David Cameron Wraith

Immune reset can be achieved by drastic disruption of the immune system. For example, myeloablative or non-myeloablative conditioning of patients followed by autologous human stem cell transplantation (aHSCT) can lead to sustained improvement in autoimmune diseases such as multiple sclerosis (Farge et al., 2010). However, this does not produce remission in all patients and remains associated with non-relapse mortality at a rate of ~1/30 (Snowden et al., 2017). As a result, clinical use of aHSCT for treatment of autoimmune diseases is only available in specialised clinics and does not have regulatory approval in most countries. Alemtuzumab, anti-CD52, antibody treatment leads to ablation of most white blood cells with sustained depletion of CD4 + cells for many months (Hill-Cawthorne et al., 2012). Treatment of patients with relapsing multiple sclerosis with a course of alemtuzumab has a dramatic impact on disease progression (Coles et al., 2012). However, treatment is associated with development of unrelated autoimmune conditions including Graves' disease and immune thrombocytopenic purpura in treated individuals as the immune system recovers from T cell depletion (Willis and Robertson, 2015). It seems likely that development of such unrelated autoimmune diseases is due to the impact of the depleting antibody on regulatory T cell populations (Baker et al., 2017).Results from studies with alemtuzumab warn us that non-discriminate depletion of T cells should be avoided.Most current immune reset approaches involve depletion of B cells. This seems sensible in antibody-mediated autoimmune conditions such as myasthenia gravis, Graves' and SLE; however, recent studies have shown that B cell depletion can have a dramatic impact on cellmediated conditions such as MS (Hauser et al., 2017). It is still not clear why antibodies targeting CD20 should have such an impact on MS. In theory, this could be due to a) depletion of antigen presenting cells (APCs), given the ability of CD20 + B cells to present antigens (Takemura et al., 2001;Rastogi et al., 2022) and b) depletion of EBV infected B cells, based on recent evidence that EBV infection has a role in initiation and / or propagation of MS-related immune pathology (Bjornevik et al., 2022). Anti-CD20 treatment has been approved for RA, pemphigus vulgaris and ANCA-positive vasculitis (Kaegi et al., 2019). While anti-CD20 was not effective in SLE and lupus nephritis, anti-CD19 CAR-T cell treatment has shown efficacy in rituximab-resistant patients (Muller et al., 2024). CAR-T cells have the added advantage of targeting B cells in lymphoid tissues (Tur et al., 2025). These results emphasise the need to understand the role of distinct B cell subsets in different diseases. More recently, bispecific antibodies targeting CD3 + T cells to CD19 + B cells have been used to treat blood cancers and have been tested in autoimmune diseases (Bucci et al., 2024). These, along with T cell engaging agents targeting T cells to alternative B cell surface antigens, such as B-cell maturation antigen (BCMA), are being developed (Hagen et al., 2024). Does B cell depletion lead to immune reset? The aim of immune reset is to provide restoration of a stable, self-tolerant immune system. However, a single cycle of B cell depletion rarely provides sustained clinical control: anti-B cell approaches generally require continuous treatment for effective disease control (Chen and Cohen, 2012). It should be noted that longterm B cell lymphopenia increases the risk of microbial infections (Varley and Winthrop, 2021) and hampers effective vaccination, as evidenced in the recent COVID-19 pandemic (Shields et al., 2022). Alternative approaches are required and here we propose the combined use of B cell depletion plus antigen-specific immunotherapy for stable control of autoimmune diseases.Antigen-specific immunotherapy (ASIT) has been used to control allergic diseases for over a century (Noon, 1911). However, despite clear evidence that this approach is effective in controlling experimental models of autoimmune diseases, it has been slow to translate to the clinic (Richardson and Wraith, 2021). The aim of ASIT is to 'switch off' pathogenic CD4 + T cells in a specific disease while simultaneously boosting self-antigen specific immunoregulatory T cells. This can be achieved through administration of self-antigens or their CD4 + T cell epitopes (Wraith, 2018). A variety of administration routes and modes of delivery are in development; in essence, these different approaches all aim to target the self-antigens or self-epitopes to tolerance promoting cells, such as steady state dendritic cells (ssDC) in lymphoid organs or tolerance promoting immune environments such as the liver. Most importantly, the ASIT approaches being developed do not rely on B cells for their functional effect. For example, work from our laboratory has shown that antigenic epitopes designed to function as highly soluble, antigen processing independent peptides (PIPs) selectively bind ssDC in lymphoid organs following injection (Shepard et al., 2021). PIPs preferentially bind to class II MHC molecules on ssDC since these cells do not load class II with peptide epitopes efficiently resulting in expression of unstable or peptide receptive class II MHC molecules at the cell surface (Santambrogio et al., 1999). This means that peptide epitopes designed to bind MHC II in the appropriate conformation (PIPs) will bind to MHC II on ssDC rather than B cells or monocytes since the latter cells load MHC II efficiently and have stable MHC II at their cell surface.Critically, ssDC express low levels of costimulatory molecules (Hubo et al., 2013); therefore, recognition of MHC II-peptide complexes on ssDC results in the induction of anergy in cognate, CD4 + helper cells and the propagation of antigen-specific regulatory T cells (Shepard et al., 2021). Most importantly, tolerance induction with PIPs does not depend on B cells....

Mechano‐modulation of cell surface proteins to influence cell activation has been shown as a promising new advanced therapy for regenerative medicine applications. These strategies rely on the manipulation of mechanosensitive cell surface receptors to initiate intracellular signal transduction. The cell surface receptor of T lymphocytes (TCR), which recognises peptide‐MHC molecules central to driving the adaptive immune response, has recently been suggested to be mechano‐responsive. Despite this advance, little is known as to whether the TCR can be mechanically modulated to achieve TCR signalling and subsequent T‐cell activation, and whether these characteristics can be exploited for immunotherapies. Here, we describe a magnetic particle‐based platform for mechanical modulation of the TCR and outline how this platform can be utilised to achieve CD4⁺ T‐cell activation. We demonstrate that mechanical manipulation of the TCR induces cell surface clustering of the TCR and downstream TCR signalling, leading to eventual TCR downregulation and T‐cell activation. We investigate the temporal relationship between mechanical modulation of the TCR and subsequent T‐cell activation, thereby identifying that accumulation of signalling events within the NFAT pathway is required to reach the threshold required for CD4⁺ T‐cell activation, outlining an axis which controls the CD4⁺ T‐cell response to external mechanical cues. These findings identify how CD4⁺ T cells can modulate their function in response to such cues while also outlining a remote‐magnetic particle‐based platform that may be used for the control of T‐cell responses.

Background: Peptides 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.Methods: Potential 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.Results: HDM-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.Conclusion: A 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.

Dysregulation and loss of immune tolerance toward pancreatic β-cell autoantigens are features of type 1 diabetes (T1D). Until recently, life-long insulin injection was the only approved treatment for T1D, but it does not address the underlying disease pathology. The aim for antigen-specific immunotherapy (ASI) is to restore tolerance. ASI holds potential as a new therapeutic strategy for treating autoimmune diseases with well-characterized antigens. Peptide ASI using processing-independent CD4+ T-cell epitopes (PIPs) shows promising results in several autoimmune diseases. Here, we successfully applied the principles of PIP design to the T1D autoantigen glutamate decarboxylase 65 (GAD65). Peptides spanning GAD65 predicted to be pan–HLA-DR binding were selected. Peptide 10 (P10) displayed enriched responses in peripheral blood mononuclear cells from people with T1D. The minimal epitope of the P10 peptide was fine mapped using T-cell hybridomas generated from HLA-DRB1*04:01 transgenic mice. This minimal epitope, P10Sol, was demonstrated, using a novel activation-induced marker assay, to induce tolerance to the parent peptide in the transgenic mice. Finally, we show that GAD65 P10Sol PIP is recognized by CD4+ T cells from people with T1D who possess a range of HLA-DR alleles and, therefore, can be defined as a pan–DR-binding peptide with therapeutic potential. Article Highlights: There are currently no approved antigen-specific immunotherapies (ASIs) for people with type 1 diabetes (T1D). We aimed to develop a peptide for ASI for T1D based on the T1D-associated auto-antigen glutamate decarboxylase 65 (GAD65). A minimal and soluble peptide derived from GAD65 was demonstrated to induce tolerance in an HLA transgenic mouse. Our data suggest this peptide derived from the GAD65 islet protein should be tested for therapeutic potential in people with T1D who have residual β-cell function.

/Summary: Mechano-modulation of cell surface proteins to influence cell activation has been shown as a promising new advanced therapy for regenerative medicine applications. These strategies rely on the manipulation of mechanosensitive cell surface receptors to initiate intracellular signal transduction. The cell surface receptor of T lymphocytes (TCR), which recognises peptide-MHC molecules central to driving the adaptive immune response, has recently been suggested to be mechano-responsive. Despite this advance, little is known as to whether the TCR can be mechanically modulated to achieve TCR signalling and subsequent T cell activation, and whether these characteristics can be exploited for immunotherapies. Here, we describe a magnetic particle-based platform for mechanical modulation of the TCR and outline how this platform can be utilised to achieve CD4 ⁺ T cell activation. We demonstrate that mechanical manipulation of the TCR induces cell surface clustering of the TCR and downstream TCR signalling, leading to eventual TCR downregulation and T cell activation. We investigate the temporal relationship between mechanical modulation of the TCR and subsequent T cell activation, hereby identifying that accumulation of signalling events within the NFAT-pathway is required to reach the threshold required for CD4 ⁺ T cell activation, outlining an axis which controls the CD4 ⁺ T cell response to external mechanical cues. These findings identify how CD4 ⁺ T cells can modulate their function in response to such cues, whilst also outlining a remote-magnetic particle-based platform that may be used for the control of T cell responses.

Summary: Strong T-cell receptor (TCR) and IL-27 signalling influence type-1 regulatory (Tr1) T-cell development but whether other signals determine their differentiation is unclear. Utilising 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 bona fide Tr1 cell protein and mRNA signatures. Kinetic transcriptional analysis revealed that the Tr1 cell module was transient and preceded by a burst of Ifng transcription in CD4+ T-cells. Neutralisation of IFNγ reduced Tr1 cell frequency and strong TCR signalling markers, which was correlated with reduced macrophage activation. Antibody depletion experiments inferred that T-cells – but not NK cells – provided the relevant source of IFNγ. Additionally, we show that blocking IL-27 in combination with IFNγ neutralisation additively reduced Tr1 cell frequency in vivo . These findings reveal that during strong tolerogenic TCR signalling IFN-γ has a non-redundant regulatory role in augmenting the differentiation of Tr1 cells in vivo .

Post-acute cardiac sequelae, following SARS-CoV-2 infection, are well recognised 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 analysed 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 patients. Furthermore, we find that DSG2 autoantibody levels are increased significantly in convalescent sera following severe COVID-19 but not in hospitalised 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 revealed disruption of the intercalated disc between cardiomyocytes that was consistent with separation of the DSG2 protein homodimer. Our results reveal the potential for DSG2 protein and autoimmunity to DSG2 to contribute to unexpected pathologies associated with COVID-19 infection.

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.