Sarah Christina Sasson

Acute lung injury (ALI) is characterised by hypoxia and inflammation that derives from diverse aetiologies. Delineating the molecular pathogeneses may identify treatable subgroups and inform clinical trials. Using a multi-omics approach, we analysed trauma-associated ALI (n = 9), COVID-19 ALI (n = 11), ICU controls (n = 13) using scRNA-seq, serum cytokine profiling, and bronchoalveolar lavage (BAL) microbiome analysis. We identified a population of activated CD4⁺ T cells expressing Toll-like receptor 4 (TLR4) and downstream NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome complex, predominantly in trauma-associated ALI (23% of CD45⁺ Cells) compared to COVID-19 ALI (14%) and ICU controls (15%). Serum IL-1RA and IL-6 were upregulated in trauma ALI, along with TLR4-binding ligand hyaluronan. TLR4 expression on CD4⁺ T cells was confirmed by spectral cytometry. Lung microbiome analysis revealed distinct diversity in trauma-associated ALI compared to COVID-19 ALI. These findings highlight TLR4-mediated inflammation in trauma-associated ALI and suggest potential precision medicine approaches for ALI management.

Neutralizing antibodies are a correlate of protection (CoP) of SARS-CoV-2 vaccine efficacy. However, data informing CoPs often explicitly exclude people on immunosuppressive therapies who are at an increased risk of symptomatic and severe COVID-19, delayed viral clearance, and death. Investigating the relationship between antibodies and protection for people on immunosuppressive therapies could provide insights into mechanisms of protection. We performed a systematic search to identify studies reporting SARS-CoV-2 binding antibody levels after mRNA vaccination in people with hematological malignancies, neurological disorders, rheumatic diseases, and mixed immune-mediated inflammatory disorders (IMIDs) on immunosuppressive therapies including B-cell depletion, JAK inhibitors, and S1P inhibitors, as well as healthy controls. Binding antibody levels varied between cohorts receiving different immunosuppressive treatments. Compared to healthy controls the lowest antibody levels were observed in subjects treated with B-cell depletion (54.5-fold reduction, 95% CI: 34.5-86.2) and S1P inhibitors (24.8-fold reduction, 95% CI: 13.2-46.7). To assess the association between antibody level and protection, we used data from a previous study of COVID-19 vaccine effectiveness in people with the same underlying conditions. We linked vaccine effectiveness estimates with the predicted antibody level in people on immunosuppressive therapies (using antibody data including this meta-analysis of binding antibody levels). Predicted neutralizing antibody levels were correlated with vaccine effectiveness both for infection and hospitalization (p<0.0001 and p<0.0001, respectively). However, we found that for any given antibody level, people who are immunosuppressed had lower protection against infection and hospitalization than a healthy population.

Introduction Immunosuppression is associated with an increased risk of delayed SARS-CoV-2 viral clearance, severe COVID-19 and related death. This heterogeneous group of affected patients includes but is not limited to those with a haematological malignancy, people on immunosuppressive therapy for the treatment of autoimmune/inflammatory diseases and those following bone marrow transplantation (BMT). Immunosuppression is associated with decreased rates of anti-spike IgG seroconversion following COVID-19 vaccination. While clinical guidelines have been established to guide vaccination pre-splenectomy and post-BMT, there are limited data to guide timing of COVID-19 or other booster vaccines in adults commencing new or intensified moderate to severe immunosuppression. The comparison of immunity-boosting regimens for COVID-19 upon initiation of immunosuppressive therapy (CIRCUIT) study was designed to address this knowledge gap. CIRCUIT investigates whether administration of a third (or subsequent) COVID-19 booster vaccine ≤2 weeks prior to immunosuppression provides greater anti-spike IgG-mediated immunity than a booster given 24 weeks after new or intensified immunosuppression, that is, week 24 timepoint (Group 1; n=280). Additionally, the research will investigate whether giving a fourth post-BMT COVID-19 booster vaccine at 9 months post-transplant provides greater anti-spike IgG-mediated immunity than a booster given 15 months post-transplant (Group 2; n=40). Methods and analysis The CIRCUIT study is an open-label, multicentre randomised clinical trial. Participants will be randomised 1:1 to receive either an additional COVID-19 booster ≤2 weeks pre-immunosuppression and a diphtheria/tetanus toxoids (DT) booster at 24 weeks following new or intensified immunosuppression (week 24 timepoint) or receive a DT booster ≤2 weeks pre-immunosuppression and an additional COVID-19 booster at week 24 (Group 1). Group 2 participants who underwent autologous or allogenic BMT in the last 9 months will be randomised 1:1 to either receive a fourth post-BMT COVID-19 booster at 9 or 15 months post-transplant. The primary outcome will be the integrated time-weighted area under the curve anti-SARS-CoV-2 neutralising antibody (NAb) response over 12 months from a SARS-CoV-2 booster as assessed by a high-throughput SARS-CoV-2 NAb platform assay. Key secondary outcomes of the CIRCUIT randomised control trial will include safety and generation of SARS-CoV-2 antigen specific T and B cell responses. Ethics and dissemination The research protocol was approved by the Western Sydney Local Health District Human Research Ethics Committee on 25 August 2022 (Ref no. 2022/PID00782 – 20022/ETH0069). Study results will be published in peer-reviewed medical journals and presented at local and international conferences. All findings regardless of the outcome will be reported. Trial registration number NCT05415267.

Toll-like receptor 4 (TLR4) is a pattern recognition receptor that binds to pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Binding of exogenous or endogenous ligands activates the TLR4 pathway and induces the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β and type 1 interferons including IFN-α and IFN-β. TLR4 plays a vital role in host defense against Gram-negative bacterial infections by recognizing lipopolysaccharides (LPS) and inducing inflammatory mediators to clear infection. However, there is emerging evidence that excessive TLR4 activation may be pathogenic in human diseases affecting the central nervous system, cardiovascular, respiratory and metabolic systems, thereby promoting inflammation and autoimmunity. In some diseases, the is conflicting evidence regarding pathogenic versus protective roles. Several TLR4 targeted therapeutics have been developed and studied in animal models, however many of these therapeutics including Eritoran and TAK-242 did not prove to be effective in clinical trials. Overall, TLR4 exhibits context-dependent protective and pathogenic roles across infectious and non-infectious diseases, reflecting the complexity of its signaling in human health and disease.

Background: Oesophageal adenocarcinoma (OAC) outcomes remain poor, with a 5-year survival rate of ~20%. Recent evidence suggests that human papillomavirus (HPV) is associated with ~35% of OAC. HPV-positive OAC patients have higher survivorship compared to HPV-negative patients, however current management remains uniform regardless of HPV status. Although the tumour microenvironment (TME), particularly intra-tumoural T-cell infiltration, is associated with improved outcomes in numerous cancers, characterisation of T-cell subsets in HPV-driven and HPV-independent OAC have not been explored. Methods: Differences in T-cell subsets within OAC biopsies of HPV-positive ( n = 15) and HPV-negative ( n = 22) were studied by multiplex immunofluorescence microscopy, in a retrospective cross-sectional study. Statistical analyses were conducted to assess differences in T-cell quantification, and to assess the correlation with patient prognosis. Results: Increased densities of FoxP3+ regulatory T-cells (Treg) and reduced CD8+:Treg ratios were evident in HPV-negative OAC compared to HPV-positive OAC. Other CD4+ and CD8+ subset densities were comparable. Univariate analyses demonstrated shorter overall survival (OS) in patients with high Treg proportions and low CD8+:Treg ratios. Multivariate analyses demonstrated a low total CD8+:Treg ratio to be independently associated with poor prognosis (HR 3.06; 95% CI 1.05–10.27; p = 0.050), along with metastasis and absence of HPV infection. Conclusion: We report novel evidence of an immunoregulatory TME in HPV-negative OAC, as indicated by high Treg proportions and low CD8+:Treg ratios. Future work may lead to improved risk stratification by the presence or absence of HPV, as well as utilising the CD8+:Treg ratio in the local TME as a predictive biomarker. Additionally, a more in-depth understanding of the TME would assist in the development of novel targeted immunotherapies, for example by modulating Tregs or CD8+ T-cells.

BACKGROUND: Checkpoint inhibitor–associated autoimmune diabetes mellitus (CIADM) is a rare but life-altering complication of immune checkpoint inhibitor (ICI) therapy. Biomarkers that predict type 1 diabetes (T1D) are unreliable for CIADM. AIM: In the present study, we sought to identify biomarkers for the prediction of CIADM. METHODS: From our prospective biobank, we identified 14 patients with CIADM who had metastatic melanoma treated with anti–programed antibody death 1 (anti–PD-1) with or without anti–cytotoxic T lymphocyte–associated antibody protein 4 (anti-CTLA4). Controls were selected from the same biobank, matched 2:1. Pretreatment, on-ICI, and post-CIADM serum and PBMCs were analyzed. Serum was analyzed for T1D autoantibodies, C-peptide, glucose, and cytokines. PBMCs were profiled using flow cytometry. Pancreatic volume was measured using CT volumetry. RESULTS: Before treatment, patients with CIADM had smaller pancreatic volume (27% reduction, P = 0.044) and higher anti–glutamic acid decarboxylase autoantibody (anti-GAD) titers (median 2.9 vs. 0, P = 0.01). They had significantly higher baseline proportions of Th17 cells ( P = 0.03), higher CD4 ⁺ central memory cells ( P = 0.04), and lower naive CD4 ⁺ T cells ( P = 0.01). With ICI treatment, greater declines in pancreatic volume were seen in patients with CIADM ( P < 0.0001). Activated CD4 ⁺ T cell subsets increased significantly in CIADM and controls with immune-related adverse effects (IRAEs) but not in controls without IRAEs. Using only pretreatment results, we found that pancreatic volume, anti-GAD antibody titers, and the baseline immune flow profile were highly predictive of CIADM development, with an AUC of greater than 0.96. CONCLUSIONS: People who develop CIADM are immunologically predisposed and have antecedent pancreatic and immunological changes that accurately predict disease with excellent sensitivity. These biomarkers could be used to guide ICI use, particularly when planning treatment for low-risk tumors. FUNDING: National Health and Medical Research Council (NHMRC) Investigator grants 2033228, 2009476, and 2007839.

The immune–epithelial–stromal interactions underpinning intestinal damage in celiac disease (CD) are incompletely understood. To address this, we performed single-cell transcriptomics (RNA sequencing; 86,442 immune, parenchymal and epithelial cells; 35 participants) and spatial transcriptomics (20 participants) on CD intestinal biopsy samples. Here we show that in CD, epithelial populations shifted toward a progenitor state, with interferon-driven transcriptional responses, and perturbation of secretory and enteroendocrine populations. Mucosal T cells showed numeric and functional changes in regulatory and follicular helper-like CD4⁺ T cells, intraepithelial lymphocytes, CD8⁺ and γδ T cell subsets, with skewed T cell antigen receptor repertoires. Mucosal changes remained detectable despite treatment, representing a persistent immune–epithelial ‘scar’. Spatial transcriptomics defined transcriptional niches beyond those captured in conventional histological scores, including CD-specific lymphoid aggregates containing T cell–B cell interactions. Receptor–ligand spatial analyses integrated with disease susceptibility gene expression defined networks of altered chemokine and morphogen signaling, and provide potential therapeutic targets for CD prevention and treatment.

Lymphopenia is a common feature of acute COVID-19 and is associated with increased disease severity and 30-day mortality. Here we aim to define the demographic and clinical characteristics that correlate with lymphopenia in COVID-19 and determine if lymphopenia is an independent predictor of poor clinical outcome. We analysed the ENTER-COVID (Epidemiology of hospitalized in-patient admissions following planned introduction of Epidemic SARS-CoV-2 to highly vaccinated COVID-19 naïve population) dataset of adults (N = 811) admitted for COVID-19 treatment in South Australia in a retrospective registry study, categorizing them as (a) lymphopenic (lymphocyte count < 1 × 10⁹/L) or (b) non-lymphopenic at hospital admission. Comorbidities and laboratory parameters were compared between groups. Multiple regression analysis was performed using a linear or logistic model. Intensive care unit (ICU) patients and non-survivors exhibited lower median lymphocyte counts than non-ICU patients and survivors respectively. Univariate analysis revealed that low lymphocyte counts associated with hypertension and correlated with haemoglobin, platelet count and negatively correlated with urea, creatinine, bilirubin, and aspartate aminotransferase (AST). Multivariate analysis identified age, male, haemoglobin, platelet count, diabetes, creatinine, bilirubin, alanine transaminase, c-reactive protein (CRP) and lactate dehydrogenase (LDH) as independent predictors of poor clinical outcome in COVID-19, while lymphopenia did not emerge as a significant predictor.

People with immunocompromising conditions are at increased risk of SARS-CoV-2 infection and mortality, however early in the pandemic it was challenging to collate data on this heterogenous population. We conducted a registry study of immunocompromised individuals with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection from March–October 2020 in Sydney, Australia to understand clinical and laboratory outcomes in this population prior to the emergence of the Delta variant. 27 participants were enrolled into the study including people with a haematologic oncologic conditions (n = 12), secondary immunosuppression (N = 8) and those with primary or acquired immunodeficiency (i.e. HIV; N = 7). All participants had symptomatic COVID-19 with the most common features being cough (64%), fever (52%) and headache (40%). Five patients demonstrated delayed SARS-CoV-2 clearance lasting three weeks to three months. The mortality rate in this study was 7% compared to 1.3% in the state of New South Wales Australia during the same period. This study provides data from the first eight months of the pandemic on COVID-19 outcomes in at-risk patient groups.