Clinical and Experimental Immunology

Background and AimsAutoimmune hepatitis (AIH) is a severe disease characterized by elevated immunoglobin levels. However, the role of autoantibodies in the pathophysiology of AIH remains uncertain. MethodsPhage Immunoprecipitation-Sequencing (PhIP-seq) was employed to identify autoantibodies in the serum of patients with AIH (n = 115), compared to patients with other liver diseases (metabolic associated steatotic liver disease (MASH) n = 178, primary biliary cholangitis (PBC), n = 26, or healthy controls, n = 94). ResultsLogistic regression using PhIP-seq enriched peptides as inputs yielded a classification AUC of 0.81, indicating the presence of a predictive humoral immune signature for AIH. Embedded within this signature were disease relevant targets, including SLA/LP, the target of a well-recognized autoantibody in AIH, disco interacting protein 2 homolog A (DIP2A), and the relaxin family peptide receptor 1 (RXFP1). The autoreactive fragment of DIP2A was a 9-amino acid stretch nearly identical to the U27 protein of human herpes virus 6 (HHV-6). Fine mapping of this epitope suggests the HHV-6 U27 sequence is preferentially enriched relative to the corresponding DIP2A sequence. Antibodies against RXFP1, a receptor involved in anti-fibrotic signaling, were also highly specific to AIH. The enriched peptides are within a motif adjacent to the receptor binding domain, required for signaling and serum from AIH patients positive for anti-RFXP1 antibody was able to significantly inhibit relaxin-2 singling. Depletion of IgG from anti-RXFP1 positive serum abrogated this effect. ConclusionsThese data provide evidence for a novel serological profile in AIH, including a possible functional role for anti-RXFP1, and antibodies that cross react with HHV6 U27 protein.

BackgroundVaccination against SARS-CoV-2 is a highly effective strategy to protect against infection, which is predominantly mediated by vaccine-induced antibodies. Postvaccination antibodies are robustly produced by those with inflammatory bowel disease (IBD) even on immune-modifying therapies but are blunted by anti-TNF therapy. In contrast, T-cell response which primarily determines long-term efficacy against disease progression,, is less well understood. We aimed to assess the post-vaccination T-cell response and its relationship to antibody responses in patients with inflammatory bowel disease (IBD) on immune-modifying therapies. MethodsWe evaluated IBD patients who completed SARS-CoV-2 vaccination using samples collected at four time points (dose 1, dose 2, 2 weeks after dose 2, 8 weeks after dose 2). T-cell clonal analysis was performed by T-cell Receptor (TCR) immunosequencing. The breadth (number of unique sequences to a given protein) and depth (relative abundance of all the unique sequences to a given protein) of the T-cell clonal response were quantified using reference datasets and were compared to antibody responses. ResultsOverall, 303 subjects were included (55% female; 5% with prior COVID) (Table). 53% received BNT262b (Pfizer), 42% mRNA-1273 (Moderna) and 5% Ad26CoV2 (J&J). The Spike-specific clonal response peaked 2 weeks after completion of the vaccine regimen (3- and 5-fold for breadth and depth, respectively); no changes were seen for non-Spike clones, suggesting vaccine specificity. Reduced T-cell clonal depth was associated with chronologic age, male sex, and immunomodulator treatment. It was preserved by non-anti-TNF biologic therapies, and augmented clonal depth was associated with anti-TNF treatment. TCR depth and breadth were associated with vaccine type; after adjusting for age and gender, Ad26CoV2 (J&J) exhibited weaker metrics than mRNA-1273 (Moderna) (p=0.01 for each) or BNT262b (Pfizer) (p=0.056 for depth). Antibody and T-cell responses were only modestly correlated. While those with robust humoral responses also had robust TCR clonal expansion, a substantial fraction of patients with high antibody levels had only a minimal T-cell clonal response. ConclusionAge, sex and select immunotherapies are associated with the T-cell clonal response to SARS-CoV-2 vaccines, and T-cell responses are low in many patients despite high antibody levels. These factors, as well as differences seen by vaccine type may help guide reimmunization vaccine strategy in immune-impaired populations. Further study of the effects of anti-TNF therapy on vaccine responses are warranted.

BackgroundSerological and clinical features with similarities to systemic autoimmunity have been reported in severe COVID-19, but there is a lack of studies that include contemporaneous controls who do not have COVID-19. MethodsObservational cohort study of adult patients admitted to an intensive care unit with acute respiratory failure. Patients were divided into COVID+ and COVID- based on SARS-CoV-2 PCR from nasopharyngeal swabs and/or endotracheal aspirates. No COVID-19 specific interventions were given. The primary clinical outcome was death in the ICU within 3 months; secondary outcomes included in-hospital death and disease severity measures. Measurements including autoantibodies, were done longitudinally. ANOVA and Fishers exact test were used with =0.05, with a false discovery rate of q=0.05. Bayesian analysis was performed to provide credible estimates of the possible states of nature compatible with our results. Results22 COVID+ and 20 COVID- patients were recruited, 69% males, median age 60.5 years. Overall, 64% had anti-nuclear antibodies, 38% had antigen-specific autoantibodies, 31% had myositis related autoantibodies, and 38% had high levels of anti-cytokine autoantibodies. There were no statistically significant differences between COVID+ and COVID- for any of the clinical or autoantibody parameters. A specific pattern of anti-nuclear antibodies was associated with worse clinical severity for both cohorts. ConclusionsSevere COVID+ patients have similar humoral autoimmune features as comparably ill COVID- patients, suggesting that autoantibodies are a feature of critical illness regardless of COVID-19 status. The clinical significance of autoimmune serology and the correlation with severity in critical illness remains to be elucidated.

Immune responses in people with multiple sclerosis (pwMS) on disease-modifying therapies (DMTs) have been of significant interest throughout the COVID-19 pandemic. Lymphocyte-targeting immunotherapies including anti-CD20 treatments and sphingosine-1-phosphate receptor (S1PR) modulators attenuate antibody responses after vaccination. Evaluation of cellular responses after vaccination is therefore of particular importance in these populations. In this study, we analysed CD4 and CD8 T cell functional responses to SARS-CoV-2 spike peptides in healthy controls and pwMS on five different DMTs by flow cytometry. Although pwMS on anti-CD20 and S1PR therapies had low antibody responses after both 2 and 3 vaccine doses, T cell responses in pwMS on anti-CD20 therapies were preserved after a third vaccination, even when additional anti-CD20 treatment was administered between vaccine doses 2 and 3. PwMS taking S1PR modulators had low detectable T cell responses in peripheral blood. CD4 and CD8 T cell responses to SARS-CoV-2 variants of concern Delta and Omicron were lower than to the ancestral Wuhan-Hu-1 variant. Our results indicate the importance of assessing both cellular and humoral responses after vaccination and suggest that even in the absence of robust antibody responses vaccination can generate immune responses in pwMS.

Expansion and pathogenicity of CD19+/CD20+/CD11c+/T-bet+ atypical B cells (ABCs) are hallmarks of numerous autoimmune disorders and chronic infections. In many such cases Epstein-Barr virus (EBV) is another associated or etiologic factor, though EBV involvement in these diseases remains poorly understood. Notably, the expansion of pro-inflammatory ABCs and a putative causal role for EBV have been identified independently in multiple sclerosis (MS). A common precipitating event in MS onset is Clinically Isolated Syndrome (CIS), a neuroinflammatory demyelinating condition of which 60-80% of cases progress to relapsing-remitting MS (RRMS). Here we report single-cell gene and surface protein expression (scRNA/CITE-seq) in peripheral B cells collected longitudinally from patients with CIS during the Immune Tolerance Network STAyCIS Trial. We focus on the transcriptomic signatures of ABCs from this cohort, publicly available scRNA-seq datasets from six other autoimmune and chronic infectious diseases, and in vitro EBV infection. Conservation of an expanded ABC expression profile across diseases establishes ABC dysregulation as a feature of CIS. Critically, we also observed transcriptomic features that distinguished CIS and de novo EBV-infected ABCs from those found in healthy controls and other disease contexts. Outcome stratification of CIS samples revealed a rare yet distinctive pro-inflammatory ABC subset that was significantly underrepresented in long-term non-progressor (LTNP) versus cases with RRMS activity ([~]5-fold difference). Collectively, this study provides evidence for altered ABC regulation - possibly arising from niche-specific responses to EBV infection - preceding MS onset. SUMMARYSingle-cell transcriptomics establishes an EBV-associated signature in T-bet+ atypical B cells in CIS and a pro-inflammatory phenotype underrepresented in patients with no disease progression.

BackgroundAutoimmune hepatitis (AIH) is a chronic immune-mediated liver disease associated with substantial morbidity and mortality. Diagnosis and monitoring typically rely on liver biopsy, despite its invasiveness and limitations. Liquid biopsies hold potential as non-invasive alternatives, but their utility in AIH remains underexplored. ObjectiveTo evaluate whether plasma-based chromatin immunoprecipitation followed by sequencing (cfChIP-seq) can detect hepatocyte-specific transcriptional activity in AIH and serve as a diagnostic and monitoring biomarker. DesignWe profiled circulating cell-free nucleosomes marked by H3K4me3--a histone modification marking active and poised promoters--in plasma samples from AIH patients (43 pediatric, 49 adult), healthy controls (6 pediatric, 1240 adults), and patients diagnosed with other liver diseases (15 pediatric, 175 adult). ResultscfChIP-seq revealed immune-related hepatocyte transcriptional signatures representing intrahepatic activity unique to AIH patients. RNA-seq of matched liver biopsies further corroborates these findings. A score trained on cfChIP-seq profiles from AIH and metabolically associated steatohepatitis (MASH) patients discriminated liver autoimmune diseases (including AIH) from other liver conditions, including MASH and drug-induced liver injury (DILI), with high accuracy on independent validation cases (AUC = 0.94; 95% CI 0.84-1). Combined with a second cfChIP-seq-based classifier for AIH vs. PSC/PBC, we distinguish between AIH and biliary autoimmune diseases (AUC = 0.92; 95% CI 0.83-1). ConclusionPlasma cfChIP-seq captures hepatocyte disease-specific gene activity in AIH patients and offers a non-invasive, accurate method for diagnosing and monitoring AIH. This approach has the potential to reduce the reliance on liver biopsy, improve diagnostic precision, and provide novel insights into AIH pathogenesis. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSAutoimmune hepatitis (AIH) is a chronic liver disease diagnosed and monitored primarily through liver biopsy, an invasive procedure with associated risks and sampling errors. Non-invasive alternatives are urgently needed but have shown limited disease specificity to date. What this study addsThis study shows that cfChIP-seq,a plasma-based assay detecting gene activity in turned over cells including from liver, can accurately distinguish AIH from other liver conditions. The cfChIP-seq signal in AIH patients reflects intrahepatic immune activity and correlates with findings from liver biopsy. How this study might affect research, practice or policyBy capturing disease-specific intrahepatic disease activity, cfChIP-seq provides positive indication of disease etiology. As such, cfChIP-seq has the potential to reduce the need for liver biopsies in AIH by providing a specific, non-invasive diagnostic tool. Its application in clinical practice could improve patient monitoring, support earlier intervention, and guide personalized management.

Systemic Lupus Erythematosus (SLE) is characterized by a prominent increase in expression of type-I interferon (IFN)-regulated genes in 50-75% of patients. Here we investigate the presence of autoantibodies (auto-Abs) against type I IFN in SLE patients and their possible role in controlling disease severity. We report that out of 491 SLE patients, 66 had detectable anti-IFN-auto-Abs. The presence of neutralizing anti-IFN-auto-Abs correlates with lower levels of circulating IFN protein, inhibition of IFN down-stream signalling molecules and gene signatures and with an inactive global disease score. Previously reported B cell frequency abnormalities, found to be involved in SLE pathogenesis, including increased levels of immature, double negative and plasmablast B cell populations were partially normalized in patients with neutralising anti-IFN-auto-Abs compared to other patient groups. We also show that sera from SLE patients with neutralising anti-IFN-auto-Abs biases in vitro B cell differentiation towards classical memory phenotype, while sera from patients without anti-IFN-Abs drives plasmablasts differentiation. Our findings support a role for neutralising anti-IFN-auto-Abs in controlling SLE pathogenesis and highlight their potential efficacy as novel therapy.

Background/ObjectiveSeptiCyte RAPID is a transcriptional host response assay that discriminates between sepsis and non-infectious systemic inflammation (SIRS) with a one-hour turnaround time. The overall performance of this test in a cohort of 419 patients has recently been described [Balk et al., J Clin Med 2024, 13, 1194]. In this study we present results from a detailed stratification analysis in which SeptiCyte RAPID performance was evaluated in the same cohort across patient groups and subgroups encompassing different demographics, comorbidities and disease, sources and types of pathogens, interventional treatments, and clinically defined phenotypes. The aims were to identify variables that might affect the ability of SeptiCyte RAPID to discriminate between sepsis and SIRS, and to determine if any patient subgroups appeared to present a diagnostic challenge for the test. Methods1) Subgroup analysis, with subgroups defined by individual demographic or clinical variables, using conventional statistical comparison tests. 2) Principal component analysis and k-means clustering analysis, to investigate phenotypic subgroups defined by unique combinations of demographic and clinical variables. ResultsNo significant differences in SeptiCyte RAPID performance were observed between most groups and subgroups. One notable exception involved an enhanced SeptiCyte RAPID performance for a phenotypic subgroup defined by a combination of clinical variables suggesting a septic shock response. ConclusionsWe conclude that for this patient cohort SeptiCyte RAPID performance was largely unaffected by key variables associated with heterogeneity in patients suspected of sepsis.

Background and ObjectivePeople with multiple sclerosis (pwMS) receiving B cell-depleting therapies have impaired antibody responses to vaccination. In a proportion of individuals, repeat vaccination against COVID-19 leads to seroconversion. We sought to describe the immune phenotype of pwMS on ocrelizumab, and identify clinical and immunological determinants of an effective vaccine response. MethodsThis was a single-centre, prospective cohort study. Peripheral blood samples were collected from pwMS receiving ocrelizumab (n = 38) pre and post administration of a third dose of mRNA COVID-19 vaccine. Immunogenicity was measured by T cell IFN{gamma} ELISpot, antibody titres, and live virus neutralisation. Humoral immunity was benchmarked against pwMS receiving natalizumab (n = 15), and against a correlate of real-world protection (50% reduction in incidence of infection) from SARS-CoV-2 ancestral and omicron BA.5 variants. The peripheral immune phenotype was comprehensively assessed by flow cytometry, and potential clinical and phenotypic determinants of response to vaccination identified. ResultsImmune cell populations relevant to disease and vaccine response were altered in pwMS receiving ocrelizumab versus natalizumab treatment, including depleted CD20-expressing B cell, T cell and NK cell populations, and elevated CD27+CD38+ T cell and NK8 cell frequencies. Following a third vaccine dose, 51% of pwMS on ocrelizumab were seropositive for SARS-CoV-2 receptor-binding-domain IgG, and 25% and 14% met the threshold for effective neutralisation of live SARS-CoV-2 ancestral and omicron BA.5 virus, respectively. B cell frequency at the time of vaccination, but not time since ocrelizumab infusion, was positively correlated with antibody response, while a strong negative correlation was observed between CD56bright NK cell frequency and antibody response in the ocrelizumab group. In this exploratory cohort, CD3-CD20+ B cells (% of lymphocytes; OR=3.92) and CD56bright NK cells (% of NK cells; OR=0.94) were predictive of an effective neutralising antibody response in second dose non-responders (AUC: 0.98). DiscussionOcrelizumab treatment was associated with an altered immune phenotype, including recently described T cell and NK populations with potential roles in disease pathogenesis. However, seroconversion was severely impaired by ocrelizumab, and less than half of those who seroconverted following a third vaccine dose demonstrated effective immunity against SARS-CoV-2 ancestral or omicron BA.5. B cell frequency was associated with an effective antibody response, while immunomodulatory CD56bright NK cells were identified as a potential negative determinant of response in those with inadequate B cell numbers. Immune phenotype rather than time since ocrelizumab infusion may help to stratify individuals for prophylaxis.

Rationale: Sepsis is a life-threatening syndrome causing significant morbidity and mortality especially in the aging population. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition of clonal expansion of hematopoietic stem cells harboring somatic mutations associated with increased incidence of chronic illness and all-cause mortality. Objective: Evaluate the association of pre-illness CHIP with mortality and morbidity in patients admitted to the ICU with sepsis. Methods: We performed a retrospective study using a de-identified electronic health record linked with a DNA biorepository. We identified adult patients with sepsis who had DNA collected prior to ICU admission. We tested the association between CHIP status, determined from whole-genome sequencing, and ICU mortality, organ support-free days, and long-term survival adjusting for age, sex, race and Sequential Organ Failure Assessment (SOFA) score on ICU admission. Measurements and Main Results: Pre-illness CHIP was associated with increased sepsis mortality (OR = 1.54, 95% CI 1.13 to 2.07, P = 0.005) and fewer days alive and free of organ support (-1.7 days, 95% CI -3.2 to -0.2, P = 0.028) after adjusting for age, sex, race, and SOFA score. In sepsis survivors, CHIP was also associated with increased long-term mortality after discharge (HR 1.40, 95% CI 1.01 to 1.93, P = 0.041). Conclusions: Pre-illness CHIP was independently associated with increased mortality and morbidity in critically-ill adults with sepsis. These findings suggest that CHIP is a risk factor for sepsis severity. Elucidating the mechanism underlying this association could uncover new therapeutic interventions for sepsis.

Red blood cell (RBC) alloimmunization is a clinically significant complication in transfused patients whose immunological determinants remain incompletely understood. Type I interferon (IFN-I) signaling drives RBC alloimmunization in murine models, and systemic lupus erythematosus (SLE) is characterized by constitutive IFN-I hyperactivation alongside elevated alloimmunization rates. We analyzed three publicly available SLE RNA-seq cohorts (GSE72509, GSE112087, GSE122459; whole blood and PBMC; total n = 150 SLE) in a pre-specified discovery-replication-validation design. A 14-gene IFN-I signature score was computed per sample; differential expression, gene set enrichment analysis, and Spearman correlation were performed independently per cohort. IFN-I scores were significantly elevated in SLE versus healthy controls in all three cohorts (p < 0.01 each). IFN-high SLE patients showed 665 differentially expressed genes, with enrichment of alloimmunization-associated and plasmablast differentiation gene sets confirmed by GSEA. The alloimmunization signature score correlated significantly with IFN-I score across all three independent cohorts ({rho} = +0.77, +0.51, +0.60; all FDR q < 0.05); Tfh differentiation showed no association in any cohort. To our knowledge, this represents the first human transcriptomic evidence that IFN-I pathway activity in SLE is coupled to alloimmunization-associated immune programs in vivo. These findings identify IFN-I score as a candidate biomarker of alloimmunization susceptibility in SLE and provide translational rationale for prospective studies incorporating transfusion outcome data.

ADCC (antibody-dependent cell-mediated cytotoxicity) is dependent on the varying capacity of NK cells to kill, the affinities of FCGR3A-encoded CD16A receptors for antibody, and the presence of antigen-specific antibodies. In vivo ADCC depends on the number of CD16A receptor-positive NK cells in blood. We hypothesized that low ADCC cell function or low effector cell numbers could be biomarkers or risk factors for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We measured NK cell ADCC lytic capacity and antibody recognition, CD16Apositive NK cells/{micro}l blood, and FCGR3A homozygosity for the F allele that encodes low affinity CD16A antibody receptors. ME/CFS patients met the Fukuda 1994 diagnostic criteria. In this pilot report, we examined 5 families, each with 2 to 5 ME/CFS patients, and compared 11 patients, 22 family members without ME/CFS, and 16 unrelated healthy controls. ADCC was measured as CX1:1 cytotoxic capacity (the percentage of 51Cr-Daudi tumors with obinutuzumab anti-CD20 antibody that were killed at a 1:1 ratio of CD16Apos NKs to Daudis) and CX-slope. Individual CX1:1 capacities varied from 16.2% to 81.8% and were comparable between patients and unaffected family members, while the ADCC of both family groups was lower than the unrelated healthy controls. The lack of difference between patients and their unaffected family members indicates that low ADCC is unsuitable as a diagnostic biomarker for ME/CFS. Familial CD16Apos NK blood cell counts were lower than unrelated healthy controls. The potential for synergistic effects of combined low CX1:1 and low effector cell counts occurring in the same individual was 24-fold greater for CFS family members than for unrelated controls. FCGR3A of the families was predominantly F/F homozygous, correlating with the observed low EC50 for NK recognition of target cell-bound antibody. In summary, low ADCC is unsuitable as a biomarker, but could be a familial risk factor, for ME/CFS.

Severe forms of inflammation-induced acute and chronic myocarditis have a poor prognosis. Promising therapeutic efforts focused on monoclonal antibodies (mAbs) inhibiting inflammation-inducing molecules. However, most mAbs target only one or a limited number of such molecules. Since inflammation involves multiple redundant pathways, we postulated that an mAb inhibiting multiple inflammatory pathways would be a potent therapeutic agent. We initially tested the commercially available anti-natural killer (NK) cell mAb (anti-NK1.1), which binds a receptor expressed on NK cells and depletes them. Since NK cells are key cellular orchestrators of inflammation, by reducing their number, we aimed to inhibit multiple inflammatory pathways. Our initial studies demonstrated that administration of this antibody significantly improved myocardial outcomes in mouse models of acute myocardial infarction and of heart failure. Since NK1.1 is not expressed in human cells, we built on these promising preclinical results by developing a novel mAb targeting CD160 on human NK cells for evaluation as an immunosuppressive therapy. We found that the anti-CD160 mAb depletes both murine and human NK cells. We also found that, while CD160+ cells were largely present in the NK population, they also occurred among CD8+ and {gamma}/{delta} T cell subsets in human cells. Anti-CD160 therapy entirely prevented the deterioration of the myocardial function of mice with autoimmune-induced acute myocarditis. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases. Key PointsO_ST_ABSQuestionC_ST_ABSCan the depletion of CD160+ cells prevent autoimmune-induced myocarditis? FindingsIn this study we found that CD160 is expressed by mouse and human natural killer cells and other subtypes of cytotoxic T cells, and that a monoclonal antibody targeting CD160 depletes NK cells. In a preclinical model of experimental autoimmune myocarditis, administration of the anti-CD160 monoclonal antibody prevented myocardial dysfunction and systemic inflammation. MeaningOur results are compatible with the hypothesis that early autoimmune-induced myocardial dysfunction is promoted by CD160+ cells, which elevate inflammation-induced circulating factors (or factors released by tissue-resident cytotoxic immune cells) that cause myocardial dysfunction in the absence of myocardial necrosis or fibrosis, and further, that targeting CD160+cells with a mAb that depletes NK cells (and probably CD160 expressing cytotoxic T cells) entirely prevents the deterioration of myocardial function in such mice. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases.

BackgroundSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients has generally relied on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision. MethodsAn ex vivo whole blood enzyme-linked immunosorbent (ELISpot) assay for cellular production of interferon-{gamma} (IFN-{gamma}) was evaluated in 107 septic and 68 non-septic patients from five academic health centers using blood samples collected on days 1, 4 and 7 following ICU admission. ResultsCompared with 46 healthy subjects, unstimulated and stimulated whole blood IFN{gamma} expression were either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole blood IFN{gamma} expression was significantly reduced on ICU days 1, 4 and 7 (all p<0.05), due to both significant reductions in total number of IFN{gamma}-producing cells and amount of IFN{gamma} produced per cell (all p<0.05). Importantly, IFN{gamma} total expression on day 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6 and procalcitonin. Septic patients with low IFN{gamma} expression were older and had lower ALC and higher sPD-L1 and IL-10 concentrations, consistent with an immune suppressed endotype. ConclusionsA whole blood IFN{gamma} ELISpot assay can both identify septic patients at increased risk of late mortality, and identify immune-suppressed, sepsis patients. Trial RegistryBecause the study is a prospective observational study, and not a clinical trial, registration with clinical trials.gov is not required.

BackgroundRecent advances in xenotransplantation in living and decedent humans using pig xenografts have laid promising groundwork towards future emergency use and first in human trials. Major obstacles remain though, including a lack of knowledge of the genetic incompatibilities between pig donors and human recipients which may led to harmful immune responses against the xenograft or dysregulation of normal physiology. In 2022 two pig heart xenografts were transplanted into two brain-dead human decedents with a minimized immunosuppression regime, primarily to evaluate onset of hyper-acute antibody mediated rejection and sustained xenograft function over 3 days. MethodsWe performed multi-omic profiling to assess the dynamic interactions between the pig and human genomes in the first two pig heart-xenografts transplants into human decedents. To assess global and specific biological changes that may correlate with immune-related outcomes and xenograft function, we generated transcriptomic, lipidomic, proteomic and metabolomics datasets, across blood and tissue samples collected every 6 hours over the 3-day procedures. ResultsSingle-cell datasets in the 3-day pig xenograft-decedent models show dynamic immune activation processes. We observe specific scRNA-seq, snRNA-seq and geospatial transcriptomic changes of early immune-activation leading to pronounced downstream T-cell activity and hallmarks of early antibody mediated rejection (AbMR) and/or ischemia reperfusion injury (IRI) in the first xenograft recipient. Using longitudinal multiomic integrative analyses from blood in addition to antigen presentation pathway enrichment, we also observe in the first xeno-heart recipient significant cellular metabolism and liver damage pathway changes that correlate with profound physiological dysfunction whereas, these signals are not present in the other xenograft recipient. ConclusionsSingle-cell and multiomics approaches reveal fundamental insights into early molecular immune responses indicative of IRI and/or early AbMR in the first human decedent, which was not evident in the conventional histological evaluations.

ObjectivesPatients with Systemic lupus erythematosus (SLE) who carry a high genetic burden often experience more severe disease. To understand the molecular consequences of polygenic risk, we analyzed single-cell gene expression profiles in SLE patients stratified by genetic risk. MethodsSingle-cell RNA sequencing (scRNA-seq) was performed on fresh peripheral blood mononuclear cells (PBMCs) from 16 female SLE patients, stratified by a weighted polygenic risk score (PRS), and 6 healthy controls (HCs). All patients were in low disease activity (LLDAS) and treated with antimalarials only. We assessed differential gene expression, interferon (IFN) signatures, transcription factor (TF) activity, and pathway enrichment across groups. ResultsPatients with High-PRS had significantly elevated IFN scores compared to HCs (p<0.001), whereas no significant difference was observed between Low-PRS patients and HCs (p>0.05) This pattern held across multiple immune cell types, including T cells, NK cells, and monocytes. Notable genes with increased expression in High-PRS patients included ISG15 and USP18 in plasmacytoid dendritic cells (pDCs), and IFI27 and RSAD2 in monocytes. IFN-related pathways were enriched in pDCs and monocytes in High-PRS patients, and only in monocytes in Low-PRS patients. TF analysis identified IRF7 and BATF3 as key candidate regulators in High-PRS of both cell types. ConclusionsHigh polygenic risk in SLE is associated with persistent activation of IFN signaling pathways, indicating that antimalarial treatment alone is insufficient to fully suppress IFN activity, even during remission or low disease activity.

Celiac disease (CeD) is a heterogeneous autoimmune disorder influenced by genetic, environmental, and socioeconomic factors. However, little is known about clinical manifestations and genetic risks in minority populations. Using data from the All of Us Research Program, we analyzed 3,040 CeD patients, referred to as the AoU-CeD cohort, to identify clinical and genetic differences across racial and ethnic groups in the United States. CeD prevalence was highest among White individuals (1.08%) and significantly lower among Hispanic (0.36%) and Black (0.16%) populations. The majority of CeD patients were female (78.4%) and diagnosed between the ages of 18 and 64. Minority groups reported poorer physical and mental quality of life (QoL) and higher levels of pain. Ancestry-specific patterns emerged in CeD-associated conditions, with minorities more likely to report diarrhea and non-infectious gastroenteritis but less likely to have osteoporosis, hypothyroidism, chronic fatigue, or a family history of CeD. Compared to previously reported data showing that over 90% of CeD patients carry the HLA-DQ2.5 haplotype, genetic analysis revealed that only 49% of patients in the AoU-CeD cohort carried the high-risk HLA-DQ2.5 haplotype. Additionally, 16.5% lacked known HLA-DQ risk haplotypes, suggesting potential diagnostic or reporting inaccuracies. Minority groups exhibited higher rates of atypical symptoms, lower frequencies of the DQ2.5 haplotype, and distinct distributions of HLA-DQ genotypes. A long haplotype block spanning HLA-A1, B8, C7 and HLA-DQ2.5 was found in Europeans but absent in other ancestries. A genome-wide association study (GWAS) using over 11 million variants from whole-genome sequencing data identified 1,651 significant single-nucleotide polymorphisms (SNPs), primarily within the MHC locus, with the strongest signals observed predominantly among individuals of European ancestry. A predictive model incorporating HLA-DQ genotype, family history, and clinical features achieved 83% accuracy for identifying seropositive CeD. These results highlight the importance of ancestry-specific clinical presentations and genetic features in CeD.

BackgroundSepsis-induced immunosuppression, characterized by lymphopenia, is associated with adverse outcomes. We aimed to identify distinct lymphocyte recovery patterns in patients with sepsis, evaluate their association with mortality, and develop a machine learning model to enhance prediction MethodsThis retrospective cohort study included adult patients with sepsis and initial lymphopenia (Absolute Lymphocyte Count [ALC] < 1.0 x 10L/L) from the Medical Information Mart for Intensive Care (MIMIC)-IV database. We defined three lymphocyte recovery patterns: "Persistent Suppression," "Partial Recovery," and "Complete Recovery." The primary outcome was 90-day all-cause mortality. Multivariable Cox models were used to assess the association between recovery patterns and mortality. An Extreme Gradient Boosting (XGBoost) model was developed to predict 90-day mortality. Results90-day mortality was highest in the Persistent Suppression group (49.1%) versus Partial (41.3%) and Complete Recovery (35.7%) groups (p<0.001). Persistent Suppression remained an independent predictor of mortality (adjusted Hazard Ratio 1.31, 95% CI 1.10-1.55). The XGBoost model achieved superior discrimination (Area Under the Receiver Operating Characteristic Curve [AUROC]=0.767) over traditional scores. SHAP analysis confirmed that dynamic features, including lymphocyte recovery metrics, were key model drivers. The model also demonstrated robust performance across various clinical subgroups (e.g., age, disease severity). ConclusionsThe trajectory of lymphocyte recovery following sepsis onset is an independent prognostic marker. Failure to restore lymphocyte counts is strongly associated with increased long-term mortality. Integrating this dynamic immunological feature into machine learning algorithms significantly enhances predictive accuracy, offering a promising tool for real-time risk stratification.

The human IgA response is composed of two structurally different subclasses termed IgA1 and IgA2. Compared to IgA1, IgA2 has a shorter hinge region, which makes it more resistant to bacterial proteases. IgA1 is produced both systemically and in mucosal surfaces, whereas IgA2 is mostly confined to the intestines. While the overall IgA response is known to be involved in intestinal homeostasis, the specific contribution of IgA1 and IgA2 remains largely unknown (Chen 2020). Selective IgA deficiency (SIgAD) is the most prevalent primary immune deficiency. About half of SIgAD cases are associated with heterogeneous but generally mild clinical manifestations. Anecdotal evidence of IgA2 deficiency is available (van Loghem 1983, Ozawa 1986, Engstrom 1990), however no associations with clinical manifestations have been reported. Here, we describe the occurrence of a selective IgA2 deficiency in a patient (CD068) with small intestinal Crohns disease (CD). The patient had undetectable IgA2+ cells and secreted IgA2 antibody in both intestine and circulation. Among other features, patient CD068 presented with duodenal and ileal inflammation. To our knowledge, this is the first case of IgA2 deficiency with a potential link to IBD, which might shed new insights into potential IgA2-specific functions.

BackgroundInflammation contributes to the increased risk of cardiovascular disease (CVD) observed in people with rheumatoid arthritis (RA), with increased prevalence observed from time of diagnosis. Subclinical vascular and myocardial abnormalities can be detected with cardiovascular magnetic resonance (CMR) imaging in otherwise low risk individuals. Identifying associated circulating markers could enable diagnostics and implicate biological pathways of RA-CVD. The studys objective was to identify blood-based proteins associated with CMR measures of vascular and myocardial abnormality and associated inflammatory and/or cardio-metabolic pathways in a new-onset RA cohort. MethodsSerum samples (baseline/pre-treatment, N = 75 and year 1, N = 71) from CADERA (Coronary Artery Disease Evaluation in Rheumatoid Arthritis) participants, a subgroup of a randomised controlled trial, who underwent CMR, were used to measure 334 proteins across 4 pre-defined Olink panels (Inflammation, Cardiovascular-II, Cardiovascular-III, Cardiometabolic). Bayesian mixed effects regression analyses, unsupervised hierarchical clustering and protein network analyses were applied. Normalised protein expression from 334 Olink proteins were used as exposures in the regression analyses. CMR measures of vascular stiffness (aortic distensibility and stiffness index) and myocardial tissue characteristics (native T1, myocardial extracellular volume and late gadolinium enhancement) were each used as individual outcomes in regression analyses. Associations were considered significant at 95% threshold for credible intervals. An expanded physical protein-protein interaction (PPI) network created using the significant (seed) proteins was subjected to topological and enrichment analysis to identify enriched biological pathways. Results54/334 proteins were associated significantly with CMR measures at baseline (7 - vascular; 48 - myocardial tissue characteristics; Coagulation factor 11 with both). Two proteins, TNFSF13B (B-cell activating factor) and CRTAC1, were associated with CMR measures at baseline, were sensitive to change over time and co-varied with changes in CMR measure. Topological analysis of expanded PPI network revealed GRB2 as most connected (degree score=64; closeness score=118.83) and four key signalling pathways, including JAK-STAT and EGFR tyrosine kinase inhibitor resistance, emerged as significant. ConclusionsThis first proteomic study of treatment-naive early RA and subclinical cardiovascular pathology identifies proteins that could aid diagnostic test development and implicates signalling pathways in the RA-cardiovascular axis to inform on our understanding of the basis of RA-CVD.