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Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Discovered 30 years ago, CD40L antagonists are proving to be powerful autoimmune drugs.

Immune checkpoints limit the activation of the immune system and serve an important homeostatic function but can also restrict immune responses against tumors. Inhibition of specific immune checkpoint proteins such as the B7:CD28 family members programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) has transformed the treatment of various cancers by promoting the anti-tumor activation of immune cells. In contrast to these effects, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) regulates the steady state of the resting immune system and promotes homeostasis by mechanisms distinct from PD-1 and CTLA-4. The effects of VISTA blockade have been shown to include a decrease in myeloid suppression coupled with proinflammatory changes by mechanisms that are separate and distinct from other immune checkpoint proteins; in some preclinical studies these immune effects appear synergistic. Given the potential benefits of VISTA blockade in the context of cancer therapy, the second Annual VISTA Symposium was convened virtually on September 23, 2022, to review new research from investigators and immuno-oncology experts. Discussions in the meeting extended the knowledge of VISTA biology and the effects of VISTA inhibition, particularly on cells of the myeloid lineage and resting T cells, as three candidate anti-VISTA antibodies are in, or nearing, clinical development., Competing Interests: JB-K is the Chief Scientific Officer at, and has ownership interest including patents in, Hummingbird Bioscience. TG is the Chief Scientific Officer at, and has ownership interest including patents in, Kineta Inc. SL receives research support from the NIH. LL has served on advisory boards and as a consultant for Curis, Inc. and has received research support from Curis, Inc; and reports additional research support from AbbVie, AstraZeneca, and Bristol Myers Squibb, has served on a data safety monitoring board for G1 Therapeutics, and is the vice chair of the pharmacogenomics and population pharmacology committee of the Alliance for Clinical Trials in Oncology. JLL has patents issued and licensed to Curis, Inc. through ImmuNext. KM has received research support from the United States Department of Defense, Kidney Cancer Association, NextPoint Therapeutics, and Bristol Myers Squibb. RM is Head of Research and Development and has ownership interest at Curis, Inc. DN receives research support from the NCI and NIDCR. RN is employed by ImmuNext, has served on advisory boards and as a consultant for Curis, Inc, and reports research support from the NIH. AS has received research support to his institution from Curis, Inc.; and additional research support to his institution from Medimmune, AVID, Telix Pharmaceuticals, ITM, Fusion, Cyclotek, Adalta, TheraMyc, Humanigen, Antengene, National Health and Medical Research Council, National Imaging Facility, National Breast Cancer Foundation, Medical Research Future Fund, Australian Cancer Research Foundation, Victorian Cancer Agency. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Noelle, Lines, Lewis, Martell, Guillaudeux, Lee, Mahoney, Vesely, Boyd-Kirkup, Nambiar and Scott.)

Radiation therapy (RT) has recently demonstrated promise at stimulating an enhanced immune response. The recent success of immunotherapies, such as checkpoint inhibitors, CART cells, and other immune modulators, affords new opportunities for combination with radiation. The aim of this study is to evaluate whether and to what extent blockade of VISTA, an immune checkpoint, can potentiate the tumor control ability of radiation therapy. Our study is novel in that it is the first comparison of two VISTA-blocking methods (antibody inhibition and genetic knockout) in combination with RT. VISTA was blocked either through genetic knockout (KO) or an inhibitory antibody and combined with RT in two syngeneic murine flank tumor models (B16 and MC38). Selected mRNA, immune cell infiltration, and tumor growth delay were used to assess the biological effects. When combined with a single 15Gy radiation dose, VISTA blockade via genetic knockout in the B16 model and via anti-VISTA antibodies in the MC38 model significantly improved survival compared to RT alone by an average of 5.5 days and 6.3 days, respectively ( p < 0.05). The gene expression data suggest that the mechanism behind the enhanced tumor control is primarily a result of increased apoptosis and immune-mediated cytotoxicity. VISTA blockade significantly enhances the anti-tumor effect of a single dose of 15Gy radiation through increased expression and stimulation of cell-mediated apoptosis pathways. These results suggest that VISTA is a biologically relevant immune promoter that has the potential to enhance the efficacy of a large single radiation dose in a synergic manner.

V-domain Ig suppressor of T-cell activation (VISTA) is a B7 family member that plays key roles in maintaining T cell quiescence and regulation of myeloid cell populations, which together establish it as a novel immunotherapy target for solid tumors. Here we review the growing literature on VISTA expression in relation to various malignancies to better understand the role of VISTA and its interactions with both tumor cells and immune cells expressing other checkpoint molecules within the tumor microenvironment (TME). The biology of VISTA creates several mechanisms to maintain the TME, including supporting the function of myeloid-derived suppressor cells, regulating natural killer cell activation, supporting the survival of regulatory T cells, limiting antigen presentation on antigen-presenting cells and maintaining T cells in a quiescent state. Understanding these mechanisms is an important foundation of rational patient selection for anti-VISTA therapy. We provide a general framework to describe distinct patterns of VISTA expression in correlation with other known predictive immunotherapy biomarkers (programmed cell death ligand 1 and tumor-infiltrating lymphocytes) across solid tumors to facilitate investigation of the most efficacious TMEs for VISTA-targeted treatment as a single agent and/or in combination with anti-programmed death 1/anti-cytotoxic T lymphocyte antigen-4 therapies., Competing Interests: AM reports research support from the NIH. MM has served on advisory boards and as a consultant for Curis, Inc. and for ImmuNext, and has intellectual property for VISTA antibodies. AU was employed by Curis, Inc. RR is employed by Curis, Inc. RN employed by ImmuNext, and has served on advisory boards and as a consultant for Curis, Inc, and reports research support from the NIH. LL has served on advisory boards and as a consultant for Curis, Inc. and has received research support from Curis, Inc; and reports additional research support from AbbVie, AstraZeneca, and Bristol Meyers Squibb, has served on a data safety monitoring board for G1 Therapeutics, and is the vice chair of the pharmacogenomics and population pharmacology committee of the Alliance for Clinical Trials in Oncology. MJ has received research support from Curis, Inc.; and reports additional research support to institution from AbbVie, Acerta, Adaptimmune, Amgen, Apexigen, Arcus Biosciences, Array BioPharma, Artios Pharma, AstraZeneca, Atreca, BeiGene, BerGenBio, BioAtla, Boehringer Ingeheim, Calithera Biosciences, Checkpoint Therapeutics, Corvus Pharmaceuticals, Cytomx, Daiichi Sankyo, Dracen Pharmaceuticals, Dynavax, Lilly, EMD Serono, Erasca, Exelixis, Fate Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Guardant Health, Harpoon, Helsinn Healthcare, Hengrui Therapeutics, Hutchison MediPharma, IDEAYA Biosciences, IGM Biosciences, Immunocore, Incyte, Janssen, Jounce Therapeutics, Kadmon Pharmaceuticals, Loxo Oncology, Lycera, Memorial Sloan-Kettering, Merck, Merus, NeoImmune Tech, Neovia Oncology, Novartis, Numab Therapeutics, Nuvalent, OncoMed Pharmaceuticals, Pfizer, PMV Pharmaceuticals, RasCal Therapeutics, Regeneron Pharmaceuticals, Relay Therapeutics, Revolution Medicine, Ribon Therapeutics, Rubius Therapeutics, Sanofi, Seven and Eight Biopharmaceuticals / Birdie Biopharmaceuticals, Shattuck Labs, Silicon Therapeutics, Stem CentRx, Syndax Pharmaceuticals, Takeda Pharmaceuticals, Tarveda, TCR2 Therapeutics, Tempest Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, Turning Point Therapeutics, University of Michigan, Vyriad, WindMIL, Y-mAbs Therapeutics, Black Diamond, Carisma Therapeutics, Elicio Therapeutics, EQRx, Impact, Kartos Therapeutics, Mirati Therapeutics, Palleon Pharmaceuticals, and Rain Therapeutics, and has served as a consultant for AbbVie, Amgen, Astellas, AstraZeneca, Axelia Oncology, Black Diamond, Calithera Biosciences, Checkpoint Therapeutics, CytomX Therapeutics, Daiichi Sankyo, EcoR1, Editas Medicine, Eisai, EMD Serono, G1 Therapeutics, Genentech/Roche, Genmab, Genocea Biosciences, GlaxoSmithKline, Gritstone Oncology, Ideaya Biosciences, iTeos, Janssen, Lilly, Merck, Mirati Therapeutics, Molecular Axiom, Novartis, Oncorus, Regeneron Pharmaceuticals, Ribon Therapeutics, Sanofi-Aventis, Turning Point Therapeutics, and VBL Therapeutics. LR has served on advisory boards and as a consultant for Curis, Inc. RM is employed by Curis, Inc. This manuscript received medical writing support funded by Curis, Inc. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martin, Molloy, Ugolkov, von Roemeling, Noelle, Lewis, Johnson, Radvanyi and Martell.)

The suppressive function of T-regulatory cells (Tregs) can have a detrimental effect on immune responses against tumor cells. Within the Treg cells subset, a new non-classical population has been reported, which expresses high levels of CD49b molecule and, depending on their activation status, can also express the canonical Tregs transcription factor Foxp3. In this report, we sought to characterize Tregs subsets in a murine melanoma model and disrupt the CD49b/CD29 axis by administering an anti-CD29 antibody in tumor-bearing mice. Our data shows that whereas in the draining lymph nodes, the Tr1 cells subset composes <5% of CD4+ T cells, in the tumor, they reach ∼30% of CD4+ T cells. Furthermore, Tr1 cells share the expression of suppressive molecules, such as Nrp-1, PD-1, and CD73, which are highly expressed on Tr1 cells found in tumor-infiltrating leukocytes (TILs). Regardless of the phenotypic similarities with cTreg cells, Tr1 cells display a low proliferative activity, as shown in the kinetics and the incorporation of 5-bromodeoxyuridine (BrdU) experiments. With the intent to impact on Tr1 cells, we administered anti-CD29 antibody into tumor mice, observing that the treatment effectively inhibits tumor growth. This effect is at least mediated by the enrichment of pro-inflammatory T cells, including IFN-γ+ cTreg and IFN-γ+ Tr1 cells (with reduced expression of IL-10), plus Th1 and Tc cells. In this study, we present Tr1 cell characterization in tumor-bearing animals and introduce CD29 as a target for tumor therapy, supported by a meta-analysis indicating that CD29 is present in human biopsies., Competing Interests: Author FA-M was employed by Cells for Cells. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Contreras-Kallens, Gálvez-Jirón, De Solminihac, Elhusseiny, González-Arriagada, Alcayaga-Miranda, Noelle and Pino-Lagos.)

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner., (© 2022. The Author(s), under exclusive licence to CSI and USTC.)

Background and Objectives: Costimulation by CD40 and its ligand CD40L (CD154) is important for the functional differentiation of T cells. Preclinical studies have recognized the importance of this costimulatory interaction in the pathogenesis of experimental models of multiple sclerosis (MS). To determine safety, pharmacokinetics, and immune effect of a humanized monoclonal antibody (mAb) against CD40 ligand (toralizumab/IDEC-131) in patients with relapsing-remitting MS (RRMS)., Methods: This single-institution open-label dose-escalation study (phase I) enrolled 12 patients with RRMS to receive 4 doses of 1, 5, 10, or 15 mg/kg of humanized αCD40L (toralizumab) IV infusion every other week. Patients were followed up to 18 weeks, annually, and finally at 5 years. In addition to safety and pharmacokinetics, other secondary and exploratory measurements are immune effects, clinical, MRI, laboratory, and neuropsychological evaluations., Results: Fifteen adverse events, all of mild to moderate severity, were considered to be of possible or of unknown relationship to treatment. No serious adverse events, including thromboembolic events, occurred during the 18-week defined study period. Annual and long-term follow-up at 5 years revealed no delayed toxicity. Pharmacokinetics were nonlinear between the 5 and 10 mg/kg dose groups. The serum half-life of toralizumab was consistent between the dose groups with a mean of 15.3 days (SD = 1.9). Flow cytometry revealed no depletion of lymphocyte subsets. An increase in the CD25+/CD3+ and CD25+/CD4+ ratio and a shift toward an anti-inflammatory cytokine response were seen after treatment., Discussion: Our study suggests that blocking CD40L is safe and well tolerated in patients with RRMS while increasing CD25 + T cells and anti-inflammatory cytokine profile. These findings support further studies to assess the efficacy of blocking CD40L as a potential treatment of RRMS., Classification of Evidence: This study provides Class IV evidence on the safety, pharmacokinetics, and immune effects of an mAb to CD40L in patients with RRMS., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) that is involved in T-cell quiescence, inhibition of T-cell activation, and in myeloid cells regulates cytokine production, chemotaxis, phagocytosis, and tolerance induction. In the central nervous system (CNS), VISTA is expressed by microglia, the resident macrophage of the parenchyma, and expression is decreased during neuroinflammation; however, the function of VISTA in microglia is unknown. Here, we extensively analyzed VISTA expression in different MS lesion stages and characterized the function of VISTA in the CNS by deleting VISTA in microglia. VISTA is differentially expressed in distinct MS lesion stages. In mice, VISTA deletion in Cx3Cr1-expressing cells induced a more amoeboid microglia morphology, indicating an immune-activated phenotype. Expression of genes associated with cell cycle and immune-activation was increased in VISTA KO microglia. In response to LPS and during experimental autoimmune encephalomyelitis (EAE), VISTA KO and WT microglia shared similar transcriptional profiles and VISTA deletion did not affect EAE disease progression or microglia responses. VISTA KO in microglia in vitro decreased the uptake of myelin. This study demonstrates that VISTA is involved in microglia function, which likely affects healthy CNS homeostasis and neuroinflammation.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity., Competing Interests: The authors have declared that no competing interests exist.

Following their exit from the thymus, T cells are endowed with potent effector functions but must spare host tissue from harm. The fate of these cells is dictated by a series of checkpoints that regulate the quality and magnitude of T cell-mediated immunity, known as tolerance checkpoints. In this Perspective, we discuss the mediators and networks that control the six main peripheral tolerance checkpoints throughout the life of a T cell: quiescence, ignorance, anergy, exhaustion, senescence and death. At the naive T cell stage, two intrinsic checkpoints that actively maintain tolerance are quiescence and ignorance. In the presence of co-stimulation-deficient T cell activation, anergy is a dominant hallmark that mandates T cell unresponsiveness. When T cells are successfully stimulated and reach the effector stage, exhaustion and senescence can limit excessive inflammation and prevent immunopathology. At every stage of the T cell's journey, cell death exists as a checkpoint to limit clonal expansion and to terminate unrestrained responses. Here, we compare and contrast the T cell tolerance checkpoints and discuss their specific roles, with the aim of providing an integrated view of T cell peripheral tolerance and fate regulation.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic., (© 2021 by The American Society of Hematology.)

In recent years, the success of immunotherapy targeting immunoregulatory receptors (immune checkpoints) in cancer have generated enthusiastic support to target these receptors in a wide range of other immune related diseases. While the overwhelming focus has been on blockade of these inhibitory pathways to augment immunity, agonistic triggering via these receptors offers the promise of dampening pathogenic inflammatory responses. V-domain Ig suppressor of T cell activation (VISTA) has emerged as an immunoregulatory receptor with constitutive expression on both the T cell and myeloid compartments, and whose agonistic targeting has proven a unique avenue relative to other checkpoint pathways to suppress pathologies mediated by the innate arm of the immune system. VISTA agonistic targeting profoundly changes the phenotype of human monocytes towards an anti-inflammatory cell state, as highlighted by striking suppression of the canonical markers CD14 and Fcγr3a (CD16), and the almost complete suppression of both the interferon I (IFN-I) and antigen presentation pathways. The insights from these very recent studies highlight the impact of VISTA agonistic targeting of myeloid cells, and its potential therapeutic implications in the settings of hyperinflammatory responses such as cytokine storms, driven by dysregulated immune responses to viral infections (with a focus on COVID-19) and autoimmune diseases. Collectively, these findings suggest that the VISTA pathway plays a conserved, non-redundant role in myeloid cell function., Competing Interests: RN is an inventor on patent applications (10035857, 9631018, 9217035, 8501915, 8465740, 8236304, and 8231872) submitted by Dartmouth College, and patent applications (9890215 and 9381244) submitted by Kings College London and Dartmouth College and a co-founder of ImmuNext, a company involved in the development of VISTA-related assets. These applications cover the use of VISTA targeting for modulation of the immune response. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 ElTanbouly, Zhao, Schaafsma, Burns, Mabaera, Cheng and Noelle.)

We present the novel finding that V-domain Ig suppressor of T cell activation (VISTA) negatively regulates innate inflammation through the transcriptional and epigenetic re-programming of macrophages. Representative of VISTA re-programming is the ability of VISTA agonistic antibodies to augment LPS tolerance and reduce septic shock lethality in mice. This anti-inflammatory effect of anti-VISTA was mimicked in vitro demonstrating that anti-VISTA treatment caused a significant reduction in LPS-induced IL-12p40, IL-6, CXCL2, and TNF; all hallmark pro-inflammatory mediators of endotoxin shock. Even under conditions that typically "break" LPS tolerance, VISTA agonists sustained a macrophage anti-inflammatory profile. Analysis of the proteomic and transcriptional changes imposed by anti-VISTA show that macrophage re-programming was mediated by a composite profile of mediators involved in both macrophage tolerance induction (IRG1, miR221, A20, IL-10) as well as transcription factors central to driving an anti-inflammatory profile (e.g., IRF5, IRF8, NFKB1). These findings underscore a novel and new activity of VISTA as a negative checkpoint regulator that induces both tolerance and anti-inflammatory programs in macrophages and controls the magnitude of innate inflammation in vivo ., (Copyright © 2020 ElTanbouly, Schaafsma, Smits, Shah, Cheng, Burns, Blazar, Noelle and Mabaera.)

Negative checkpoint regulators (NCR) are intensely pursued as targets to modulate the immune response in cancer and autoimmunity. A large variety of NCR is expressed by central nervous system (CNS)-resident cell types and is associated with CNS homeostasis, interactions with peripheral immunity and CNS inflammation and disease. Immunotherapy blocking NCR affects the CNS as patients can develop neurological issues including encephalitis and multiple sclerosis (MS). How these treatments affect the CNS is incompletely understood, since expression and function of NCR in the CNS are only beginning to be unravelled. V-type immunoglobulin-like suppressor of T cell activation (VISTA) is an NCR that is expressed primarily in the haematopoietic system by myeloid and T cells. VISTA regulates T cell quiescence and activation and has a variety of functions in myeloid cells including efferocytosis, cytokine response and chemotaxis. In the CNS, VISTA is predominantly expressed by microglia and macrophages of the CNS. In this review, we summarize the role of NCR in the CNS during health and disease. We highlight expression of VISTA across cell types and CNS diseases and discuss the function of VISTA in microglia and during CNS ageing, inflammation and neurodegeneration. Understanding the role of VISTA and other NCR in the CNS is important considering the adverse effects of immunotherapy on the CNS, and in view of their therapeutic potential in CNS disease.

The immune response is governed by a highly complex set of interactions among cells and mediators. T cells may be rendered dysfunctional by the presence of high levels of antigen in the absence of co-stimulation while myeloid cells may be programmed towards an immunosuppressive state that promotes cancer growth and metastasis while deterring tumor immunity. In addition, inhibitory programs driven by immune checkpoint regulators dampen anti-tumor immunity. The ideal cancer immunotherapy treatment will improve both cross-priming in the tumor microenvironment and relieve suppression by the inhibitory checkpoints. Recently, blockade of programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) has elicited impressive results, but not in all patients, so additional targets are under investigation. V-set immunoglobulin domain suppressor of T cell activation (VISTA) is a novel immunoregulatory receptor that is broadly expressed on cells of the myeloid and lymphoid lineages, and is frequently implicated as a poor prognostic indicator in multiple cancers. Importantly, antibody targeting of VISTA uniquely engages both innate and adaptive immunity. This, combined with the expression of VISTA and its non-redundant activities compared to other immune checkpoint regulators, qualifies VISTA to be a promising target for improving cancer immunotherapy., (© 2020 British Society for Immunology.)

Negative checkpoint regulators (NCRs) temper the T cell immune response to self-antigens and limit the development of autoimmunity. Unlike all other NCRs that are expressed on activated T lymphocytes, V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is expressed on naïve T cells. We report an unexpected heterogeneity within the naïve T cell compartment in mice, where loss of VISTA disrupted the major quiescent naïve T cell subset and enhanced self-reactivity. Agonistic VISTA engagement increased T cell tolerance by promoting antigen-induced peripheral T cell deletion. Although a critical player in naïve T cell homeostasis, the ability of VISTA to restrain naïve T cell responses was lost under inflammatory conditions. VISTA is therefore a distinctive NCR of naïve T cells that is critical for steady-state maintenance of quiescence and peripheral tolerance., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The role of negative checkpoint regulators (NCRs) in human health and disease cannot be overstated. V-domain Ig-containing Suppressor of T-cell Activation (VISTA) is an Ig superfamily protein predominantly expressed within the hematopoietic compartment and has been studied for its role in the negative regulation of T cell responses. The findings presented in this study show that, unlike all other NCRs, VISTA deficiency dramatically impacts on macrophage cytokine and chemokine production, as well as the chemotactic response of VISTA-deficient macrophages. A select group of inflammatory chemokines, including CCL2, CCL3, CCL4, and CCL5, was strikingly elevated in culture supernatants from VISTA KO macrophages. VISTA deficiency also altered chemokine receptor recycling and profoundly disrupted myeloid chemotaxis. The impact of VISTA deficiency on chemotaxis in vivo was apparent with the reduced ability of both KO macrophages and MDSCs to migrate to the tumor microenvironment. This is the first demonstration of an NCR impacting on myeloid mediator production and chemotaxis, and will guide the use of anti-VISTA therapeutics to manipulate the chemotaxis of inflammatory macrophages or immunosuppressive MDSCs in inflammatory diseases and cancer., (Copyright © 2019 Broughton, ElTanbouly, Schaafsma, Deng, Sarde, Croteau, Li, Nowak, Mabaera, Smits, Kuta, Noelle and Lines.)

Tumor hypoxia is a negative prognostic factor that is implicated in oncogenic signal activation, immune escape, and resistance to treatment. Identifying the mechanistic role of hypoxia in immune escape and resistance to immune-checkpoint inhibitors may aid the identification of therapeutic targets. We and others have shown that V-domain Ig suppressor of T-cell activation (VISTA), a negative checkpoint regulator in the B7 family, is highly expressed in the tumor microenvironment in tumor models and primary human cancers. In this study, we show that VISTA and HIF1α activity are correlated in a cohort of colorectal cancer patients. High VISTA expression was associated with worse overall survival. We used the CT26 colon cancer model to investigate the regulation of VISTA by hypoxia. Compared with less hypoxic tumor regions or draining lymph nodes, regions of profound hypoxia in the tumor microenvironment were associated with increased VISTA expression on tumor-infiltrating myeloid-derived suppressor cells (MDSC). Using chromatin immunoprecipitation and genetic silencing, we show that hypoxia-inducible factor (HIF)-1α binding to a conserved hypoxia response element in the VISTA promoter upregulated VISTA on myeloid cells. Further, antibody targeting or genetic ablation of VISTA under hypoxia relieved MDSC-mediated T-cell suppression, revealing VISTA as a mediator of MDSC function. Collectively, these data suggest that targeting VISTA may mitigate the deleterious effects of hypoxia on antitumor immunity., (©2019 American Association for Cancer Research.)

Recent studies have underscored the critical role of retinoic acid (RA) in the development of lineage-committed CD4 and CD8 T cells in vivo. We have shown that under acute graft-versus-host disease (GVHD) inflammatory conditions, RA is upregulated in the intestine and is proinflammatory, as GVHD lethality was attenuated when donor allogeneic T cells selectively expressed a dominant negative RA receptor α that blunted RA signaling. RA can function in an autocrine and paracrine fashion, and as such, the host cell lineage responsible for the production of RA metabolism and the specific RA-metabolizing enzymes that potentiate GVHD severity are unknown. In this study, we demonstrate that enhancing RA degradation in the host and to a lesser extent donor hematopoietic cells by overexpressing the RA-catabolizing enzyme CYP26A1 reduced GVHD. RA production is facilitated by retinaldehyde isoform-2 (RALDH2) preferentially expressed in dendritic cells (DCs). Conditionally deleted RA-synthesizing enzyme RALDH2 in host or to a lesser extent donor DCs reduced GVHD lethality. Improved survival in recipients with RALDH2-deleted DCs was associated with increased T cell death, impaired T effector function, increased regulatory T cell frequency, and augmented coinhibitory molecule expression on donor CD4 + T cells. In contrast, retinaldehydrogenase isoform-1 (RALDH1) is dominantly expressed in intestinal epithelial cells. Unexpectedly, conditional host intestinal epithelial cells RALDH1 deletion failed to reduce GVHD. These data demonstrate the critical role of both donor and especially host RALDH2 + DCs in driving murine GVHD and suggest RALDH2 inhibition or CYP26A1 induction as novel therapeutic strategies to prevent GVHD., (Copyright © 2019 by The American Association of Immunologists, Inc.)

V-domain Ig suppressor of T cell activation (VISTA) is a novel checkpoint regulator with limited homology to other B7 family members. The constitutive expression of VISTA on both the myeloid and T lymphocyte lineages coupled to its important role in regulating innate and adaptive immune responses, qualifies VISTA to be a promising target for immunotherapeutic intervention. Studies have shown differential impact of agonistic and antagonistic targeting of VISTA, providing a unique landscape for influencing the outcome of cancer and inflammatory diseases., (Published by Elsevier Ltd.)

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell-mediated immunity. Expression changes of other NCRs (PD-1, PD-L1/L2, CTLA-4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1 Δ/- mouse model. After LPS injection, decreased VISTA expression in mouse microglia was accompanied by decreased acetylation of lysine residue 27 in histone 3 in both its promoter and enhancer region. ATAC-sequencing indicated a potential regulation of VISTA expression by Pu.1 and Mafb, two transcription factors crucial for microglia function. Finally, our data suggested that VISTA expression was decreased in microglia in multiple sclerosis lesion tissue, whereas it was increased in Alzheimer's disease patients. This study is the first to demonstrate that in the CNS, VISTA is expressed by microglia, and that VISTA is differentially expressed in CNS pathologies., (© 2018 The Authors. Glia published by Wiley Periodicals, Inc.)

Adaptive immune responses contribute to the pathogenesis of melanoma by facilitating immune evasion. V-domain Ig suppressor of T-cell activation (VISTA) is a potent negative regulator of T-cell function and is expressed at high levels on monocytes, granulocytes, and macrophages, and at lower densities on T-cell populations within the tumor microenvironment. In this study, 85 primary melanoma specimens were selected from pathology tissue archives and immunohistochemically stained for CD3, PD-1, PD-L1, and VISTA. Pearson's correlation coefficients identified associations in expression between VISTA and myeloid infiltrate (r = 0.28, p = 0.009) and the density of PD-1+ inflammatory cells (r = 0.31, p = 0.005). The presence of VISTA was associated with a significantly worse disease-specific survival in univariate analysis (hazard ratio = 3.57, p = 0.005) and multivariate analysis (hazard ratio = 3.02, p = 0.02). Our findings show that VISTA expression is an independent negative prognostic factor in primary cutaneous melanoma and suggests its potential as an adjuvant immunotherapeutic intervention in the future.

V-domain Ig suppressor of T-cell activation (VISTA) is a critical negative checkpoint molecule involved in regulating the immune response. Targeting the pathway with an antagonist anti-VISTA antibody designated 13F3 has been shown to enhance disease severity in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. To determine if VISTA plays a role in murine lupus, New Zealand Black × New Zealand White (BWF 1 ) mice were treated with 13F3 or control hamster Ig and disease monitored. Onset of proteinuria was earlier and renal damage more profound in mice treated with 13F3. Cell subset analysis showed an increase of activated splenic T cells and inflammatory splenic myeloid cells, but no effect on B cells, in mice receiving 13F3. Examination of the kidney showed an increase in inflammatory myeloid cell infiltration with 13F3 treatment. This study along with previous EAE data, suggests that interventions that enhance VISTA regulatory activity may be effective for the treatment of autoimmune disease.

Background: In addition to activated T cells, the immune checkpoint inhibitor "V domain-containing Ig suppressor of T-cell activation" (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated., Methods: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages., Results: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn)., Conclusions: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.

HSV type 1 (HSV-1) is a prevalent human pathogen that infects >3.72 billion individuals worldwide and can cause potentially blinding recurrent corneal herpetic disease. HSV-1 establishes latency within sensory neurons of trigeminal ganglia (TG), and TG-resident CD8 + T cells play a critical role in preventing its reactivation. The repertoire, phenotype, and function of protective CD8 + T cells are unknown. Bolstering the apparent feeble numbers of CD8 + T cells in TG remains a challenge for immunotherapeutic strategies. In this study, a comprehensive panel of 467 HLA-A*0201-restricted CD8 + T cell epitopes was predicted from the entire HSV-1 genome. CD8 + T cell responses to these genome-wide epitopes were compared in HSV-1-seropositive symptomatic individuals (with a history of numerous episodes of recurrent herpetic disease) and asymptomatic (ASYMP) individuals (who are infected but never experienced any recurrent herpetic disease). Frequent polyfunctional HSV-specific IFN-γ + CD107 a/b+ CD44 high CD62L low CD8 + effector memory T cells were detected in ASYMP individuals and were primarily directed against three "ASYMP" epitopes. In contrast, symptomatic individuals have more monofunctional CD44 high CD62L high CD8 + central memory T cells. Furthermore, therapeutic immunization with an innovative prime/pull vaccine, based on priming with multiple ASYMP epitopes (prime) and neurotropic TG delivery of the T cell-attracting chemokine CXCL10 (pull), boosted the number and function of CD44 high CD62L low CD8 + effector memory T cells and CD103 high CD8 + tissue-resident T cells in TG of latently infected HLA-A*0201-transgenic mice and reduced recurrent ocular herpes following UV-B-induced reactivation. These findings have profound implications in the development of T cell-based immunotherapeutic strategies to treat blinding recurrent herpes infection and disease., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: The targeting of negative checkpoint regulators as a means of augmenting antitumor immune responses is now an increasingly used and remarkably effective approach to the treatment of several human malignancies. The negative checkpoint regulator VISTA (V-domain Ig-containing suppressor of T cell activation; also known as programmed death 1 homolog or as death domain 1α) suppresses T cell responses and regulates myeloid activities. We proposed that exploitation of the VISTA pathway is a novel strategy for the treatment of human autoimmune disease, and therefore we undertook this study to determine the impact of VISTA genetic deficiency on lupus development in a lupus-prone mouse strain., Methods: To evaluate whether genetic deficiency of VISTA affects the development of lupus, we interbred VISTA-deficient mice with Sle1.Sle3 mice, a well-characterized model of systemic lupus erythematosus (SLE)., Results: We demonstrated that the development of proteinuria and glomerulonephritis in these mice, designated Sle1.Sle3 VISTA -/- mice, was greatly accelerated and more severe compared to that in Sle1.Sle3 and C57BL/6 VISTA -/- mice. Analysis of cells from Sle1.Sle3 VISTA -/- mice showed enhanced activation of splenic CD4+ T cells and myeloid cell populations. No increase in titers of autoantibodies was seen in Sle1.Sle3 VISTA -/- mice. Most striking was a significant increase in proinflammatory cytokines, chemokines, and interferon (IFN)-regulated genes associated with SLE, such as IFNα, IFNγ, tumor necrosis factor, interleukin-10, and CXCL10, in Sle1.Sle3 VISTA -/- mice., Conclusion: This study demonstrates for the first time that loss of VISTA in murine SLE exacerbates disease due to enhanced myeloid and T cell activation and cytokine production, including a robust IFNα signature, and supports a strategy of enhancement of the immunosuppressive activity of VISTA for the treatment of human lupus., (© 2016, American College of Rheumatology.)

Utilization of negative checkpoint regulators (NCRs) for cancer immunotherapy has garnered significant interest with the completion of clinical trials demonstrating efficacy. While the results of monotherapy treatments are compelling, there is increasing emphasis on combination treatments in an effort to increase response rates to treatment. One of the most recently discovered NCRs is VISTA (V-domain Ig-containing Suppressor of T cell Activation). In this review, we describe the functions of this molecule in the context of cancer immunotherapy. We also discuss factors that may influence the use of anti-VISTA antibody in combination therapy and how genomic analysis may assist in providing indications for treatment., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Initially, a role for the interaction between CD40, expressed on B cells, and gp39 (CD40L), expressed on activated T cells, has been defined in humoral immunity. CD40-CD40L interaction is an essential signal for B cell proliferation, expression of activation markers, immunoglobulin production, and isotype switching. CD40-CD40L interaction is also required for formation of B memory cells and germinal centers, and signaling through CD40 prevents apoptosis of germinal center B cells. Defective expression of CD40L in humans leads to an inability to produce isotypes other than IgM (hyper IgM syndrome), and to an absence of germinal centers. More recent evidence indicates an expansion of the role of the CD40-CD40L axis in cellular interactions beyond antibody formation. Induced expression of CD40 on monocytes can lead to CD40L-activated monocyte effector mechanisms. In addition, CD40-CD40L interactions are crucially involved in development of autoimmune disease in a number of animal models. CD40-CD40L interactions also impact on growth regulation of certain carcinomas. Manipulation of CD40L has also been used to develop novel strategies for long-term antigen-specific tolerization of peripheral T cells. Finally, the CD40-CD40L axis is involved in thymic selection. Following is a comprehensive overview of CD40L-CD40 interactions in physiological and pathogenic cellular responses and a discussion of the therapeutic ramifications of these interactions.

Retinoic acid (RA) plays an important role in the balance of inflammation and tolerance in T cells. Furthermore, it has been demonstrated that RA facilitates IgA isotype switching in B cells in vivo . However, it is unclear whether RA has a direct effect on T-independent B cell responses in vivo . To address this question, we generated a mouse model where RA signaling is specifically silenced in the B cell lineage. This was achieved through the overexpression of a dominant negative receptor α for RA (dnRARα) in the B cell lineage. In this model, we found a dramatic reduction in marginal zone (MZ) B cells and accumulation of transitional 2 B cells in the spleen. We also observed a reduction in B1 B cells in the peritoneum with a defect in the T-independent B cell response against 2,4,6-trinitrophenyl. This was not a result of inhibited development of B cells in the bone marrow, but likely the result of both defective expression of S1P 1 in MZ B cells and a defect in the development of MZ and B1 B cells. This suggests that RARα expression in B cells is important for B cell frequency in the MZ and peritoneum, which is crucial for the generation of T-independent humoral responses.

Negative checkpoint regulators function to restrain T cell responses to maintain tolerance and limit immunopathology. However, in the setting of malignancy, these pathways work in concert to promote immune-mediate escape leading to the development of a clinically overt cancer. In the recent years, clinical trials demonstrating the efficacy of blocking antibodies against these molecules have invigorated the field of immunotherapy. In this review, we discuss the current understanding on established NCR blockade and how strategic combination therapy with anti-VISTA antibody can be used to target multiple non-redundant NCR pathways.

Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM + IgD lo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM + IgD lo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM + IgD lo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM + B cells in the spleen and peritoneal cavity., Competing Interests: The authors of this manuscript have no conflicts of interest to disclose as described by the Journal of Immunology., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Several molecules have been described as CD4+ T cells differentiation modulators and among them retinoic acid (RA) and more recently, IL-33, have been studied. Due to the similarities in T helper cell skewing properties between RA and IL-33, we asked whether IL-33 intersects, directly or indirectly, the RA signaling pathway. Total CD4+ T cells from DR5-luciferase mice were activated in the presence of RA with or without IL-33, and RA signaling was visualized using ex vivo imaging. Our results demonstrate that IL-33 itself is able to trigger RA signaling on CD4+ T cells, which is highly increased when IL-33 is added in conjunction with RA. This study presents IL-33 as a potential player that may synergize with RA in controlling T cell differentiation, and suggests that IL-33 may be an attractive target in controlling T cell differentiation in vivo., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Beyond their eminent role in hemostasis and thrombosis, platelets are recognized as mediators of inflammation. Platelet cluster of differentiation 40 (CD40) ligand (CD40L and CD154) plays a key role in mediating platelet-induced inflammation in atherosclerosis. CD40, the receptor for CD40L, is present on platelets; however, the role of CD40 on this cell type is until now undefined., Approach and Results: We found that in both mice and humans, platelet CD40 mediates the formation of platelet-leukocyte aggregates and the release of chemokine (C-X-C motif) ligand 4. Leukocytes were also less prone to adhere to CD40-deficient thrombi. However, platelet CD40 was not involved in platelet aggregation. Activated platelets isolated from Cd40(-/-)Apoe(-/-) mice adhered less to the endothelium upon injection into Apoe(-/-) mice when compared with CD40-sufficient platelets. Furthermore, lack of CD40 on injected platelets led to reduced leukocyte recruitment to the carotid artery as assayed by intravital microscopy. This was accompanied by a decrease in endothelial vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule, VE-cadherin, and P-selectin expression. To investigate the effect of platelet CD40 in atherosclerosis, Apoe(-/-) mice received thrombin-activated Apoe(-/-) or Cd40(-/-)Apoe(-/-) platelets every 5 days for 12 weeks, starting at the age of 17 weeks, when atherosclerotic plaques had already formed. When compared with mice that received Apoe(-/-) platelets, those receiving Cd40(-/-)Apoe(-/-) platelets exhibited a >2-fold reduction in atherosclerosis. Plaques of mice receiving CD40-deficient platelets were less advanced, contained less macrophages, neutrophils, and collagen, and displayed smaller lipid cores., Conclusions: Platelet CD40 plays a crucial role in inflammation by stimulating leukocyte activation and recruitment and activation of endothelial cells, thereby promoting atherosclerosis., (© 2016 American Heart Association, Inc.)

Retinoid x receptors (RXRs) are master regulators that control cell growth, differentiation, and survival and form heterodimers with many other family members. Here we show that treatment with the RXR agonist IRX4204 enhances the differentiation of CD4(+) T cells into inducible regulatory T cells (iTreg) and suppresses the development of T helper (Th) 17 cells in vitro. Furthermore in a murine model of multiple sclerosis (experimental autoimmune encephalomyelitis (EAE)), treatment with IRX4204 profoundly attenuates both active and Th17-mediated passive disease. In the periphery, treatment with IRX4204 is associated with decreased numbers of CD4(+) T cells that produce pro-inflammatory cytokines. In addition, CD4(+) T cells express decreased levels of Ki-67 and increased expression of CTLA-4. Our findings demonstrate IRX4204 treatment during EAE results in immune modulation and profound attenuation of disease severity.

Inhibition of T-cell responses in tumor microenvironments by myeloid-derived suppressor cells (MDSCs) is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-cell, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T cells by inducible nitric oxide synthase (iNOS)/nitric oxide (NO), but uniquely, inhibition of B cells was ~50% dependent each on iNOS/NO and the MDSC-expressed negative-checkpoint regulator VISTA. Blockade with a combination of iNOS/NO and VISTA caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy.

Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA(+) Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Although donor-specific transfusion (DST) plus CD154 blockade represents a robust protocol for inducing transplantation tolerance, the underlying mechanisms are incompletely understood. In a murine T-cell adoptive transfer model, we have visualized alloantigen-specific, TCR-transgenic for H2-A(b) /H2-K(d) 54-68 epitope (TCR75) CD4(+) T cells with indirect allospecificity during the course of tolerance induction. Three main observations were made. First, although the majority of TCR75 CD4(+) T cells were deleted following DST plus CD154 blockade, the surviving TCR75 CD4(+) T cells were capable of making IL-2, upregulating CD44, and undergoing cell division, suggesting that they were functionally active. Indeed, residual TCR75 CD4(+) T cells reisolated from the primary recipients given DST plus CD154 blockade were fully capable of rejecting allografts upon secondary transfer. Second, in tolerant mice, TCR75 CD4(+) T cells were not induced to express Foxp3 in the graft-draining lymph node. TCR75 CD4(+) T cells were also absent in accepted graft tissues in which endogenous Treg cells were enriched. Finally, DST plus CD154 blockade resulted in an abortive expansion of TCR75 CD4(+) T cells, a process that required the presence of endogenous Treg cells. Collectively, surviving TCR75 CD4(+) T cells are immunocompetent but kept in check by an endogenous immunosuppressive network induced by DST plus CD154 blockade., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora.

The importance of vitamin A for host defense is undeniable and the study of its mechanisms is paramount. Of the estimated 250 million preschool children who are vitamin A-deficient (VAD), 10% will die from their increased susceptibility to infectious disease. Vitamin A supplementation was established in the 1980s as one of the most successful interventions in the developing world. Understanding how vitamin A controls immunity will help curb the mortality and morbidity associated with vitamin A deficiency and exploit the immune-enhancing capacity of vitamin A to heighten host resistance to infectious disease. The discoveries that retinoic acid (RA) imprints the homing of leukocytes to the gut and enhances the induction of regulatory T cells, highlighted a potential role for RA in mucosal tolerance. However, more recently emerging data tell of a more profound systemic impact of RA on leukocyte function and commitment. In animal models using genetic manipulation of RA signaling, we learned when and how RA controls T cell fate. Here, we review the role for RA as a critical checkpoint regulator in the differentiation of CD4(+) T cells within the immune system., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD4(+) T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4(+) T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)