Unique properties of tissue-resident memory T cells in the lungs: implications for COVID-19 and other respiratory diseases

Abstract

Tissue-resident memory T (T_RM) cells were originally identified as a tissue-sequestered population of memory T cells that show lifelong persistence in non-lymphoid organs. That definition has slowly evolved with the documentation of T_RM cells having variable terms of tissue residency combined with a capacity to return to the wider circulation. Nonetheless, reductionist experiments have identified an archetypical population of T_RM cells showing intrinsic permanent residency in a wide range of non-lymphoid organs, with one notable exception: the lungs. Despite the fact that memory T cells generated during a respiratory infection are maintained in the circulation, local T_RM cell numbers in the lung decline concomitantly with a decay in T cell-mediated protection. This Perspective describes the mechanisms that underpin long-term T cell lodgement in non-lymphoid tissues and explains why residency is transient for select T_RM cell subsets. In doing so, it highlights the unusual nature of memory T cell egress from the lungs and speculates on the broader disease implications of this process, especially during infection with SARS-CoV-2.

In this Perspective, Francis Carbone considers the unique characteristics of the tissue-resident memory T (T_RM) cell populations that develop in the lungs. He discusses how the different properties of lung T_RM cells may affect immunity to lung infections, including SARS-CoV-2.

Introduction

Memory T cells can show a range of persistence within non-lymphoid compartments. Many lymphocytes move freely through the various organs unimpeded before exiting the tissue via the draining lymphatic vessels^1–3. Recognition of antigen leads to their transient retention⁴ while physical barriers may slow the return of cells to the circulation⁵. Finally, a subset of T cells is specialized for purely localized patterns of immune surveillance^6,7 and only poorly exits the tissues, if at all⁸. These tissue-resident memory T (T_RM) cells⁹ have a cell-autonomous limitation in their recirculation capacity^10–12 and show a superior ability to control localized infections in a number of settings^13–15. In this Perspective, I detail the transcription networks that define sequestered T_RM cells, identifying CD103⁺CD8⁺ memory T cells as the key population of memory CD8⁺ T cells that encompasses all the hallmarks of permanent tissue residency. Finally, I describe how these archetypical T_RM cells show an unusual pattern of egress from the lungs and discuss how this impacts the course of respiratory infections, including SARS-CoV-2.

Identifying tissue-resident memory T cells

T_RM cells were initially identified as a distinct, sessile T cell subset that coexisted alongside tissue-emigrating T cells⁸. This was a break from the prevailing understanding of tissue T cells based on early lymphocyte circulation experiments^5,16,17. At that time, the widely accepted view was that these T cells were simply recirculating memory cells that either happened to be found in non-lymphoid tissues in large numbers^18–20 or, alternatively, were trapped by some sort of gating mechanisms or by structural barriers such as the basement membrane that lines epithelia⁵. The identification of a unique T_RM cell subset meant that non-lymphoid tissues contained at least two populations of memory T cells, each with its own distinct phenotype and functional properties. One was a recirculating subset that at the time was thought to comprise effector memory T (T_EM) cells¹⁷ and the other, the newly identified permanently resident T_RM cell population.

A major challenge from that point onwards has been distinguishing non-migrating T_RM cells from recirculating memory T cells, largely because of the phenotypic overlap between these populations. For example, T_RM cells do not express CC-chemokine receptor 7 (CCR7) — a receptor required for entry into lymphoid tissues and the marker that was originally used to differentiate T_EM cells (identified as CCR7-negative) from lymphoid-tissue-constrained central memory T (T_CM) cells (identified as CCR7-positive)¹⁷. Separately, CD69 had been proposed to be a pan-T_RM cell identifier²¹, yet it is upregulated by both antigen-specific and nonspecific stimuli²² and a substantial fraction of migratory T cells express this molecule once in the tissues²³. Compounding the confusion is the extensive heterogeneity seen in both circulating and tissue-resident memory T cell populations^24–27, expanded by a history of natural infection²⁸. Therefore, although combinations of surface markers can cover a range of T_RM-like tissue cells, it would be fair to say that to date there remains no unifying phenotypic identifier for this population.

CD103⁺CD8⁺ T_RM cells: the archetypical T_RM cell

Although it has proven difficult to identify T_RM cells by definitive phenotypic means, therapeutic and experimental interventions can eliminate all circulating T cells from the blood, leaving long-term tissue residents as the only memory T cells remaining in non-lymphoid compartments. Two approaches have proven particularly useful in this regard. The first exploits T cell responses against a transplantation mismatch to selectively eliminate cells in the circulation^29–31 whereas the second uses a more versatile cytolytic antibody-based technique for the same purpose^27,32,33. Of additional importance is the in vivo infusion of labelling antibodies before tissue analyses to exclude cells that are simply in the vasculature³⁴. This technique eliminates confounding contributions by blood-borne cells and is critical when examining highly vascularized organs such as the lung, although it does not identify T_RM cells per se.

One of the striking features of the mouse T_RM cells that remain after circulating T cells are depleted from the tissues is the dominance of a population of CD8⁺ T cells expressing the CD103 (also known as αE integrin) subunit of the αEβ7 integrin complex^23,33. CD103⁺ T_RM cells are highly enriched in the environmentally exposed epithelia of the skin, small intestine and female reproductive tract^8,10,35. At these epithelial sites, interaction between αEβ7 and its abundantly expressed target ligand E-cadherin³⁶ probably plays a role in cell adhesion and retention. However, CD103⁺CD8⁺ T_RM cells are also found in non-epithelial tissues such as the brain^12,37, and although CD103 has variously been implicated as being important for T_RM cell development^38–40, its expression is not ubiquitous³⁷ and therefore not mandatory for all forms of T cell residency. Nonetheless, tissue-lodged CD103⁺CD8⁺ memory T cells are highly resistant to equilibration across parabiotic pairs⁴¹, are uniquely spared from elimination by the approaches mentioned above^23,30, selectively survive for prolonged periods in transplanted tissues in mice^8,33 as well as in humans^42,43 and persist independently of antigen recognition^15,37. Moreover, CD103⁺CD8⁺ memory T cells are usually not found in secondary lymphoid organs^15,44 — with one striking exception^45,46 to be described in detail below. Thus, although not all T_RM cells express CD103, the balance of evidence argues that CD103⁺CD8⁺ tissue T cells are true T_RM cells, making this an easily identifiable archetypical population and an ideal reductionist means for delineating tissue residency mechanisms.

RUNX3 and TGFβ in CD8⁺ T_RM cell development

Early experiments in mice comparing the transcriptomes of CD103⁺CD8⁺ T_RM cells isolated from a range of organs with those of their circulating counterparts provided some of the first insights into the transcription networks critical for T_RM cell development and survival^10,39. Not surprisingly, genes associated with tissue egress were found to be downregulated in T_RM cells, including Ccr7 and the genes encoding the sphingosine-1-phosphate receptors S1PR1 and S1PR5^10,11. Without the downregulation of these receptors, the precursors of T_RM cells return to the circulation, thereby dampening T_RM cell development^11,47. Other genes that come into play are those involved in dealing with local metabolite availability^7,48,49 and those that prolong T cell survival²³, with both sets of genes necessary to maintain a long-lived sequestered T cell population. Further experiments fleshed out how transcription factors control the various networks, such as the involvement of KLF2, which modulates the expression of S1PR1 and CCR7¹¹. Following this, key upstream gene regulators were identified, such as T-bet and EOMES^23,50 as well as BLIMP1 and the BLIMP1 homologue HOBIT^51,52; of note, BLIMP1 and HOBIT are also involved in the development of innate-like lymphocytes that permanently reside in mouse tissues, such as natural killer cells and natural killer T cells⁵¹. Most recently, an overarching transcription factor has come into focus. RUNX3 has been identified as contributing to T_RM cell formation, and it directly or indirectly regulates BLIMP1 and KLF2 expression as well as modulating downstream retention components⁵³. This contribution is particularly striking as RUNX3 is a pivotal player in CD8⁺ T cell development and functionality^54,55.

As the network analyses evolved, one commonality to emerge was the involvement of TGFβ in T_RM cell development and survival in a range of tissues and organs^{23,40,56–58}. TGFβ appears to use a non-canonical signalling pathway⁵⁹ that controls much of the CD8⁺ T_RM cell gene expression signature⁶⁰. It has been shown to facilitate tissue entry via selectin upregulation⁶¹ and can regulate a broad range of transcription regulators and cytokine-driven survival factors during CD8⁺ T_RM cell development^11,23,50. Combined, there is now a wealth of data regarding the tenets of transcriptional control of T_RM cell formation, which largely pivots around a TGFβ–RUNX3 axis, at least in the case of the mouse CD8⁺ T_RM cell subset.

T_RM cell re-entry into the circulation

Although T_RM cells were originally shown to persist in non-lymphoid organs in quasi-perpetuity⁸, there have been subsequent descriptions of T_RM cell egress with the resultant ‘ex-T_RM cells’ ultimately being incorporated into the circulation^33,41,62. CD8⁺ T_RM cell numbers show an intrinsic decline in organs such as the lung and liver^30,41, but not in tissues such as the skin and small intestine, where the cells effectively remain in place for life once lodged^8,41. However even for these fixed populations, T_RM cells can be forced to leave using in situ antigen stimulation via peptide challenge^33,44. Such active dislodgement is not universal, with CD103⁺CD8⁺ T_RM cells sometimes remaining resident in the tissue even after multiple rounds of cell division initiated by local infection^8,63,64. Perhaps tellingly, when CD103⁺CD8⁺ T_RM cells are selectively dislodged by intervention, the resultant ex-T_RM cells appear to adopt a phenotype intermediate between those of upstream resident memory T cells and conventional recirculating memory T cell populations, with a CD103 expression status that is either undefined or reported to be transient^33,44. Moreover, when these same cells are directly isolated from non-lymphoid compartments, they are inferior in their capacity to enter the circulation compared to counterparts extracted from lymphoid organs^33,65.

It remains difficult to reconcile these conflicting results, but studies on CD8⁺ T_RM cells in the liver and recent revelations regarding the basis for CD4⁺ T cell residency provide valuable insight that might explain egress variability. Although much more is known about CD8⁺ T_RM cells, there are many examples of CD4⁺ T_RM cell-type counterparts^13,27,66. Comparisons make it clear that the two are unrelated in terms of mechanistic underpinnings and they can exhibit quite distinct patterns of tissue residency even in the same organ^29,67. As noted above, the archetypical CD103⁺CD8⁺ T_RM cells use a set of TGFβ-driven transcriptional networks to shut down tissue egress, upregulate survival factors and tailor metabolic pathways. By contrast, few of these networks have been associated with CD4⁺ T_RM cell residency, which instead relies on retention mechanisms variously operating via cell aggregation, antigen-specific T cell activation and chemotactic agents^68,69 (Fig. 1). The reason why CD4⁺ and CD8⁺ T_RM cells are likely to differ at the mechanistic level is the pivotal role RUNX3 plays in T_RM cell development and survival⁵³. This transcription factor is repressed in CD4⁺ T cells by the opposing gene regulator ThPOK (also known as ZBT7B), which is itself a lineage-determining factor^70,71. Although natural RUNX3 upregulation can convert CD4⁺ T cells to an unconventional CD8αα⁺ intraepithelial regulatory T cell population with CD8⁺ T_RM cell-like qualities⁷², the intrinsic paucity of RUNX3 expression in conventional CD4⁺ T_RM cells results in low CD103 levels in these cells and more transient tissue residency as a direct consequence of their inability to access the RUNX3-mediated pathways downstream of TGFβ signalling^53,73.

Fig. 1. Subtypes of tissue-resident memory T cells based on transcription profiles.

The mechanism promoting permanent residency in non-lymphoid tissues for the CD103⁺CD8⁺ tissue-resident memory T (T_RM) cell population involves a RUNX3-driven transcriptional network that is downstream of TGFβ receptor signalling^53,60. This transcription programme is missing in CD4⁺ T_RM cells as a consequence of deficiencies in RUNX3 expression⁷³. Instead, these populations use a combination of cell aggregation and extrinsic chemokine networks for tissue retention^68,69. The typical CD103⁺CD8⁺ T_RM cell transcription programme is also missing in CD103⁻ liver-like T_RM cells because of deficiencies in TGFβ engagement⁶⁵.

Somewhat analogous to their CD4⁺ tissue-resident T cell counterparts, mouse liver CD8⁺ T_RM cells are also deficient in CD103 expression⁷⁴. These cells show medium-to-long-term tissue residency⁷⁴, but not the almost lifelong persistence of T_RM cells in organs such as skin and small intestine^41,65. Although the liver T cells are fully capable of responding to TGFβ, local requirements negate this capacity, resulting in an immature or less differentiated CD103⁻ T_RM cell population (Fig. 1) with an inferior term of residency combined with more flexible reprograming compared to mature CD103⁺ T_RM cell counterparts⁶⁵. Collectively, the results show that although CD103⁻ T_RM cells can reside in tissues for a considerable period, they can exhibit a range of spontaneous egress and reprogramming capabilities because of deficiencies in TGFβ-mediated maturation. Given the heterogeneous nature of tissue-resident T cells, including variable CD103⁺ T cell content across different organs³⁷ and the known recruitment of recirculating T cells by the peptide stimulation used for T_RM cell dislodgement³², it is possible that less differentiated populations analogous to the liver CD103⁻ T_RM cells may preferentially contribute to the egress process. Regardless, although some T_RM cells can leave the tissues and enter the circulation, the balance of data argues that for the archetypical CD103⁺CD8⁺ T_RM cells, this process is not constitutive and when it does happen, it usually results in cells that do not phenocopy their direct upstream antecedents.

T_RM cells in the lungs

From the discussion above, it can be reasonably argued that because they fully engage the TGFβ–RUNX3 residency programme, mouse CD103⁺CD8⁺ tissue T cells fit the original T_RM cell definition⁸; specifically, they form a distinct subset of memory T cells that remains lodged in peripheral compartments in virtual perpetuity. However, there is one organ where the CD103⁺CD8⁺ T cells do not abide by this rule, and its uniqueness has important disease implications. Unlike the situation in other tissues, CD103⁺CD8⁺ T_RM cells in the lung do not require local antigen stimulation for dislodgement⁴⁵. Also unusually, the egressing memory T cells retain cell surface expression of CD103 post-exit, meaning that the lung-draining lymph nodes are unique in having a substantive subset of memory CD8⁺ T cells with this marker^45,46. Lung CD103⁺CD8⁺ T_RM cells are fully mature and unremarkable in terms of their TGFβ requirement for development and survival⁵⁸. They also express the gene signatures associated with tissue residency^10,12, including a cluster of T_RM cell-associated transcription factors, namely HOBIT, NR4A1, aryl hydrocarbon receptor (AhR) and BHLHE40^7,51,75,76. In all critical aspects, they resemble T_RM cells from other tissues, meaning that their exit from the lung is probably an organ-specific feature rather than due to a cell-intrinsic programme. Such a mechanistic distinction is important, as it would suggest that the egress process would probably capture T_RM cells beyond the archetypical CD103⁺CD8⁺ subset that was used to define this phenomenon and would do so regardless of where they fall on a maturation and term-of-residency continuum.

Exacting experiments by Stolley and colleagues⁴⁵ proved that the resultant draining lymph node-resident memory T cells were indeed constitutively derived from upstream lung tissue counterparts, possibly dislodged as a consequence of virus-induced tissue damage^77,78 or the interruption of tonic TGFβ signalling needed to retain T_RM cells in tissues⁷⁹. Although the resultant lymph node accumulation offers an additional avenue to maintain regional protection^45,80, memory T cell exit helps to explain one of the intriguing conundrums associated with immune protection in the lungs. It has long been known that T cell immunity in the lung wanes over time, with this first reported for respiratory infections with influenza virus and Sendai virus in mice^81,82. This decline in lung-based immunity occurs despite virus-specific memory cells persisting in the circulation^30,82–84. Non-T_RM cell-based mechanisms were originally proposed to describe the behaviour of lung T cell populations^81,85–87, variously confounded by blood-borne cells that are particularly problematic when dealing with this highly vascularized organ³⁴. More recently, it was shown that the waning local immunity correlates with declining lung T_RM cell numbers in mouse after influenza virus infection^83,84 and in humans after respiratory syncytial virus challenge⁸⁸. Although other mechanisms have been posited to account for this T_RM cell attrition, such as the selective death of lung T_RM cells^30,84 or the disappearance of structures associated with focal damage⁶⁷, none exclude concurrent tissue egress. Once lost, lung T_RM cells are difficult to replace in the absence of renewed infection owing to the strict antigen recognition requirements for effective lodgement^67,83,89, which are optional in many other tissues³⁷ including the upper respiratory tract⁹⁰. Overall, a range of mechanistic overlays would imply that losing T_RM cells over time is important for this organ — for example, to limit ongoing damage to its delicate oxygen-exchange architecture⁹¹.

Finally, the natural decay of lung T_RM cells stands in stark contrast to what is seen elsewhere in the body, where CD103⁺CD8⁺ T_RM cell populations can remain tightly contained (Fig. 2). CD103⁺CD8⁺ T_RM cells show long-term persistence in organs such as the brain, skin and cervicovaginal tissue^8,39,92, despite the loss of their CD103⁻ counterparts. The extent to which these spatial and temporal restrictions can operate was dramatically illustrated by experiments that lodged CD103⁺CD8⁺ T_RM cells in a small patch of skin, thus confining effective protection to just that location while leaving the remainder of the torso under the inferior control of memory cells in the blood^8,63. By contrast, lung T_RM cell residency is unstable and transient, resulting in surveillance that is increasingly dependent on recirculating populations over time, with a concomitant decline in local T cell immunity.

Fig. 2. Selective and constitutive egress of lung CD103⁺CD8⁺ T_RM cells.

Inflammation associated with infection of tissues such as skin, small intestine and reproductive tract (left panels) and lung (right panels) leads to the recruitment of a variety of CD4⁺ and CD8⁺ T cells that combat the invading pathogens (part a). These populations include effector memory T (T_EM) cells that continuously recirculate between non-lymphoid organs and blood as well as tissue-resident memory T (T_RM) cell precursors (not shown). Following resolution of the infection (part b), most of the recruited T cells exit or die, leaving local immunosurveillance to recirculating T_EM cells and the more potent T_RM cells. Over time, some T_RM cell subsets selectively disappear (part c, left panel), resulting in a resident population highly enriched in long-lived CD103⁺CD8⁺ T_RM cells that afford long-term local immunity against re-infection (part d, left panel). In the lungs, CD103⁺CD8⁺ T_RM cells are gradually lost after the infection has resolved and instead accumulate in the proximal draining lymph nodes (part c, right panel) leaving the lower respiratory tract deficient in CD103⁺CD8⁺ T_RM cells and thus susceptible to re-infection (part d, right panel).

T_RM cell lung egress and immunity to SARS-CoV-2

At the time of this writing and nearly three years since the emergence of the SARS-CoV-2 virus in late 2019^93,94, the COVID-19 pandemic continues to be a major challenge in many parts of the world. Despite reports showing that circulating antiviral T cell immunity can be cross-reactive against emerging variants⁹⁵, long lived^96,97 and associated with better disease outcomes^98,99, immunity from combinations of COVID-19 vaccination and SARS-CoV-2 infection has been found to steadily decline^100,101. One possible contributor may be that anti-SARS-CoV-2 tissue-resident T cells that are pivotal for immune protection show the same type of numerical decay as reported for mouse CD103⁺CD8⁺ T_RM cells. Employing strategies that slow T_RM cell loss¹⁰² could be advantageous, as might approaches that circumvent the lung altogether. The upper respiratory tract, especially the nasal mucosa, is a prime target with respect to the latter possibility as it does not show the T_RM cell decline that is intrinsic to the lung⁹⁰. Alternatively, it may be that T_RM cells are actually counterproductive, leading to tissue damage. This is especially poignant because repeated antigen encounters extend the durability of CD103⁺CD8⁺ T_RM cells in the lung¹⁰², yet a recent report found that experiencing successive SARS-CoV-2 infections progressively increases the risk of adverse health outcomes¹⁰³. In terms of their potential to contribute to tissue damage, TRM cells have an innate immune alarm and recruitment function^32,104, and the innate response has been shown to be a key mediator of COVID-19-associated lung pathology^105,106.

Conclusion

Overall, T_RM cells provide superior protection against tissue-localized infection, primarily because of constraints in their migration capabilities. Despite proving to be long-lived and effective in a range of different infectious diseases, lung T_RM cells have an unusual propensity for tissue exit reflected in a decay in local T cell immunity. Such a feature may have evolved to protect this organ against long-term damage or may simply be a by-product of some unique anatomical feature intrinsic to lung function. Given the ability of T_RM cells to respond to infection with an immediacy unmatched by the blood-based memory populations, there is a need to focus on their deposition in the different compartments of the respiratory system, especially in settings or sub-regions that support their long-term survival.

Peer review

Peer review information

Nature Reviews Immunology thanks J. Harty, M. Hassert and J. Schenkel for their contribution to the peer review of this work.

Competing interests

The author declares no competing interests.