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Cytomegalovirus (CMV) is one of the most common and relevant opportunistic pathogens in people who are immunocompromised, such as kidney transplant recipients (KTRs). The exact mechanisms underlying the disability of cytotoxic T cells to provide sufficient protection against CMV in people who are immunosuppressed have not been identified yet. Here, we performed in-depth metabolic profiling of CMV-specific CD8+ T cells in patients who are immunocompromised and show the development of metabolic dysregulation at the transcriptional, protein, and functional level of CMV-specific CD8+ T cells in KTRs with noncontrolled CMV infection. These dysregulations comprise impaired glycolysis and increased mitochondrial stress, which is associated with an intensified expression of the nicotinamide adenine dinucleotide nucleotidase (NADase) CD38. Inhibiting NADase activity of CD38 reinvigorated the metabolism and improved cytokine production of CMV-specific CD8+ T cells. These findings were corroborated in a mouse model of CMV infection under conditions of immunosuppression. Thus, dysregulated metabolic states of CD8+ T cells could be targeted by inhibiting CD38 to reverse hyporesponsiveness in individuals who fail to control chronic viral infection.

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.)

E-cadherin is a key regulator of epithelial cell-cell adhesion, the loss of which accelerates tumor growth and invasion. E-cadherin is also expressed in hematopoietic cells as well as epithelia. The function of hematopoietic E-cadherin is, however, mostly elusive. In this study, we explored the validity of mouse models to functionally investigate the role of hematopoietic E-cadherin in human hematopoiesis. We generated a hematopoietic-specific E-cadherin knockout mouse model. In mice, hematopoietic E-cadherin is predominantly expressed within the basophil lineage, the expression of which is dispensable for the generation of basophils. However, neither E-cadherin mRNA nor protein were detected in human basophils. In contrast, human hematopoietic E-cadherin marks the erythroid lineage. E-cadherin expression in hematopoiesis thereby revealed striking evolutionary differences between the basophil and erythroid cell lineage in humans and mice. This is remarkable as E-cadherin expression in epithelia is highly conserved among vertebrates including humans and mice. Our study therefore revealed that the mouse does not represent a suitable model to study the function of E-cadherin in human hematopoiesis and an alternative means to study the role of E-cadherin in human erythropoiesis needs to be developed.

The PD-L1/2-PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often poorly defined. In this study, using genome-wide CRISPR activation screening, we identified three factors that upregulate PD-L1 expression: GATA2, MBD6, and transcription cofactor vestigial-like protein 3 (VGLL3). VGLL3 acts as a transcriptional regulator, and its expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN-γ-induced PD-L1/2 expression in human keratinocytes. Mechanistically, by performing a second screen to identify proteins acting in concert with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional complex controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of proinflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8 + T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8 + T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8 + effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8 + T cell response leading to protection against otherwise lethal SARS-CoV-2 infection., (© 2022. The Author(s).)

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 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 T (T RM ) cells mediate potent local innate and adaptive immune responses and provide long-lasting protective immunity. T RM cells localize to many different tissues, including barrier tissues, and play a crucial role in protection against infectious and malignant disease. The formation and maintenance of T RM cells are influenced by numerous factors, including inflammation, antigen triggering, and tissue-specific cues. Emerging evidence suggests that these signals also contribute to heterogeneity within the T RM cell compartment. Here, we review the phenotypic and functional heterogeneity of CD8 + T RM cells at different tissue sites and the molecular determinants defining CD8 + T RM cell subsets. We further discuss the possibilities of targeting the unique cell surface molecules, cytokine and chemokine receptors, transcription factors, and metabolic features of T RM cells for therapeutic purposes. Their crucial role in immune protection and their location at the frontlines of the immune defense make T RM cells attractive therapeutic targets. A better understanding of the possibilities to selectively modulate T RM cell populations may thus improve vaccination and immunotherapeutic strategies employing these potent immune cells.

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.)

Background: Adenoviral vectors emerged as important platforms for cancer immunotherapy. Vaccination with adenoviral vectors is promising in this respect, however, their specific mechanisms of action are not fully understood. Here, we assessed the development and maintenance of vaccine-induced tumor-specific CD8 + T cells elicited upon immunization with adenoviral vectors., Methods: Adenoviral vaccine vectors encoding the full-length E7 protein from human papilloma virus (HPV) or the immunodominant epitope from E7 were generated, and mice were immunized intravenously with different quantities (10 7 , 10 8 or 10 9 infectious units). The magnitude, kinetics and tumor protection capacity of the induced vaccine-specific T cell responses were evaluated., Results: The adenoviral vaccines elicited inflationary E7-specific memory CD8 + T cell responses in a dose-dependent manner. The magnitude of these vaccine-specific CD8 + T cells in the circulation related to the development of E7-specific CD8 + tissue-resident memory T (T RM ) cells, which were maintained for months in multiple tissues after vaccination. The vaccine-specific CD8 + T cell responses conferred long-term protection against HPV-induced carcinomas in the skin and liver, and this protection required the induction and accumulation of CD8 + T RM cells. Moreover, the formation of CD8 + T RM cells could be enhanced by temporal targeting CD80/CD86 costimulatory interactions via CTLA-4 blockade early after immunization., Conclusions: Together, these data show that adenoviral vector-induced CD8 + T cell inflation promotes protective T RM cell populations, and this can be enhanced by targeting CTLA-4., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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.)

A fundamental benefit of immunological memory is the ability to respond in an enhanced manner upon secondary encounter with the same pathogen. Tissue-resident memory CD8 T (T RM ) cells contribute to improved protection against reinfection through the generation of immediate effector responses at the site of pathogen entry. Key to the potential of T RM cells to develop rapid recall responses is their location within the epithelia of the skin, lungs, and intestines at prime entry sites of pathogens. T RM cells are among the first immune cells to respond to pathogens that have been previously encountered in an antigen-specific manner. Upon recognition of invading pathogens, T RM cells release IFN-γ and other pro-inflammatory cytokines and chemokines. These effector molecules activate the surrounding epithelial tissue and recruit other immune cells including natural killer (NK) cells, B cells, and circulating memory CD8 T cells to the site of infection. The repertoire of T RM effector functions also includes the direct lysis of infected cells through the release of cytotoxic molecules such as perforin and granzymes. The mechanisms enabling T RM cells to respond in such a rapid manner are gradually being uncovered. In this review, we will address the signals that instruct T RM generation and maintenance as well as the underlying transcriptional network that keeps T RM cells in a deployment-ready modus. Furthermore, we will discuss how T RM cells respond to reinfection of the tissue and how transcription factors may control immediate and proliferative T RM responses.

Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations., (Copyright © 2016, American Association for the Advancement of Science.)