The Roles of Tissue-Resident Memory T Cells in Lung Diseases

The unique environment of the lungs is protected by complex immune interactions. Human lung tissue-resident memory T cells (TRM) have been shown to position at the pathogen entry points and play an essential role in fighting against viral and bacterial pathogens at the frontline through direct mechanisms and also by orchestrating the adaptive immune system through crosstalk. Recent evidence suggests that TRM cells also play a vital part in slowing down carcinogenesis and preventing the spread of solid tumors. Less beneficially, lung TRM cells can promote pathologic inflammation, causing chronic airway inflammatory changes such as asthma and fibrosis. TRM cells from infiltrating recipient T cells may also mediate allograft immunopathology, hence lung damage in patients after lung transplantations. Several therapeutic strategies targeting TRM cells have been developed. This review will summarize recent advances in understanding the establishment and maintenance of TRM cells in the lung, describe their roles in different lung diseases, and discuss how the TRM cells may guide future immunotherapies targeting infectious diseases, cancers and pathologic immune responses.


INTRODUCTION
Tissue-resident memory T (T RM ) cells comprise a recently identified lymphocyte lineage that occupies tissues without recirculating. They reside in epithelial barrier tissues, such as lung, gastrointestinal tract, reproductive tract, and skin, and in some non-barrier tissues, such as brain, kidney, and joint (1)(2)(3). T RM cells are transcriptionally, functionally and phenotypically distinct from circulating effector memory T cells (4).
The human lung is continuously exposed to environmental and microbial antigens (2). T RM cells in lung tissues play a crucial role in both innate and adaptive immune responses to lung infections, such as Respiratory Syncytial Virus (RSV), SARS-CoV-2, Brucella and Mycobacterium tuberculosis (5)(6)(7). Growing evidence has revealed that the T RM cells reside in tissues in the absence of antigens and may provide rapid on-site immune protection against previously exposed pathogens in peripheral tissues to accelerate pathogen clearance (5).
Recently, T RM cells have been found to participate in antitumor immunity as well. T RM cells can promote intra-tumoral cytotoxic T lymphocytes (CTLs) responses and are correlated with overall survival in lung cancer patients (8)(9)(10). Induction of T RM cells can enhance the efficacy of cancer vaccines (11) and increase the response rate when using anti-PD-1 antibodies to reverse tumor-induced T cell exhaustion in NSCLC patients (12,13).
In addition to the protective roles against diseases, evidence suggests that T RM cells also become activated after sensitization to self-antigens. Aberrantly activated T RM cells can induce autoimmune disorders, such as autoimmune hepatitis and psoriasis (4,14). In the respiratory system, T RM specifically activated by environmental allergens might underlie the development and worsening of allergic asthma and other airway diseases (15)(16)(17)(18). Pathogenic T RM cells may contribute to chronic pulmonary inflammation and fibrosis (2). T RM cells are also recognized as the primary mediator of acute cellular rejection (ACR) after lung transplantation.
This review aims to comprehensively summarize the current understandings of the biology of T RM cells, including the distinguishing molecular markers, regulators, and functions of T RM cells, discuss the contributions of T RM cells to lung diseases, especially infectious diseases and tumors, and highlight potential T RM -related therapeutic strategies for respiratory diseases.

DEFINING LUNG T RM CELLS
Human memory T cells can be broadly categorized into three subsets: central memory T cells (T CM ), effector memory T cells (T EM ), and tissue-resident memory T cells (T RM ). T CM cells are memory T cells that recirculate through secondary lymphoid organs, whereas T EM cells recirculate through nonlymphoid tissues. T RM cells, by contrast, typically reside in specific tissues, especially mucus organs, such as lungs and gastrointestinal tracts, defending against pathogens in peripheral nonlymphoid tissues. Compared with T CM and T EM cells, commitment to the tissue of residence is a defining characteristic of T RM cells, which has been described in almost all organs (19). Most memory T cells in non-lymphoid tissues are T RM cells, either CD4+ or CD8+ (9). T RM cells can be further fractionated by their functional characteristics into epithelial and stromal T RM cells (20,21).
T RM cells are widely distributed throughout the body, including the skin, lungs, lymphoid organs, etc. However, T RM cells in different organs display distinct properties (21). In healthy human skin, most of the T RM cells are dermal CD4+ CD69+CD103− cells, which express high levels of the cutaneous lymphocyte antigen (CLA) and specific chemokine receptors like CCR4 (22). With increased expression of T cell factor-1 (TCF-1) and lymphoid enhancer factor-1 (LEF-1), human lymph node CD8+ T RM cells exhibit a phenotype of tissue residency as well as an organ-specific signature (23). Human lymph node-specific profile of memory CD8+ T cells is defined by expression of CXCR5 and TCF-1 and high proliferative capacity, which accordingly indicates that human lymph node memory CD8+ T cells display higher proliferative capacity than their counterparts in other tissues (24). Similarly, T RM cells in non-small-cell lung cancer (NSCLC) may be identified by CD39 and CD103 (25). In mice, the formation and maintenance of skin T RM cells is mediated by chemokine receptors like CXCR3, CXCR6, and CCR10 (26). Genetic knockout studies of mice have shown that CD69 deficiency reduces the retention of CD4+ T cells in the bone marrow (27).
Eomes and T-bet are T-box transcription factors (TFs) that restrict the formation of CD103+T RM cells, indicating that downregulation of both transcription factors is crucial for the generation of CD103+ T RM cells. Eomes TF is significantly downregulated in CD8+CD103+T RM cells compared to circulating T EM or T CM cells. The residual T-bet expression upregulates interleukin-15 receptor (IL-15R) b-chain (CD122) expression, which is essential for long-term T RM cell survival. The coordinated downregulation of both T-box TFs optimizes cytokine transforming growth factor-beta (TGF-b) signaling, leading to the efficient development of CD8+CD103+T RM cells (28).
Both CD69 and CD103 are expressed in CD8+ T RM cells and less frequently, CD103 is expressed in CD4+T RM cells (29). CD69, which is an early activation marker involved in lymphocyte proliferation and retention, plays a key role in distinguishing T R M cells from those in circulation. Additionally, CD103 is a key to recognize most CD4+and CD8+T RM cells (30). The expression of CD103 helps T RM cells dock to the E-cadherin-expressed on epithelial cells and prevents them from re-circulating in the blood (31). It is generally accepted that TGF-b is an upstream regulator of T RM transformation. Increasing TGF-b in vivo appears to significantly increase the number of local T RM cells (32,33). It has been revealed that the function and expression of CD103 greatly depends on the TGF-b, indicating that the T EM and other T cells might lack TGF-b cytokines and thus fail to upregulate CD103 (34). CD39 is also highly expressed in T RM cells and is associated with higher T RM cells activities and quantity. CD39 could protect cells from apoptosis induced by adenosine triphosphate (ATP). As a transmembrane glycoprotein and extracellular nucleosidase, CD39 is also present in many biological processes such as adenosine regulation, proliferation, and resident transduction signals (28). While most human CD4+T cells express CD69, a portion of them express CD103+at the same time, especially in the lung. The majority of lung CD4+ T EM phenotype cells express the canonical T RM marker CD69. Comparing the gene expression patterns of CD103+T RM cells in lung and T EM cells in the blood, human lung CD4+CD103+T RM cells express higher levels of ITGAE (which encodes CD103), CTLA4, KLRC1 (which encodes the inhibitory receptor NKG2A) (35,36) and ICOS (31). CD4+CD103+T RM cells express deployment-ready mRNAs encoding effector molecules that rapidly respond to pathogens. Human lung T RM cells express lower levels of S1PR1 (which encodes the S1P receptor) (37), lymph node-homing molecules, SELL (which encodes the lymph node-homing receptor CD62L), KLRG1 (which encodes the activation marker KLRG1) (35), KLF2 (which drives expression of S1PR1) and CCR7 (31). Additionally, some genes have different expressions between CD103+T RM cells and peripheral T EM cells, including genes that encode heat-shock proteins (HSPA1A, HSPA7, HSPA2, and HSPD1), transcription factors (EGR2, FOSB, ATF3, RBPJ, EPAS1, and BATF) (31,35), anti-apoptotic factors (PHLDA1 and BIRC3), the tumor necrosis factor (TNF) receptor signaling family (TRAF1 and TANK) (31), chemokine (XCL1), solute carrier family members mediating amino acid transport (SLC7A5 and SLC1A5), chemokine receptor (CXCR6), transforming growth factor (TGF-b1), interleukin (IL-21R), the ligand for the death receptor Fas (FASLG), adhesion G-proteincoupled receptor (CD97), interferon-g receptors (IFNGR) (35), and fatty-acid-binding protein (FABP5) (38). All the genes mentioned above have higher expression levels in lung CD103+ T RM cells. Characteristically, lung CD4+CD103+T RM cells exhibit high levels of various integrins and adhesion molecules (31,39). Compared with blood-derived T EM cells, differences in the molecular expression of lung CD103+T RM cells are summarized in Table 1.
Several transcription factors play critical roles in the proliferation of T RM cells. The activation of the programmed cell death protein 1(PD-1) signal pathway downregulates the expression of Bhlhe40, which supports mitochondria and chromatin production in T RM cells and is thus essential to the proliferation and maintenance of T RM cells (40). Transcription factors such as BATF, NAB1, and NAB2 are also highly expressed in T RM cells. These factors can regulate T cell metabolisms to maintain their survival and reduce the expression of inhibitory phenotypes (41).

LUNG T RM CELLS AND PROTECTION AGAINST RESPIRATORY INFECTION
CD8+T RM cells are considered as the first line of defense in peripheral tissues against pathogens. Many studies suggest that some risk factors may interfere with circulating memory CD8+T cell function ( Table 2 and Figure 1). Reticular fibroblasts located near T cells around the infection site can transmit long-lasting activation signals to CD8+T cells by upregulating ICOS ligand (ICOSL), CD40, and interleukin-6 (IL-6), which promotes the preferential differentiation of T cells into T RM cells (55). CD8+T RM cells can produce chemokines after local tissue activation and recruit non-antigen-specific T cells, exerting natural effector functions (56,57). T RM cells promote the production of IL-2 and some pro-inflammatory cytokines, effectively mobilizing inflammatory responses and exert immune defenses (58). At the same time, T RM cells produce IL-10 and express inhibitory receptors, thus inhibit excessive inflammatory response and limit tissue damage caused by inflammation (59). CD4+ T RM cells in non-lymphoid tissues, such as lung, skin, and genital mucosa, can influence the immune reaction of various pathogenic microorganisms (60-62).

SARS-CoV-2 infection
tissue-resident memory-like Th1 cells and tissue-resident memory-like Th17 cells Natural Th17 cells were recruited to the infected site by CCL20 on lung epithelial cells stimulated by IL-17A and expanded in the presence of IL-23, which then were converted to T RM cells, existing as ex-Th17 cells and exerting Th1-like immunity in the event of SARS-CoV-2.
Pulmonary Inflammation

CD69hiCD103loCD4+T RM cells
Enhance the secretion of IL-5 and IL-13 which can cause pulmonary inflammation and fibrosis.
CD69hiCD103hiCD4+T RM cells Improve the fibrosis reaction caused by pulmonary inflammation and reduce lung injury.
Asthma Th2-T RM cells Th2-T RM cells expressing high levels of CD44 and ST2 can reside in lung tissues and retain allergen memory. Once re-exposed to an allergen, Th2-T RM cells proliferate near the airway, producing type 2 cytokines that enhance eosinophil activation and promote peribronchial inflammation.
(18, 53, 54) FIGURE 1 | CD8+ T RM cells in lung infection and immunopathology. CD8+ T RM cells are considered as the first line of defense in peripheral tissues against earlier exposure to antigens. CD8+ T RM cells located in the lung parenchyma could rapidly synthesize IFN-g following the inhalation of pathogens, driven by exposure to IL-12/IL-18. Fibroblast reticular cells located near T cells around the focus can transmit long-lasting activation signals to CD8+T cells, by upregulating ICOSL, CD40, and IL-6. Additionally, CD8+ T RM cells promote the production of IL-2, mobilizing inflammatory response. At the same time, T RM cells can produce IL-10, thus inhibiting the excessive inflammatory response and limiting tissue damage caused by inflammation. However, CD8+ T RM cells can be abnormally deposited in the lung due to overexpression of TGF-b-related genes, which may damage normal tissues by releasing IFN-g, GZMB, and perforin, leading to lung emphysema or fibrosis.

Antivirus Effect
In lungs, follicular tissue-resident CD4+ T helper cells contribute to the defense against virus in conjunction with CD8+ T cells relying on IL-21 (63). These helper T cells can also induce antiviral B cell reactions in bronchus lymphoid tissue in flu virus infection, indicating that T RM cells could promote the protective response of B cells and CD8+T cells in lung infections (63). CD8+T RM cells in the lung act as protective agents against viruses through interferon-g (IFN-g) ( (66). COVID-19 prevents the T RM cells from remaining ex-Th17 cells and exerting Th1-like immunity effects (42). Previous studies showed in RSV-immune mice, T RM cells enhanced respiratory syncytial virus clearance, indicating CD8+T RM cells can enhance resistance against secondary RSV infection (43,44). In RSVimmune mice, CD69 co-expressed heavily with CD38, consistent with its role as an early activation marker. Some CD4+CD69+T cells also expressed integrin CD103, and permanent memory CD4+T cells were enriched in airways. As the infection progressed, these T RM cells were enriched in infection site with increased CXCR3 expression (45). Similarly, T RM cells protect the human body from influenza viruses by producing large amounts of IFN-g and TNF-a (50). When encountered with influenza A virus, dendritic cells with high expression of IRF4 can effectively promote the production of CD8+T RM cells, thus reducing infection severity (67). PPAR-g expression accelerates the establishment of CD8+TRM cells, suggesting that PPAR-g is a positive regulatory factor for T RM cells. Moreover, PPAR-g deficiency reduces the number of alveolar macrophages residing in tissues during pulmonary infections, indicating that alveolar macrophages might be negative regulators of CD8+T RM cells and could limit the establishment of T RM cells (51).

Antibacterial Effect
Non-homologous bystander activation can trigger the sensory and alerting functions of lung CD8+T RM cells (68). Unlike memory CD8+T cells in circulating blood, CD8+T RM cells located in the lung parenchyma can rapidly synthesize IFN-g after the inhalation of heat-killed bacteria or bacterial products, a process-driven by exposure to IL-12/IL-18 (69). Bacterial infection of respiratory tract leads to bystander activation of pulmonary T RM cells, enhancement of the recruitment of neutrophils to the airway and reduction of the severity of bacterial pneumonia (69). These activations suggest that T RM cells innately amplify inflammatory responses and participate in non-homologous responses to bacterial infections (68). Lung CD4+T RM cells remodel epithelial responses to accelerate neutrophil recruitment during pneumonia. During heterotypic immunity, CD4+T cells upregulate CXCL5 and drive neutrophil recruitment in the lung (70). In mice infected with Bordetella pertussis, T RM cells produced IL-17 and IFN-g, recruiting neutrophils and preventing nasal colonization (46)(47)(48)(49). In addition, uninfected mice acquired immunity after receiving adoptive transferred CD4 T cells isolated from either lungs or spleens of convalescent mice (71). Following mucosal znBAZ vaccination, lung CD8+ T RM cells exhibit superior protection against Brucella infections. Mucosal znBAZ immunization induces CD103+ and CD103-CD8+ T RM cells expressing CXCR3 lo and CXCR3 hi phenotypes in the lung parenchyma and airways, respectively. These CXCR3-expressing CD103+ and CD103-CD8+T RM cells are not depleted by anti-CD8 mAb treatment (52).

Other Effect
T RM cells also increase resistance to parasite invasions. Both the percentage and absolute numbers of lung CD4+ and CD8+ cells increase after Schistosoma. japonicum infection (72). CD103expressing pulmonary CD4+ and CD8+ T cells play essential roles in mediating granulomatous inflammation induced by S. japonicum infection (72).

LUNG T RM CELLS IN ANTI-TUMOR IMMUNITY
Approximately 85% of lung cancers worldwide are non-small cell lung cancer (NSCLC), of which lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the most common (73). Growing evidence suggested that in human solid tumors, tumor-associated lymphocytes in NSCLC may comprise the function of T RM cells (25). CD8+T RM cells in the tumor microenvironment (TME) are a homogeneous CD103+ CD49+CD69+ population expressing T-bet, porylated (p) STAT-3, and Aiolos transcription factors and a subset of these cells produces IFN-g and IL-17. In patients with NSCLC, CD8+ T RM cells overexpress several T cell inhibitory receptors and exhaustion surface markers and co-express PD-1 and CD39, implying that they are enriched in activated tumor-antigen reactive T cells (74). Cytotoxic CD8+T lymphocytes (CTLs) in NSCLC with a high level of CD103 display enhanced cytotoxicity and proliferation, suggesting a robust anti-tumor immune response in human lung cancer (41). Compared to T cells from adjacent and tumor-free lung tissues, these cells exhibit more significant heterogeneity in the expression of molecules associated with T cell antigen receptor (TCR) activation and immune checkpoints such as 4-1BB, PD-1, TIM-3. However, in human lung cancer, far from being exhausted, PD-1-expressing T RM cells in tumors are clonally expanded and enriched for transcripts linked to cell proliferation and cytotoxicity (12).
The origination, infiltration, and differentiation of T RM in NSCLC is still unclear. O'Brien and colleagues' model speculated that in patients with early-stage NSCLC, T EM cells encountered antigens during tumor formation and were converted to CD103+ T RM cells that exerted anti-tumor activity (75). However, due to a variety of factors associated with tumor growth in TME, the tumors might not be eliminated. These factors, combined with the chronic antigen stimulation, may trigger an exhaustion program characterized by increased Eomes and CD39 expression. The presence of B7-H4 in tumors or other TME stromal cells might upregulate Eomes expression in T cells. As a tumor grows, this exhaustion program may dominate T RM cells, causing increasing TILs hypofunctionality.
In addition, transcription factors may play a role. Patients suffering from advanced-stage NSCLC exhibit a progressive decrease of NFATc1 in tumor cells and TILs decrease progressively (76). Some CD103+T RM cells may lead to decreased function and cytotoxicity of CD8+T cells, a phenomenon observed in the lungs of tumor-bearing NFATc1 DCD4 mice, likely promoted by decreased IL-2 in the absence of NFATc1 (77). Runx3 also plays an important part in the differentiation of T RM in NSCLC (78). The Runx3 is required for optimal T RM cell differentiation in the lung parenchyma and maximal expression of granzyme B in T RM cells. Several tissueresidency signature genes are upregulated in Runx3overexpressing cells and downregulated in Runx3-deficient cells. Conversely, circulating memory cell signatures are enriched in Runx3-deficient cells and depleted from Runx3overexpressing cells.
Other immune cells such as M1 hot tumor-associated macrophages (TAM) can boost the infiltration and survival of T RM cells in patients with NSCLC (32). M1 hot TAMs may recruit T RM cells via CXCL9 expression and sustain them by producing more essential fatty acids on which T RM cells depend. Monocytes acquire the ability to prime T RM cells via IL-10-mediated TGF-b release. IL-10 plays a negative regulatory role in the immune system per classical theory, but it has been found that CD103 was significantly upregulated, and T cells transform into T RM cells when influenced by IL-10 (33). Significantly upregulated CD103 leads to more T cells transforming into T RM cells. Therefore, IL-10-mediated TGF-b signaling may have a critical role in postvaccination T RM generation (33).
Moreover, chemokine receptors and cytoskeleton proteins contribute to T RM cell infiltration. The focal adhesion-associated protein paxillin binding to the CD103 cytoplasmic tail triggers aEb7 integrin outside-in signaling that promotes the migration and functions of CD8+T cells (79). This binding process explains the more favorable prognosis associated with more retention of T RM cells in TME (79). In both mice and human lungs, CXCR6 is expressed on the surface of T RM cells with the action of intrapulmonary antigens, aiding the migration of T RM cells from pulmonary interstitium to TME and maintaining the T RM cell pool (80), whereas memory CD8+T cells of the spleen do not express this receptor (81).
Duhen et al. proposed a model in human solid tumors associating T RM cells with tumor growth: CD8+ T cells are primed by dendritic cells presenting tumor antigens within the tumor-draining lymph nodes and then migrate to the tumor where they recognize the cognate antigens then clonally expand (25). The consequence of this TCR activation in a TGF-b-rich environment is the upregulation of CD39 and CD103 on CD8+ TILs. CD103 expressed on some T RM cells may promote immunologic synapse by binding to E-cadherin on tumor cells (82). Activation of these cells also leads to the downregulation of the proteins that are essential for T-cell recirculation, retaining CD8+ TILs within the tumor. In human lung cancer, there are many important anti-tumor costimulatory molecules such as SIRPG and KIR2DL4 on the surface of T RM cells, which help the CTLs kill tumor cells (41). In human, CD103+ T RM cells can also produce granzyme B (GZMB) and IFN-g, which can restrict tumor cell growth and metastasis by inducing fibronectin production, make antigens available to prime for the priming of new tumor-specific T cells, and enhance recruitment of monocytes, NK cells, and XCR1+ cDC1 to the tumor site (9). However, repetitive TCR stimulations of the CD8+ TILs impair effector function, mediate immune escape, and ultimately tumor progression. CD103+CD39+CD8+T RM cells efficiently kill autologous tumor cells in an MHC-class I-dependent manner. Additionally, the content of cytokine and receptor function influence immune functions ( Figure 2).
The infiltration of CD8+ T cells in solid tumors is a favorable prognostic marker (83). In the tumors that exhibited a high level of infiltrated CD8+ T cells, the proliferation of CD103+ T cells was correlated with improved long-term survival, indicating that infiltration of CD8+CD103+ T RM cells is a favorable prognostic marker (84). Another study suggested that high intratumoral but not stromal CD103+ TILs were associated with better overall survival in patients with resected LUSC, another significant prognostic implication of CD103 expression in TILs in human LUSC (85). Moreover, CD103 and E-cadherin interaction play a vital role in granule polarization and exocytosis, enhancing recruitment and retention of tumor-antigen-specific TILs in human NSCLC. In human NSCLC, CD28H is mainly expressed in T RM cells and is thus associated with improved tumor prognosis (86). However, other studies conversely demonstrated opposite results that B7-H5 (the ligand of CD28H) was expressed in more than 60% of cases of NSCLC and was associated with worse prognosis. Hence, the expression patterns of CD103 in TILs of NSCLC and the associated prognostic implications are significant and merit further investigation.
In T RM cells in human NSCLC tissues, there are several dysfunctional subtypes, such as NKG2A+CD8+ T cells (87).

NKG2A is an inhibitory receptor of both T cells and natural killer (NK) cells. Persistent activation causes T cells and NK cells to express NKG2A and may lead to chronic infection and cancer.
Tumor-infiltrating NKG2A+CD8+T cells form the predominant subset of NKG2A+cells in human lung cancer (87). Blockading NKG2A may promote anti-tumor immunity by unleashing dysfunctional CD8+T cells in tumors, and targeting NKG2A+ CD8+T cells is a promising approach for future anti-lung cancer immunotherapy.
While numerous studies have reported that the presence of T RM -like CD8+T cells in human NSCLC is a favorable prognosis (77), the role of CD4+TILs with a shared phenotype remains unclear. As CD4+T RM cells exhibit phenotypic and functional heterogeneity, different subsets are expected to play different and even opposite roles in TME. CD4+T RM cells are known to be essential for cytotoxic programming of CD8+T cells, and they can also suppress tumor growth through secretion of IFN-g or direct killing tumor cells in human NSCLC (88).

POTENTIAL THERAPEUTIC STRATEGIES BASED ON T RM CELLS
Given the remarkable roles of T RM cells in lung diseases, increasing T RM production or reactivating suppressed T RM cells may be a valuable therapeutic strategy ( Table 3). It is believed that in lung diseases without medical intervention, T RM cells play a less critical role because their function is inhibited and disabled in the focal microenvironment (88). Therefore, current researches focus on reactivating T RM cells that have adapted to the disease microenvironment, increasing the load of T RM cells in the lesions, and mediating immune responses such as cytotoxicity and conditioning, to kill pathogens or slow down disease progression ( Table 4).

T RM Cells and Neoadjuvant Chemotherapy
T RM cells may be involved in varied NSCLC therapies. Neoadjuvant chemotherapy was one modality in the treatment of resectable NSCLC. The beneficial effects of neoadjuvant chemotherapy might be mediated partially by CD8+CD103+ mediated tumor cell killing (13). With neoadjuvant chemotherapy, more infiltration of CD4+CD103+PD-1 T RM cells at the time of surgery was associated with longer overall survival. Moreover, T RM cells could be of great importance in TME and in cancer immune checkpoint blockade immunotherapy. In both mice model and human, dual anti-PD-L1/anti-4-1BB immunotherapy increased the number of intratumoral CD103+ CD8+ T cells and altered their distribution (90). Administration of PD-L1 mAb and 4-1BB mAb further increased the cytolytic capacity of CD103+CD8+T cells. Collectively, infiltrated CD103+ CD8+T cells served as a potential effector T cell population.  Combining 4-1BB agonism with PD-L1 blockade may increase tumor-infiltrated CD103+CD8+T cells, facilitating tumor regression. It is also reported that CD103+CD8+T RM cells could be considered potential biomarkers when selecting patients that may benefit from immune checkpoint blockade immunotherapy in patients with multiple primary lung adenocarcinoma after neoadjuvant immunotherapy (91). Yet more evidence is required to determine the clinical practice of potential the therapeutic strategies based on T RM cells, as a more favorable indicator of prognosis or a target of immune therapy. Several treatments may potentially activate or increase the number of T RM cells. One possible treatment involves promoting the separation and trans-differentiation of T cells to T RM cells in TME, which could inhibit tumor progression. At the same time, in murine models, apoptosis induced by IR increases the number of newly infiltrated T cells and converts them into T RM cells, producing an inflammation-like effect that may assist immunotherapy (92).

T RM Cells and Radiotherapy
Since T RM cells have a unique survival advantage in radiotherapy, a RT-PD1-MerTK triple therapy based on radiotherapy may also be effective. Promoting T RM cell production from other sources such as traditional radiotherapy may be an equally valuable potential treatment. T RM cells have stronger radiation resistance than tumor cells, and efficient infrared irradiation (IR) makes preexisting T RM cells survive and mediates the anti-tumor effect of T RM cells (93). In murine models, compared with traditional radiotherapy, anti-PD-1 therapy relieves the inhibitory effect on immune cells such as T RM cells, while anti-MerTK can transform apoptosis caused by radiation into cell necrosis and turn macrophages near tumors from M2 to M1 and reduce tumor load (94). This triple therapy could increase the content of CTLs and promote the differentiation of T RM cells, improving the antitumor effect. Adding anti-PD1 and anti-MerTK to radiation could significantly upregulate CD8+CD103+TRM at the abscopal tumors, suppress the abscopal tumor growth and extended the survival rate (95). As for epigenetic and metabolic regulation, a treatment scheme for TA/AC may be considered. TA, or microtubule inhibitor A, is a histone deacetylase inhibitor that can promote the production of IFN-g in Bhlhe40+CD8+T RM cells (96). Acetic acid (AC) can be used as the substrate of acetyl-CoA synthesis, which is independent from the tricarboxylic acid (TCA) cycle and promotes histone acetylation and cytokine production in Bhlhe40+CD8+ T RM cells (97). The combination of TA and AC can promote tissue retention and functional differentiation of T RM cells (40). TA/Ac treatment not only enhances Bhlhe40−/− CD8+ T cell effector and resident gene expression but also promotes the expression of these genes in WT CD8+ T cells, indicating appropriate combinations of epigenetic modifiers with certain metabolites may represent promising approaches for maximally reinvigorating tissue or tumor-resident CD8+ T cell antiviral or antitumor activities.

T RM Cells and Vaccines
Another treatment approach involves the induction of persistent T RM cells by vaccines. Phosphatidylserine liposomes are excellent antigen carriers, which can be combined with polyconic adjuvants for the development of new BCG vaccines (71). Because anti-cytomegalovirus response is one of the most powerful and persistent cellular immune responses observed in human bodies, cytomegalovirus is a possible effective T RM -cellinducing vaccine vector (98). Murine models show that other peptide nanofibers with strong immunogenicity may likewise improve the immune response (99), particularly with combined polypeptide antigen and adjuvant (33). Continuous stimulation with local homologous antigens can increase T RM cell population, and zymosan used as an adjuvant could transform CD8+T cells into T RM cells in the absence of antigens. Mice models indicate that adding zymosan adjuvant to a possible vaccine may moderate local inflammation as well as greatly enhance the production of T RM cells (100). Similarly, combining ovalbumin antigen and CpG DNA adjuvant hybridized pH-responsive substances can increase the T RM cells response range and lifespan. This combination can also activate antigen-presenting cells (APCs), and stimulate continuous T RM cell production in mice (101).
Intranasal vaccine administration induces T RM cells in the lung (11). Triggering an appropriate inflammatory response in the immune process may allow T RM cells to bypass antigen recognition. Lung T RM cells are most effectively induced at the memory stage of basic vaccines in murine models (99). In-depth analysis of the phenotypes of the locally induced CD8+T cells showed that after vaccination, T RM cells and CD8+T cells coexist as effector phenotypes and that T RM cells play an important role. Indeed, at the peak of the local immune response, concentrations of T RM are 10-fold higher than those of effector CD8+T cells, and only the T RM cells population persist locally after 30 days. Even when effector CD8+T cells are no longer detectable, tumor resistance is still observed (11). The CXCR6-CXCL16 axis demonstrably governs the growth of NSCLC in the migration of CD8+ resident memory T cells in lung mucosa after vaccination. CXCR6 deficiency impairs cancer vaccine efficacy and CD8+ resident memory T-cell recruitment in lung tumors (80). Interestingly, intranasal vaccination induces higher and more sustained concentrations of CXCL16 than intramuscular vaccination, particularly compared with other chemokines in the bronchoalveolar lavage fluid and pulmonary parenchyma in both mice and human (81).
Despite it is observed that vaccines can promote the T RM cells population, retention and function and then enhance the antitumor immunity both in mice and human, the efficiency and safety of tumor-related vaccines remains unclear thus require further investigations.

LUNG T RM AND IMMUNOPATHOLOGY
In certain conditions, lung T RM cells may cause excessive inflammatory responses and tissue fibrosis ( Table 4). After acute influenza infection, abnormal reactivation of T RM cells in the lung may likewise cause lung tissue changes and fibrosis (102). In elderly mice, CD8+ T RM cells can be abnormally deposited in the lung due to overexpression of TGF-b-related genes (103). Low responsive T RM cells not only fail to perform an immune function but may also lead to chronic inflammation and the sequelae of fibrosis (89). CD69 hi CD103 lo CD4+ T RM cells produce effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus (15). Studies have shown that pathogenic immune cells like CD69 hi CD103 lo CD4+T RM cells enhance the secretion of IL-5 and IL-13, which can cause excessive pulmonary inflammation and fibrosis. By contrast, CD69 hi CD103 hi CD4+ TRM cells can improve the fibrosis reaction caused by pulmonary inflammation and reduce lung injury, indicating that lung CD4 + T RM cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive T RM cell subpopulations in the the lung (15).

Association With Asthma
Th2-T RM cells are associated with asthma. These cells release cytokines that recruit eosinophils and sustain mast cells in the airway, leading to an inflammatory response. Th2-T RM cells expressed with high levels of CD44 and ST2 have been observed in lung tissues and can retain allergen memory throughout the life of a host organism (53). Re-exposure to a known allergen causes Th2-T RM cells to proliferate near the airway, producing type 2 cytokines that enhance eosinophil activation and promote peribronchial inflammation (104). Together with circulating memory Th2 cells, they perform non-redundant functions in asthma induction (18,53,54). Although these T RM cells eliminate invasive pathogens, the release of pro-inflammatory factors (such as IFN-g, perforin, and granulose) may damage normal cells, leading to lung damage, emphysema, or fibrosis.

Participation in Acute Cellular Rejection (ACR) After Lung Transplantation
T cells are mediators of acute cellular rejection (ACR) after lung transplantation (105). The role of pulmonary T RM cells in ACR in lung transplantation remains uncertain. Longitudinal analysis of lung transplant recipients has indicated that T RM cells from recipients gradually formed T RM phenotypes approximating healthy people after 6 months allograft, while donor T cells persisted in the form of T RM . The increase in the proportion of recipient-derived T RM cells was associated with ACR, suggesting that T RM cells may influence the inflammatory environment of lung allograft after transplantation (2).

CONCLUSION
Human lung T RM cells, whether CD8+ or CD4+, persist in lung tissues for decades of human life. The essential role of lung T RM cells is maintaining tissue homeostasis when facing viruses, antigens, and pathogens encountered through respiration, and may also be important in tumor surveillance. Lung T RM cells can also promote pathologic inflammation, inducing chronic airway inflammatory changes leading to asthma and fibrosis. Similarly, lung T RM cells from infiltrating recipient T cells in transplantation may mediate allograft immunopathology and promote lung damage. More comprehensive understanding of the induction and maintenance of T RM cells by cancer vaccines or other immunotherapeutic approaches may provide insights into the innovation of immunotherapies of lung diseases.