Tissue-resident memory T (TRM) cells: Front-line workers of the immune system

Tissue-resident memory T (T_RM) cells play a vital role in local immune protection against infection and cancer. The location of T_RM cells within peripheral tissues at sites of pathogen invasion allows for the rapid detection and elimination of microbes, making their generation an attractive goal for the development of next-generation vaccines. Here, we discuss differential requirements for CD8⁺ T_RM cell development across tissues with implications for establishing local prophylactic immunity, emphasizing the role of tissue-derived factors, local antigen, and adjuvants on T_RM cell generation in the context of vaccination.     

Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

Tissue‐resident memory T (T_RM) cells are CD8⁺ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in T_RM cells and correlate it with their functional potential. Fifty‐three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in T_RM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in‐vitro stimulation were used to evaluate T_RM cell phenotypes. We discovered that tumour T_RM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin‐expressing T_RM cells. Surprisingly, programmed cell death 1 (PD‐1) expression is high in tumour T_RM cells, suggesting exhaustion. Following interleukin‐15 and T cell receptor stimulation, perforin and T‐bet expressions are enhanced, indicating that T_RM cells from tumours are not terminally exhausted. Moreover, a high number of T_RM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, T_RM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies T_RM cells as potential new targets for cancer immunotherapy.     

Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization

The generation of tissue-resident memory T cells (T_RM) is an essential aspect of immunity at mucosal surfaces, and it has been suggested that preferential generation of T_RM is one of the principal advantages of mucosally administered vaccines. We have previously shown that antigen-specific, IL-17-producing CD4⁺ T cells can provide capsular antibody-independent protection against nasal carriage of Streptococcus pneumoniae; but whether pneumococcus-responsive T_RM are localized within the nasal mucosa and are sufficient for protection from carriage has not been determined. Here, we show that intranasal administration of live or killed pneumococci to mice generates pneumococcus-responsive IL-17A-producing CD4⁺ mucosal T_RM. Furthermore, we show that these cells are sufficient to mediate long-lived, neutrophil-dependent protection against subsequent pneumococcal nasal challenge. Unexpectedly, and in contrast with the prevailing paradigm, we found that parenteral administration of killed pneumococci also generates protective IL-17A⁺CD4⁺ T_RM in the nasal mucosa. These results demonstrate a critical and sufficient role of T_RM in prevention of pneumococcal colonization, and further that these cells can be generated by parenteral immunization. Our findings therefore have important implications regarding the generation of immune protection at mucosal surfaces by vaccination.     

Lung niches for the generation and maintenance of tissue-resident memory T cells

The extent to which tissue-specific viral infections generate memory T cells specifically adapted to and maintained within the target infection site is unknown. Here, we show that respiratory virus-specific memory T cells in mice and humans are generated and maintained in compartmentalized niches in lungs, distinct from populations in lymphoid tissue or circulation. Using a polyclonal mouse model of influenza infection combined with an in vivo antibody labeling approach and confocal imaging, we identify a spatially distinct niche in the lung where influenza-specific T-cell responses are expanded and maintained long term as tissue-resident memory (T_RM) CD4 and CD8 T cells. Lung T_RM are further distinguished from circulating memory subsets in lung and spleen based on CD69 expression and persistence independent of lymphoid stores. In humans, influenza-specific T cells are enriched within the lung T_RM subset, whereas memory CD8 T cells specific for the systemic virus cytomegalovirus are distributed in both lung and spleen, suggesting that the site of infection affects T_RM generation. Our findings reveal a precise spatial organization to virus-specific T-cell memory, determined by the site of the initial infection, with important implications for the development of targeted strategies to boost immunity at appropriate tissue sites.     

Induction of vaginal-resident HIV-specific CD8 T cells with mucosal prime–boost immunization

Tissue-resident memory (T_RM) CD8 T cells survey a range of non-lymphoid mucosal tissues where they rapidly mediate clearance of viral infections at the entry portals. Vaccines that establish CD8 T_RM cells in the cervicovaginal mucosa hold promise for effective immunity against sexually transmitted HIV. We demonstrate that HIV-specific CD8 T_RM cells can be established in the murine vaginal mucosa using a combined intranasal and intravaginal mucosal immunization with recombinant influenza-HIV vectors. Using in situ tetramer immunofluorescence microscopy, we found that this mucosally administered prime–boost immunization also resulted in the durable seeding of CD8 T cells in the frontline vaginal epithelial compartment as opposed to the vaginal submucosa. Upon cognate antigen recognition within the vaginal mucosa, these HIV-specific CD8 T_RM cells rapidly initiated a tissue-wide state of immunity. The activation of HIV-specific CD8 T_RM cells resulted in the upregulation of endothelial vessel addressin expression and substantial recruitment of both adaptive and innate immune cells in the vaginal mucosa. These findings suggest that the epithelial localization of HIV-specific CD8 T_RM cell populations and their capacity to rapidly activate both arms of the immune system could significantly augment frontline defenses against vaginal HIV infection.     

Long-term maintenance of lung resident memory T cells is mediated by persistent antigen

Tissue-resident memory T cells (T_RM) in the lungs are pivotal for protection against repeated infection with respiratory viruses. However, the gradual loss of these cells over time and the associated decline in clinical protection represent a serious limit in the development of efficient T cell based vaccines against respiratory pathogens. Here, using an adenovirus expressing influenza nucleoprotein (AdNP), we show that CD8 T_RM in the lungs can be maintained for at least 1 year post vaccination. Our results reveal that lung T_RM continued to proliferate in situ 8 months after AdNP vaccination. Importantly, this required airway vaccination and antigen persistence in the lung, as non-respiratory routes of vaccination failed to support long-term lung T_RM maintenance. In addition, parabiosis experiments show that in AdNP vaccinated mice, the lung T_RM pool is also sustained by continual replenishment from circulating memory CD8 T cells that differentiate into lung T_RM, a phenomenon not observed in influenza-infected parabiont partners. Concluding, these results demonstrate key requirements for long-lived cellular immunity to influenza virus, knowledge that could be utilized in future vaccine design.     

Development of Plasmodium-specific liver-resident memory CD8+ T cells after heat-killed sporozoite immunization in mice

Malaria remains a major cause of mortality in the world and an efficient vaccine is the best chance of reducing the disease burden. Vaccination strategies for the liver stage of disease that utilise injection of live radiation-attenuated sporozoites (RAS) confer sterile immunity, which is mediated by CD8⁺ memory T cells, with liver-resident memory T cells (T_RM) being particularly important. We have previously described a TCR transgenic mouse, termed PbT-I, where all CD8⁺ T cells recognize a specific peptide from Plasmodium. PbT-I form liver T_RM cells upon RAS injection and are capable of protecting mice against challenge infection. Here, we utilize this transgenic system to examine whether nonliving sporozoites, killed by heat treatment (HKS), could trigger the development of Plasmodium-specific liver T_RM cells. We found that HKS vaccination induced the formation of memory CD8⁺ T cells in the spleen and liver, and importantly, liver T_RM cells were fewer in number than that induced by RAS. Crucially, we showed the number of T_RM cells was significantly higher when HKS were combined with the glycolipid α-galactosylceramide as an adjuvant. In the future, this work could lead to development of an antimalaria vaccination strategy that does not require live sporozoites, providing greater utility.     

Pulmonary antigen encounter regulates the establishment of tissue-resident CD8 memory T cells in the lung airways and parenchyma

Resident memory CD8 T (T_RM) cells in the lung parenchyma (LP) and airways provide heterologous protection against influenza virus challenge. However, scant knowledge exists regarding factors necessary to establish and maintain lung CD8 T_RM. Here we demonstrate that, in contrast to mechanisms described for other tissues, airway, and LP CD8 T_RM establishment requires cognate antigen recognition in the lung. Systemic effector CD8 T cells could be transiently pulled into the lung in response to localized inflammation, however these effector cells failed to establish tissue residency unless antigen was present in the pulmonary environment. The interaction of effector CD8 T cells with cognate antigen in the lung resulted in increased and prolonged expression of the tissue-retention markers CD69 and CD103, and increased expression of the adhesion molecule VLA-1. The inability of localized inflammation alone to establish lung T_RM resulted in decreased viral clearance and increased mortality following heterosubtypic influenza challenge, despite equal numbers of circulating memory CD8 T cells. These findings demonstrate that pulmonary antigen encounter is required for the establishment of lung CD8 T_RM and may inform future vaccine strategies to generate robust cellular immunity against respiratory pathogens.     

Bystander activation of Bordetella pertussis-induced nasal tissue-resident memory CD4 T cells confers heterologous immunity to Klebsiella pneumoniae

Tissue-resident memory CD4 T (T_RM) cells induced by infection with Bordetella pertussis persist in respiratory tissues and confer long-term protective immunity against reinfection. However, it is not clear how they are maintained in respiratory tissues. Here, we demonstrate that B. pertussis-specific CD4 T_RM cells produce IL-17A in response to in vitro stimulation with LPS or heat-killed Klebsiella pneumoniae (HKKP) in the presence of dendritic cells. Furthermore, IL-17A-secreting CD4 T_RM cells expand in the lung and nasal tissue of B. pertussis convalescent mice following in vivo administration of LPS or HKKP. Bystander activation of CD4 T_RM cells was suppressed by anti-IL-12p40 but not by anti-MHCII antibodies. Furthermore, purified respiratory tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A following direct stimulation with IL-23 and IL-1β or IL-18. Intranasal immunization of mice with a whole-cell pertussis vaccine induced respiratory CD4 T_RM cells that were reactivated following stimulation with K. pneumoniae. Furthermore, the nasal pertussis vaccine conferred protective immunity against B. pertussis but also attenuated infection with K. pneumoniae. Our findings demonstrate that CD4 T_RM cells induced by respiratory infection or vaccination can undergo bystander activation and confer heterologous immunity to an unrelated respiratory pathogen.     

Tissue‐resident memory T cells: Local guards of the thymus

T‐cell surveillance of nonlymphoid tissues has traditionally been ascribed to recirculating memory T cells that continuously patrol the body. Extending this concept, recent evidence suggests that T cells also exist as nonmigratory memory cells that provide local immune protection in a broad range of peripheral tissues, including barrier locations such as skin and mucosa. In this issue of the European Journal of Immunology, Pircher and colleagues [Eur. J. Immunol. 2013. 43: 2295–2304] demonstrate, for the first time, the existence of such permanently tissue‐resident CD8⁺ memory T (T_RM) cells in a primary lymphoid organ, the thymus. T_RM cells in this location provide potent local immunity, which may help to preserve thymic integrity and normal T‐cell development in the face of infection with thymus‐invading pathogens.     

α4β1 integrin promotes accumulation of tissue‐resident memory CD8+ T cells in salivary glands

The salivary glands (SGs) of virus‐immune mice contain substantial numbers of tissue‐resident memory CD8⁺ T cells (T_RM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus‐specific CD8⁺ T cells to the SGs have not yet been defined. Here, we found that polyinosinic‐polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR‐transgenic CD8⁺ T_RM cells in SGs in an α₄β₁ integrin‐dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 T_RM cells in SGs and intestine but not in kidney was also α₄ integrin dependent. Blockade of α₄β₇ by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus‐induced accumulation of P14 T_RM cells in the intestine but not in SGs. In conclusion, our data reveal that α₄β₁ integrin mediates CD8⁺ T_RM accumulation in SGs and that poly(I:C) can be used to direct activated CD8⁺ T cells to this organ.     

Circulating memory CD8+ T cells are limited in forming CD103+ tissue-resident memory T cells at mucosal sites after reinfection

Tissue-resident memory CD8⁺ T cells (T_RM) localize to barrier tissues and mediate local protection against reinvading pathogens. Circulating central memory (T_CM) and effector memory CD8⁺ T cells (T_EM) also contribute to tissue recall responses, but their potential to form mucosal T_RM remains unclear. Here, we employed adoptive transfer and lymphocytic choriomeningitis virus reinfection models to specifically assess secondary responses of T_CM and T_EM at mucosal sites. Donor T_CM and T_EM exhibited robust systemic recall responses, but only limited accumulation in the small intestine, consistent with reduced expression of tissue-homing and -retention molecules. Murine and human circulating memory T cells also exhibited limited CD103 upregulation following TGF-β stimulation. Upon pathogen clearance, T_CM and T_EM readily gave rise to secondary T_EM. T_CM also formed secondary central memory in lymphoid tissues and T_RM in internal tissues, for example, the liver. Both T_CM and T_EM failed to substantially contribute to resident mucosal memory in the small intestine, while activated intestinal T_RM, but not liver T_RM, efficiently reformed CD103⁺ T_RM. Our findings demonstrate that circulating T_CM and T_EM are limited in generating mucosal T_RM upon reinfection. This may pose important implications on cell therapy and vaccination strategies employing memory CD8⁺ T cells for protection at mucosal sites.     

Tissue-resident memory T cells in the skin

Purpose

Tissue-resident memory T (T_RM) cells are a newly described subset of memory T cells. The best characterized T_RM cells are CD8+ and express CD103 and CD69. These cells are non-recirculating and persist long term in tissues, providing immediate protection against invading pathogens. However, their inappropriate activation might contribute to the pathogenesis of autoimmune and inflammatory disorders. In the skin, these cells have been described in psoriasis, vitiligo, and melanoma among other diseases.

Methods

Literature review was done to highlight what is currently known on the phenotype and function of T_RM cells and summarizes the available data describing their role in various cutaneous conditions.

Results

Resolved psoriatic skin and disease-naïve non-lesional skin contain a population of IL-17-producing T_RM cells with shared receptor sequences that recognize common antigens and likely contribute to disease recurrence after cessation of therapy. In vitiligo, T_RM cells produce perforin, granzyme B, and interferon-γ following stimulation by interleukin-15 and collaborate with recirculating memory T cells to maintain disease. In melanoma, increased accumulation of T_RM cells was recently shown to correlate with improved survival in patients undergoing therapy with immune checkpoint inhibitors.

     

Skin resident memory CD8+ T cells are phenotypically and functionally distinct from circulating populations and lack immediate cytotoxic function

The in‐depth understanding of skin resident memory CD8⁺ T lymphocytes (T_RM) may help to uncover strategies for their manipulation during disease. We investigated isolated T_RM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8⁺ T cell populations from the same donors. There were significantly increased proportions of CD8⁺CD45RA^–CD27^– T cells in the skin that expressed low levels of killer cell lectin‐like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8⁺ T_RM in skin were therefore phenotypically distinct from circulating CD8⁺CD45RA^–CD27^– T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8⁺ T_RM with T cell receptor (TCR)/CD28 or interleukin (IL)‐2 or IL‐15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)‐1 further boosted granzyme B expression. A unique feature of some CD8⁺ T_RM cells was their ability to secrete high levels of tumour necrosis factor (TNF)‐α and IL‐2, a cytokine combination that was not seen frequently in circulating CD8⁺ T cells. The cutaneous CD8⁺ T_RM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous T_RM appears to be stringently controlled by environmental signals in situ .     

Pulmonary monocytes interact with effector T cells in the lung tissue to drive TRM differentiation following viral infection

Lung resident memory CD8 T cells (T_RM) are critical for protection against respiratory viruses, but the cellular interactions required for their development are poorly understood. Herein we describe the necessity of classical monocytes for the establishment of lung T_RM following influenza infection. We find that, during the initial appearance of lung T_RM, monocytes and dendritic cells are the most numerous influenza antigen-bearing APCs in the lung. Surprisingly, depletion of DCs after initial T cell priming did not impact lung T_RM development or maintenance. In contrast, a monocyte deficient pulmonary environment in CCR2^−/− mice results in significantly less lung T_RM following influenza infection, despite no defect in the antiviral effector response or in the peripheral memory pool. Imaging shows direct interaction of antigen-specific T cells with antigen-bearing monocytes in the lung, and pulmonary classical monocytes from the lungs of influenza infected mice are sufficient to drive differentiation of T cells in vitro. These data describe a novel role for pulmonary monocytes in mediating lung T_RM development through direct interaction with T cells in the lung.