Deletion of Vß3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vß3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vß3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vß3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vß repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

From Snoot to Tail: A Brief Review of Influenza Virus Infection and Immunity in Pigs.

Pigs play an important role in influenza A virus (IAV) epidemiology because they support replication of human, avian, and swine origin viruses and act as an IAV reservoir for pigs and other species, including humans. Moreover, novel IAVs with human pandemic potential may be generated in pigs. To minimize the threat of IAVs to human and swine health, it is crucial to understand host defense mechanisms that restrict viral replication and pathology in pigs. In this article, we review IAV strains circulating in the North American swine population, as well as porcine innate and acquired immune responses to IAV, including recent advances achieved through immunological tools developed specifically for swine. Furthermore, we highlight unique aspects of the porcine pulmonary immune system, which warrant consideration when developing vaccines and therapeutics to limit IAV in swine or when using pigs to model human IAV infections.

Die Kämpfe únd schláchten-the struggles and battles of innate-like effector T lymphocytes with microbes.

The large majority of lymphocytes belong to the adaptive immune system, which are made up of B2 B cells and the αß T cells; these are the effectors in an adaptive immune response. A multitudinous group of lymphoid lineage cells does not fit the conventional lymphocyte paradigm; it is the unconventional lymphocytes. Unconventional lymphocytes-here called innate/innate-like lymphocytes, include those that express rearranged antigen receptor genes and those that do not. Even though the innate/innate-like lymphocytes express rearranged, adaptive antigen-specific receptors, they behave like innate immune cells, which allows them to integrate sensory signals from the innate immune system and relay that umwelt to downstream innate and adaptive effector responses. Here, we review natural killer T cells and mucosal-associated invariant T cells-two prototypic innate-like T lymphocytes, which sense their local environment and relay that umwelt to downstream innate and adaptive effector cells to actuate an appropriate host response that confers immunity to infectious agents.

Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza.

Evaluating α-galactosylceramide as an adjuvant for live attenuated influenza vaccines in pigs.

Natural killer T (NKT) cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses. Several studies have used this approach to adjuvant inactivated and subunit influenza A virus (IAV) vaccines, including to enhance cross-protective influenza immunity. However, less is known about whether α-GalCer can enhance live attenuated influenza virus (LAIV) vaccines, which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating influenza vaccines. The current study used the swine influenza challenge model to assess whether α-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine (TX98ΔNS1) encoding a truncated NS1 protein. In one study, weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0, 10, 50, and 100 µg/kg doses of α-GalCer, and subsequently challenged with a heterologous H3N2 virus. All treatment groups were protected from infection. However, the addition of α-GalCer appeared to suppress nasal shedding of the LAIV vaccine. In another experiment, pigs vaccinated with the H3N2 LAIV, with or without 50 µg/kg of α-GalCer, were challenged with the heterosubtypic pandemic H1N1 virus. Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways, and significantly decreased virus shedding. On the other hand, combining the vaccine with α-GalCer reduced cross-protective cellular and antibody responses, and resulted in higher virus titers in respiratory tissues. These findings suggest that: (i) high doses of α-GalCer impair the replication and nasal shedding of the LAIV vaccine; and (ii) α-GalCer might interfere with heterosubtypic cross-protective immune responses. This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines. Supplementary Information: The online version contains supplementary material available at 10.1186/s44149-022-00051-x.

A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs.

Many aspects of the porcine immune system remain poorly characterized, which poses a barrier to improving swine health and utilizing pigs as preclinical models. Here, we employ single-cell RNA sequencing (scRNA-seq) to create a cell atlas of the early-adolescent pig thymus. Our data show conserved features as well as species-specific differences in cell states and cell types compared with human thymocytes. We also describe several unconventional T cell types with gene expression profiles associated with innate effector functions. This includes a cell census of more than 11,000 differentiating invariant natural killer T (iNKT) cells, which reveals that the functional diversity of pig iNKT cells differs substantially from the iNKT0/1/2/17 subset differentiation paradigm established in mice. Our data characterize key differentiation events in porcine thymopoiesis and iNKT cell maturation and provide important insights into pig T cell development.

Future of biomedical, agricultural, and biological systems research using domesticated animals.

Increased knowledge of reproduction and health of domesticated animals is integral to sustain and improve global competitiveness of U.S. animal agriculture, understand and resolve complex animal and human diseases, and advance fundamental research in sciences that are critical to understanding mechanisms of action and identifying future targets for interventions. Historically, federal and state budgets have dwindled and funding for the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) competitive grants programs remained relatively stagnant from 1985 through 2010. This shortage in critical financial support for basic and applied research, coupled with the underappreciated knowledge of the utility of non-rodent species for biomedical research, hindered funding opportunities for research involving livestock and limited improvements in both animal agriculture and animal and human health. In 2010, the National Institutes of Health and USDA NIFA established an interagency partnership to promote the use of agriculturally important animal species in basic and translational research relevant to both biomedicine and agriculture. This interagency program supported 61 grants totaling over $107 million with 23 awards to new or early-stage investigators. This article will review the success of the 9-year Dual Purpose effort and highlight opportunities for utilizing domesticated agricultural animals in research.

Chronic heat stress delays immune system development and alters serotonin signaling in pre-weaned dairy calves.

Exposure to heat stress can alter the development and immune system function in dairy calves. Serotonin is an immunomodulatory biogenic amine that functions as a neurotransmitter and as a stress-response mediator. Our objectives were to characterize the patterns of serum serotonin concentrations and the pattern of serotonin-related genes expressed by immune cells of calves exposed to chronic heat stress or heat stress abatement during early life, and to explore whether these might relate to immune system development. Dairy calves were exposed to chronic heat stress (HS; n = 6) or heat stress abatement (cooling, CL; n = 6) across the prenatal (late gestation, last 46 d) and postnatal (from birth to weaning, 56 d) developmental windows. Blood samples were collected to harvest serum (weekly, from d 1 to 49), to isolate of circulating leukocyte mRNA (at 1, 21 and 42 d of age) and characterize immune cell populations by flow cytometry (at 21 and 47 d of age). Calves exposed to chronic heat stress pre- and postnatally had lower red blood cell counts and lower circulating serotonin, immunoglobulin G, and B-lymphocytes compared to CL calves. Circulating blood leukocyte mRNA expression of serotonin receptors -1A, -1F, -4 and -5 was greater, while heat shock protein 70 and immune-related genes (i.e., TBX21, TLR4, and TGFß) were lower in HS relative to CL calves. Peripheral blood leukocytes from all calves secreted serotonin and interleukin-6 after in-vitro lipopolysaccharide stimulation. However, the HS calves produced more serotonin and less interleukin-6 than CL calves when activated in-vitro. Together, our data suggest that providing heat stress abatement to dairy calves across prenatal and postnatal developmental windows might modulate the serotonin synthesis pathway in ways that may benefit humoral immunity against microbial pathogens.

Unaltered influenza disease outcomes in swine prophylactically treated with α-galactosylceramide.

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice.

Modulation of Immune Responses to Influenza A Virus Vaccines by Natural Killer T Cells.

Influenza A viruses (IAVs) circulate widely among different mammalian and avian hosts and sometimes give rise to zoonotic infections. Vaccination is a mainstay of IAV prevention and control. However, the efficacy of IAV vaccines is often suboptimal because of insufficient cross-protection among different IAV genotypes and subtypes as well as the inability to keep up with the rapid molecular evolution of IAV strains. Much attention is focused on improving IAV vaccine efficiency using adjuvants, which are substances that can modulate and enhance immune responses to co-administered antigens. The current review is focused on a non-traditional approach of adjuvanting IAV vaccines by therapeutically targeting the immunomodulatory functions of a rare population of innate-like T lymphocytes called invariant natural killer T (iNKT) cells. These cells bridge the innate and adaptive immune systems and are capable of stimulating a wide array of immune cells that enhance vaccine-mediated immune responses. Here we discuss the factors that influence the adjuvant effects of iNKT cells for influenza vaccines as well as the obstacles that must be overcome before this novel adjuvant approach can be considered for human or veterinary use.

CD70 Inversely Regulates Regulatory T Cells and Invariant NKT Cells and Modulates Type 1 Diabetes in NOD Mice.

The CD27-CD70 costimulatory pathway is essential for the full activation of T cells, but some studies show that blocking this pathway exacerbates certain autoimmune disorders. In this study, we report on the impact of CD27-CD70 signaling on disease progression in the NOD mouse model of type 1 diabetes (T1D). Specifically, our data demonstrate that CD70 ablation alters thymocyte selection and increases circulating T cell levels. CD27 signaling was particularly important for the thymic development and peripheral homeostasis of Foxp3+Helios+ regulatory T cells, which likely accounts for our finding that CD70-deficient NOD mice develop more-aggressive T1D onset. Interestingly, we found that CD27 signaling suppresses the thymic development and effector functions of T1D-protective invariant NKT cells. Thus, rather than providing costimulatory signals, the CD27-CD70 axis may represent a coinhibitory pathway for this immunoregulatory T cell population. Moreover, we showed that a CD27 agonist Ab reversed the effects of CD70 ablation, indicating that the phenotypes observed in CD70-deficient mice were likely due to a lack of CD27 signaling. Collectively, our results demonstrate that the CD27-CD70 costimulatory pathway regulates the differentiation program of multiple T cell subsets involved in T1D development and may be subject to therapeutic targeting.

Next Generation Sequencing of the Pig αß TCR Repertoire Identifies the Porcine Invariant NKT Cell Receptor.

Swine represent the only livestock with an established invariant NKT (iNKT) cell-CD1d system. In this study, we exploited the fact that pig iNKT cells can be purified using a mouse CD1d tetramer reagent to establish their TCR repertoire by next generation sequencing. CD1d tetramer-positive pig cells predominantly expressed an invariant Vα-Jα rearrangement, without nontemplate nucleotide diversity, homologous to the Vα24-Jα18 and Vα14-Jα18 rearrangements of human and murine iNKT cells. The coexpressed ß-chain used a Vß segment homologous to the semivariant Vß11 and Vß8.2 segments of human and murine iNKT cell receptors. Molecular modeling found that contacts within CD1d and CDR1α that underlie fine specificity differences between mouse and human iNKT cells are conserved between pigs and humans, indicating that the response of porcine and human iNKT cells to CD1d-restricted Ags may be similar. Accordingly, pigs, which are an important species for diverse fields of biomedical research, may be useful for developing human-based iNKT cell therapies for cancer, infectious diseases, and other disorders. Our study also sequenced the expressed TCR repertoire of conventional porcine αß T cells, which identified 48 Vα, 50 Jα, 18 Vß, and 18 Jß sequences, most of which correspond to human gene segments. These findings provide information on the αß TCR usage of pigs, which is understudied and deserves further attention.

The Role of Autophagy in iNKT Cell Development.

CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T cells that express an invariant T cell receptor (TCR) α-chain and recognize self and foreign glycolipid antigens. They can rapidly respond to agonist activation and stimulate an extensive array of immune responses. Thymic development and function of iNKT cells are regulated by many different cellular processes, including autophagy, a self-degradation mechanism. In this mini review, we discuss the current understanding of how autophagy regulates iNKT cell development and effector lineage differentiation. Importantly, we propose that iNKT cell development is tightly controlled by metabolic reprogramming.

The Role of NOD Mice in Type 1 Diabetes Research: Lessons from the Past and Recommendations for the Future.

For more than 35 years, the NOD mouse has been the primary animal model for studying autoimmune diabetes. During this time, striking similarities to the human disease have been uncovered. In both species, unusual polymorphisms in a major histocompatibility complex (MHC) class II molecule confer the most disease risk, disease is caused by perturbations by the same genes or different genes in the same biological pathways and that diabetes onset is preceded by the presence of circulating autoreactive T cells and autoantibodies that recognize many of the same islet antigens. However, the relevance of the NOD model is frequently challenged due to past failures translating therapies from NOD mice to humans and because the appearance of insulitis in mice and some patients is different. Nevertheless, the NOD mouse remains a pillar of autoimmune diabetes research for its usefulness as a preclinical model and because it provides access to invasive procedures as well as tissues that are rarely procured from patients or controls. The current article is focused on approaches to improve the NOD mouse by addressing reasons why immune therapies have failed to translate from mice to humans. We also propose new strategies for mixing and editing the NOD genome to improve the model in ways that will better advance our understanding of human diabetes. As proof of concept, we report that diabetes is completely suppressed in a knock-in NOD strain with a serine to aspartic acid substitution at position 57 in the MHC class II Aß. This supports that similar non-aspartic acid substitutions at residue 57 of variants of the human class II HLA-DQß homolog confer diabetes risk.

Harnessing Invariant NKT Cells to Improve Influenza Vaccines: A Pig Perspective.

Invariant natural killer T (iNKT) cells are an "innate-like" T cell lineage that recognize glycolipid rather than peptide antigens by their semi-invariant T cell receptors. Because iNKT cells can stimulate an extensive array of immune responses, there is considerable interest in targeting these cells to enhance human vaccines against a wide range of microbial pathogens. However, long overlooked is the potential to harness iNKT cell antigens as vaccine adjuvants for domestic animal species that express the iNKT cell-CD1d system. In this review, we discuss the prospect of targeting porcine iNKT cells as a strategy to enhance the efficiency of swine influenza vaccines. In addition, we compare the phenotype and tissue distribution of porcine iNKT cells. Finally, we discuss the challenges that must be overcome before iNKT cell agonists can be contemplated for veterinary use in livestock.

Detection of Alphacoronavirus vRNA in the Feces of Brazilian Free-Tailed Bats (Tadarida brasiliensis) from a Colony in Florida, USA.

Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats.

Characterizing porcine invariant natural killer T cells: A comparative study with NK cells and T cells.

CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T cells that share phenotypic characteristics of both NK and conventional T cells (Tconv). Although iNKT cells have been well characterized in mice and humans, functional CD1d and CD1d-restricted iNKT cells are not universally expressed in mammals. Swine express iNKT cells that can be detected using α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. In the present study, we characterized iNKT cells from the blood, spleen, lymph node, lung and liver of commercial mixed-breed pigs, and compared their phenotype to NK cells and Tconv. The principal findings are that pig iNKT cells are CD8α and CD44 positive and CD11b and Nkp46 negative. Most are also negative for the CD4 co-receptor, which is used to distinguish functionally distinct mouse and human iNKT cells subsets. The frequency of IFN-γ-producing CD8αbright iNKT cells was 3-4-fold higher than CD8αdull iNKT cells, suggesting that CD8α expression identifies iNKT cells with a unique functional role in immune responses. Finally, large variability was detected among pigs in interactions between iNKT cells and monocytes when iNKT cells were activated with α-GalCer, which raises a cautionary note about manipulating iNKT cells for immunotherapy. Collectively, our study provides important phenotypic and functional information about porcine iNKT cells that will be useful for understanding how iNKT cells contribute to immune responses in swine, with potential implications for human health.

Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes.

Type 1 diabetes development in the NOD mouse model is widely reported to be dependent on high-level production by autoreactive CD4+ and CD8+ T cells of interferon-γ (IFN-γ), generally considered a proinflammatory cytokine. However, IFN-γ can also participate in tolerance-induction pathways, indicating it is not solely proinflammatory. This study addresses how IFN-γ can suppress activation of diabetogenic CD8+ T cells. CD8+ T cells transgenically expressing the diabetogenic AI4 T-cell receptor adoptively transferred disease to otherwise unmanipulated NOD.IFN-γnull , but not standard NOD, mice. AI4 T cells only underwent vigorous intrasplenic proliferation in NOD.IFN-γnull recipients. Disease-protective IFN-γ could be derived from any lymphocyte source and suppressed diabetogenic CD8+ T-cell responses both directly and through an intermediary nonlymphoid cell population. Suppression was not dependent on regulatory T cells, but was associated with increased inhibitory STAT1 to STAT4 expression levels in pathogenic AI4 T cells. Importantly, IFN-γ exposure during activation reduced the cytotoxicity of human-origin type 1 diabetes-relevant autoreactive CD8+ T cells. Collectively, these results indicate that rather than marking the most proinflammatory lymphocytes in diabetes development, IFN-γ production could represent an attempted limitation of pathogenic CD8+ T-cell activation. Thus, great care should be taken when designing possible diabetic intervention approaches modulating IFN-γ production.

Rapid control of pandemic H1N1 influenza by targeting NKT-cells.

Swine influenza A viruses (IAV) are a major cause of respiratory disease in pigs and humans. Currently approved anti-influenza therapies directly target the virus, but these approaches are losing effectiveness as new viral strains quickly develop drug resistance. To over come this challenge, there is an urgent need for more effective antiviral drugs. Here we tested the anti-influenza efficacy of the invariant natural killer T (NKT) cell superagonist, α-galactosylceramide (α-GalCer), which stimulates a wide array of anti-viral immune responses. We show that intranasal but not systemic administration of α-GalCer to piglets infected with pandemic A/California/04/2009 (CA04) H1N1 IAV ameliorated disease symptoms and resulted in the restoration of weight gain to the level of uninfected pigs. Correspondingly, viral titers in the upper-and lower-respiratory tract were reduced only in piglets that had received intranasal α-GalCer. Most significantly, lung inflammation as a consequence of virus persistence was largely prevented when NKT-cells were targeted via the respiratory route. Thus, targeting mucosal NKT-cells may provide a novel and potent platform for improving the course of disease in swine infected with seasonal and pandemic influenza viruses, and leads to the suggestion that this may also be true in humans and therefore deserves further study.

Suppressors of Cytokine Signaling in Sickness and in Health of Pancreatic ß-Cells.

Suppressors of cytokine signaling (SOCS) are a family of eight proteins that negatively regulate Janus kinase and signal transducers and activators of transcription signaling in cells that utilize this pathway to respond to extracellular stimuli. SOCS are best known for attenuating cytokine signaling in the immune system. However, they are also expressed in many other cell types, including pancreatic ß-cells, where there is considerable interest in harnessing SOCS molecules to prevent cytokine-mediated apoptosis during diabetes and allogeneic transplantation. Apart from their potential as therapeutic targets, SOCS molecules play a central role for regulating important functions in ß-cells, including growth, glucose sensing, and insulin secretion. This review will discuss SOCS proteins as central regulators for diverse cellular processes important for normal ß-cell function as well as their protective anti-apoptotic effects during ß-cell stress.