BackgroundPeptides were designed to induce immune tolerance to the major antigen associated with house dust mite (HDM) allergy, Der p 1. HDM is commonly associated with allergic responses in allergic rhinitis and asthma, with Der p 1 specific T-cells implicated in ongoing disease. Tolerogenic peptide immunotherapy can induce tolerance in pathogenic T-cells, bypass mast cell activation and hence reduce the risk of anaphylaxis. A pan-DR binding epitope of Der p 1, covering the broad population, was tested for efficacy in HLA-DR transgenic mice.MethodsPotential pan-HLA-DR binding tolerogenic T-cell epitopes from Der p 1 were predicted in silico and manufactured (synthetic peptides A-E). Participants included HDM sensitised (allergic rhinitis/asthma, n=25), non-HDM sensitised (atopic controls sensitised to ≥1 other aero-allergens, n=10) and non-atopic healthy controls, n=10). Peripheral blood mononuclear cells (PBMC) were collected and screened for immune responses to Der p 1 or test peptides A-E. Mapping of minimal T-cell epitopes, apitope (antigen-processing independent epitope) validation and tolerance induction were tested in HLA-DR transgenic mice.ResultsHDM-sensitised subjects have an elevated response to pan-DR binding peptide D 30mer. Peptide analogue D121B, containing the minimal epitope and optimised for solubility, was verified as a tolerogenic apitope and induced tolerance against Der p 1 antigens in HLA-DR4 transgenic mice in vivo.ConclusionA tolerogenic peptide, apitope D121B, reduces T-cell immune responses to Der p 1 and is a promising candidate for further development as an immunotherapy for HDM-associated allergic rhinitis and asthma.

Strong T cell receptor (TCR) and interleukin (IL)-27 signaling influence type 1 regulatory (Tr1) T cell development, but whether other signals determine their differentiation is unclear. Utilizing Tg4 TCR transgenic mice, we established a model for rapid Tr1 cell induction. A single high dose of [4Y]-MBP peptide drove the differentiation of Il10+ T cells with Tr1 cell mRNA and protein signatures. Kinetic transcriptional and phenotypic analyses revealed that the Tr1 cell module was transient and preceded by Ifng transcription in other CD4+ T cells. Changes in Tr1 cell frequency correlated with altered macrophage activation, while neutralization of interferon (IFN)γ reduced Tr1 cell frequency and the TCR signal strength markers Nur77, inducible T cell costimulator (ICOS), and OX40. Antibody depletion experiments inferred that the relevant source of IFNγ was not natural killer (NK) cell derived. Additionally, blocking IL-27 in combination with IFNγ neutralization additively reduced Tr1 cell frequency in vivo. These findings reveal that IFNγ has a non-redundant role in augmenting Tr1 cell differentiation in vivo.

Current therapies for autoimmune diseases do not address the underlying cause of disease, the failure of immune tolerance to self. The non-specific therapies currently used to treat autoimmune diseases increase the risk of infections and cancers. Antigen-specific therapies target pathogenic autoreactive lymphocytes while preserving protective immune responses. This short synthesis will review our laboratory's work on the design and development of antigen-specific immunotherapies for multiple sclerosis and other autoimmune diseases.

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.

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of antibodies to SARS-CoV-2 from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the subject group from whom the sera were obtained. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending the disease status of the subject and on the specific antigen targeted.

AbstractObjectiveTo assess whether there is an association between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) infection and the incidence of immune mediated inflammatory diseases (IMIDs).DesignMatched cohort study.SettingPrimary care electronic health record data from the Clinical Practice Research Datalink Aurum database.ParticipantsThe exposed cohort included 458,147 adults aged 18 years and older with a confirmed SARS CoV-2 infection by reverse transcriptase polymerase chain reaction (RT-PCR) or lateral flow antigen test, and no prior diagnosis of IMIDs. They were matched on age, sex, and general practice to 1,818,929 adults in the unexposed cohort with no diagnosis of confirmed or suspected SARS CoV-2 infection and no prior diagnosis of IMIDs.Main Outcome MeasuresThe primary outcome measure was a composite of the incidence of any of the following IMIDs: 1. autoimmune thyroiditis, 2. coeliac disease, 3. inflammatory bowel disease (IBD), 4. myasthenia gravis, 5. pernicious anaemia, 6. psoriasis, 7. rheumatoid arthritis (RA), 8. Sjogren’s syndrome, 9. systemic lupus erythematosus (SLE), 10. type 1 diabetes mellitus (T1DM), and 11. vitiligo. The secondary outcomes were the incidence of each of these conditions separately. Cox proportional hazards models were used to estimate adjusted hazard ratios (aHR) and 95% confidence intervals (CI) for the primary and secondary outcomes comparing the exposed to the unexposed cohorts, and adjusting for age, sex, ethnic group, smoking status, body mass index, relevant infections, and medications.Results537 patients (0.11%) in the exposed cohort developed an IMID during the follow-up period over 0.29 person years, giving a crude incidence rate of 3.54 per 1000 person years. This was compared 1723 patients (0.09%) over 0.29 person years in the unexposed cohort, with an incidence rate of 2.82 per 1000 person years. Patients in the exposed cohort had a 22% relative increased risk of developing an IMID, compared to the unexposed cohort (aHR 1.22, 95% CI 1.10 to 1.34). The incidence of three IMIDs were statistically significantly associated with SARS CoV-2 infection. These were T1DM (aHR 1.56, 95% CI 1.09 to 2.23), IBD (1.52, 1.23 to 1.88), and psoriasis (1.23, 1.05 to 1.42).ConclusionsSARS CoV-2 was associated with an increased incidence of IMIDs including T1DM, IBD and psoriasis. Further research is needed to replicate these findings in other populations and to measure autoantibody profiles in cohorts of individuals with COVID-19, including Long COVID and matched controls.Summary BoxWhat is already known on this topicA subsection of the population who tested positive for SARS CoV-2 is suffering from post-Covid-19 condition or long COVID.Preliminary findings, such as case reports of post-COVID-19 IMIDs, increased autoantibodies in COVID-19 patients, and molecular mimicry of the SARS-CoV-2 virus have given rise to the theory that long COVID may be due in part to a deranged immune response.What this study addsCOVID-19 exposure was associated with a 22% relative increase in the risk of developing certain IMIDs, including type 1 diabetes mellitus, inflammatory bowel disease, and psoriasis.These findings provide further support to the hypothesis that a subgroup of Long Covid may be caused by immune mediated mechanisms.

Lymphocyte receptors confer antigen specificity on the adaptive immune response. Increasing evidence points to the role of adaptive tuning particularly amongst CD4+ T cell responses. This review summarises how T cell tuning impacts on critically important aspects of immune regulation including thymic selection, the immune response to chronic antigen exposure and antigen-specific immunotherapy of autoimmune conditions. Recent work has revealed a novel mechanism for T cell anergy and regulatory type 1 T cell differentiation through a limitation of T cell receptor mediated signalling combined with epigenetic priming of tolerance associated genes.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a range of persistent symptoms impacting everyday functioning, known as post-COVID-19 condition or long COVID. We undertook a retrospective matched cohort study using a UK-based primary care database, Clinical Practice Research Datalink Aurum, to determine symptoms that are associated with confirmed SARS-CoV-2 infection beyond 12 weeks in non-hospitalized adults and the risk factors associated with developing persistent symptoms. We selected 486,149 adults with confirmed SARS-CoV-2 infection and 1,944,580 propensity score-matched adults with no recorded evidence of SARS-CoV-2 infection. Outcomes included 115 individual symptoms, as well as long COVID, defined as a composite outcome of 33 symptoms by the World Health Organization clinical case definition. Cox proportional hazards models were used to estimate adjusted hazard ratios (aHRs) for the outcomes. A total of 62 symptoms were significantly associated with SARS-CoV-2 infection after 12 weeks. The largest aHRs were for anosmia (aHR 6.49, 95% CI 5.02–8.39), hair loss (3.99, 3.63–4.39), sneezing (2.77, 1.40–5.50), ejaculation difficulty (2.63, 1.61–4.28) and reduced libido (2.36, 1.61–3.47). Among the cohort of patients infected with SARS-CoV-2, risk factors for long COVID included female sex, belonging to an ethnic minority, socioeconomic deprivation, smoking, obesity and a wide range of comorbidities. The risk of developing long COVID was also found to be increased along a gradient of decreasing age. SARS-CoV-2 infection is associated with a plethora of symptoms that are associated with a range of sociodemographic and clinical risk factors.

Objective To determine clinical and ethnodemographic correlates of serological responses against the SARS-CoV-2 spike glycoprotein following mild-to-moderate COVID-19. Design A retrospective cohort study of healthcare workers who had self-isolated due to COVID-19. Setting University Hospitals Birmingham NHS Foundation Trust, UK (UHBFT). Participants 956 healthcare workers were recruited by open invitation via UHBFT trust email and social media between 27 April 2020 and the 8 June 2020. Intervention Participants volunteered a venous blood sample that was tested for the presence of anti-SARS-CoV-2 spike glycoprotein antibodies. Results were interpreted in the context of the symptoms of their original illness and ethnodemographic variables. Results Using an assay that simultaneously measures the combined IgG, IgA and IgM response against the spike glycoprotein (IgGAM), the overall seroprevalence within this cohort was 46.2% (n=442/956). The seroprevalence of immunoglobulin isotypes was 36.3%, 18.7% and 8.1% for IgG, IgA and IgM, respectively. IgGAM identified serological responses in 40.6% (n=52/128) of symptomatic individuals who reported a negative SARS-CoV-2 PCR test. Increasing age, non-white ethnicity and obesity were independently associated with greater IgG antibody response against the spike glycoprotein. Self-reported fever and fatigue were associated with greater IgG and IgA responses against the spike glycoprotein. The combination of fever and/or cough and/or anosmia had a positive predictive value of 92.3% for seropositivity in self-isolating individuals a time when Wuhan strain SARS-CoV-2 was predominant. Conclusions and relevance Assays employing combined antibody detection demonstrate enhanced seroepidemiological sensitivity and can detect prior viral exposure even when PCR swabs have been negative. We demonstrate an association between known ethnodemographic risk factors associated with mortality from COVID-19 and the magnitude of serological responses in mild-to-moderate disease.

Summary 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 as a digital read-out of NFAT pathway activity, we identify the rapid quantitative and qualitative changes that occur in CD4 + T cells in response to a range of TCR signalling strengths. We demonstrate that the time and dose dependent programming of distinct co-inhibitory receptors rapidly re-calibrates T cell activation thresholds. By developing a new in vivo model, we analyse the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 but not anti-Lag3 immunotherapy leads to an increased TCR signal strength. We define a strong TCR signal metric of five genes specifically upregulated by anti-PD1 in T cells (TCR.strong), which can stratify clinical outcomes during anti-PD1 monotherapy in melanoma patients. Our study therefore reveals how analysis of TCR signal strength – and its manipulation – can provide powerful metrics for monitoring outcomes to immunotherapy. Key Points TCR signal strength-dependent programming of CD4 + T cells revealed over time in vivo Inhibitory receptor expression is dynamic, TCR signal strength dependent, and rapidly re-calibrates T cell activation thresholds PD1 but not Lag3 blockade leads to a unique and increased TCR signal strength signature (coined TCR.strong) TCR.strong metric stratifies melanoma patient survival in response to Nivolumab (anti-PD1) therapy

Summary Targeted restoration of immunological tolerance to self-antigens or innocuous environmental allergens represents the ultimate aim of treatment options in autoimmune and allergic disease. Antigen-specific immunotherapy (ASI) is the only intervention that has proven disease-modifying efficacy as evidenced by induction of long-term remission in a number of allergic conditions. Mounting evidence is now indicating that specific targeting of pathogenic T cells in autoinflammatory and autoimmune settings enables effective restoration of immune homeostasis between effector and regulatory cells and alters the immunological course of disease. Here, we discuss the key lessons learned during the development of antigen-specific immunotherapies and how these can be applied to inform future interventions. Armed with this knowledge and current high-throughput technology to track immune cell phenotype and function, it may no longer be a matter of ‘if’ but ‘when’ this ultimate aim of targeted tolerance restoration is realised.

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.

Haemophilia A (HA) is a blood clotting disorder caused by various genetic deficiencies in the factor VIII (FVIII) encoding F8 gene. Patients receiving FVIII replacement therapy are at risk of developing neutralizing antibodies (FVIII inhibitors) rendering the FVIII replacement therapy ineffective. Immunological tolerance towards FVIII can be achieved through immune tolerance induction (ITI) protocols in some patients but this is a lengthy and costly desensitization programme. Long-term eradication of inhibitors in HA patients could be achieved by antigen-specific immunotherapy targeting CD4+ T cells since formation of FVIII inhibitors is T cell dependent. Here, we report a peptide-based, antigen-specific immunotherapy designed to specifically re-establish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified two FVIII immunodominant peptides in immunised human leukocyte antigen (HLA) DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimised for tolerogenicity. These modified peptide analogues were initially screened for recognition using FVIII-specific T cell hybridoma clones from FVIII-immunised HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1*haplotypes. The combination of these two FVIII apitopes (ATX-F8-117), administered according to a dose escalation protocol, promoted T cell tolerance towards FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates both anti-FVIII T cell and B cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to both suppress and treat inhibitor formation in susceptible HA patients.

Current treatments for autoimmune diseases do not address the immune pathology underlying their initiation and progression and too often rely on non-specific immunosuppressive drugs for control of symptoms. Antigen-specific immunotherapy aims to induce tolerance selectively among the cells causing the disease while leaving the rest of the adaptive immune system capable of protecting against infectious diseases and cancers. Here we describe how novel approaches for antigen-specific immunotherapy are designed to manipulate antigen presentation and promote tolerance to specific self-antigens. This analysis points to liver antigen presenting cells, targeted by carrier particles, and steady-state dendritic cells, to which antigen-processing independent T-cell epitopes (apitopes) bind directly, as the principal targets for antigen-specific immunotherapy. Delivery of antigens to these cells holds great promise for effective control of this rapidly expanding group of diseases.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

Immunotherapy with antigen-processing independent T cell epitopes (apitopes) targeting autoreactive CD4+ T cells has translated to the clinic and been shown to modulate progression of both Graves’ disease and multiple sclerosis. The model apitope (Ac1-9[4Y]) renders antigen-specific T cells anergic while repeated administration induces both Tr1 and Foxp3+ regulatory cells. Here we address why CD4+ T cell epitopes should be designed as apitopes to induce tolerance and define the antigen presenting cells that they target in vivo. Furthermore, we reveal the impact of treatment with apitopes on CD4+ T cell signaling, the generation of IL-10-secreting regulatory cells and the systemic migration of these cells. Taken together these findings reveal how apitopes induce tolerance and thereby mediate antigen-specific immunotherapy of autoimmune diseases.

Summary 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.

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.