An Exercise Immune Fitness Test to Unravel Disease Mechanisms-A Proof-of-Concept Heart Failure Study.

Background: Cardiorespiratory fitness positively correlates with longevity and immune health. Regular exercise may provide health benefits by reducing systemic inflammation. In chronic disease conditions, such as chronic heart failure and chronic fatigue syndrome, mechanistic links have been postulated between inflammation, muscle weakness, frailty, catabolic/anabolic imbalance, and aberrant chronic activation of immunity with monocyte upregulation. We hypothesize that (1) temporal changes in transcriptome profiles of peripheral blood mononuclear cells during strenuous acute bouts of exercise using cardiopulmonary exercise testing are present in adult subjects, (2) these temporal dynamic changes are different between healthy persons and heart failure patients and correlate with clinical exercise-parameters and (3) they portend prognostic information. Methods: In total, 16 Heart Failure (HF) patients and 4 healthy volunteers (HV) were included in our proof-of-concept study. All participants underwent upright bicycle cardiopulmonary exercise testing. Blood samples were collected at three time points (TP) (TP1: 30 min before, TP2: peak exercise, TP3: 1 h after peak exercise). We divided 20 participants into 3 clinically relevant groups of cardiorespiratory fitness, defined by peak VO2: HV (n = 4, VO2 ≥ 22 mL/kg/min), mild HF (HF1) (n = 7, 14 < VO2 < 22 mL/kg/min), and severe HF (HF2) (n = 9, VO2 ≤ 14 mL/kg/min). Results: Based on the statistical analysis with 20-100% restriction, FDR correction (p-value 0.05) and 2.0-fold change across the three time points (TP1, TP2, TP3) criteria, we obtained 11 differentially expressed genes (DEG). Out of these 11 genes, the median Gene Expression Profile value decreased from TP1 to TP2 in 10 genes. The only gene that did not follow this pattern was CCDC181. By performing 1-way ANOVA, we identified 8/11 genes in each of the two groups (HV versus HF) while 5 of the genes (TTC34, TMEM119, C19orf33, ID1, TKTL2) overlapped between the two groups. We found 265 genes which are differentially expressed between those who survived and those who died. Conclusions: From our proof-of-concept heart failure study, we conclude that gene expression correlates with VO2 peak in both healthy individuals and HF patients, potentially by regulating various physiological processes involved in oxygen uptake and utilization during exercise. Multi-omics profiling may help identify novel biomarkers for assessing exercise capacity and prognosis in HF patients, as well as potential targets for therapeutic intervention to improve VO2 peak and quality of life. We anticipate that our results will provide a novel metric for classifying immune health.

Integrated transcriptomic analysis reveals immune signatures distinguishing persistent versus resolving outcomes in MRSA bacteremia.

Introduction: Staphylococcus aureus bacteremia (SAB) is a life-threatening infection particularly involving methicillin-resistant S. aureus (MRSA). In contrast to resolving MRSA bacteremia (RB), persistent MRSA bacteremia (PB) blood cultures remain positive despite appropriate antibiotic treatment. Host immune responses distinguishing PB vs. RB outcomes are poorly understood. Here, integrated transcriptomic, IL-10 cytokine levels, and genomic analyses sought to identify signatures differentiating PB vs. RB outcomes. Methods: Whole-blood transcriptomes of propensity-matched PB (n=28) versus RB (n=30) patients treated with vancomycin were compared in one independent training patient cohort. Gene expression (GE) modules were analyzed and prioritized relative to host IL-10 cytokine levels and DNA methyltransferase-3A (DNMT3A) genotype. Results: Differential expression of T and B lymphocyte gene expression early in MRSA bacteremia discriminated RB from PB outcomes. Significant increases in effector T and B cell signaling pathways correlated with RB, lower IL-10 cytokine levels and DNMT3A heterozygous A/C genotype. Importantly, a second PB and RB patient cohort analyzed in a masked manner demonstrated high predictive accuracy of differential signatures. Discussion: Collectively, the present findings indicate that human PB involves dysregulated immunity characterized by impaired T and B cell responses associated with excessive IL-10 expression in context of the DNMT3A A/A genotype. These findings reveal distinct immunologic programs in PB vs. RB outcomes, enable future studies to define mechanisms by which host and/or pathogen drive differential signatures and may accelerate prediction of PB outcomes. Such prognostic assessment of host risk could significantly enhance early anti-infective interventions to avert PB and improve patient outcomes.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

Background: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Results: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Conclusions: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Integrated longitudinal multiomics study identifies immune programs associated with acute COVID-19 severity and mortality.

BACKGROUNDPatients hospitalized for COVID-19 exhibit diverse clinical outcomes, with outcomes for some individuals diverging over time even though their initial disease severity appears similar to that of other patients. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity.METHODSWe performed deep immunophenotyping and conducted longitudinal multiomics modeling, integrating 10 assays for 1,152 Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) study participants and identifying several immune cascades that were significant drivers of differential clinical outcomes.RESULTSIncreasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, formation of neutrophil extracellular traps, and T cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma Igs and B cells and dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to failure of viral clearance in patients with fatal illness.CONCLUSIONOur longitudinal multiomics profiling study revealed temporal coordination across diverse omics that potentially explain the disease progression, providing insights that can inform the targeted development of therapies for patients hospitalized with COVID-19, especially those who are critically ill.TRIAL REGISTRATIONClinicalTrials.gov NCT04378777.FUNDINGNIH (5R01AI135803-03, 5U19AI118608-04, 5U19AI128910-04, 4U19AI090023-11, 4U19AI118610-06, R01AI145835-01A1S1, 5U19AI062629-17, 5U19AI057229-17, 5U19AI125357-05, 5U19AI128913-03, 3U19AI077439-13, 5U54AI142766-03, 5R01AI104870-07, 3U19AI089992-09, 3U19AI128913-03, and 5T32DA018926-18); NIAID, NIH (3U19AI1289130, U19AI128913-04S1, and R01AI122220); and National Science Foundation (DMS2310836).

Tensor modeling of MRSA bacteremia cytokine and transcriptional patterns reveals coordinated, outcome-associated immunological programs.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a common and life-threatening infection that imposes up to 30% mortality even when appropriate therapy is used. Despite in vitro efficacy determined by minimum inhibitory concentration breakpoints, antibiotics often fail to resolve these infections in vivo, resulting in persistent MRSA bacteremia. Recently, several genetic, epigenetic, and proteomic correlates of persistent outcomes have been identified. However, the extent to which single variables or their composite patterns operate as independent predictors of outcome or reflect shared underlying mechanisms of persistence is unknown. To explore this question, we employed a tensor-based integration of host transcriptional and cytokine datasets across a well-characterized cohort of patients with persistent or resolving MRSA bacteremia outcomes. This method yielded high correlative accuracy with outcomes and immunologic signatures united by transcriptomic and cytokine datasets. Results reveal that patients with persistent MRSA bacteremia (PB) exhibit signals of granulocyte dysfunction, suppressed antigen presentation, and deviated lymphocyte polarization. In contrast, patients with resolving bacteremia (RB) heterogeneously exhibit correlates of robust antigen-presenting cell trafficking and enhanced neutrophil maturation corresponding to appropriate T lymphocyte polarization and B lymphocyte response. These results suggest that transcriptional and cytokine correlates of PB vs. RB outcomes are complex and may not be disclosed by conventional modeling. In this respect, a tensor-based integration approach may help to reveal consensus molecular and cellular mechanisms and their biological interpretation.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Advancing precision in histocompatibility and immunogenetics: a comprehensive review of the UCLA exchange program.

Precise typing of human leukocyte antigens (HLA) is crucial for clinical hematopoietic stem cell and solid organ transplantations, transfusion medicine, HLA-related disease association, and drug hypersensitivity analysis. The UCLA Cell Exchange program has played a vital role in providing educational and proficiency testing surveys to HLA laboratories worldwide for the past 5 decades. This article highlights the significant contribution of the UCLA Cell and DNA Exchange Programs in advancing HLA antibody testing, genotyping, crossmatches, and, more recently, virtual crossmatches. Additionally, we discuss future directions of the UCLA Cell Exchange program to support histocompatibility testing to adapt to the fast-evolving field of immunotherapy, tolerance and xenotransplantation.

Features of acute COVID-19 associated with post-acute sequelae of SARS-CoV-2 phenotypes: results from the IMPACC study.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

Myeloid spatial and transcriptional molecular signature of ischemia-reperfusion injury in human liver transplantation.

BACKGROUND: Ischemia-reperfusion injury (IRI) is a significant clinical concern in liver transplantation, with a key influence on short-term and long-term allograft and patient survival. Myeloid cells trigger and sustain tissue inflammation and damage associated with IRI, but the mechanisms regulating these activities are unknown. To address this, we investigated the molecular characteristics of intragraft myeloid cells present in biopsy-proven IRI- and IRI+ liver transplants. METHODS: RNA-sequencing was performed on 80 pre-reperfusion and post-reperfusion biopsies from 40 human recipients of liver transplantation (23 IRI+, 17 IRI-). We used transcriptional profiling and computational approaches to identify specific gene coexpression network modules correlated with functional subsets of MPO+, lysozyme+, and CD68+ myeloid cells quantified by immunohistochemistry on sequential sections from the same patient biopsies. RESULTS: A global molecular map showed gene signatures related to myeloid activation in all patients regardless of IRI status; however, myeloid cell subsets differed dramatically in their spatial morphology and associated gene signatures. IRI- recipients were found to have a natural corticosteroid production and response profile from pre-reperfusion to post-reperfusion, particularly among monocytes/macrophages. The pre-reperfusion signature of IRI+ recipients included acute inflammatory responses in neutrophils and increased translation of adaptive immune-related genes in monocytes/macrophages coupled with decreased glucocorticoid responses. Subsequent lymphocyte activation at post-reperfusion identified transcriptional programs associated with the transition to adaptive immunity found only among IRI+ recipients. CONCLUSIONS: Myeloid subset-specific genes and related signaling pathways provide targets for the development of therapeutic strategies aimed at limiting IRI in the clinical setting of liver transplantation.

Longitudinal analysis of influenza vaccination implicates regulation of RIG-I signaling by DNA methylation.

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection.

Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFß-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.

Non-HLA Antibodies to G Protein-coupled Receptors in Pediatric Kidney Transplant Recipients: Short- and Long-term Clinical Outcomes.

BACKGROUND: Angiotensin II type 1 receptor antibodies (AT1R-Abs) and endothelin-type A receptor antibodies (ETAR-Abs) are G protein-coupled receptor activating autoantibodies associated with antibody-mediated rejection, vascular pathology, increased cytokines, allograft dysfunction, and allograft loss in pediatric kidney transplant recipients in the first 2 y posttransplantation. The impact of AT1R-Ab and ETAR-Ab positivity on longer-term 5-y transplant outcomes is unknown. METHODS: One hundred pediatric kidney transplant recipients were tested for ETAR-Ab and AT1R-Ab on serially collected blood samples in the first 2 y posttransplant. Biopsies were collected per protocol and 6, 12, and 24 mo posttransplant and at any time during the 5-y follow-up period for clinical indication. Clinical outcomes, including renal dysfunction, rejection, HLA donor-specific antibodies, and allograft loss, were assessed through 5 y posttransplantation. RESULTS: AT1R-Ab or ETAR-Ab were positive in 59% of patients. AT1R-Ab or ETAR-Ab positivity was associated with greater declines in estimated glomerular filtration rate, and de novo AT1R-Ab or ETAR-Ab was associated with allograft loss in the first 2 y posttransplant. There was no association between antibody positivity and rejection, antibody-mediated rejection, or allograft loss in the first 5 y posttransplant. In a model controlled for age, sex, immunosuppression, and HLA mismatch, AT1R-Ab or ETAR-Ab positivity was significantly associated with the development of HLA donor-specific antibodies at 5 y posttransplant (odds ratio 2.87, P = 0.034). CONCLUSIONS: Our findings suggest temporally distinct clinical complications associated with AT1R-Ab or ETAR-Ab positivity in pediatric patients; these injury patterns are of significant interest for developing effective treatment strategies.

Proteome Analysis for Inflammation Related to Acute and Convalescent Infection.

Infectious diseases are a significant burden in global healthcare. Pathogens engage with different host defense mechanisms. However, it is currently unknown if there are disease-specific immune signatures and/or if different pathogens elicit common immune-associated molecular entities to common therapeutic interventions. We studied patients enrolled through the Human Immunology Project Consortium (HIPC), which focuses on immune responses to various infections. Blood samples were collected and analyzed from patients during infection and follow-up time points at the convalescent stage. The study included samples from patients with Lyme disease (LD), tuberculosis (TB), malaria (MLA), dengue virus (DENV), and West Nile virus (WNV), as well as kidney transplant patients with cytomegalovirus (CMV) and polyomavirus (BKV) infections. Using an antibody-based assay, we quantified ~ 350 cell surface markers, cytokines, and chemokines involved in inflammation and immunity. Unique protein signatures were identified specific to the acute phase of infection irrespective of the pathogen type, with significant changes during convalescence. In addition, tumor necrosis factor receptor superfamily member 6 (TNR6), C-C Motif Chemokine Receptor 7 (CCR7), and C-C motif chemokine ligand-1 (CCL1) were increased in the acute and convalescent phases across all viral, bacterial, and protozoan compared to blood from healthy donors. Furthermore, despite the differences between pathogens, proteins were enriched in common biological pathways such as cell surface receptor signaling pathway and response to external stimulus. In conclusion, we demonstrated that irrespective of the pathogen type, there are common immunoregulatory and proinflammatory signals.

Report of the 2022 Banff Heart Concurrent: Focus on non-human leukocyte antigen antibodies in rejection and the pathology of "mixed" rejection.

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system.

Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants.

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

Immune Features of Disparate Liver Transplant Outcomes in Female Hispanics With Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is a severe immune-mediated stage of nonalcoholic fatty liver disease that is rapidly becoming the most common etiology requiring liver transplantation (LT), with Hispanics bearing a disproportionate burden. This study aimed to uncover the underlying immune mechanisms of the disparities experienced by Hispanic patients undergoing LT for NASH. Methods: We enrolled 164 LT recipients in our institutional review board-approved study, 33 of whom presented with NASH as the primary etiology of LT (20%), with 16 self-reported as Hispanic (48%). We investigated the histopathology of prereperfusion and postreperfusion biopsies, clinical liver function tests, longitudinal soluble cytokines via 38-plex Luminex, and immune cell phenotypes generated by prereperfusion and postreperfusion blood using 14-color flow cytometry and enzyme-linked immunosorbent assay. Results: Hispanic LT recipients transplanted for NASH were disproportionately female (81%) and disproportionately suffered poor outcomes in the first year posttransplant, including rejection (26%) and death (38%). Clinically, we observed increased pro-inflammatory and apoptotic histopathological features in biopsies, increased AST/international normalized ratio early posttransplantation, and a higher incidence of presensitization to mismatched HLA antigens expressed by the donor allograft. Experimental investigations revealed that blood from female Hispanic NASH patients showed significantly increased levels of leukocyte-attracting chemokines, innate-to-adaptive switching cytokines and growth factors, HMGB1 release, and TLR4/TLR8/TLR9/NOD1 activation, and produced a pro-inflammatory, pro-apoptotic macrophage phenotype with reduced CD14/CD68/CD66a/TIM-3 and increased CD16/CD11b/HLA-DR/CD80. Conclusions: A personalized approach to reducing immunological risk factors is urgently needed for this endotype in Hispanics with NASH requiring LT, particularly in females.

Relationship of Heterologous Virus Responses and Outcomes in Hospitalized COVID-19 Patients.

The clinical trajectory of COVID-19 may be influenced by previous responses to heterologous viruses. We examined the relationship of Abs against different viruses to clinical trajectory groups from the National Institutes of Health IMPACC (Immunophenotyping Assessment in a COVID-19 Cohort) study of hospitalized COVID-19 patients. Whereas initial Ab titers to SARS-CoV-2 tended to be higher with increasing severity (excluding fatal disease), those to seasonal coronaviruses trended in the opposite direction. Initial Ab titers to influenza and parainfluenza viruses also tended to be lower with increasing severity. However, no significant relationship was observed for Abs to other viruses, including measles, CMV, EBV, and respiratory syncytial virus. We hypothesize that some individuals may produce lower or less durable Ab responses to respiratory viruses generally (reflected in lower baseline titers in our study), and that this may carry over into poorer outcomes for COVID-19 (despite high initial SARS-CoV-2 titers). We further looked at longitudinal changes in Ab responses to heterologous viruses, but found little change during the course of acute COVID-19 infection. We saw significant trends with age for Ab levels to many of these viruses, but no difference in longitudinal SARS-CoV-2 titers for those with high versus low seasonal coronavirus titers. We detected no difference in longitudinal SARS-CoV-2 titers for CMV seropositive versus seronegative patients, although there was an overrepresentation of CMV seropositives among the IMPACC cohort, compared with expected frequencies in the United States population. Our results both reinforce findings from other studies and suggest (to our knowledge) new relationships between the response to SARS-CoV-2 and Abs to heterologous viruses.

Disulfide-HMGB1 signals through TLR4 and TLR9 to induce inflammatory macrophages capable of innate-adaptive crosstalk in human liver transplantation.

Ischemia-reperfusion injury (IRI) during orthotopic liver transplantation (OLT) contributes to graft rejection and poor clinical outcomes. The disulfide form of high mobility group box 1 (diS-HMGB1), an intracellular protein released during OLT-IRI, induces pro-inflammatory macrophages. How diS-HMGB1 differentiates human monocytes into macrophages capable of activating adaptive immunity remains unknown. We investigated if diS-HMGB1 binds toll-like receptor (TLR) 4 and TLR9 to differentiate monocytes into pro-inflammatory macrophages that activate adaptive immunity and promote graft injury and dysfunction. Assessment of 106 clinical liver tissue and longitudinal blood samples revealed that OLT recipients were more likely to experience IRI and graft dysfunction with increased diS-HMGB1 released during reperfusion. Increased diS-HMGB1 concentration also correlated with TLR4/TLR9 activation, polarization of monocytes into pro-inflammatory macrophages, and production of anti-donor antibodies. In vitro, healthy volunteer monocytes stimulated with purified diS-HMGB1 had increased inflammatory cytokine secretion, antigen presentation machinery, and reactive oxygen species production. TLR4 inhibition primarily impeded cytokine/chemokine and costimulatory molecule programs, whereas TLR9 inhibition decreased HLA-DR and reactive oxygen species production. diS-HMGB1-polarized macrophages also showed increased capacity to present antigens and activate T memory cells. In murine OLT, diS-HMGB1 treatment potentiated ischemia-reperfusion-mediated hepatocellular injury, accompanied by increased serum alanine transaminase levels. This translational study identifies the diS-HMGB1/TLR4/TLR9 axis as potential therapeutic targets in OLT-IRI recipients.