Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength.

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

Regulatory T Cell Migration Is Dependent on Glucokinase-Mediated Glycolysis.

Migration of activated regulatory T (Treg) cells to inflamed tissue is crucial for their immune-modulatory function. While metabolic reprogramming during Treg cell differentiation has been extensively studied, the bioenergetics of Treg cell trafficking remains undefined. We have investigated the metabolic demands of migrating Treg cells in vitro and in vivo. We show that glycolysis was instrumental for their migration and was initiated by pro-migratory stimuli via a PI3K-mTORC2-mediated pathway culminating in induction of the enzyme glucokinase (GCK). Subsequently, GCK promoted cytoskeletal rearrangements by associating with actin. Treg cells lacking this pathway were functionally suppressive but failed to migrate to skin allografts and inhibit rejection. Similarly, human carriers of a loss-of-function GCK regulatory protein gene-leading to increased GCK activity-had reduced numbers of circulating Treg cells. These cells displayed enhanced migratory activity but similar suppressive function, while conventional T cells were unaffected. Thus, GCK-dependent glycolysis regulates Treg cell migration.

Incidence of immune-mediated inflammatory diseases following COVID-19: a matched cohort study in UK primary care.

BACKGROUND: Some patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) go on to experience post-COVID-19 condition or long COVID. Preliminary findings have given rise to the theory that long COVID may be due in part to a deranged immune response. In this study, we assess whether there is an association between SARS-CoV-2 infection and the incidence of immune-mediated inflammatory diseases (IMIDs). METHODS: Matched cohort study using primary care electronic health record data from the Clinical Practice Research Datalink Aurum database. The exposed cohort included 458,147 adults aged 18 years and older with a confirmed SARS-CoV-2 infection and no prior diagnosis of IMIDs. They were matched on age, sex, and general practice to 1,818,929 adults with no diagnosis of confirmed or suspected SARS-CoV-2 infection. The primary outcome was a composite of any of the following IMIDs: autoimmune thyroiditis, coeliac disease, inflammatory bowel disease (IBD), myasthenia gravis, pernicious anaemia, psoriasis, rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1DM), and vitiligo. The secondary outcomes were each of these conditions separately. Cox proportional hazard models were used to estimate adjusted hazard ratios (aHR) and 95% confidence intervals (CI) for the primary and secondary outcomes, adjusting for age, sex, ethnic group, smoking status, body mass index, relevant infections, and medications. RESULTS: Six hundred and nighty six (0.15%) and 2230 (0.12%) patients in the exposed and unexposed cohort developed an IMID during the follow-up period over 0.29 person-years, giving a crude incidence rate of 4.59 and 3.65 per 1000 person-years, respectively. Patients in the exposed cohort had a 22% increased risk of developing an IMID, compared to the unexposed cohort (aHR 1.22, 95% CI 1.12 to 1.33). The incidence of three IMIDs was significantly associated with SARS-CoV-2 infection. These were T1DM (aHR 1.56, 1.09 to 2.23), IBD (aHR 1.36, 1.18 to 1.56), and psoriasis (1.23, 1.05 to 1.42). CONCLUSIONS: SARS-CoV-2 was associated with an increased incidence of IMIDs including T1DM, IBD and psoriasis. However, these findings could be potentially due to ascertainment bias. Further research is needed to replicate these findings in other populations and to measure autoantibody profiles in cohorts of individuals with COVID-19.

SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection.

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognized as complications of COVID-19. We have previously shown the persistence of autoantibodies against antigens in skin, muscle, and heart in individuals following severe COVID-19; the most common staining on skin tissue displayed an inter-cellular cement pattern consistent with antibodies against desmosomal proteins. Desmosomes play a critical role in maintaining the structural integrity of tissues. For this reason, we analyzed desmosomal protein levels and the presence of anti-desmoglein (DSG) 1, 2, and 3 antibodies in acute and convalescent sera from patients with COVID-19 of differing clinical severity. We find increased levels of DSG2 protein in sera from acute COVID-19 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalized patients recovering from influenza infection or healthy controls. Levels of autoantibody in sera from patients with severe COVID-19 were comparable to levels in patients with non-COVID-19-associated cardiac disease, potentially identifying DSG2 autoantibodies as a novel biomarker for cardiac damage. To determine if there was any association between severe COVID-19 and DSG2, we stained post-mortem cardiac tissue from patients who died from COVID-19 infection. This confirmed DSG2 protein within the intercalated discs and disruption of the intercalated disc between cardiomyocytes in patients who died from COVID-19. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.

Development of a high-sensitivity ELISA detecting IgG, IgA and IgM antibodies to the SARS-CoV-2 spike glycoprotein in serum and saliva.

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Establishing the prevalence of common tissue-specific autoantibodies following severe acute respiratory syndrome coronavirus 2 infection.

Coronavirus 19 (COVID-19) has been associated with both transient and persistent systemic symptoms that do not appear to be a direct consequence of viral infection. The generation of autoantibodies has been proposed as a mechanism to explain these symptoms. To understand the prevalence of autoantibodies associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we investigated the frequency and specificity of clinically relevant autoantibodies in 84 individuals previously infected with SARS-CoV-2, suffering from COVID-19 of varying severity in both the acute and convalescent setting. These were compared with results from 32 individuals who were on the intensive therapy unit (ITU) for non-COVID reasons. We demonstrate a higher frequency of autoantibodies in the COVID-19 ITU group compared with non-COVID-19 ITU disease control patients and that autoantibodies were also found in the serum 3-5 months post-COVID-19 infection. Non-COVID patients displayed a diverse pattern of autoantibodies; in contrast, the COVID-19 groups had a more restricted panel of autoantibodies including skin, skeletal muscle and cardiac antibodies. Our results demonstrate that respiratory viral infection with SARS-CoV-2 is associated with the detection of a limited profile of tissue-specific autoantibodies, detectable using routine clinical immunology assays. Further studies are required to determine whether these autoantibodies are specific to SARS-CoV-2 or a phenomenon arising from severe viral infections and to determine the clinical significance of these autoantibodies.

Preclinical models of arthritis for studying immunotherapy and immune tolerance.

Increasingly earlier identification of individuals at high risk of rheumatoid arthritis (RA) (eg, with autoantibodies and mild symptoms) improves the feasibility of preventing or curing disease. The use of antigen-specific immunotherapies to reinstate immunological self-tolerance represent a highly attractive strategy due to their potential to induce disease resolution, in contrast to existing approaches that require long-term treatment of underlying symptoms.Preclinical animal models have been used to understand disease mechanisms and to evaluate novel immunotherapeutic approaches. However, models are required to understand critical processes supporting disease development such as the breach of self-tolerance that triggers autoimmunity and the progression from asymptomatic autoimmunity to joint pain and bone loss. These models would also be useful in evaluating the response to treatment in the pre-RA period.This review proposes that focusing on immune processes contributing to initial disease induction rather than end-stage pathological consequences is essential to allow development and evaluation of novel immunotherapies for early intervention. We will describe and critique existing models in arthritis and the broader field of autoimmunity that may fulfil these criteria. We will also identify key gaps in our ability to study these processes in animal models, to highlight where further research should be targeted.

Peptide allergen-specific immunotherapy for allergic airway diseases-State of the art.

Allergen-specific immunotherapy (AIT) is the only means of altering the natural immunological course of allergic diseases and achieving long-term remission. Pharmacological measures are able to suppress the immune response and/or ameliorate the symptoms but there is a risk of relapse soon after these measures are withdrawn. Current AIT approaches depend on the administration of intact allergens, often comprising crude extracts of the allergen. We propose that the challenges arising from current approaches, including the risk of serious side-effects, burdensome duration of treatment, poor compliance and high cost, are overcome by application of peptides based on CD4+ T cell epitopes rather than whole allergens. Here we describe evolving approaches, summarize clinical trials involving peptide AIT in allergic rhinitis and asthma, discuss the putative mechanisms involved in their action, address gaps in evidence and propose future directions for research and clinical development.

SARS-CoV-2 seroprevalence and asymptomatic viral carriage in healthcare workers: a cross-sectional study.

OBJECTIVE: To determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers. DESIGN: A cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020. SETTING: University Hospitals Birmingham NHS Foundation Trust (UHBFT), UK. PARTICIPANTS: 545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded. INTERVENTION: Participants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked. MAIN OUTCOME MEASURE: Proportion of participants demonstrating infection and positive SARS-CoV-2 serology. RESULTS: The point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02). CONCLUSIONS AND RELEVANCE: We identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.

A humanized HLA-DR4 mouse model for autoimmune myocarditis.

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment.

Myeloid-derived suppressor cells mediate tolerance induction in autoimmune disease.

In multiple sclerosis (MS) T cells aberrantly recognize self-peptides of the myelin sheath and attack the central nervous system (CNS). Antigen-specific peptide immunotherapy, which aims to restore tolerance while avoiding the use of non-specific immunosuppressive drugs, is a promising approach to combat autoimmune disease, but the cellular mechanisms behind successful therapy remain poorly understood. Myeloid-derived suppressor cells (MDSCs) have been studied intensively in the field of cancer and to a lesser extent in autoimmunity. Because of their suppressive effect on the immune system in cancer, we hypothesized that the development of MDSCs and their interaction with CD4+ T cells could be beneficial for antigen-specific immunotherapy. Hence, changes in the quantity, phenotype and function of MDSCs during tolerance induction in our model of MS were evaluated. We reveal, for the first time, an involvement of a subset of MDSCs, known as polymorphonuclear (PMN)-MDSCs, in the process of tolerance induction. PMN-MDSCs were shown to adopt a more suppressive phenotype during peptide immunotherapy and inhibit CD4+ T-cell proliferation in a cell-contact-dependent manner, mediated by arginase-1. Moreover, increased numbers of tolerogenic PMN-MDSCs, such as observed over the course of peptide immunotherapy, were demonstrated to provide protection from disease in a model of experimental autoimmune encephalomyelitis.

Glycogen synthase kinase-3 controls IL-10 expression in CD4(+) effector T-cell subsets through epigenetic modification of the IL-10 promoter.

The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Extra-thymically induced T regulatory cell subsets: the optimal target for antigen-specific immunotherapy.

Antigen-specific immunotherapy aims to selectively restore tolerance to innocuous antigens in cases of autoimmune or allergic disease, without the need for general immune suppression. Although the principle of antigen-specific immunotherapy was discovered more than a century ago, its clinical application to date is limited, particularly in the control of autoimmunity. This has resulted mainly from a lack of in-depth understanding of the underlying mechanism. More recently, the differentiation of extra-thymically induced T regulatory (Treg) cell subsets has been shown to be instrumental in peripheral tolerance induction. Two main types of inducible Treg cells, interleukin-10-secreting or Foxp3(+) , have now been described, each with distinct characteristics and methods of therapeutic induction. It is crucial, therefore, to identify the suitability of either subset in the control of specific immune disorders. This review explores their natural function, the known mechanisms of therapeutic differentiation of either subset as well as their in vivo functionality and discusses new developments that may aid their use in antigen-specific immunotherapy, with a focus on autoimmune disease.

Nanoparticle-based autoantigen delivery to Treg-inducing liver sinusoidal endothelial cells enables control of autoimmunity in mice.

BACKGROUND & AIMS: It is well-known that the liver can induce immune tolerance, yet this knowledge could, thus far, not be translated into effective treatments for autoimmune diseases. We have previously shown that liver sinusoidal endothelial cells (LSECs) could substantially contribute to hepatic tolerance through their ability to induce CD4+ Foxp3+ regulatory T cells (Tregs). Here, we explored whether the Treg-inducing potential of LSECs could be harnessed for the treatment of autoimmune disease. METHODS: We engineered a polymeric nanoparticle (NP) carrier for the selective delivery of autoantigen peptides to LSECs in vivo. In the well-characterized autoimmune disease model of experimental autoimmune encephalomyelitis (EAE), we investigated whether administration of LSEC-targeting autoantigen peptide-loaded NPs could protect mice from autoimmune disease. RESULTS: We demonstrate that NP-based autoantigen delivery to LSECs could completely and permanently prevent the onset of clinical EAE. More importantly, in a therapeutic approach, mice with already established EAE improved rapidly and substantially following administration of a single dose of autoantigen peptide-loaded NPs, whereas the control group deteriorated. Treatment efficacy seemed to depend on Tregs. The Treg frequencies in the spleens of mice treated with autoantigen peptide-loaded NPs were significantly higher than those in vehicle-treated mice. Moreover, NP-mediated disease control was abrogated after Treg depletion by repeated administration of Treg-depleting antibody. CONCLUSION: Our findings provide proof of principle that the selective delivery of autoantigen peptides to LSECs by NPs can induce antigen-specific Tregs and enable effective treatment of autoimmune disease. These findings highlight the importance of Treg induction by LSECs for immune tolerance.

Selection and fine-tuning of the autoimmune T-cell repertoire.

The immune system must avoid aggressive T-cell responses against self-antigens. But, paradoxically, exposure to self-peptides seems to have an important role in positive selection in the thymus and the maintenance of a broad T-cell repertoire in the periphery. Recent experiments have highlighted situations that allow high-avidity self-reactive T cells to avoid negative selection in the thymus. Accumulating evidence indicates that other, non-deleting mechanisms control the avidity with which T cells recognize self-antigens--a phenomenon that is known as 'tuning'. This might maximize the peripheral T-cell repertoire by allowing the survival of T cells that can respond to self, but only at concentrations that are not normally reached in vivo.

TGF-ß-dependent induction of CD4⁺CD25⁺Foxp3⁺ Tregs by liver sinusoidal endothelial cells.

BACKGROUND & AIMS: CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. METHODS: To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3(-) non-Tregs into Foxp3(+) Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-ß was tested in Treg conversion assays using T cells with reduced TGF-ß sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). RESULTS: All tested liver cell types were capable of inducing Foxp3(+) Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-ß. LSECs featured membrane-bound LAP/TGF-ß and the anchor molecule GARP, which is required for tethering LAP/TGF-ß to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. CONCLUSIONS: We demonstrate that LSECs are the major liver cell type responsible for TGF-ß dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-ß to their membrane.