The adhesion G protein-coupled receptor GPR56/ADGRG1 in cytotoxic lymphocytes.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor connected to brain development, haematopoiesis, male fertility, and tumorigenesis. Nevertheless, expression of GPR56 is not restricted to developmental processes. Studies over the last years have demonstrated a marked presence of GPR56 in human cytotoxic NK and T cells. Expression of GPR56 in these cells is driven by the transcription factor HOBIT, corresponds with the production of cytolytic mediators and the presence of CX3 CR1 and CD57, indicates a state of terminal differentiation and cellular exhaustion, and disappears upon cellular activation. Functional studies indicate that GPR56 regulates cell migration and effector functions and thereby acts as an inhibitory immune checkpoint. We here discuss the current state of knowledge regarding GPR56 in cytotoxic lymphocytes.

Memory CD8+ T cells upregulate glycolysis and effector functions under limiting oxygen conditions.

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.

Hyperglycemia and Not Hyperinsulinemia Mediates Diabetes-Induced Memory CD8 T-Cell Dysfunction.

Type 2 diabetes (T2D) causes an increased risk of morbidity and mortality in response to viral infection. T2D is characterized by hyperglycemia and is typically associated with insulin resistance and compensatory hyperinsulinemia. CD8 T cells express the insulin receptor, and previously, we have shown that insulin is able to directly modulate effector CD8 T-cell function. We therefore hypothesized that memory CD8 T-cell responsiveness in the context of T2D is negatively impacted by hyperinsulinemia or hyperglycemia. Using a mouse model for T2D, we could show that memory CD8 T-cell function was significantly reduced in response to rechallenge by viral infection or with melanoma cells. Basal insulin injection of mice increased GLUT-1 expression and glucose uptake in memory CD8 T-cell precursors early after infection, which was prevented when these cells were deficient for the insulin receptor. However, neither insulin injection nor insulin receptor deficiency resulted in a difference in metabolism, memory formation, cytokine production, or recall responses of memory CD8 T cells compared with controls. Importantly, in context of obesity, insulin receptor deficiency on CD8 T cells did not affect the functional capacity of memory CD8 T cells. In contrast, we could show in vitro and in vivo that hyperglycemia significantly impairs the antiviral capacity of memory CD8 T cells. Our findings indicate that obesity impairs the memory CD8 T-cell response against viral infection and cancer through the detrimental effects of hyperglycemia rather than hyperinsulinemia.

Tissue-resident memory T cells in the urogenital tract.

Our understanding of T cell memory responses changed drastically with the discovery that specialized T cell memory populations reside within peripheral tissues at key pathogen entry sites. These tissue-resident memory T (TRM) cells can respond promptly to an infection without the need for migration, proliferation or differentiation. This rapid and local deployment of effector functions maximizes the ability of TRM cells to eliminate pathogens. TRM cells do not circulate through peripheral tissues but instead form isolated populations in the skin, gut, liver, kidneys, the reproductive tract and other organs. This long-term retention in the periphery might allow TRM cells to fully adapt to the local conditions of their environment and mount customized responses to counter infection and tumour growth in a tissue-specific manner. In the urogenital tract, TRM cells must adapt to a unique microenvironment to confer protection against potential threats, including cancer and infection, while preventing the onset of auto-inflammatory disease. In this Review, we discuss insights into the diversification of TRM cells from other memory T cell lineages, the adaptations of TRM cells to their local environment, and their enhanced capacity to counter infection and tumour growth compared with other memory T cell populations, especially in the urogenital tract.

The Inhibitory Receptor GPR56 (Adgrg1) Is Specifically Expressed by Tissue-Resident Memory T Cells in Mice But Dispensable for Their Differentiation and Function In Vivo.

Tissue-resident memory T (TRM) 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 TRM 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 TRM cells during acute infection and is upregulated by the TRM cell-inducing cytokine TGF-ß and the TRM 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, TRM cells specifically acquire the inhibitory receptor GPR56, but the impact of this receptor on TRM cells after acute infection does not appear essential to regulate effector functions of TRM cells.

The Potential of Tissue-Resident Memory T Cells for Adoptive Immunotherapy against Cancer.

Tissue-resident memory T cells (TRM) comprise an important memory T cell subset that mediates local protection upon pathogen re-encounter. TRM populations preferentially localize at entry sites of pathogens, including epithelia of the skin, lungs and intestine, but have also been observed in secondary lymphoid tissue, brain, liver and kidney. More recently, memory T cells characterized as TRM have also been identified in tumors, including but not limited to melanoma, lung carcinoma, cervical carcinoma, gastric carcinoma and ovarian carcinoma. The presence of these memory T cells has been strongly associated with favorable clinical outcomes, which has generated an interest in targeting TRM cells to improve immunotherapy of cancer patients. Nevertheless, intratumoral TRM have also been found to express checkpoint inhibitory receptors, such as PD-1 and LAG-3. Triggering of such inhibitory receptors could induce dysfunction, often referred to as exhaustion, which may limit the effectiveness of TRM in countering tumor growth. A better understanding of the differentiation and function of TRM in tumor settings is crucial to deploy these memory T cells in future treatment options of cancer patients. The purpose of this review is to provide the current status of an important cancer immunotherapy known as TIL therapy, insight into the role of TRM in the context of antitumor immunity, and the challenges and opportunities to exploit these cells for TIL therapy to ultimately improve cancer treatment.

T-cell memory in tissues.

Immunological memory equips our immune system to respond faster and more effectively against reinfections. This acquired immunity was originally attributed to long-lived, memory T and B cells with body wide access to peripheral and secondary lymphoid tissues. In recent years, it has been realized that both innate and adaptive immunity to a large degree depends on resident immune cells that act locally in barrier tissues including tissue-resident memory T cells (Trm). Here, we will discuss the phenotype of these Trm in mice and humans, the tissues and niches that support them, and their function, plasticity, and transcriptional control. Their unique properties enable Trm to achieve long-lived immunological memory that can be deposited in nearly every organ in response to acute and persistent infection, and in response to cancer. However, Trm may also induce substantial immunopathology in allergic and autoimmune disease if their actions remain unchecked. Therefore, inhibitory and activating stimuli appear to balance the actions of Trm to ensure rapid proinflammatory responses upon infection and to prevent damage to host tissues under steady state conditions.

Peripheral and systemic antigens elicit an expandable pool of resident memory CD8+ T cells in the bone marrow.

BM has been put forward as a major reservoir for memory CD8+  T cells. In order to fulfill that function, BM should "store" memory CD8+ T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8+ T (TRM ) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8+ TRM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8+ TRM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8+ T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8+ T cells, which develop in response to a large variety of peripheral antigens.

Tissue-resident memory T cells at the center of immunity to solid tumors.

Immune responses in tissues are constrained by the physiological properties of the tissue involved. Tissue-resident memory T cells (TRM cells) are a recently discovered lineage of T cells specialized for life and function within tissues. Emerging evidence has shown that TRM cells have a special role in the control of solid tumors. A high frequency of TRM cells in tumors correlates with favorable disease progression in patients with cancer, and studies of mice have shown that TRM cells are necessary for optimal immunological control of solid tumors. Here we describe what defines TRM cells as a separate lineage and how these cells are generated. Furthermore, we discuss the properties that allow TRM cells to operate in normal and transformed tissues, as well as implications for the treatment of patients with cancer.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

Molecular characterization of HCMV-specific immune responses: Parallels between CD8(+) T cells, CD4(+) T cells, and NK cells.

CD8(+) T cells are important for immunity against human cytomegalovirus (HCMV). The HCMV-specific CD8(+) T-cell response is characterized by the accumulation of terminally differentiated effector cells that have downregulated the costimulatory molecules CD27 and CD28. These HCMV-specific CD8(+) T cells maintain high levels of cytotoxic molecules such as granzyme B and rapidly produce the inflammatory cytokine IFN-γ upon activation. Remarkably, HCMV-specific CD8(+) T cells are able to persist long term as fully functional effector cells, suggesting a unique differentiation pathway that is distinct from the formation of memory CD8(+) T cells after infection with acute viruses. In this review, we aim to highlight the most recent developments in HCMV-specific CD8(+) T-cell differentiation, maintenance, tissue distribution, metabolism and function. HCMV also induces the differentiation of effector CD4(+) T cells and NK cells, which share characteristics with HCMV-specific CD8(+) T cells. We propose that the overlap in differentiation of NK cells, CD4(+) and CD8(+) T cells after HCMV infection may be regulated by a shared transcriptional machinery. A better understanding of the molecular framework of HCMV-specific CD8(+) T-cell responses may benefit vaccine design, as these cells uniquely combine the capacity to rapidly respond to infection with long-term survival.

Enhanced CD8 T cell responses through GITR-mediated costimulation resolve chronic viral infection.

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

Pro-apoptotic protein Noxa regulates memory T cell population size and protects against lethal immunopathology.

Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple clones with slightly different affinities, thereby creating T cell memory with a certain degree of diversity. Currently, the mechanisms that control size, diversity, and cross-reactivity of the memory T cell pool are incompletely defined. Previously, we established a role for apoptosis, mediated by the BH3-only protein Noxa, in controlling diversity of the effector T cell population. This function might positively or negatively impact T cell memory in terms of function, pool size, and cross-reactivity during recall responses. Therefore, we investigated the role of Noxa in T cell memory during acute and chronic infections. Upon influenza infection, Noxa(-/-) mice generate a memory compartment of increased size and clonal diversity. Reinfection resulted in an increased recall response, whereas cross-reactive responses were impaired. Chronic infection of Noxa(-/-) mice with mouse CMV resulted in enhanced memory cell inflation, but no obvious pathology. In contrast, in a model of continuous, high-level T cell activation, reduced apoptosis of activated T cells rapidly led to severe organ pathology and premature death in Noxa-deficient mice. These results establish Noxa as an important regulator of the number of memory cells formed during infection. Chronic immune activation in the absence of Noxa leads to excessive accumulation of primed cells, which may result in severe pathology.

The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.

CD70-driven costimulation induces survival or Fas-mediated apoptosis of T cells depending on antigenic load.

Apoptosis plays an essential role in the removal of activated CD8 T cells that are no longer required during or postinfection. The Bim-dependent intrinsic pathway of apoptosis removes effector CD8 T cells upon clearance of viral infection, which is driven by withdrawal of growth factors. Binding of Fas ligand to Fas mediates activation-induced T cell death in vitro and cooperates with Bim to eliminate CD8 T cells during chronic infection in vivo, but it is less clear how this pathway of apoptosis is initiated. In this study, we show that the costimulatory TNFR CD27 provides a dual trigger that can enhance survival of CD8 T cells, but also removal of activated CD8 T cells through Fas-driven apoptosis. Using in vitro stimulation assays of murine T cells with cognate peptide, we show that CD27 increases T cell survival after stimulation with low doses of Ag, whereas CD27 induces Fas-driven T cell apoptosis after stimulation with high doses of Ag. In vivo, the impact of constitutive CD70-driven stimulation on the accumulation of memory and effector CD8 T cells is limited by Fas-driven apoptosis. Furthermore, introduction of CD70 signaling during acute infection with influenza virus induces Fas-dependent elimination of influenza-specific CD8 T cells. These findings suggest that CD27 suppresses its costimulatory effects on T cell survival through activation of Fas-driven T cell apoptosis to maintain T cell homeostasis during infection.

BH3-only protein Noxa regulates apoptosis in activated B cells and controls high-affinity antibody formation.

The efficiency of humoral immune responses depends on the selective outgrowth of B cells and plasma cells that produce high affinity antibodies. The factors responsible for affinity maturation of B cell clones in the germinal center (GC) have been well established but selection mechanisms that allow clones to enter the GC are largely unknown. Here we identify apoptosis, regulated by the proapoptotic BH3-only member Noxa (Pmaip1), as a critical factor for the selection of high-affinity clones during B cell expansion after antigen triggering. Noxa is induced in activated B cells, and its ablation provides a survival advantage both in vitro and in vivo. After immunization or influenza infection, Noxa(-/-) mice display enlarged GCs, in which B cells with reduced antigen affinity accumulate. As a consequence, Noxa(-/-) mice mount low affinity antibody responses compared with wild-type animals. Importantly, the low affinity responses correlate with increased immunoglobulin diversity, and cannot be corrected by booster immunization. Thus, normal elimination of low affinity cells favors outgrowth of the remaining high-affinity clones, and this is mandatory for the generation of proper antibody responses. Manipulation of this process may alter the breadth of antibody responses after immunization.

The costimulatory molecule CD27 maintains clonally diverse CD8(+) T cell responses of low antigen affinity to protect against viral variants.

CD70 and CD27 are costimulatory molecules that provide essential signals for the expansion and differentiation of CD8(+) T cells. Here, we show that CD27-driven costimulation lowered the threshold of T cell receptor activation on CD8(+) T cells and enabled responses against low-affinity antigens. Using influenza infection to study in vivo consequences, we found that CD27-driven costimulation promoted a CD8(+) T cell response of overall low affinity. These qualitative effects of CD27 on T cell responses were maintained into the memory phase. On a clonal level, CD27-driven costimulation established a higher degree of variety in memory CD8(+) T cells. The benefit became apparent when mice were reinfected, given that CD27 improved CD8(+) T cell responses against reinfection with viral variants, but not with identical virus. We propose that CD27-driven costimulation is a strategy to generate memory clones that have potential reactivity to a wide array of mutable pathogens.

Apoptosis threshold set by Noxa and Mcl-1 after T cell activation regulates competitive selection of high-affinity clones.

The adaptive immune system generates protective T cell responses via a poorly understood selection mechanism that favors expansion of clones with optimal affinity for antigen. Here we showed that upon T cell activation, the proapoptotic molecule Noxa (encoded by Pmaip1) and its antagonist Mcl-1 were induced. During an acute immune response against influenza or ovalbumin, Pmaip1(-/-) effector T cells displayed decreased antigen affinity and functionality. Molecular analysis of influenza-specific T cells revealed persistence of many subdominant clones in the Pmaip1(-/-) effector pool. When competing for low-affinity antigen, Pmaip1(-/-) TCR transgenic T cells had a survival advantage in vitro, resulting in increased numbers of effector cells in vivo. Mcl-1 protein stability was controlled by T cell receptor (TCR) affinity-dependent interleukin-2 signaling. These results establish a role for apoptosis early during T cell expansion, based on antigen-driven competition and survival of the fittest T cells.

Eosinophil differentiation in the bone marrow is inhibited by T cell-derived IFN-gamma.

To explore whether and how T cells can affect myelopoiesis, we investigated myeloid differentiation in a model for T cell-mediated immune activation. We found that CD70-transgenic (CD70TG) mice, which have elevated numbers of interferon-γ (IFN-γ)-producing effector T cells in the periphery and bone marrow, are almost devoid of eosinophilic granulocytes. Induction of allergic airway inflammation in these mice failed to induce eosinophilia as well as airway hyperresponsiveness. CD70TG mice also have strongly reduced numbers of eosinophil lineage-committed progenitors, whereas granulocyte/macrophage progenitors from these mice are unable to generate eosinophils in vitro. We found that granulocyte/macrophage progenitors express IFN-γR1 and that IFN-γ is sufficient to inhibit eosinophil differentiation of both murine and human progenitor cells in vitro. We demonstrate that inhibition of eosinophil development in CD70TG mice is IFN-γ-dependent and that T cell-derived IFN-γ is sufficient to inhibit eosinophil formation in vivo. Finally, we found that IFN-γ produced on anti-CD40 treatment and during viral infection can also suppress eosinophil formation in wild-type mice. These data demonstrate that IFN-γ inhibits the differentiation of myeloid progenitors to eosinophils, indicating that the adaptive immune system plays an important role in orchestrating the formation of the appropriate type of myeloid cells during immune activation.