TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes.

Introduction: Chronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function. Methods: The phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody. Results: We show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. Discussion: These results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes.

LAG-3, TIM-3, and TIGIT: Distinct functions in immune regulation.

LAG-3, TIM-3, and TIGIT comprise the next generation of immune checkpoint receptors being harnessed in the clinic. Although initially studied for their roles in restraining T cell responses, intense investigation over the last several years has started to pinpoint the unique functions of these molecules in other immune cell types. Understanding the distinct processes that these receptors regulate across immune cells and tissues will inform the clinical development and application of therapies that either antagonize or agonize these receptors, as well as the profile of potential tissue toxicity associated with their targeting. Here, we discuss the distinct functions of LAG-3, TIM-3, and TIGIT, including their contributions to the regulation of immune cells beyond T cells, their roles in disease, and the implications for their targeting in the clinic.

Ovarian cancer G protein-coupled receptor 1 deficiency exacerbates crystal deposition and kidney injury in oxalate nephropathy in female mice.

Ovarian cancer G protein-coupled receptor 1 (OGR1) (Gpr68) and G protein-coupled receptor 4 (GPR4) (Gpr4) are proton-activated G protein-coupled receptors that are stimulated upon increased extracellular acidity. These receptors have various physiological and pathophysiological roles in renal acid-base physiology, tissue inflammation, and fibrosis among others. Their function in injured renal tissue, however, remains mostly unclear. To address this, we investigated their role in crystalline nephropathy by increasing the oxalate intake of GPR4 KO and OGR1 KO mice. After 10 days of high-oxalate intake and 4 days of recovery, renal crystal content, histopathology, filtration function, and inflammation were assessed. While GPR4 deficiency did not show major alterations in disease progression, OGR1 KO mice had higher urinary calcium levels and exacerbated crystal accumulation accompanied by decreased creatinine clearance and urea excretion and a decreased presence of regulatory T (Treg) cells in kidney tissue. When lowering the severity of the kidney injury, OGR1 KO mice were more prone to develop crystalline nephropathy. In this setting, OGR1 KO mice displayed an increased activation of the immune system and a higher production of proinflammatory cytokines by T cells and macrophages. Taken together, in the acute setting of oxalate-induced nephropathy, the lack of the proton-activated G protein-coupled receptor (GPCR) GPR4 does not influence disease. OGR1 deficiency, however, increases crystal deposition leading to impaired kidney function. Thus, OGR1 may be important to limit kidney crystal deposition, which might subsequently be relevant for the pathophysiology of oxalate kidney stones or other crystallopathies.

Treg-selective IL-2 starvation synergizes with CD40 activation to sustain durable responses in lymphoma models.

BACKGROUND: Roughly half of all diffuse large B-cell lymphomas (DLBCLs) are infiltrated by large numbers of regulatory T-cells (Tregs). Although the presence of 'effector' Tregs in particular is associated with an inferior prognosis in patients on standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy, the role of this cell type during lymphoma initiation and progression is poorly understood. METHODS: Here, we use tissue microarrays containing prospectively collected DLBCL patient specimens, as well as data from publicly available cohorts to explore the mutational landscape of Treg-infiltrated DLBCL. We further take advantage of a model of MYC-driven lymphoma to mechanistically dissect the contribution of Tregs to lymphoma pathogenesis and to develop a strategy of Treg-selective interleukin-2 (IL-2) starvation to improve immune control of MYC-driven lymphoma. RESULTS: We find that all genetic DLBCL subtypes, except for one characterized by co-occurring MYD88/CD79 mutations, are heavily infiltrated by Tregs. Spectral flow cytometry and scRNA-sequencing reveal the robust expression of functional and immunosuppressive markers on Tregs infiltrating MYC-driven lymphomas; notably, we find that intratumoral Tregs arise due to local conversion from naïve CD4+ precursors on tumor contact. Treg ablation in Foxp3iDTR mice, or by antibody-mediated Treg-selective blockade of IL-2 signaling, strongly reduces the lymphoma burden. We identify lymphoma B-cells as a major source of IL-2, and show that the effects of Treg depletion are reversed by the simultaneous depletion of Foxp3-negative CD4+ T-cells, but not CD8+ T-cells or natural killer (NK) cells. The inhibition of ATP hydrolyzation and adenosine production by Tregs at least partly phenocopies the effects of Treg depletion. Treg depletion further synergizes with pro-apoptotic CD40 activation to sustain durable responses. CONCLUSION: The combined data implicate Tregs as a potential therapeutic target in DLBCL, especially in combination with other immunotherapies.

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity.

Adaptive immune repertoires are composed by the ensemble of B and T-cell receptors within an individual, reflecting both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Here, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following lymphocytic choriomeningitis virus (LCMV) infection, CD8+ T cells with binding specificity restricted to two distinct LCMV peptides, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T-cell inflation, and regulation. Together, this dataset provides a resource for immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

Nematode microRNAs can Individually Regulate Interferon Regulatory Factor 4 and mTOR in Differentiating T Helper 2 Lymphocytes and Modulate Cytokine Production in Macrophages.

Parasitic nematodes are masterful immunomodulators. This class of pathogens has evolved a spectrum of sophisticated strategies to regulate and evade host immune responses, mediated through the release of various molecules. In this context, the release of microRNAs (miRNAs), short post-transcriptional regulators of gene expression, has been of particular interest in the host-parasite interplay. Evidence that parasite-derived miRNAs modulate host innate and adaptive immune responses has become increasingly compelling. However, since miRNAs are usually contained in extracellular vesicles containing other mediators, it is difficult to assign an observed effect on host cells to miRNAs specifically. Here, the effects of some abundantly secreted miRNAs by nematodes used as models of gastrointestinal infections (Heligmosomoides polygyrus bakeri, Trichuris muris and Ascaris suum) were evaluated, addressing the potential of parasite miRNAs to impair in vitro differentiation of two important types of immune cells in the context of helminth infections, Th2 lymphocytes and macrophages. Mimicking a continuous exposure to low concentrations of nematode miRNAs, the interferon gamma signaling, the IL-2/STAT5 signaling, and the mTOR signaling pathways were identified as downregulated by Hpo-miR-71-5p. Interferon regulatory factor 4 (Irf4) was validated as a target of Hpo-miR-71-5p, while Mtor is targeted by Asu-miR-791-3p, abundant in the T. muris secretions. By trend, Hpo-miR-71-5p impacts mildly but consistently on the amounts of inflammatory cytokines in unpolarized macrophages but leads to slightly increased IL-10 level in alternatively activated cells. In addition, our data suggests that transfected miRNAs remain for days in recipient cells, and that Hpo-miR-71-5p can incorporate into mouse Argonaute protein complexes. Nematode miRNAs can impair both innate and adaptive arms of host immunity. Hpo-miR-71-5p in particular, absent in mammals, interacts with host genes and pathways with crucial involvement in anthelmintic immune responses. This report brings new insights into the dynamics of miRNA-driven immunomodulation and highlights putative targeted pathways. Although the absolute repression is subtle, it is expected that the dozens of different miRNAs released by nematodes may have a synergistic effect on surrounding host cells.

Moving to the Outskirts: Interplay Between Regulatory T Cells and Peripheral Tissues.

Regulatory T cells (Tregs) restrain excessive immune responses and dampen inflammation. In addition to this classical immune suppressive role, Tregs in non-lymphoid tissues also promote tissue homeostasis, regeneration and repair. In this review, we outline our current understanding of how Tregs migrate to peripheral tissues and the factors required for their maintenance at these sites. We discuss the tissue-specific adaptations of Tregs at barrier and immuno-privileged sites and the mechanisms that regulate their function within these organs. Furthermore, we outline what is known about the interactions of Tregs with non-immune cells in the different peripheral tissues at steady state and upon challenge or tissue damage. A thorough understanding of the tissue-specific adaptations and functions of Tregs will potentially pave the way for therapeutic approaches targeting their regenerative role.

Alkali therapy protects renal function, suppresses inflammation, and improves cellular metabolism in kidney disease.

Chronic kidney disease (CKD) affects approximately 10-13% of the population worldwide and halting its progression is a major clinical challenge. Metabolic acidosis is both a consequence and a possible driver of CKD progression. Alkali therapy counteracts these effects in CKD patients, but underlying mechanisms remain incompletely understood. Here we show that bicarbonate supplementation protected renal function in a murine CKD model induced by an oxalate-rich diet. Alkali therapy had no effect on the aldosterone-endothelin axis but promoted levels of the anti-aging protein klotho; moreover, it suppressed adhesion molecules required for immune cell invasion along with reducing T-helper cell and inflammatory monocyte invasion. Comparing transcriptomes from the murine crystallopathy model and from human biopsies of kidney transplant recipients (KTRs) suffering from acidosis with or without alkali therapy unveils parallel transcriptome responses mainly associated with lipid metabolism and oxidoreductase activity. Our data reveal novel pathways associated with acidosis in kidney disease and sensitive to alkali therapy and identifies potential targets through which alkali therapy may act on CKD and that may be amenable for more targeted therapies.

Dampening antiviral immunity can protect the host.

Viral infections are very common, and in most cases, the virus is well controlled and eliminated by the immune system. Nevertheless, in some cases, damage of the host tissue inflicted by the virus itself or by the elicited immune response may result in severe disease courses. Thus, regulatory mechanisms are necessary to control virus-induced and immune pathology. This ensures immune responses are elicited in a potent but controlled manner. In this review, we will outline how immune regulation may contribute to this process. We focus on regulatory T cells and co-inhibitory receptors and outline how these two regulatory immune components allow for and may even promote potent but not pathologic immune responses. By enabling a balanced immune response, regulatory mechanisms can thus contribute to pathogen control as well as tissue and host protection.

Asymmetric cell division shapes naive and virtual memory T-cell immunity during ageing.

Efficient immune responses rely on heterogeneity, which in CD8+ T cells, amongst other mechanisms, is achieved by asymmetric cell division (ACD). Here we find that ageing, known to negatively impact immune responses, impairs ACD in murine CD8+ T cells, and that this phenotype can be rescued by transient mTOR inhibition. Increased ACD rates in mitotic cells from aged mice restore the expansion and memory potential of their cellular progenies. Further characterization of the composition of CD8+ T cells reveals that virtual memory cells (TVM cells), which accumulate during ageing, have a unique proliferation and metabolic profile, and retain their ability to divide asymmetrically, which correlates with increased memory potential. The opposite is observed for naive CD8+ T cells from aged mice. Our data provide evidence on how ACD modulation contributes to long-term survival and function of T cells during ageing, offering new insights into how the immune system adapts to ageing.

CD85k Contributes to Regulatory T Cell Function in Chronic Viral Infections.

Regulatory T cells (Tregs) prevent excessive immune responses and limit immune pathology upon infections. To fulfill this role in different immune environments elicited by different types of pathogens, Tregs undergo functional specialization into distinct subsets. During acute type 1 immune responses, type 1 Tregs are induced and recruited to the site of ongoing Th1 responses to efficiently control Th1 responses. However, whether a similar specialization process also takes place following chronic infections is still unknown. In this study, we investigated Treg specialization in persistent viral infections using lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV) infection as models for chronic and latent infections, respectively. We identify CD85k as a Th1-specific co-inhibitory receptor with sustained expression in persistent viral infections and show that recombinant CD85k inhibits LCMV-specific effector T cells. Furthermore, expression of the CD85k ligand ALCAM is induced on LCMV-specific and exhausted T cells during chronic LCMV infection. Finally, we demonstrate that type 1 Tregs arising during chronic LCMV infection suppress Th1 effector cells in an ALCAM-dependent manner. These results extend the current knowledge of Treg specialization from acute to persistent viral infections and reveal an important functional role of CD85k in Treg-mediated suppression of type 1 immunity.

Checkpoint Receptor TIGIT Expressed on Tim-1+ B Cells Regulates Tissue Inflammation.

Tim-1, a phosphatidylserine receptor expressed on B cells, induces interleukin 10 (IL-10) production by sensing apoptotic cells. Here we show that mice with B cell-specific Tim-1 deletion develop tissue inflammation in multiple organs including spontaneous paralysis with inflammation in the central nervous system (CNS). Transcriptomic analysis demonstrates that besides IL-10, Tim-1+ B cells also differentially express a number of co-inhibitory checkpoint receptors including TIGIT. Mice with B cell-specific TIGIT deletion develop spontaneous paralysis with CNS inflammation, but with limited inflammation in other organs. Our findings suggest that Tim-1+ B cells are essential for maintaining self-tolerance and restraining tissue inflammation, and that Tim-1 signaling-dependent TIGIT expression on B cells is essential for maintaining CNS-specific tolerance. A possible critical role of aryl hydrocarbon receptor (AhR) in regulating the B cell function is discussed, as we find that AhR is among the preferentially expressed transcription factors in Tim-1+ B cells and regulates their TIGIT and IL-10 expression.

Preceding Viral Infections Do Not Imprint Long-Term Changes in Regulatory T Cell Function.

Regulatory T cells (Tregs) maintain peripheral self-tolerance and limit immune mediated pathology. Like effector T cells, Tregs can specialize in TH1-dominated immune responses and co-express T-bet together with Foxp3. This allows for expression of CXCR3 and efficient homing to sites of TH1 responses. However, whether such functional specialization is paralleled by memory generation among Tregs is unknown. In this study, we investigated the ability of polyclonal Tregs to form functional memory in response to viral infection. Using adoptive transfer models to compare infection-experienced Tregs generated upon acute Lymphocytic Choriomeningitis Virus (LCMV) WE and Vaccinia Virus (VV) infections with naive Tregs, we observed no differences in their phenotype or their in vivo maintenance. When comparing functional properties of infection-experienced and naive Tregs, we found no differences in in vitro suppressive capacity nor in their ability to limit the effector response upon homologous, systemic or local re-challenge in vivo. Our results suggest that no functional Treg memory is generated in the context of systemic LCMV or VV infection, but we cannot rule out the possibility that the generation of Treg memory may be possible in other contexts.

Rapid expansion of Treg cells protects from collateral colitis following a viral trigger.

Foxp3+ regulatory T (Treg) cells are essential for maintaining peripheral tolerance and preventing autoimmunity. While genetic factors may predispose for autoimmunity, additional environmental triggers, such as viral infections, are usually required to initiate the onset of disease. Here, we show that viral infection with LCMV results in type I IFN-dependent Treg cell loss that is rapidly compensated by the conversion and expansion of Vß5+ conventional T cells into iTreg cells. Using Vß5-deficient mice, we show that these Vß5+ iTreg cells are dispensable for limiting anti-viral immunity. Rather, the delayed replenishment of Treg cells in Vß5-deficient mice compromises suppression of microbiota-dependent activation of CD8+ T cells, resulting in colitis. Importantly, recovery from clinical symptoms in IBD patients is marked by expansion of the corresponding Vß2+ Treg population in humans. Collectively, we provide a link between a viral trigger and an impaired Treg cell compartment resulting in the initiation of immune pathology.

TIGIT limits immune pathology during viral infections.

Co-inhibitory pathways have a fundamental function in regulating T cell responses and control the balance between promoting efficient effector functions and restricting immune pathology. The TIGIT pathway has been implicated in promoting T cell dysfunction in chronic viral infection. Importantly, TIGIT signaling is functionally linked to IL-10 expression, which has an effect on both virus control and maintenance of tissue homeostasis. However, whether TIGIT has a function in viral persistence or limiting tissue pathology is unclear. Here we report that TIGIT modulation effectively alters the phenotype and cytokine profile of T cells during influenza and chronic LCMV infection, but does not affect virus control in vivo. Instead, TIGIT has an important effect in limiting immune pathology in peripheral organs by inducing IL-10. Our data therefore identify a function of TIGIT in limiting immune pathology that is independent of viral clearance.

Chronic viral infections impinge on naive bystander CD8 T cells.

INTRODUCTION: Epidemiological data suggest that persistent viral infections impair immune homeostasis and immune responsiveness. Previous studies showed that chronic virus infections negatively impact bystander T-cell differentiation and memory formation but there is limited knowledge of how chronic virus infections impinge on heterologous naive T-cell populations. METHODS: We used adoptive transfer of naive CD8 T cells with defined nonviral specificity into hosts, which were subsequently chronically infected with lymphocytic choriomeningitis virus, followed by analyses of numeric, phenotypic, and functional changes provoked in the chronically infected host. RESULTS: We demonstrate that chronic virus infections have a profound effect on the number and phenotype of naive bystander CD8 T cells. Moreover, primary expansion upon antigen encounter was severely compromised in chronically infected hosts. However, when naive bystander CD8 T cells were transferred from the chronically infected mice into naive hosts, they regained their expansion potential. Conversely, when chronically infected hosts were supplied with additional antigen-presenting cells (APCs), primary expansion of the naive CD8 T cells was restored to levels of the uninfected hosts. CONCLUSIONS: Our results document numeric, phenotypic, and functional adaptation of bystander naive CD8 T cells during nonrelated chronic viral infection. Their functional impairment was only evident in the chronically infected host, indicating that T-cell extrinsic factors, in particular the quality of priming APCs, are responsible for the impaired function of naive bystander T cells in the chronically infected hosts.

Role of Co-stimulatory Molecules in T Helper Cell Differentiation.

CD4+ T cells play a central role in orchestrating the immune response to a variety of pathogens but also regulate autoimmune responses, asthma, allergic responses, as well as tumor immunity. To cover this broad spectrum of responses, naïve CD4+ T cells differentiate into one of several lineages of T helper cells, including Th1, Th2, Th17, and TFH, as defined by their cytokine pattern and function. The fate decision of T helper cell differentiation integrates signals delivered through the T cell receptor, cytokine receptors, and the pattern of co-stimulatory signals received. In this review, we summarize the contribution of co-stimulatory and co-inhibitory receptors to the differentiation and maintenance of T helper cell responses.

Infection History Determines Susceptibility to Unrelated Diseases.

Epidemiological data suggest that previous infections can alter an individual's susceptibility to unrelated diseases. Nevertheless, the underlying mechanisms are not completely understood. Substantial research efforts have expanded the classical concept of immune memory to also include long-lasting changes in innate immunity and antigen-independent reactivation of adaptive immunity. Collectively, these processes provide possible explanations on how acute infections might induce long-term changes that also affect immunity to unrelated diseases. Here, we review lasting changes the immune compartment undergoes upon infection and how infection experience alters the responsiveness of immune cells towards universal signals. This heightened state of alert enhances the ability of the immune system to combat even unrelated infections but may also increase susceptibility to autoimmunity. At the same time, infection-induced changes in the regulatory compartment may dampen subsequent immune responses and promote pathogen persistence. The concepts presented here outline how infection-induced changes in the immune system may affect human health.

Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression.

Controlled immune activation in response to commensal microbes is critical for the maintenance of stable colonization and prevention of microbial overgrowth on epithelial surfaces. Our understanding of the host mechanisms that regulate bacterial commensalism has increased substantially, however, much less data exist regarding host responses to members of the fungal microbiota on colonized surfaces. Using a murine model of oropharyngeal candidiasis, we have recently shown that differences in immune activation in response to diverse natural isolates of Candida albicans are associated with different outcomes of the host-fungal interaction. Here we applied a genome-wide transcriptomic approach to show that rapid induction of a strong inflammatory response characterized by neutrophil-associated genes upon C. albicans colonization inversely correlated with the ability of the fungus to persist in the oral mucosa. Surprisingly, persistent fungal isolates showed no signs of a compensatory regulatory immune response. By combining RNA-seq data, genetic mouse models, and co-infection experiments, we show that attenuation of the inflammatory response at the onset of infection with a persistent isolate is not a consequence of enhanced immunosuppression. Importantly, depletion of regulatory T cells or deletion of the immunoregulatory cytokine IL-10 did not alter host-protective type 17 immunity nor did it impair fungal survival in the oral mucosa, indicating that persistence of C. albicans in the oral mucosa is not a consequence of suppressed antifungal immunity.