Defining the effects of CD28 superagonist and TGF-β on T cell function and metabolism

Immunomodulatory monoclonal antibodies (mAbs) indicated for the treatment of cancer, inflammatory and autoimmune diseases or to prevent organ transplant rejection, mostly target cell surface molecules expressed by immune cells including T cells. The presence of immunosuppressive molecules during disease progression can hinder the activity of immune cells. Tumour-induced immunosuppression enables disease progression and various strategies are being developed to enhance the anti-tumour responses of cytotoxic T cells. Activation of effective effector responses require resetting of metabolic activity to fit energy and anabolic needs. While the benefits of exploiting immunomodulatory mAbs for therapy are substantial, it is also clear that use of these biologics may be accompanied by adverse effects such as cytokine release, immunosuppression, infections and autoimmunity. Significant focus in recent times has been on assessing the potential of immunomodulatory mAbs to induce enhanced cytokine release but much less attention has been paid to other aspects of T cell biology including non-physiological activation phenotype/functions, migration characteristics and metabolism. An improved understanding of these parameters may assist in accurately predicting the propensity of new mAbs to induce serious adverse effects. Superagonistic CD28-specific monoclonal antibody (CD28SA) is one such immunomodulatory monoclonal antibody which is a potent stimulator of T cells, originally intended for the management of B cell chronic lymphocytic leukaemia and rheumatoid arthritis. Human volunteers who received humanized CD28SA (TGN1412) as part of a first-in-man trial experienced life-threatening cytokine release syndrome. Follow-up studies revealed aberrant activation of effector memory T cells (TEM) contributed towards the adverse reaction. The biopharmaceutical industry is actively pursuing development of T cell immunostimulatory mAbs and there is a significant need to improve the understanding of and accurately predict the propensity of a new mAb to drive excessive T cell activation. Therefore, one of the main aims of the study discussed in this thesis was to determine mechanism/s underlying the hyperactive phenotype of CD28SA-activated TEM. Observations in the current study revealed activation of TEM by CD28SA upregulated the expression of activation markers such as CD137 and HLA-DR, but failed to express co-inhibitory receptor, PD-1. This led to the lack of PD-1-mediated regulation of aberrant TEM activation. In addition, CD28SA-activated TEM expressed elevated levels of LFA-1 and CCR5 receptors, and displayed increased migratory capacity. Subsequent studies highlighted increased metabolic demand of CD28SA-activated TEM. The hyperactive cells with increased proliferative capacity exhibited distinct metabolic profile characterized by increased glycolysis and lipogenesis. These findings have profound implications for strategies aiming at understanding and predicting the safety profile of immunostimulatory mAbs. Deployment of immunosuppressive strategies, including TGF-β secretion by tumours to render immunostimulatory mAbs-mediated anti-tumour responses ineffective is well studied. The other study discussed in this thesis aimed to delineate the effects of oxidative stress in TGF-β-induced suppression of antigen-specific cytotoxic T cell responses. This study showed antigen-specific T cells exposed to TGF-β down regulate CD25 and LAG-3 (co-inhibitory T cell receptor) expression, secrete lower levels of IL-2 and IFN-γ, and reduced glycolysis. In addition, mitochondrial reactive oxygen species (MitoROS) scavenger rescued the effector functions such as proliferation and IFN-γ secretion of stimulated T cells that were inhibited by TGF-β. Our findings demonstrate that relief of TGF-β-induced oxidative stress restores the effector function of CD8+ cytotoxic T cells. Based on the current findings it would be potentially beneficial to supplement immunotherapies with antioxidants to counteract the immunosuppressive effects of tumour-derived TGF-β to help restore CD8+ T cell-mediated anti-tumour function. The findings presented in this thesis may help with defining key T cell biomarkers based of efficacy and hazard associated with immunomodulation

Inflammatory profiles across the spectrum of disease reveal a distinct role for GM-CSF in severe COVID-19

While it is now widely accepted that host inflammatory responses contribute to lung injury, the pathways that drive severity and distinguish coronavirus disease 2019 (COVID-19) from other viral lung diseases remain poorly characterized. We analyzed plasma samples from 471 hospitalized patients recruited through the prospective multicenter ISARIC4C study and 39 outpatients with mild disease, enabling extensive characterization of responses across a full spectrum of COVID-19 severity. Progressive elevation of levels of numerous inflammatory cytokines and chemokines (including IL-6, CXCL10, and GM-CSF) were associated with severity and accompanied by elevated markers of endothelial injury and thrombosis. Principal component and network analyses demonstrated central roles for IL-6 and GM-CSF in COVID-19 pathogenesis. Comparing these profiles to archived samples from patients with fatal influenza, IL-6 was equally elevated in both conditions whereas GM-CSF was prominent only in COVID-19. These findings further identify the key inflammatory, thrombotic, and vascular factors that characterize and distinguish severe and fatal COVID-19

Evaluation of intensive community care services for young people with psychiatric emergencies: study protocol for a multi-centre parallel-group, single-blinded randomized controlled trial with an internal pilot phase

Background: Over 3000 young people under the age of 18 are admitted to Tier 4 Child and Adolescent Mental Health Services (CAMHS) inpatient units across the UK each year. The average length of hospital stay for young people across all psychiatric units in the UK is 120 days. Research is needed to identify the most effective and efficient ways to care for young people (YP) with psychiatric emergencies. This study aims to evaluate the clinical effectiveness and cost-effectiveness of intensive community care service (ICCS) compared to treatment as usual (TAU) for young people with psychiatric emergencies. Methods: This is a multicentre two-arm randomized controlled trial (RCT) with an internal pilot phase. Young people aged 12 to < 18 considered for admission at participating NHS organizations across the UK will be randomized 1:1 to either TAU or ICCS. The primary outcome is the time to return to or start education, employment, or training (EET) at 6 months post-randomization. Secondary outcomes will include evaluations of mental health and overall well-being and patient satisfaction. Service use and costs and cost-effectiveness will also be explored. Intention-to-treat analysis will be adopted. The trial is expected to be completed within 42 months, with an internal pilot phase in the first 12 months to assess the recruitment feasibility. A process evaluation using visual semi-structured interviews will be conducted with 42 young people and 42 healthcare workers. Discussion: This trial is the first well-powered randomized controlled trial evaluating the clinical and cost-effectiveness of ICCS compared to TAU for young people with psychiatric emergencies in Great Britain. Trial registration: ISRCTN ISRCTN42999542, Registration on April 29, 202

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics

Elevated antiviral, myeloid and endothelial inflammatory markers in severe COVID-19

Introductory paragraph The mechanisms that underpin COVID-19 disease severity, and determine the outcome of infection, are only beginning to be unraveled. The host inflammatory response contributes to lung injury, but circulating mediators levels fall below those in classical ‘cytokine storms’. We analyzed serial plasma samples from 619 patients hospitalized with COVID-19 recruited through the prospective multicenter ISARIC clinical characterization protocol U.K. study and 39 milder community cases not requiring hospitalization. Elevated levels of numerous mediators including angiopoietin-2, CXCL10, and GM-CSF were seen at recruitment in patients who later died. Markers of endothelial injury (angiopoietin-2 and von-Willebrand factor A2) were detected early in some patients, while inflammatory cytokines and markers of lung injury persisted for several weeks in fatal COVID-19 despite decreasing antiviral cytokine levels. Overall, markers of myeloid or endothelial cell activation were associated with severe, progressive, and fatal disease indicating a central role for innate immune activation and vascular inflammation in COVID-19