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Resident memory T (T RM ) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that T RM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro T RM expansion to apply in an immunotherapy setting. However, it has also been shown that T RM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how T RM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that T RM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded T RM retained their phenotype, including the expression of key T RM markers CD69 and CD103 (ITGAE). The optimal culture of T RM required low O 2 pressure to maintain the expression of these and other T RM -associated molecules. Expanded T RM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed T RM profile retention, including expression of T RM -related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of T RM cells that maintain their T RM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of T RM ., (© 2024 The Authors. European Journal of Immunology published by Wiley‐VCH GmbH.)

Memory CD8 + T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8 + T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8 + T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8 + T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8 + T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8 + T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8 + T cells with superior effector functions., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Tissue-resident memory T cells (Trm) are retained in peripheral tissues after infection for enhanced protection against secondary encounter with the same pathogen. We have previously shown that the transcription factor Hobit and its homolog Blimp-1 drive Trm development after viral infection, but how and when these transcription factors mediate Trm formation remains poorly understood. In particular, the major impact of Blimp-1 in regulating several aspects of effector T-cell differentiation impairs study of its specific role in Trm development. Here, we used the restricted expression of Hobit in the Trm lineage to develop mice with a conditional deletion of Blimp-1 in Trm, allowing us to specifically investigate the role of both transcription factors in Trm differentiation. We found that Hobit and Blimp-1 were required for the upregulation of CD69 and suppression of CCR7 and S1PR1 on virus-specific Trm precursors after LCMV infection, underlining a role in their retention within tissues. The early impact of Hobit and Blimp-1 favored Trm formation and prevented the development of circulating memory T cells. Thus, our findings highlight a role of Hobit and Blimp-1 at the branching point of circulating and resident memory lineages by suppressing tissue egress of Trm precursors early during infection., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Tissue-resident memory T (T RM ) cells with potent antiviral and antibacterial functions protect the epithelial and mucosal surfaces of our bodies against infection with pathogens. The strong proinflammatory activities of T RM cells suggest requirement for expression of inhibitory molecules to restrain these memory T cells under steady state conditions. We previously identified the adhesion G protein-coupled receptor GPR56 as an inhibitory receptor of human cytotoxic lymphocytes that regulates their cytotoxic effector functions. Here, we explored the expression pattern, expression regulation, and function of GPR56 on pathogen-specific CD8 + T cells using two infection models. We observed that GPR56 is expressed on T RM cells during acute infection and is upregulated by the T RM cell-inducing cytokine TGF-β and the T RM cell-associated transcription factor Hobit. However, GPR56 appeared dispensable for CD8 + T-cell differentiation and function upon acute infection with LCMV as well as Listeria monocytogenes . Thus, T RM cells specifically acquire the inhibitory receptor GPR56, but the impact of this receptor on T RM cells after acute infection does not appear essential to regulate effector functions of T RM cells.

Innate lymphoid cells (ILCs) participate in tissue homeostasis, inflammation, and early immunity against infection. It is unclear how ILCs acquire effector function and whether these mechanisms differ between organs. Through multiplexed single-cell mRNA sequencing, we identified cKit + CD127 hi TCF-1 hi early differentiation stages of T-bet + ILC1s. These cells were present across different organs and had the potential to mature toward CD127 int TCF-1 int and CD127 - TCF-1 - ILC1s. Paralleling a gradual loss of TCF-1, differentiating ILC1s forfeited their expansion potential while increasing expression of effector molecules, reminiscent of T cell differentiation in secondary lymphoid organs. The transcription factor Hobit was induced in TCF-1 hi ILC1s and was required for their effector differentiation. These findings reveal sequential mechanisms of ILC1 lineage commitment and effector differentiation that are conserved across tissues. Our analyses suggest that ILC1s emerge as TCF-1 hi cells in the periphery and acquire a spectrum of organ-specific effector phenotypes through a uniform Hobit-dependent differentiation pathway driven by local cues., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Tissue-resident memory CD8 + T cells (T RM ) constitute a noncirculating memory T cell subset that provides early protection against reinfection. However, how T RM arise from antigen-triggered T cells has remained unclear. Exploiting the T RM -restricted expression of Hobit, we used T RM reporter/deleter mice to study T RM differentiation. We found that Hobit was up-regulated in a subset of LCMV-specific CD8 + T cells located within peripheral tissues during the effector phase of the immune response. These Hobit + effector T cells were identified as T RM precursors, given that their depletion substantially decreased T RM development but not the formation of circulating memory T cells. Adoptive transfer experiments of Hobit + effector T cells corroborated their biased contribution to the T RM lineage. Transcriptional profiling of Hobit + effector T cells underlined the early establishment of T RM properties including down-regulation of tissue exit receptors and up-regulation of T RM -associated molecules. We identified Eomes as a key factor instructing the early bifurcation of circulating and resident lineages. These findings establish that commitment of T RM occurs early in antigen-driven T cell differentiation and reveal the molecular mechanisms underlying this differentiation pathway., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2020 Wiley-VCH GmbH.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4 + T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients., (© 2020 Wiley-VCH GmbH.)

Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tissue-resident memory CD8 + T cells (T RM cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall responses is unclear. We developed a unique lineage tracer mouse model exploiting the T RM -defining transcription factor homolog of Blimp-1 in T cells (Hobit) to fate map the T RM progeny in secondary responses. After reinfection, a sizeable fraction of secondary memory T cells in the circulation developed downstream of T RM cells. These tissue-experienced ex-T RM cells shared phenotypic properties with the effector memory T cell population but were transcriptionally and functionally distinct from other secondary effector memory T cell cells. Adoptive transfer experiments of T RM cells corroborated their potential to form circulating effector and memory cells during recall responses. Moreover, specific ablation of primary T RM cell populations substantially impaired the secondary T cell response, both locally and systemically. Thus, T RM cells retain developmental plasticity and shape both local and systemic T cell responses on reinfection.

Tissue-resident memory CD8 + T (T RM ) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8 + T RM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8 + T cells. In contrast to CD8 + T RM cells at these sites, CD8 + T RM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8 + T RM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8 + T RM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8 + T RM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8 + T RM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8 + T RM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8 + T RM cells and inhibited the differentiation of central memory CD8 + T (T CM ) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8 + T RM cells in the lungs, potentially through control of the lineage choice between T CM and T RM cells during the differentiation of influenza-specific CD8 + T cells.

Tissue-resident memory T cells (T RM ) are noncirculating immune cells that contribute to the first line of local defense against reinfections. Their location at hotspots of pathogen encounter frequently exposes T RM to tissue damage. This history of danger-signal exposure is an important aspect of T RM -mediated immunity that has been overlooked so far. RNA profiling revealed that T RM from liver and small intestine express P2RX7, a damage/danger-associated molecular pattern (DAMP) receptor that is triggered by extracellular nucleotides (ATP, NAD + ). We confirmed that P2RX7 protein was expressed in CD8 + T RM but not in circulating T cells (T CIRC ) across different infection models. Tissue damage induced during routine isolation of liver lymphocytes led to P2RX7 activation and resulted in selective cell death of T RM P2RX7 activation in vivo by exogenous NAD + led to a specific depletion of T RM while retaining T CIRC The effect was absent in P2RX7-deficient mice and after P2RX7 blockade. TCR triggering down-regulated P2RX7 expression and made T RM resistant to NAD-induced cell death. Physiological triggering of P2RX7 by sterile tissue damage during acetaminophen-induced liver injury led to a loss of previously acquired pathogen-specific local T RM in wild-type but not in P2RX7 KO T cells. Our results highlight P2RX7-mediated signaling as a critical pathway for the regulation of T RM maintenance. Extracellular nucleotides released during infection and tissue damage could deplete T RM locally and free niches for new and infection-relevant specificities. This suggests that the recognition of tissue damage promotes persistence of antigen-specific over bystander T RM in the tissue niche., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA + effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)