Clinical and research updates on the VISTA immune checkpoint: immuno-oncology themes and highlights.

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.

VISTA Targeting of T-cell Quiescence and Myeloid Suppression Overcomes Adaptive Resistance.

V domain immunoglobulin suppressor of T-cell activation (VISTA) is a premier target for cancer treatment due to its broad expression in many cancer types and enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colorectal cancer model, monotherapy of small tumors with anti-VISTA resulted in slowed tumor growth. In a combination therapy setting, large CT26 tumors showed complete adaptive resistance to anti-PD-1/CTLA-4, but inclusion of anti-VISTA led to rejection of half the tumors. Mechanisms of enhanced antitumor immunity were investigated using single-cell RNA sequencing (scRNA-seq), multiplex image analysis, and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated antigen presentation pathways and reduced myeloid-mediated suppression. Imaging revealed an anti-VISTA stimulated increase in contacts between T cells and myeloid cells, further supporting the notion of increased antigen presentation. scRNA-seq of tumor-specific CD8+ T cells revealed that anti-VISTA therapy induced T-cell pathways highly distinct from and complementary to those induced by anti-PD-1 therapy. Whereas anti-CTLA-4/PD-1 expanded progenitor exhausted CD8+ T-cell subsets, anti-VISTA promoted costimulatory genes and reduced regulators of T-cell quiescence. Notably, this is the first report of a checkpoint regulator impacting CD8+ T-cell quiescence, and the first indication that quiescence may be a target in the context of T-cell exhaustion and in cancer. This study builds a foundation for all future studies on the role of anti-VISTA in the development of antitumor immunity and provides important mechanistic insights that strongly support use of anti-VISTA to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4. See related Spotlight by Wei, p. 3.

Secondary polycythaemia with elevated carbon monoxide levels due to hookah pipe smoking: A public health concern.

Hookah pipe (HP) smoking is perceived as a harmless activity, enjoyed by young adults and high school-going children. Awareness of the health impact of recreational habits, and their intersection with new social norms in the COVID-era, requires critical review. We describe a case series of young HP smokers presenting with secondary polycythaemia with significant clinical sequelae necessitating extensive work-up. HP smoking may lead to acute and chronic carbon monoxide intoxication, with resultant secondary polycythaemia and complications including provoked thrombosis.

VISTA: Coming of age as a multi-lineage immune checkpoint.

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.

VISTA is a checkpoint regulator for naïve T cell quiescence and peripheral tolerance.

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.

Defining the Signature of VISTA on Myeloid Cell Chemokine Responsiveness.

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.

VISTA: a novel immunotherapy target for normalizing innate and adaptive immunity.

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.

Hypoxia-Induced VISTA Promotes the Suppressive Function of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

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.

Immunoregulatory functions of VISTA.

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.

Regulatory T Cells Are Critical for Clearing Influenza A Virus in Neonatal Mice.

We previously reported that neonatal mice infected with influenza A virus (IAV) develop interstitial pneumonia characterized by reduced lung cytokine and chemokine responses. The failure of T cells to infiltrate the airways of neonates correlated with delayed clearance of sublethal IAV infections compared to adults. We hypothesized that negative regulators in the neonatal lungs such as cytokines or T regulatory (Treg) cells are responsible for these differences. Neonates either deficient in interleukin-10 (IL-10) or with T cells unresponsive to transforming growth factor-ß signaling due to absence of SMAD family member 4 (Smad4) had similar IAV clearance kinetics to wild-type pups and no difference in T-cell responses. In contrast, functional depletion of Treg cells with anti-CD25 monoclonal antibody resulted in increased proportions of activated CD4(+) T cells in the lungs, but failure to clear IAV. Similarly, scurfy pups (mutation in forkhead box P3 [Foxp3] rendering them deficient in Treg cells) had increased proportions of activated T cells in the lungs compared to littermate controls. Scurfy pups also had increased proportions of IL-13-producing CD4(+) T cells. Interestingly, like anti-CD25-treated pups, scurfy pups had significantly elevated viral loads compared to controls. Based on these data, we conclude that Tregs are critical for clearance of IAV in neonatal mice.

Beyond CTLA-4 and PD-1, the Generation Z of Negative Checkpoint Regulators.

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.

Humane harvesting and slaughter of farmed fish.

The overwhelming majority of farmed fish produced throughout the world are killed with little or no consideration for their welfare. Fasting periods can be excessive, transport stressful and killing inhumane. At the time of writing, the salmon industry is the only sector in which consideration of the welfare of the fish at slaughter has resulted in significant improvements throughout most of the industry. There are signs of interest in the use of more humane slaughter methods for some other fish species. This is mostly initiated by the demand for higher standards from European fish retailers. For most species, the humane killing options are limited to percussive stunning and electrical stunning. However, even these methods can have a poor welfare outcome if insufficient consideration is given to the needs of the fish or if the equipment has not been properly designed. The use of food-grade anaesthetics to assist with the harvest has significant potential for improving welfare and their wider use should be investigated further.

VISTA is a novel broad-spectrum negative checkpoint regulator for cancer immunotherapy.

In the past few years, the field of cancer immunotherapy has made great progress and is finally starting to change the way cancer is treated. We are now learning that multiple negative checkpoint regulators (NCR) restrict the ability of T-cell responses to effectively attack tumors. Releasing these brakes through antibody blockade, first with anti-CTLA4 and now followed by anti-PD1 and anti-PDL1, has emerged as an exciting strategy for cancer treatment. More recently, a new NCR has surfaced called V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA). This NCR is predominantly expressed on hematopoietic cells, and in multiple murine cancer models is found at particularly high levels on myeloid cells that infiltrated the tumors. Preclinical studies with VISTA blockade have shown promising improvement in antitumor T-cell responses, leading to impeded tumor growth and improved survival. Clinical trials support combined anti-PD1 and anti-CTLA4 as safe and effective against late-stage melanoma. In the future, treatment may involve combination therapy to target the multiple cell types and stages at which NCRs, including VISTA, act during adaptive immune responses.

VISTA is an immune checkpoint molecule for human T cells.

V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T-cell function that is expressed on hematopoietic cells. VISTA levels are heightened within the tumor microenvironment, in which its blockade can enhance antitumor immune responses in mice. In humans, blockade of the related programmed cell death 1 (PD-1) pathway has shown great potential in clinical immunotherapy trials. Here, we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells and blunted the production of T-cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T-cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer.

Characteristics of electronic training collars for dogs.

A wide range of electronic dog training collars (e-collars) is available in the UK, but information enabling purchasers to compare the important characteristics of these collars is not available. In this research, the electrical characteristics of 13 e-collar models were examined, and an approach to ranking the strength of the electrical stimuli was developed. To achieve this, the electrical impedance of dogs' necks were measured so that e-collars could be tested under realistic conditions. This impedance was found to be about 10 kΩ for wet dogs and 640 kΩ for dry dogs. Two replicates of eight e-collar models and single copies of a further five models were then examined. The stimuli generated by these collars comprised sequences of short high-voltage pulses. There were large differences between e-collar models in the energy, peak voltage, number of pulses and duration of the pulses, but little variation between the replicates. The peak voltage varied with the impedance, from 6000V at an impedance of 500 kΩ to 100V at 5 kΩ. The highest voltages were generated for a few millionths of a second. Stimulus energy levels at the maximum strength setting with a 50 kΩ load ranged from 3.3 mJ to 287 mJ. A stimulus strength ranking indicator was then developed to enable the strengths of e-collars with diverse electrical characteristics to be ranked. This ranking shows a wide range in the stimulus strengths of collars, and that the relationships between 'momentary' and 'continuous' stimuli for various models differ significantly.

Alveolar macrophages in neonatal mice are inherently unresponsive to Pneumocystis murina infection.

Pneumocystis pneumonia was first diagnosed in malnourished children and has more recently been found in children with upper respiratory symptoms. We previously reported that there is a significant delay in the immune response in newborn mice infected with Pneumocystis compared to adults (Garvy BA, Harmsen AG, Infect. Immun. 64:3987-3992, 1996, and Garvy BA, Qureshi M, J. Immunol. 165:6480-6486, 2000). This delay is characterized by the failure of neonatal lungs to upregulate proinflammatory cytokines and attract T cells into the alveoli. Here, we report that regardless of the age at which we infected the mice, they failed to mount an inflammatory response in the alveolar spaces until they were 21 days of age or older. Anti-inflammatory cytokines had some role in dampening inflammation, since interleukin-10 (IL-10)-deficient pups cleared Pneumocystis faster than wild-type pups and the neutralization of transforming growth factor beta (TGF-ß) with specific antibody enhanced T cell migration into the lungs at later time points. However, the clearance kinetics were similar to those of control pups, suggesting that there is an intrinsic deficiency in the ability of innate immunity to control Pneumocystis. We found, using an adoptive transfer strategy, that the lung environment contributes to association of Pneumocystis organisms with alveolar macrophages, implying no intrinsic deficiency in the binding of Pneumocystis by neonatal macrophages. Using both in vivo and in vitro assays, we found that Pneumocystis organisms were less able to stimulate translocation of NF-κB to the nucleus of alveolar macrophages from neonatal mice. These data indicate that there is an early unresponsiveness of neonatal alveolar macrophages to Pneumocystis infection that is both intrinsic and related to the immunosuppressive environment found in neonatal lungs.

Safeguarding the welfare of farmed fish at harvest.

Fish welfare at harvest is easily compromised by poor choice of handling and slaughter methods, lack of attention to detail and by unnecessary adherence to fish farming traditions. The harvest process comprises fasting the fish to empty the gut, crowding the fish, gathering and moving the fish using brails, fish pumps, and sometimes also road or boat transport and finally stunning and killing the fish. The harvesting processes commonly used for bass, bream, carp, catfish, cod, eel, halibut, pangasius, salmon, tilapia, trout, tuna and turbot are outlined. These harvesting processes are discussed; the consequences for fish welfare identified and practical tests which can be made at the harvest site highlighted. Welfare at harvest for the majority of farmed fish species can be improved by adopting and adapting existing procedures already known to be beneficial for fish welfare through their use in other fish farming systems or with other species. It is seldom necessary to develop completely new concepts or methods.

Head-only electrical stunning of poultry using a waterbath: a feasibility study.

1. The objective of this work was to investigate the feasibility of head only waterbath stunning as a means of generating immediate and long lasting unconsciousness while preventing wing flapping and avoiding carcass damage. 2. EEG measurements showed that immersion of the heads of the broilers for one second in a waterbath containing water of conductivity 2 x 5 mS/cm and a 50 Hz electric field of 10 V/cm resulted in immediate unconsciousness, and that increasing the electric field strength extended the duration of unconsciousness. 3. The passage of a 25-30 mA alternating current of frequency 2000 Hz through the broilers' bodies suppressed the wing flapping that followed a stun. 4. When the body current and electric field were applied simultaneously, wing flapping was prevented and EEG signals were suppressed for over 30 seconds indicating that the immediate unconsciousness lasted long enough to facilitate humane slaughter.

Broiler carcass quality using head-only electrical stunning in a waterbath.

1. The objective was to assess carcass quality of broilers when they were stunned by immersing their heads in a waterbath with an electric current flowing from one side of it to the other, while a second small current passed through the body to the waterbath to prevent involuntary wing flapping. 2. The prevalence of wing, shoulder and breast fillet haemorrhages and of broken bones in the pectoral region was not greater than that resulting from the normal stunning practice in that plant (63 mA, 610 Hz pDC). 3. These results imply that carcass damage using this technique will be significantly lower than that which will result from the application of higher stunning currents required by the new EU slaughter poultry slaughter regulations.