Legend of the sentinels: development of lung resident memory t cells and their roles in diseases

Introduction

The COVID-19 pandemic is ravaging the world. By the end of November 2020, there are over 60 million cumulative cases globally, and the number of deaths has exceeded one million ( 1 ). This disease is caused by SARS-CoV-2, which is mainly transmitted through air-borne droplets, leading to severe pulmonary diseases and systemic damage ( 2 ). Up to now, the treatment for COVID-19 is very limited, and no specific antiviral drug has been developed. Multiple candidate COVID-19 vaccines are undergoing clinical trials ( 3 ).

In general, most COVID-19 vaccines in clinical trials focus on humoral immunity, which exerts antibodies to prevent the virus from invading cells. However, antibodies alone may not be sufficient to prevent SARS-CoV-2 infection. One reason is that extracellular antibodies cannot completely clear the cells infected by virus ( 4 ). The final elimination of the virus depends on the supplement of cellular immunity, that is, the role of T cells, which help B cells produce neutralizing antibodies and can directly kill virus-infected cells. The second is that the memory B cell response tends to be short-lived ( 5 ), whereas the T cell response can last for many years. Recent researches have demonstrated that patients who recovered from the severe acute respiratory syndrome (SARS) still had long-lasting memory T-cells but reduced antibody responses ( 6 , 7 ). Therefore, vaccines against SARS-CoV-2 should focus on activating the adaptive branch of the immune system and explicitly focus on inducing long-term memory T cells. Given that many respiratory viruses are controlled by tissue immune cells that may not be present in the blood, the tissue-resident memory T (T _RM ) cells infiltrated in the lungs that can recognize foreign antigens locally and provide a rapid immune response will be an area of concern.

Actually, CD8+T cells retained for a long time after influenza virus infection were observed in mouse lungs as early as 2001 ( 8 ). Extensive studies in mouse models have determined that the lungs are enriched in T _RM cells against a variety of viral and bacterial antigens brought by respiratory infections or vaccination. Specific T _RM cells were also detected in the respiratory tract of patients with influenza or tuberculosis (TB) ( 9 ). These pathogen-specific T _RM cells produced by prior exposure can control acute re-infections and achieve long-term immunity. In mouse model, an intranasal recombinant vaccinia virus boosting regimen has generated SARS-CoV-specific lung resident memory CD8+T cells. When re-stimulated, these T _RM cells can effectively release a variety of effector cytokines and cytotoxic molecules that prevent extensive virus replication and limit the alveolar damage ( 10 ). Another study suggested that the administration of SARS vaccine intranasally induced CD4+ T _RM cells in the respiratory tract of mice, which offered the protective immunity against death ( 11 ). Regarding SARS-CoV-2, recent published single-cell profiles have indicated that the CD8+ T cells in bronchoalveolar lavage fluids (BALFs) of patients with severe infection exhibited a less proportion of tissue-resident phenotypes than those in moderately infected patients ( 12 ). Hence a vaccine that induces the production of lung T _RM cells is an ideal candidate for generating a strong and rapid immune response against SARS-CoV-2.

There are other T _RM cells in the lungs with different roles, including T _RM cells that may cause pathological immune responses and tumor-infiltrating T _RM cells that can enhance anti-tumor immunity in the lungs ( 13 ). These T _RM cells under different immune microenvironment in the lungs act in various roles in immune defense, immune homeostasis, and immune surveillance. An in-depth understanding of the generation and maintenance of lung T _RM cells will provide new insights for the development of novel vaccine formation and delivery strategies and lung-specific immunoregulatory therapy.

This review will focus on the definition, generation, and different roles of lung T _RM cells in infection, pathological immune responses, and cancers, and discuss T _RM cell-related vaccination strategies combined with emerging cutting-edge discoveries.

Hallmarks of T _RM Cells

T _RM cells, also known as non-circulating memory T cells, include both CD8+ and CD4+ subgroups. It refers to those memory T cells that occupy long-term residency in local tissues such as lung, intestine, and skin. Through cell labeling, parabiosis, tissue transplantation, and other methods, the circulation trajectory of cells can be observed to determine T _RM cells ( 14 – 16 ). However, it is still a challenge to clearly distinguish T _RM cells from other cells in vitro by surface markers.

In recent years, with the development of transcriptomics, T _RM cells have been found to have unique transcriptional profiles and functional characteristics. The main hallmarks of T _RM cells that distinguish it from other circulating memory T cells are the ability to adhere to peripheral tissues and the lack of homing signals. Based on the research on both mouse and humans, the most used phenotypic marker defining T _RM cell subsets is CD69. Due to the competitive protein-protein interaction between CD69 and sphingosine-1-P receptors (S1PR), it inhibits the expression of S1PR and prevents S1P-mediated egress ( 17 , 18 ). These cells also lack CD62L and CC-chemokine receptor 7 (CCR7), both of which direct cells into lymphoid tissue ( 19 ). On the flip side, CD44 up-regulated by T _RM cells is the receptor for hyaluronic acid and other ligands expressed in peripheral tissues, which can induce the retention of memory T cells in peripheral tissues ( 20 ). As another key T _RM cell marker, the integrin αE: β7 (CD103) is mainly expressed on CD8+ T _RM cells and some on CD4+ T _RM cells, which binds E-cadherin and anchors cells around epithelial cells ( 21 ). It is worth noting that T _RM cells in lungs can be defined by several major surface markers, but this subset itself is still heterogeneous in some way. The transcriptome analysis reveals the inconsistent changes in gene expression among different cells ( 19 , 22 , 23 ). Further elucidation of detailed mechanism of T _RM cell formation and maintenance will add to understanding of the phenotype of lung T _RM cells under different pathophysiological conditions.

Development of Lung T _RM Cells

The development of lung T _RM cells can be divided into several steps: 1) activation in lymphoid tissues and migration into inflammatory lung tissue guided by local cytokines, 2) expression of homing molecules and specific transcription factors and differentiation into lung resident memory T cells, 3) local maintenance in specific niches and replenishment from T _CM cells ( Figure 1 ). So far, the focus on specific transcription factors and cell surface receptors has gradually revealed details in the fate determination mechanism of lung T _RM cells.

FIGURE 1

Generation and maintenance of lung T _RM cells. During the activated phase of infection, dendritic cells present antigens to activate naïve T cells in the lymph nodes. These cells turn into effector T cells and up-regulate surface marker CXCR3, CXCR6, CCR5, which guide them into inflammatory tissues. After entering lung tissue, part of effector T cells is regulated by environmental signals including cytokines such as TGF-β and cognate antigens, and differentiate into lung T _RM cells. The rest of the effector T cells undergoes cell death or egress out of the lung. Compared with Teff cells, lung T _RM cells manipulate multiple surface markers and transcription factors that facilitate cell maintenance and survival.

Activation and Migration

The inability to recirculate between lung and lymph nodes or bloodstream is a key determinant of lung T _RM cells ( 24 , 25 ). However, these cells did not start in the lung tissue but migrated into it later. Under normal conditions, naïve T cells consecutively circulate throughout the body. When infection occurs, dendritic cells (DCs) migrate from infected respiratory sites into mediastinal lymph nodes (MdLN) and activate naïve T cells. Among these migrant DCs there are two subsets, and only airway localized CD103+ DCs can fully induce the differentiation of naïve T cells into T _eff cells ( 26 ). Once activated, the T _eff cells up-regulate the expression of CXCR3, CCR5, and CCR4, which specifically guide T _eff cells into lung tissue and help control pathogen invasion ( 27 – 31 ). For example, after TB infection, chemokine ligand IP-10 in the lung increases significantly, which binds to CXCR3 and facilitates T cell migration ( 29 ). In addition, CD8+ and CD4+ lung T _eff cells are regulated differently and tend to localize in different regions. CD8+ T _eff cells are inclined to migrate to the collagen IV-rich region and CD4+ T _eff cells are more prone to be located in areas abundant in collagen I ( 32 ). Compared with CD8+ T cells, CD4+ T cells enter the lung tissues first and direct the localization of CD8+ T cells. CD4+ T cells fine-tune chemokine gradients in the microenvironment such as TGF-β, which promotes the production of CD103 and is crucial for CD8+ T _RM cell formation ( 33 ).

Differentiation

T _eff cells will not transform into lung T _RM cells immediately after entering the lung tissues. The tissue microenvironment has an important influence on the development of lung T _RM cells. In the early stage of infection, T _eff cells that migrate into the infection site will encounter redundant inflammatory signals, which guide T _eff cells towards terminal T _eff cells ( 34 ). They reduce local inflammation, help remold the microenvironment and make it more appropriate for the differentiation of lung T _RM cells. In the later stage, CD8+ T cells are recruited into tissue damage sites, which later developed into regenerative tissues termed as repair-associated memory depots (RAMDs). RAMDs provide environmental cues that help drive CD8+ T _eff cells into CD8+ T _RM cells and later become niches for CD8+ T _RM cells ( 35 , 36 ). Predominant environmental cues include cytokines such as TGF-β, IL-33, TNF, IFN-γ, IL-15, and cognate antigens ( 18 , 33 , 37 ). TGF-β plays an important role in promoting the expression of T _RM cell marker CD103 and CD69. Together with IL-33 and TNF, TGF-β can provoke KLF2 down-regulation, which further down-regulates its target protein S1P1 and increases expression of CD69 ( 18 ). Furthermore, TGF-β down-regulate T-box transcriptional factor and promote the expression of CD103. T-box transcriptional factors are composed of eomesodermin (Eomes) and T-bet, and they vary in the degree of decline. While Eomes is effectively removed, T _RM cells maintain residual levels of T-bet which is important for T _RM cell survival ( 37 ). The decrease in production of T-box transcriptional factor is demonstrated in mature lung CD8+CD103+ T _RM cells ( 33 , 37 ). Unlike CD8+ T _RM cells in other tissues like skin and vagina, where they can be generated with only local inflammatory signals ( 38 ), lung CD8+ T _RM cells must interact with cognate antigen before differentiation. After the exposure to cognate antigen, CD8+ T _eff cells increase the expression of CD69, CD103, and collagen-binding integrin VLA-1 ( 39 ). T cell receptor (TCR) signaling can also induce Blimp-1 expression, which biased CD8+ T _eff cell differentiation towards T _RM cells rather than T _CM cells ( 40 ). It is surprising that pulmonary monocytes and type 1 regulatory T (T _reg ) cells also contribute to the differentiation. Pulmonary monocytes are the major cells to present pathogen antigens, while type 1 T _reg cells promote the bioavailability of TGF-β ( 41 , 42 ). As mentioned above, CD4+ T _RM cells have different development pathways compared with CD8+ T _RM cells. CD4+ T _RM cells express different cell markers and are affected by different cytokines ( 43 ). They have low expression of CD103, and their generation is not interfered by TGF-β, which has a great impact on the generation of CD8+ T _RM cell ( 44 , 45 ). Beyond that, IL-2 and IL-15 were found to affect the differentiation of CD4+ T _eff cells in different subsets, respectively ( 44 ). Researches on differentiation of CD4+ T _RM cells are not as thorough as those on CD8+ T _RM cells, and there are still many points to be clarified.

Maintenance

While persisting in lung tissues, CD8+ and CD4+ T _RM cells will construct different structures that contribute to long-term survival. Most CD8+ T _RM cells reside in specific niches we refer to as RAMDs, which are constructed by tissue regeneration after tissue damage. These niches are significant for lung CD8+ T _RM cells. They may present cytokines that help lung CD8+ T _RM cell maintenance. Considering that the recovery of tissue damage takes a long time, the lung CD8+ T _RM cells may protect this vulnerable part from secondary infection ( 35 , 36 ). Unlike CD8+ T _RM cells, lung CD4+ T _RM cells combine with B cells and other cells to form ectopic lymphoid tissue called inducible bronchus-associated lymphoid tissue (iBALT) that benefits cell survival. In iBALT, CD4+ T _RM cells surround B cell follicles, which facilitate rapid interaction with each other and provide a recall response toward potential infection ( 43 , 46 ). Compared with circulating T _EM cells, lung T _RM cells displayed different patterns of genes and transcription factors that regulate the expression of cytokine receptors and adhesion molecules, most of which have been mentioned above. Single-cell sequencing found an important transcription factor Notch, which controls the expression of CD103 and the basic metabolic function of lung T _RM cells ( 47 ). The absence of Notch greatly reduces the population of lung T _RM cells. Another study indicated that lung T _RM cells were programmed to express IFITM3, which can protect them from secondary infection and improve survival ( 48 ). Except for cytokines and surface molecules, M1 ^hot tumor-associated macrophages can also contribute to the maintenance of lung T _RM cells in tumor, possibly due to reduction in nutrition competition ( 49 ). In comparison with other tissue T _RM cells that may persist for a long time or even a lifetime, lung T _RM cells gradually disappear 4–5 months after infection. Lung T _RM cells that reside in the airway quickly decline due to the harsh environment, where amino acid starvation triggers the integrated stress response, leading to cell apoptosis ( 50 ). And those retained in the parenchyma decrease along with the shrink of RMADs. After full regeneration, most of the RAMDs will disappear, and only a minority of lung CD8+ T _RM cells may survive in iBALTs ( 35 , 36 ). In order to compensate for the constant loss, airway T _RM cells are replaced primarily by recruitment from lung interstitium ( 51 ), and T _RM cells in interstitium receive continuous replenishment from circulating T _EM cells. T _EM cells are recruited and transformed into lung T _RM cells under the influence of TGF-β, IL-33, and TNF but antigen-independently. However, T _EM cells gradually lose their ability to migrate and convert into lung T _RM cells after infection ( 52 ). All in all, T _RM cells can only provide a short period of protection, which leaves the lung much more susceptible to further infection. However, this may be a designed mechanism for the prevention of pathological immune response.

Lung T _RM Cells Against Infection

The lungs and respiratory tract, as part of direct access to the outside world, are easily exposed to various pathogens. Common pulmonary pathogens include influenza virus, respiratory syncytial virus (RSV), as well as Streptococcus pneumoniae, Klebsiella pneumoniae, Bordetella pertussis, and Mycobacterium tuberculosis. Under normal circumstances, the first infection caused by these pathogens will not only be cleared by the body’s immune system but also induce memory T cells, some of which settle in the lungs as T _RM cells ( Figure 2 ).

FIGURE 2

An abstract figure of the role of T _RM cells in various lung diseases. Lung T _RM cells can:(A)rapidly respond towards invasive pathogens during re-infection,(B)cause pathologic immune response after overactivated by environmental stimuli or allergen(C)infiltrate in lung tumor and express cytotoxic molecules and effector cytokines.

A large aggregation of studies has shown that the lung is rich in T _RM cells specific to a variety of pathogens such as viruses and bacteria. These T _RM cells have the potential to mediate immunity against different pathogens and protect the body from re-infection. It has been demonstrated that influenza-specific T _RM cells exhibited rapid and robust IFN-γ and TNF-α responses after restimulation in vitro ( 53 , 54 ). In human RSV challenge model, cells with T _RM phenotype can be detected in BALFs, and the higher frequency of RSV-specific CD8+ T _RM is related to the decrease in the severity of disease and the viral load ( 55 ). CD4+ T _RM cells accumulate in the lungs after Bordetella pertussis infection. These cells are pathogen-specific and can secrete IL-17 and/or IFN-γ. A research observed that mice treated with the S1P antagonist Fingolimod (FTY720) to prevent lymphocyte migration into the lungs before initial infection with Bordetella pertussis were significantly more severely affected in the later stages of infection. However, in the case of re-infection, because the tissue-infiltrated T _EM cells have partially transformed into T _RM cells in the lung, they are not affected by Fingolimod treatment and can still quickly clear the bacillus. At the same time, the adoptive transfer of CD4+ T _RM cells from the lungs of mice in convalescence to uninfected mice can protect the latter from pathogens attack ( 56 ). All these evidences indicate that T _RM cells act as a pivotal role in the rapid response of secondary infection.

However, while T _RM cells eliminate invasive pathogens, the released proinflammatory factors such as IFN-γ or perforin and granzymes may damage normal cells, cause lung injury and lead to emphysema or fibrosis, even result in ARDS. Hence, an effective immune response to these infections requires precise immune regulation to eliminate pathogens while protecting the function of normal lung tissue. Many mechanisms exist in the lung to restrict the inflammatory response to acute infection, including inhibitory receptors, immunomodulatory molecules and cells like FOXP3+CD4+ T _reg cells ( 57 ). Under stable conditions, a large number of T _reg cells is reserved in the lung and IL-10 expression is significantly increased after influenza infection ( 58 ). In RSV-infected mice, the TCR of T _reg cells can specifically recognize the viral epitope-MHC II complex. Immunization of mice with this epitope can reduce clinical manifestations and immunopathology without virus clearance defects ( 59 ). In addition, PD-L1 and PD-L2 are expressed in alveolar epithelial cells and are significantly up-regulated to control inflammation in RSV infection ( 60 ). However, some studies held that this may limit the formation and development of T _RM cells and cause negative effects ( 61 ). The detailed mechanisms of lung T _RM cell function and immune homeostasis are not yet fully understood, and future improvement in the number and stability of T _RM cell population must be carried out on the premise that prevents re-infection of the virus and does not impair the respiratory health of the host.

Lung T _RM Cells in Pathologic Immune Response

As mentioned above, sometimes T _RM cells may cease to be the protector and become part of the destructor, and thus attack normal tissue and induce chronic inflammatory diseases ( 13 ) ( Figure 2 ). After acute influenza infection, antigen deposits in the lung for 2–3 months. In young mice, the persistent presentation of the antigens may induce part of the T _RM cells to exhibit exhausted-like phenotype. This phenotype is thought to help maintain lung’s immune balance and prevent damage. If PD-L1 antibody is used to blockade PD-L1 and PD-1 interaction, exhausted-like T _RM cells would rejuvenate, express more cytokines, and enhance their heterogeneous protection against infection. But they would also cause pulmonary pathological change and fibrosis ( 62 ). In elderly mice, increased expression of TGF-β in the environment led to accumulation of T _RM cells in the lungs. However, these T _RM cells have low effector activity due to intrinsic defects and fail to enhance the protective function, but can instead lead to chronic inflammation and fibrotic sequela ( 63 ). Also, it has been discovered that T _H 2-T _RM cells are closely related to asthma ( 64 ). They release specific cytokines that recruit eosinophils and maintain mast cells in the airway, which result in the inflammatory response. Using a mouse model exposed to house dust mite (HDM), T _H 2-T _RM cells that specifically respond to HDM are identified. These T _H 2-T _RM cells are developed from HDM-specific CD4+ T _eff cells and are mediated by IL-2 signaling. IL-2 up-regulates chemokine receptors such as CCR4 and CXCR3 that improve migration into the lung, as well as programs related to tissue intention ( 64 ). A recently published paper further reports that these T _H 2-T _RM cells highly express CD44 and ST2, and can reside in lung tissue and maintain their memory towards allergen for the whole life of a mouse ( 65 ). Once re-exposed to allergen, T _H 2-T _RM cells robustly proliferate near airways, produce type 2 cytokines, enhance eosinophil activation, and promote peribronchial inflammation. They together with circulating memory T _H 2 cells perform nonredundant function in the induction of asthma ( 66 , 67 ).

Lung T _RM Cells in Anti-Tumor Immunity

Accumulating evidence suggests that T _RM cells are important in anti-tumor immunity ( Figure 2 ). It is suggested that a part of the tumor-infiltrating lymphocytes (TILs) isolated from several cancers displays a similar transcriptomic and phenotypic feature with T _RM cells. Some refer to it as T _RM -like TILs ( 9 ), but here we still call it “ lung tumor T _RM cells”, as the consensus in most articles. These lung tumor T _RM cells predict a better survival outcome in early-stage non-small-cell lung carcinoma (NSCLC) patients, as well as increased intraepithelial lymphocyte infiltration ( 68 ). Single-cell and bulk transcriptomic analysis reveals that lung tumor T _RM cells have slightly different transcriptomes compared with other lung T _RM cells. They express similar surface marker CD103, CD69, CD49a, and they also up-regulate Notch and Runx3. But lung tumor T _RM cells express more cell cycle-related genes, such as CD39, CXCL13, CCL3, and TNFSF4, indicating that they belong to a new subset ( 22 ). Comparing samples from different lung cancer patients, the T _RM cells of advanced lung cancer are mostly exhausted, while the function of early-stage lung tumor T _RM cells is relatively heterogeneous ( 69 ). Among them, CD103+CD8+ T _RM cells are found to release more cytokines, proliferate faster, and exhibit better anti-tumor performance ( 70 ). It is described that CD103 can connect with E-cadherin on tumor cells, which induces cytotoxic granule polarization at the immune synapses ( 71 , 72 ). CD103 also facilitates T _RM cells to reside near tumor tissues ( 73 ). In contrast with previous studies, lung tumor T _RM cells show the diffuse expression of inhibitory receptors, but do not exhibit the exhausted phenotype. And instead, transcription factor Eomes is found to negatively correlate with T _RM cell function ( 69 , 74 ). Single-cell analysis even discovered a PD-1+TIM-3+IL-7R- T _RM cell subset expresses high levels of inhibitory receptors, but remains the ability to proliferate rapidly in situ and displays enhanced capacity to express key cytotoxic molecules and effector cytokines ( 22 ). Since TIM-3+IL-7R- T _RM cells are the major cells expressing PD-1, and CD103+CD8+ T _RM cells show positive responses towards anti-PD-1 and anti-PD-L1 monoclonal antibodies, the researchers believe that these cells may be the major subset that reacts in anti-PD-1 therapy ( 22 , 68 , 70 ). In combination with the performance of T _RM cells in different stages of lung cancer, it has been speculated that T _eff cells were influenced by tumor antigens and cytokines such as TGF-β, up-regulate CD39 and CD103, and converted into CD103+ T _RM cells. They exercise their anti-tumor function diligently. If, for one reason or another, the tumor is not eliminated, the local microenvironment as well as the repetitive TCR stimulation may trigger their exhaustion program and they finally become hypofunctional T _RM cells ( 69 , 75 ).

Vaccination Strategies Inducing Lung T _RM Cells

The growing literature that considers T _RM cells are indispensable in eliminating infectious pathogens and controlling tumor progression has led to increasing interest in the induction of T _RM cells by vaccination for disease treatment and prevention. Compared with circulating T cells or B cells, activated T _RM cells are more focused in killing virus-infected cells in target tissues, which help complement neutralizing antibodies and reduce antibodies titer threshold needed to control virus ( 4 , 76 , 77 ).

There are two main strategies to establish T _RM cell pool within lung tissues. The first approach applies a one-step method to directly induce antigen-specific lung T _RM cells by vaccine vectors ( 78 , 79 ). For this approach, the route of immunization is very important. Direct intranasal or intrapulmonary route provides better protection compared with commonly used intraperitoneal, intramuscular, or subcutaneous administration route ( 80 , 81 ). Intranasal administration but not injection of live-attenuated influenza virus has shown the capacity to generate long-term CD4+ and CD8+ T _RM cells and provide heterosubtypic protection to nonvaccine influenza strains in mice ( 82 ). Intratracheal and intranasal rather than subcutaneous inoculation of Bacille Calmette-Guérin (BCG) also results in generation of T _EM and T _RM cells in the lung, which remedy the low efficacy of parenteral BCG vaccination to prevent pulmonary TB ( 83 ). In a preclinical head and neck cancer model, local T _RM cells can be induced and tumor growth can be controlled in mice immunized with the cancer vaccine (STxB-E7) by intranasal route ( 84 ). Another approach is a two-step method that combines conventional elicitation of systemic T cell response with the recruitment of these cells into target tissues, which are referred to as “ prime and pull” ( 85 ). Actually, in a very early stage, scientists have discovered that mucosal boosting with the same vaccine after systemic priming can elicit more CD4+ and CD8+ lung T _RM cells compared with only mucosal or systemic vaccination ( 80 ). There is also evidence indicates that compared with the original “ prime and pull” strategy used in genital tract, the pull step applied in lung disease should use pathogen antigens instead of proinflammatory chemokines. This is because only pathogen antigens can maintain the recruited T cells in airway lumen and persevere immune protection over time ( 86 ). Intranasal administration of a novel recombinant anti-TB vaccine (SeV85AB) after subcutaneous immunization with BCG uses this way to provide larger immune protection for lungs than either SeV85AB or BCG alone ( 87 ). As opposed to vaccines that directly provide the pathogen antigens like SeV85AB, recent research developed an “ antibody-targeted vaccination (ATV)” for the pull step. It connects antigen with antibody that targets lung DC cells, give raise to local antigen presentation, and improve activation of lung T _RM cells ( 88 ). Pulmonary surfactant-biomimetic liposomes containing stimulator of interferon genes that target alveolar epithelial cells give a new way to recruit CD8+ T _RM cells and provide long term wide-spectrum protection ( 89 ). These methods may also be used in inducing tumor antigen presentation and lung tumor T _RM cell function.

In summary, multiple studies have proved that T _RM cells can be induced by vaccination to make a difference in preventing pathogens or controlling tumor growth. However, many problems remained to be solved, for example, how to attract T _eff cells into target areas not close to mucosal, and how to maintain long-term lung T _RM cells ( 79 ). Systemic approaches should also be developed to evaluate the safety and efficiency of these vaccines and prevent overactivation of T _RM cells resulting in pathologic immune responses ( 90 ).

Concluding Remarks

It is now obvious that lung T _RM cells are an important part of the adaptive immune response within lung tissues. Although we have a rudimentary understanding of lung T _RM cells, they remain shrouded in mystery, waiting to be discovered more. While mentioning the migration, activation, differentiation, and maintenance of lung T _RM cells, main steps are outlined but there are still huge empties in the details. Do lung T _RM cells undergo pre-differentiation in lymph nodes before infection ( 91 )? Which cytokines, transcription factors, and surface molecules are more decisive in the migration, formation, and maintenance of lung CD4+ or CD8+ T _RM cells? Are there different subtypes of lung T _RM in different lung tissue structures (such as in interstitium and parenchyma)? To answer these questions, more advanced techniques such as single-cell RNA-sequencing that identifies cell-cell interaction and TCR lineage tracking may be used.

A better understanding of these issues will undoubtedly help better manipulate lung T _RM cells to prevent or treat disease. Therapy focusing on lung T _RM cells in tumor and pathologic immune response is still in a nascent state. Besides direct activation or transmission of tumor-specific T _RM cells, currently there are vaccines that activate antiviral lung T _RM cells near tumor tissue ( 92 ), which reverse the immunosuppressive microenvironment, and may pave the way for later cell therapy. Drugs that prevent lung T _RM cell formation or function may also be useful in suppressing the immune response to lung transplantations or preventing lung sequela after respiratory infection in the elderly ( 63 ). Of course, T _RM cells in the lungs are mostly deemed to fight off lung infections. During the COVID-19 pandemic, lung T _RM cells are particularly important in the first line of defense against re-infection of SARS-CoV-2. Actually, influenza viruses have never been conquered, not only because of its versatility, but also because the immune memory only lasts for a short time in lung. To fight them, one possible solution is to improve the “ width and depth” of the function of vaccines that induce lung T _RM cells. The width refers to the prospect that the same vaccine can induce lung T _RM cells that resist a wide range of virus strains in response to virus variability ( 88 ). The depth hopes that the induced T _RM cells can remain in the lungs for nearly lifelong, enhancing the killing effect and duration of protection of the vaccine ( 79 ). More insight and precise manipulation of the fate of lung T _RM cells will help to better develop novel immunomodulators to treat lung diseases by T _RM cells, and thus to exert the rapid and powerful action in critical illnesses such as COVID-19 pandemic.

Author Contributions

YQ and YZ contributed to the central idea and coordinated the writing of the manuscript. YQ, YZ, YL, and BL read, discussed, and revised the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by China National Funds for Distinguished Young Scientists (Grant No: 31525008), National Natural Science Foundation of China (Grant Nos: 81830051, 31700775, 31961133011), National Key Research and Development Program of China (Grant No: 2019YFA0906100), and China Postdoctoral Science Foundation (Grant No: 2017M631497).

Conflict of Interest

BL is a co-founder of Biotheus Inc and the chairman of its scientific advisory board.

The remaining authors declare that the work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The figures in this review were created with Biorender. com .

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