Location versus ID: what matters to lung-resident memory T cells?

Tissue-resident memory T cells (TRM cells) are vital for the promotion of barrier immunity. The lung, a tissue constantly exposed to foreign pathogenic or non-pathogenic antigens, is not devoid of these cells. Lung TRM cells have been considered major players in either the protection against respiratory viral infections or the pathogenesis of lung allergies. Establishment of lung TRM cells rely on intrinsic and extrinsic factors. Among the extrinsic regulators of lung TRM cells, the magnitude of the impact of factors such as the route of antigen entry or the antigen natural tropism for the lung is not entirely clear. In this perspective, we provide a summary of the literature covering this subject and present some preliminary results on this potential dichotomy between antigen location versus antigen type. Finally, we propose a hypothesis to synthesize the potential contributions of these two variables for lung TRM cell development.

CD8+ T Cells Keep Their (K+)urrency for Function.

CD8+ T-cell responses are influenced by ion abundance, which can widely vary within the tumor microenvironment. In this issue, Collier and colleagues investigated how intracellular versus extracellular potassium ion (K+) regulates intratumoral CD8+ T cells. They show that, while excessive extracellular K+ induces exhaustion, intracellular K+ is needed for protection from dysfunction. This work shows additional evidence that the regulation of CD8+ T-cell responses depends on a fine balance between intracellular and extracellular metabolite levels. See related article by Collier et al., p. 36 (2) .

T cell-specific P2RX7 favors lung parenchymal CD4+ T cell accumulation in response to severe lung infections.

CD4+ T cells are key components of the immune response during lung infections and can mediate protection against tuberculosis (TB) or influenza. However, CD4+ T cells can also promote lung pathology during these infections, making it unclear how these cells control such discrepant effects. Using mouse models of hypervirulent TB and influenza, we observe that exaggerated accumulation of parenchymal CD4+ T cells promotes lung damage. Low numbers of lung CD4+ T cells, in contrast, are sufficient to protect against hypervirulent TB. In both situations, lung CD4+ T cell accumulation is mediated by CD4+ T cell-specific expression of the extracellular ATP (eATP) receptor P2RX7. P2RX7 upregulation in lung CD4+ T cells promotes expression of the chemokine receptor CXCR3, favoring parenchymal CD4+ T cell accumulation. Our findings suggest that direct sensing of lung eATP by CD4+ T cells is critical to induce tissue CD4+ T cell accumulation and pathology during lung infections.

Amphipathic Liponecrosis Impairs Bacterial Clearance and Causes Infection During Sterile Inflammation.

BACKGROUND & AIMS: Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. METHODS: Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects-NEFA-membrane phospholipid (phosphatidylcholine) interactions-were studied on isothermal titration calorimetry. RESULTS: Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered ß-diversity; enrichment in Pseudomonadales; and increased annexin V-positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. In vivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. CONCLUSIONS: Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.

Pannexin-1 channels promote CD8+ T cell effector and memory responses through distinct intracellular pathways.

Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses to antigen, however, has not been previously addressed. Here, we report that T cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 favors memory CD8+ T cell survival primarily through ATP export and induction of mitochondrial metabolism. CD8-specific Panx1 is also crucial for the effector expansion of CD8+ T cells, however this regulation occurs independently of eATP. Instead, our results suggest a connection between Panx1-induced extracellular lactate accumulation and the complete activation of effector CD8+ T cells. In summary, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different metabolic and signaling pathways.

Tissue- and temporal-specific roles of extracellular ATP on T cell metabolism and function.

The activation and function of T cells is fundamental for the control of infectious diseases and cancer, and conversely can mediate several autoimmune diseases. Among the signaling pathways leading to T cell activation and function, the sensing of extracellular adenosine triphosphate (eATP) has been recently appreciated as an important component. Through a plethora of purinergic receptors, most prominently P2RX7, eATP sensing can induce a wide variety of processes in T cells, such as proliferation, subset differentiation, survival, or cell death. The downstream roles of eATP sensing can vary according to (a) the T cell subset, (b) the tissue where T cells are, and (c) the time after antigen exposure. In this mini-review, we revisit the recent findings on how eATP signaling pathways regulate T-cell immune responses and posit important unanswered questions on this field.

Responsiveness to interleukin-15 therapy is shared between tissue-resident and circulating memory CD8+ T cell subsets.

Interleukin-15 (IL-15) is often considered a central regulator of memory CD8+ T cells, based primarily on studies of recirculating subsets. However, recent work identified IL-15-independent CD8+ T cell memory populations, including tissue-resident memory CD8+ T cells (TRM) in some nonlymphoid tissues (NLTs). Whether this reflects the existence of IL-15-insensitive memory CD8+ T cells is unclear. We report that IL-15 complexes (IL-15c) stimulate rapid proliferation and expansion of both tissue-resident and circulating memory CD8+ T cell subsets across lymphoid and nonlymphoid tissues with varying magnitude by tissue and memory subset, in some sites correlating with differing levels of the IL-2Rß. This was conserved for memory CD8+ T cells recognizing distinct antigens and elicited by different pathogens. Following IL-15c-induced expansion, divided cells contracted to baseline numbers and only slowly returned to basal proliferation, suggesting a mechanism to transiently amplify memory populations. Through parabiosis, we showed that IL-15c drive local proliferation of TRM, with a degree of recruitment of circulating cells to some NLTs. Hence, irrespective of homeostatic IL-15 dependence, IL-15 sensitivity is a defining feature of memory CD8+ T cell populations, with therapeutic potential for expansion of TRM and other memory subsets in an antigen-agnostic and temporally controlled fashion.

P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7-/- CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Extracellular vesicle proteomic analysis leads to the discovery of HDGF as a new factor in multiple myeloma biology.

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.

The Extracellular ATP Receptor P2RX7 Imprints a Promemory Transcriptional Signature in Effector CD8+ T Cells.

Development of CD8+ central memory T (Tcm) and resident memory T (Trm) cells, which promote immunity in the circulation and in barrier tissues, respectively, is not completely understood. Tcm and Trm cells may arise from common precursors; however, their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors, which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm cell formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Mechanistically, P2RX7 negatively regulates Zeb2 expression, at least partially through TGF-ß sensing in early effector CD8+ T cells. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.

Engagement of the costimulatory molecule ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells.

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8+ tissue-resident memory (Trm) T cells. Here, we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos-/- CD8+ T cells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS deficiency or ICOS-L blockade compromised establishment of CD8+ Trm cells but not their maintenance. ICOS ligation during CD8+ T cell priming did not determine Trm induction; rather, effector CD8+ T cells showed reduced Trm differentiation after seeding into Icosl-/- mice. IcosYF/YF CD8+ T cells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos-/- Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8+ T cell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations.

Navigating in Deep Waters: How Tissue Damage and Inflammation Shape Effector and Memory CD8+ T Cell Responses.

Memory CD8+ T cells promote protective immunity against viruses or cancer. Our field has done a terrific job identifying how CD8+ T cell memory forms in response to Ag. However, many studies focused on systems in which inflammation recedes over time. These situations, while relevant, do not cover all situations in which CD8+ T cell memory is relevant. It is increasingly clear that CD8+ T cells with a memory phenotype form in response to infections with extensive or prolonged tissue inflammation, for example, influenza, herpes, and more recently, COVID-19. In these circumstances, inflammatory mediators expectedly affect forming memory CD8+ T cells, especially in tissues in which pathogens establish. Notwithstanding recent important discoveries, many outstanding questions on how inflammation shapes CD8+ T cell memory remain unanswered. We will discuss, in this review, what is already known and the next steps to understand how inflammation influences CD8+ T cell memory.

CD8+ T cell self-tolerance permits responsiveness but limits tissue damage.

Self-specific CD8+T cells can escape clonal deletion, but the properties and capabilities of such cells in a physiological setting are unclear. We characterized polyclonal CD8+ T cells specific for the melanocyte antigen tyrosinase-related protein 2 (Trp2) in mice expressing or lacking this enzyme (due to deficiency in Dct, which encodes Trp2). Phenotypic and gene expression profiles of pre-immune Trp2/Kb-specific cells were similar; the size of this population was only slightly reduced in wild-type (WT) compared to Dct-deficient (Dct-/-) mice. Despite comparable initial responses to Trp2 immunization, WT Trp2/Kb-specific cells showed blunted expansion and less readily differentiated into a CD25+proliferative population. Functional self-tolerance clearly emerged when assessing immunopathology: adoptively transferred WT Trp2/Kb-specific cells mediated vitiligo much less efficiently. Hence, CD8+ T cell self-specificity is poorly predicted by precursor frequency, phenotype, or even initial responsiveness, while deficient activation-induced CD25 expression and other gene expression characteristics may help to identify functionally tolerant cells.

Metabolic regulation of tissue-resident memory CD8+ T cells.

Intracellular metabolic adaptations help define the function and homeostasis of memory CD8+ T cells. These cells, which promote protection against infections or cancer, undergo consecutive metabolic shifts, ultimately relying on mitochondrial-related pathways. Past CD8+ T cell metabolism studies focused on circulating memory cells, which are exclusive to secondary lymphoid organs or recirculate between lymphoid and non-lymphoid organs. Yet, now there is unequivocal evidence that memory CD8+ T cells reside in many non-lymphoid organs and mediate protective immunity in barrier tissues. The metabolic adaptations occurring in forming and established tissue-resident memory CD8+ T cells are currently subject of intense research. In this review, we discuss the latest breakthroughs on the transcriptional and protein control of tissue-resident memory CD8+ T cell metabolism.

KLRG1+ Memory CD8 T Cells Combine Properties of Short-Lived Effectors and Long-Lived Memory.

CD8 effector T cells with a CD127hi KLRG1- phenotype are considered precursors to the long-lived memory pool, whereas KLRG1+CD127low cells are viewed as short-lived effectors. Nevertheless, we and others have shown that a KLRG1+CD127low population persists into the memory phase and that these T cells (termed long-lived effector cells [LLEC]) display robust protective function during acute rechallenge with bacteria or viruses. Whether these T cells represent a true memory population or are instead a remnant effector cell population that failed to undergo initial contraction has remained unclear. In this study, we show that LLEC from mice express a distinct phenotypic and transcriptional signature that shares characteristics of both early effectors and long-lived memory cells. We also find that in contrast to KLRG1+ effector cells, LLEC undergo homeostatic proliferation and are not critically dependent on IL-15 for their maintenance. Furthermore, we find that LLEC are predominantly derived from KLRG1+ effector cells when isolated at day 12 of the response. Our work challenges the concept that the KLRG1+CD127low population is dominated by short-lived cells and shows that KLRG1 downregulation is not a prerequisite to become a long-lived protective memory T cell.

Sensing of ATP via the Purinergic Receptor P2RX7 Promotes CD8+ Trm Cell Generation by Enhancing Their Sensitivity to the Cytokine TGF-ß.

Tissue-resident memory (Trm) CD8+ T cells mediate protective immunity in barrier tissues, but the cues promoting Trm cell generation are poorly understood. Sensing of extracellular adenosine triphosphate (eATP) by the purinergic receptor P2RX7 is needed for recirculating CD8+ T cell memory, but its role for Trm cells is unclear. Here we showed that P2RX7 supported Trm cell generation by enhancing CD8+ T cell sensing of TGF-ß, which was necessary for tissue residency. P2RX7-deficient Trm cells progressively decayed in non-lymphoid tissues and expressed dysregulated Trm-specific markers. P2RX7 was required for efficient re-expression of the receptor TGF-ßRII through calcineurin signaling. Forced Tgfbr2 expression rescued P2RX7-deficient Trm cell generation, and TGF-ß sensitivity was dictated by P2RX7 agonists and antagonists. Forced Tgfbr2 also rescued P2RX7-deficient Trm cell mitochondrial function. Sustained P2RX7 signaling was required for long-term Trm cell maintenance, indicating that P2RX7 signaling drives induction and CD8+ T cell durability in barrier sites.