (2) CD27 + CD161 À MAIT cells, which are less abundant in germ-free mice, suggesting that microbiomes play an important role in the development of MAIT cells; (3) Some MAIT cells enter into the peripheral circulation, while others stay in the thymus and continue to develop, both of which can form CD27 lo/+ CD161 + CD218 + MAIT cells under the mediation of PLZF, Drosha, IL-18, mir-181 and microbial antigen stimulation, and this part of MAIT cells are considered to be mature cells. The counterpart in mice also divides into three stages [22]: (1) At stage 1, the MAIT cells have acquired the ability to respond to MR1-presented microbiome metabolites, as they can be detected with MR1-tetramer linked with 5-OP-RU, but stimulation with PMA and Ionomycin in vitro cannot induce the expression of pro-inflammation cytokine. These cells only exist in the thymus of very young mice and express CD24 but not CD44. (2) Stage 2 MAIT cells take smallest percentage in all-stage MAIT cells and do not express CD24 nor CD44. MAIT cells at this stage are also not able to be stimulated by PMA and Ionomycin. (3) Similar to MAIT cells in humans, stage 3 MAIT cells in mice express the transcription factor PLZF. Mouse MAIT cells are almost depleted in lymphoid organs when PLZF is mutated, showing that PLZF is of great significance in MAIT development. MAIT cells at this stage have the ability to be stimulated by PMA and Ionomycin.
In the thymus, mature MAIT cells can further differentiate into two subsets: MAIT-1 and MAIT-17 ( Figure 1). MAIT-1 cells express the transcription factor T-bet and are able to secrete IFN-γ, IL-4, IL-10, IL-13 and GM-CSF. MAIT-17 cells, which display a CD44 hi CD62L lo memory phenotype, express RORγt and are capable of secreting IL-17A [23]. In mice, it has been shown that most MAIT cells show characteristics of MAIT-17 cells when stimulated by PMA/Ionomycin or CD3/CD28 monoclonal antibodies [23]. Although human MAIT cells also express T-bet and RORγt [24], the two subsets, MAIT-1 and MAIT-17, have not been verified in humans so far. These cells are then released into the circulation and reach the peripheral tissues for further differentiation and function.
Upon migrating to peripheral tissues, MAIT cells can be activated by various antigens presented by MR1-expressing APCs. While the mechanism of MAIT cell development in the thymus is relatively well-F I G U R E 1 Mucosa-associated invariant T cells (MAIT) development and differentiation under distinct regulation in thymus and outside the thymus. MAIT cells with a semi-invariant TCR experience three-stage development in thymus: (i) Stage 1: CD24 + CD44 À phenotype in mice and CD27 À CD161 À in human; (ii) Stage 2: CD24 À CD44 À in mice and CD27 + CD161 À in human; and (iii) Stage 3: CD24 À CD44 + in mice and CD27 lo/+ CD161 + CD218 + in human. MAIT cells express transcription factor PLZF only at stage 3. When MAIT cells develop into mature stage, they migrate out of thymus to other tissues. Then these cells can further differentiate into MAIT-1 or MAIT-17 under different stimulation by miRNA, antigens and distinct cytokines. IL-2 and IL-15 can activate corresponding receptors, which further activates downstream transcription factors T-bet and pro-inflammatory cytokines expression. Mir-155 has also been found to have a similar effect on MAIT. Antigens presented by MHC-I related molecule 1 and cytokines like IL-1β and IL-23 can activate mTORC2 in MAIT, which activates transcription factor RORγt and promotes expression of co-stimulatory receptor ICOS. This process can induce IL-17A secretion to regulate the local immune microenvironment. mTOR, mammalian target of rapamycin. understood, some reports suggest that MAIT cells do not proliferate in peripheral tissues [20,25,26]. However, MAIT cells exhibit varying abundance among different tissues, and in vitro activation of peripheral blood mononuclear cells (PBMCs) using 5-OP-RU can significantly expand the MAIT cell population. Besides, evidence has shown that MAIT cells expand significantly in the stomachs of mice infected with Helicobacter pylori [27]. These findings may suggest that MAIT cells are capable of further development in peripheral tissues, offering potential strategies to regulate MAIT cell development to fight against infections or tumours.
In addition to PLZF and microbial antigen stimulation during MAIT development, microRNAs also have important roles in the development of MAIT. In mice, mir-181a/b-1 is considered a key molecule that regulates the acquisition of transcription factors PLZF, T-bet and RORγt expressed by MAIT in the thymus [28]. In addition, it has been reported that mir-155 can also regulate the development of MAIT, and it is also a key regulatory molecule for MAIT differentiation into MAIT-1 and MAIT-17 [29]. Given the abundance of microRNAs in the gastrointestinal tract, it is possible that MAIT cells are regulated by these microRNAs in the gastrointestinal tract [30][31][32], leading to further development and differentiation. Therefore, regulating microRNAs could be a potential means of targeting and regulating MAIT cells in gastrointestinal tissues.

Phenotypes and function of MAIT in digestive tract
MAIT cells are enriched in various types of tissues, accounting for 1%-10% of T cells in peripheral blood [24], 10% in the airway [33,34] and 20%-50% in the liver [24,35]. In stomach, MAIT accounts for about 1%-3% of all T cells in the gastric mucosa, and for the abundance in the gastric antrum is higher than that in the gastric body [36,37]. In the intestinal mucosa, MAIT accounts for up to 10% of αβT cells [24]. Furthermore, MAIT is highly enriched in the duodenum, but its reactivity towards the MR1-5-OP-RU tetramer is poor [36]. Another work has also shown that up to 25% of the TRAV1-2 + cells in the gastric mucosa cannot respond to the MR1-tetramer [27], addressing the importance of the MR1-tetramer in detecting tissue MAIT cells. Among PBMCs, MAIT cells are usually dominated by the CD8 + CD4 À phenotype, and only a few are CD4, CD8 double-negative or CD4, CD8 double-positive or CD4 + [38]. In the gastrointestinal mucosa, the proportion of DN cells increased [37]. The roles of CD4 and CD8 in MAIT cell biology are not fully understood, and it is currently known that CD8 plays a role in the recognition of MR1-presented antigens by directly binding to the MR1 [24,39]. After being activated, MAIT has a strong function of secreting IFN-γ, TNF-α, GZMB and other pro-inflammatory or cytolytic cytokines.
MAIT cells can be activated in response to TCR ligation of riboflavin intermediates presented on MR1 under co-stimulatory signals from specific cytokines or toll-like receptors (TLRs). Activated cells expanded significantly, inducing rapid innate-like immune responses and effector functions, including antimicrobial cytotoxic products, inflammatory chemokines, and cytokines. After activation, MAIT cells can express markers of T cell activation, such as CD69, CD25, CD38 and HLA-DR [40]. For example, in viral infection, MAIT can be activated by cytokines without being activated in a TCR-dependent manner and then express activation markers such as CD69, CD38 and HLA-DR [41]. In the gastrointestinal tract, MAIT cells exhibit effector-memory phenotype as CD45RA À CD45RO + CD95 hi CD62L lo and can routinely express the tissue resident marker CD103 and the T cell activation marker CD69, and these tissue resident markers expressed on the T cells have been found to help T cells form defence against tumour cells. CD103 + CD69 + tissue-resident MAIT cells expressed higher levels of activation markers, such as CD25, HLA-DR, CD38, compared to CD103 À CD69 À MAIT cells [18]. Hence, MAIT cells in the gastrointestinal mucosa may be more exposed to activating ligands and was recognised to be easier to be activated.
After MAIT cells mature, they can further differentiate into MAIT-1 and MAIT-17. MAIT-1 cells are similar to Th1 cells, mediated by T-bet transcription, can produce IFN-γ, kill target cells and promote the further recruitment of immune cells; MAIT-17 cells are similar to Th17, mediated by RORγt transcription, can produce IL-17A, participate in mediating the chronic inflammatory microenvironment in tissues. Single-cell transcriptional profiling results have shown that MAIT can exhibit MAIT-1 phenotype and function during their early activation (15 h) by either 5-OP-RU or anti-CD3/CD28, with high expression of IFNG, GZMB, TNF and TBX21 [42]. These results indicate that during the early stages of activation, MAIT cells can typically exhibit a pro-inflammatory role, contributing to the initiation of immune responses against various pathogens and stimuli. Prolonged activation of MAIT cells leads to the expansion of homeostatic subpopulations while simultaneously promoting proliferative, cytotoxic, immune regulatory and exhaustion phenotypes [42]. This highlights the multifaceted nature of MAIT cell responses under conditions of sustained activation. The IL-7 receptor expressed on the surface of MAIT cells makes them accessible to IL-7 stimulation, which allows MAIT cells to be promoted to secrete IL-17A [14]. Studies have shown that the effects of different cytokines on the differentiation of MAIT subsets may be mediated by different mTOR complexes (Figure 1). For example, the application of IL-2 and IL-15 can stimulate the expression of CD122 on the surface of MAIT cells, which activates mTORC1 and upregulates the expression of the transcription factor T-bet, thereby driving the differentiation of MAIT cells into MAIT-1 cells [43]. Meanwhile, the activation of ICOS or the combined use of IL-1β and IL-23 activates mTORC2 in MAIT and upregulates RORγt expression to convert MAIT to MAIT-17 [43]. These findings show that by supplementing cytokines or regulating key signalling molecules in MAIT, the phenotype of MAIT can be transformed to regulate tumours, infections and other diseases.

INTERACTION BETWEEN MAIT CELLS AND GASTROINTESTINAL MICROBIOME
Bacteria capable of metabolising riboflavin and producing uracil like 5-OP-RU have been found to activate MAIT cells. In view of the widespread existence of such bacteria in the gastrointestinal tract, some studies believe that MAIT cells in the gastrointestinal tract, especially in the gut, are more activated and are involved in regulating the homeostasis of the gastrointestinal immune microenvironment [44]. The 5-OP-RU produced by microbiomes can not only activate MAIT cells in the local environment but also travel through the body from the intestinal mucosa and can be recognised and presented by APCs expressing MR1, thereby participating in the regulation of the development of MAIT cells in the thymus [6]. This interaction mechanism is important for maintaining host immune homeostasis. For example, in graft-versus-host disease (GVHD), MAIT cells can release a large amount of IL-17A under the action of colonic microbiomes and participate in the inhibition of GVHD inflammatory response process [45]. Similarly, the presence of microbiota in the small intestine modulates MAIT cells in allogeneic haematopoietic cell transplantation (allo-HCT), which also mediates favourable outcomes after allo-HCT [46]. These findings implicate that regulation of the gut microbiome towards MAIT cells can also mediate systemic immune response in addition to the local immune response. Based on this potential, Bozkurt et al. have further raised the question that whether regulating microbiomes can be a potential strategy for regulation of MAIT cells and cancer immunotherapy [47].
In gastrointestinal infection diseases, the frequency of MAIT cells in the peripheral blood of H. pylori-infected patients was significantly lower than that in uninfected individuals, but this was not observed in MAIT cells in gastric tissue, which indicates that H. pylori infection may exert a regulatory effect on peripheral blood MAIT. H. pylori infection can activate MAIT cells in different ways and enable them to release different cytokines. For example, H. pylori can activate MAIT in a TCRdependent manner, promoting the release of IL-17A and sustaining long-term chronic inflammation [27]. Additionally, it can induce MAIT cells to release IL-9 via the OX40 co-stimulatory molecule on the MAIT cell surface, which promotes inflammation development [48]. Apart from H. pylori infection, other microbiome alterations have been found in gastric cancer compared to pre-cancerous lesions [49,50], with the impact on tumour progression remaining uncertain. These dysbiosis phenomenon have also been found in colorectal cancer [51,52]. As MAIT cells show tissue resident memory phenotype, an interesting phenomenon has been observed that gastric microbiota alterations are associated to decreased infiltrated CD8 + tissue resident memory T cells in gastric cancer [53], which indicates that these resident memory T cells are regulated by gastrointestinal microbiomes and remodelling of dysbiosis in tumour microenvironment may benefit the antitumour response.

Role of MAIT cell in digestive autoimmune inflammation
The role of MAIT cells in gastrointestinal inflammation remains controversial, especially in auto-immune inflammation like inflammatory bowel disease (IBD). Clinical data has shown that the frequency and absolute count of circulating MAIT cells significantly reduce and are correlated with the disease activities of IBD patients, with a reduction of proportion of CD8 + MAIT in CD3 + T cells. On the contrary, MAIT cells aggregate in inflamed tissue compared to normal tissue [54][55][56], potentially due to elevated chemokine levels in inflamed areas [55]. Besides, in vitro activation revealed that circulating MAIT in IBD patients secreted higher levels of TNF-α and IL-17A [54,57] and highly expressed activation and proliferation markers [55]. Despite their activation, MAIT cells appear to play a protective role in IBD by producing lower levels of IFN-γ or higher levels of IL-22, which may attribute to the prolonged activation during chronic inflammation [54,55].
However, in murine models, MR1 deficiency significantly reduced the severity of oxazolone colitis, while administration of 6-FP, an antagonist of MR1 ligand, could also reverse the development of disease activity [58]. Another study also showed that non-specifically activated MAIT cells caused by immune-checkpoint inhibitor (ICI) with ipilimumab and nivolumab could aggravate in ICI-associated colitis, but the impact of these cells on disease development was not confirmed [59].
In order to determine the certain type of T cells involved in Crohn's disease (CD), Rosati et al. performed single-cell RNA sequencing and bulk TCR repertoire profiling, which revealed that clonotypes of Crohn associated invariant T (CAIT) cells sharing the certain group of semi-invariant CDR3 motif on TCR alpha chains were enriched in CD [60]. Further analysis suggested that most of these CAIT cells were unconventional CD8 + T cells with an overlap to the MAIT phenotype, while the latter were further confirmed to be enriched in intestinal tissues compared to the CAIT cells, and both of them express activation and pro-inflammatory genes as CD69, IFNg and TNF [60], which showed that these unconventional cells are potential targets for IBD therapy.
In general, the mechanism of MAIT cell activation in IBD remains elusive, as distinct activation pathway, either through MR1-dependent or cytokine-dependent way, can lead to different functions displayed via activation of different transcription patterns (Figure 2), and further resolution for MAIT variable function and the mechanism may provide novel therapeutic strategies for IBD.

Role of MAIT in gastrointestinal infection
Another reason to cause gastrointestinal inflammation is microbiome infection. Many of the gastrointestinal infections are acute infections caused by bacteria, necessitating the activation of MAIT cells to clear the infection. Upon activation, the release of pro-inflammatory factors may exacerbate local inflammation and potentially trigger a systemic inflammatory response ( Figure 2). As described earlier, abundant groups of microbiomes exist in the gastrointestinal tract, which takes part in the regulation of MAIT development and function [61]. Thus, dysbiosis or infection may also result in abnormal MAIT activation and drive gastrointestinal inflammation, as reported in Vibrio cholerae O1 [62], H. pylori [27,36,48], Mycobacterium tuberculosis [63], Shigella flexneri [64], S. typhimurium [65] and so forth. Alongside dysbiosis, MAIT cells can migrate and redistribute to ectopic tissues to trigger local inflammation and even systemic inflammation [66]. Abnormal activation or impaired function of MAIT can also lead to local change of homeostasis, distant lesion or systemic disease. For instance, patients suffered from alcoholic liver disease have fewer circulating MAIT with dysfunction of the remaining MAIT cells, which makes the patients susceptible to gut infection [67]. Gut yeast-mediated MAIT activation can drive it crossing the blood-brain barrier and release proinflammatory cytokines, leading to the occurrence of multiple sclerosis brain [68]. MAIT activation in an MR1-dependent manner can aggravate dysbiosis in the gut, which results in M1 macrophage polarisation and inflammation in adipose tissue, leading to metabolic dysfunction and obesity status eventually [69]. Therefore, the complex interaction between gut microbiomes and MAIT is significant to the local systemic homeostasis, and exploration is still needed to elucidate the mechanism.

MAIT PHENOTYPE AND FUNCTION IN GASTROINTESTINAL CANCER
To date, functions of MAIT cells in malignancies remain controversial. Recent studies have found that MAIT cells can play a role in regulating the tumour immune microenvironment, revealing that MAIT cells have a regulatory function in addition to their cell-killing and proinflammatory effect. These emerging evidences also indicate that MAIT cells are likely to exhibit heterogeneity, further highlighting the complexity of their involvement in cancer biology (Figure 2). The comprehensive characterisation of MAIT cell phenotypes, functions, and heterogeneity, along with the elucidation of their underlying mechanisms, will enhance our understanding of their role in tumour biology. This knowledge will pave the way for the development of strategies aimed at promoting positive tumour immune responses mediated by MAIT cells.

Tumour-eradicating effect of MAIT cells in gastrointestinal cancer
In gastrointestinal malignancies, the anti-tumour immune response capacity exerted by MAIT cells is generally attenuated. Shao et al. reported that the abundance of MAIT cells in the peripheral blood of gastric cancer patients was less than that of normal people, and their ability to secrete GZMB for cell killing was also simultaneously weakened [70]. This finding is similar to that of a study on oesophageal adenocarcinoma. Recent studies have found that MAIT cells express IFN-γ and GZMB in gastric cancer tissues [71]. They also found that the abundance of MAIT cells in distant metastases increased compared to primary tumours, but these cells have lost their functions and highly expressed KLRG1, which was a marker indicating T cell exhaustion [71]. In colon cancer, the abundance of MAIT cells compared with normal mucosal tissue remains controversial. Li et al. found that the abundance of MAIT cells in colon cancer was increased compared with normal colonic mucosa, while their ability to express cytotoxic cytokines was significantly decreased [72]. Simultaneously, the expression of CD4 and FOXP3 in MAIT cells increases in colon cancer, with CD39 and other exhaustion-related markers highly expressed in a TCR-dependent manner mediated by tumour-infiltrating bacteria, such as Fusobacterium nucleatum [72]. However, CD4 + FOXP3 + MAIT cells exhibit only a weak correlation with conventional Treg cells and are capable of secreting TNF-α, demonstrating their distinction from Treg cells [72]. This finding suggests that FOXP3 expression in CD4 + MAIT cells serves as an activation marker rather than an indicator of regulatory function. Ling et al. suggested that the abundance of MAIT cells in colon cancer decreased compared to F I G U R E 2 Mucosa-associated invariant T cells (MAIT) activation and function in normal gastrointestinal tissue (top half) and in inflamed tissue or malignancies (bottom half). MAIT can be activated by riboflavin-metabolising bacteria and a variety of cytokines in the normal gastrointestinal tissue and secrete cytotoxic or pro-inflammatory cytokines. When dysbiosis or malignancies occur in gastrointestinal tissues, both CD8 + MAIT and CD4 + MAIT functions change differently and regulates local immune microenvironment or systemic inflammation, and during the process they are able to switch the phenotype and express different surface markers and cytokines. For example, CD8 + MAIT can be over-stimulated by abnormal increase of microbiota and secrete pro-inflammatory cytokines to induce local and systemic inflammation to prevent the dysbiosis in intestine. MAIT cells can also secrete IL-22 to protect the gastrointestinal mucosa from inflammatory bowel disease. In tumour microenvironment, CD8 + MAIT cells can express CD25 to show regulatory function or CD39 to show exhausted phenotype, which indicates their cytotoxic function is impaired, while CD4 + MAIT cells express PD-1 and TIM-3, activate transcription factor Foxp3 and secrete IL-17A, which negatively regulates tumour immune microenvironment and inhibits anti-tumour response. APC, antigen-presenting cell. normal tissues [73]. Another study found that the abundance of CD4 + MAIT cells in colon cancer tissue increased, and the exhaustion-related markers such as PD-1 and TIM-3 expressed on the surface of MAIT cells also increased [74]. The above studies reveal that the tumour suppressive effect of MAIT cells is attenuated in the gastrointestinal tract, which is similar to the findings of MAIT cells in other tumours, such as liver cancer [75,76], oesophageal adenocarcinoma [77] and cholangiocarcinoma [78]. Strategies to reverse this diminished tumour suppressor effect of MAIT cells present in tumours have the potential to positively contribute to tumour immune responses.

Regulatory role of MAIT cells in gastrointestinal malignancies
Within the tumour microenvironment, MAIT cells can further differentiate and modulate the tumour immune microenvironment. For example, in colon cancer, the abundance of CD4 + MAIT cells is increased and FOXP3 is expressed. As described earlier, despite the Treg-like phenotype, Li et al. found that this part of MAIT cells also expresses TNF-α, and this function is different from traditional Treg cells [72]. Accordingly, their findings reveal that the expression of Foxp3 on the surface of tumour-infiltrating CD4 + MAIT cells is a marker of MAIT activation, and whether it has a regulatory role in the tumour microenvironment is worth exploring. The results of phenotypic and functional tracking after MAIT cells activation by single-cell RNA sequencing also showed that Foxp3 is abundantly expressed on the surface of CD4 + MAIT cells in the presence of persistent TCR-dependent stimulation [75]. Moreover, FOXP3 and CD25 expression have been observed on CD8 + MAIT cells [42], and these CD8 + CD25 + FOXP3 + cells were considered to have a strong ability to inhibit the proliferation of effector T cells in vitro [79]. In general, CD4 + and CD8 + Treg-like MAIT cells seem to be related to the inhibitory state of the tumour immune microenvironment, but their regulatory effect on the tumour immune microenvironment still needs to be further studied.
In addition to differentiating into immune cells with a Treg phenotype, MAIT cells can also develop and differentiate into MAIT-17 and release IL-17A to regulate the tumour immune microenvironment. For example, in colon cancer, the ability of MAIT cells to express IL-17A is increased, and IL-17A released by MAIT cells can inhibit the function of NK cells [80]. Another study found that deletion of MAIT cells in melanoma promotes NK cell-mediated anti-tumour immune responses [81]. Interestingly, they also found that activated MAIT cells can also promote NK cells to exert anti-tumour immune responses in vitro. These findings highlight the complex and context-dependent relationship between MAIT cells and other immune cells in the tumour microenvironment. In addition to functioning as MAIT-17 cells, MAIT cells are able to secrete Th2 cytokines, such as IL-13, when chronically stimulated. IL-13 secreted from MAIT cells can bind to the IL-13 receptor expressed on colon cancer cells and promote tumour progression and metastasis [82]. In summary, MAIT cells have the capacity to secrete cytokines that can modulate the tumour microenvironment. Gaining a deeper understanding of these mechanisms may pave the way for the development of innovative strategies in tumour immunotherapy, ultimately enhancing the effectiveness of cancer treatments.
In addition, recent studies have found that MAIT cells can express CXCR5 in human tonsils and possess PD-1 high phenotype, which is the characteristics of follicular helper T (Tfh) cells. The Tfh-like MAIT cells that reside in the tonsils are capable of releasing IL-21, and they are spatially located near germinal centre to assist B cells to produce antibodies and play an anti-infection role when challenged with V. cholerae [83]. Concurrently, Jiang et al. found that PD-1 expressing MAIT cells from tuberculous pleural effusions in tuberculosis-infected patients can produce CXCL13 [84]. These PD-1 high CXCL13 + MAIT cells produce less IFN-γ but more IL-21 [84]. CXCL13 is the ligand of CXCR5, and the interaction between CXCR5 and CXCL13 leads to the activation of Tfh cells and their migration to the germinal centre of lymphoid organs, where they support the differentiation of B cells into antibody-producing plasma cells [85]. These findings suggest that MAIT cells may regulate immune responses by promoting B cell activity. In malignancies, it remains unclear whether MAIT cells can differentiate into a Tfh-like phenotype in gastrointestinal cancer. Investigating the abundance and function of Tfhlike MAIT cells may help to better understand the diverse roles of MAIT cells in tumour development.

POTENTIAL THERAPEUTIC IMPLICATION OF MAIT CELLS
Despite impaired function, MAIT cells may still have a positive effect on tumour positive immune responses and the prognosis of tumour patients. Investigating the potential of pharmacological interventions to either inhibit or stimulate MAIT cell function or to modulate their immune response by regulating gastrointestinal microbiomes is crucial for further exploration in cancer immunotherapy. Despite the known interactions between microbes and MAIT cells, our understanding of how microbes regulate the local infiltration and functional and phenotypic changes of MAIT cells is extremely limited. Hence, further research is needed to elucidate these mechanisms and their implications in disease contexts, including cancer and autoimmune disorders. MAIT cells recognition of the antigen are dependent on the MR1 and the binding ligands. Therefore, an important therapeutic strategy to target MAIT cells is to find antigens that can bind MR1 and promote its transport to the cell membrane surface and activate MAIT cells. Vitamin B metabolites, such as pterins, uracils and lumazines, can bind to MR1 and prompt MR1 to bring them to the cell membrane surface and present them to MAIT cells [2]. Among them, uracils and lumazines have been summarised to induce the activation of MAIT cells, while pterins, exemplified by 6-FP, serve as inhibitory molecules for MAIT activation [2]. Lumazines have been found to be a weak activator of MAIT cells. 5-OP-RU is a representative uracil compound, which can not only induce the activation of MAIT cells in vitro but also induce the proliferation of MAIT cells in vivo and has a good inhibitory effect on tumour progression and metastasis of in vivo models [81]. In addition, the combination of 5-OP-RU and the TLR9 receptor agonist CpG can better induce the proliferation and activation of MAIT cells and promote its stronger tumour killing effect in vivo than 5-OP-RU alone [86]. Furthermore, the proliferation and activation of MAIT cells induced by 5-OP-RU can promote the killing effect of NK cells on tumour cells in an IFN-γ-dependent manner. However, after treatment with 5-OP-RU, tumours can also express MR1 on their surface, and activate MAIT cells in a TCR-dependent manner and promote its further differentiation into MAIT-17 cells. These cells release IL-17A, which inhibits NK cell function and has been reported to promote tumour angiogenesis and distant metastasis [87,88]. Further research is needed to investigate the mechanism of MAIT cell phenotype switching and to identify potential strategies for reversing the pro-tumour effects of MAIT cells. Therefore, while identifying a suitable stimulus to continuously activate MAIT cells and induce their local aggregation or proliferation is a potential therapeutic strategy for gastrointestinal tumours, it is also essential to find a mechanism that regulates the phenotype and function of MAIT cells to enable them to exert anti-tumour immune responses. Single-cell sequencing results reveal that MAIT cells exhibit a cytotoxic phenotype early in their immune response to the same antigen [42]. However, with prolonged antigen stimulation, more diverse phenotypes emerge, presenting immunomodulatory functions primarily mediated by CD4 + MAIT cells with a traditional Treg phenotype [42]. Investigating the mechanism of phenotypic transformation during continuous MAIT cell activation can offer new strategies for clinical regulation of MAIT function and enhancing their anti-tumour immune response.
In addition to inducing local activation and proliferation of MAIT cells within tumours, using genetic engineering to transform MAIT cells into CAR-MAIT cells in vitro and adoptive reinfusion represents a novel cancer treatment strategy. Studies demonstrate that co-culturing MAIT cells with chimeric Her2 antigen receptors and Her2-expressing breast cancer cell lines results in more effective tumour cell killing compared to CAR-CD8 + T cells [89]. Similar strategies have also been applied to treat viral infections, such as using chimeric HCV antigen peptide-specific TCR-MAIT cells to accurately and effectively recognise and clear HCV virus antigen peptide-carrying cells [90]. Moreover, in GVHD models, MAIT cells show no significant alterations in function and quantity compared to other T cells, suggesting potential alloreactivity and indicating that they may not be rejected by the body after adoptive infusion and killing, which makes them suitable candidates for chimeric antigen receptor cells [91]. These studies imply that in vitro engineered MAIT cells could offer advantages over other T cells in tumour-targeted therapeutic strategies. Currently, mature experimental protocols exist for expanding MAIT cells in vitro [42,91], with expanded MAIT cells being more mature and possessing greater cytotoxic potential. In the future, combining highthroughput sequencing results to transform MAIT cells and adoptively infusing them into cancer patients may represent a tumour-targeted therapy strategy with promising application prospects.

CONCLUDING REMARKS
After thymic selection, MAIT cells can be further differentiated in the gastrointestinal tract and can be activated by TCR-dependent and cytokine-dependent pathways to produce many cytokines. The microbiomes exist in the gastrointestinal tract metabolises riboflavin and utilise the metabolites to modulate the phenotype and function of MAIT cells residing in the gastrointestinal mucosa. While gastrointestinal infection can activate MAIT cells to clear the infection, it can also lead to over-activation of MAIT cells, resulting in the aggravation of local inflammation and even systemic inflammation. In autoimmune diseases, MAIT cells play a role in regulating immune responses and maintaining tissue homeostasis, and dysregulation of MAIT cells has been implicated in the pathogenesis of various autoimmune diseases. These findings suggest that the phenotype and function of MAIT cells differ when activated during acute and chronic inflammation, which are consistent with the results of single-cell sequencing [42]. Hence, adopting tailored regulatory strategies for MAIT cells in various disease models may represent a potential therapeutic approach to address these diverse inflammatory conditions.
In gastrointestinal malignancies, MAIT cells exhibit impaired anti-tumour function, accompanied by phenotype transformation that may promote tumour development to some extent. It has been revealed that microbiomes play a role in gastrointestinal malignancies, and antigens derived from these microbiomes may be more important in inducing tumour immune responses than antigens produced by the tumour itself because this process may influence the abundance, phenotype, and functional alterations of MAIT cells in malignancies and regulate the immune microenvironment. However, much of the underlying mechanisms remain unknown. Deep insights into these aspects of MAIT cells nature may provide a novel strategy for gastrointestinal malignancies.
In summary, MAIT cells play a changeable role in various diseases, and their phenotypes and functions can be influenced depending on the specific tumour microenvironment. To develop novel strategies for controlling diseases by regulating MAIT cells, it is important to dissect the heterogeneity of MAIT cells and further explore the underlying mechanisms that drive their phenotypic and functional changes.