A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE hi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS. </p

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.</p

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.</p

Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN–specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non–COVID-19 controls revealed a lack of type I IFN–stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN–specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN–specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms

Increased risk of severe clinical course of COVID-19 in carriers of HLA-C*04:01

Background: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing urgency to identify pathophysiological characteristics leading to severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors that affect individual immune response to SARS-CoV-2. We sought to evaluate this hypothesis by conducting a multicenter study using HLA sequencing. Methods: We analyzed the association between COVID-19 severity and HLAs in 435 individuals from Germany (n = 135), Spain (n = 133), Switzerland (n = 20) and the United States (n = 147), who had been enrolled from March 2020 to August 2020. This study included patients older than 18 years, diagnosed with COVID19 and representing the full spectrum of the disease. Finally, we tested our results by meta-analysing data from prior genome-wide association studies (GWAS). Findings: We describe a potential association of HLA-C*04:01 with severe clinical course of COVID-19. Carriers of HLA-C*04:01 had twice the risk of intubation when infected with SARS-CoV-2 (risk ratio 1.5 [95% CI 1.1-2.1], odds ratio 3.5 [95% CI 1.9-6.6], adjusted p-value = 0.0074). These findings are based on data from four countries and corroborated by independent results from GWAS. Our findings are biologically plausible, as HLA-C*04:01 has fewer predicted bindings sites for relevant SARS-CoV-2 peptides compared to other HLA alleles. Interpretation: HLA-C*04:01 carrier state is associated with severe clinical course in SARS-CoV-2. Our findings suggest that HLA class I alleles have a relevant role in immune defense against SARS-CoV-2. Funding: Funded by Roche Sequencing Solutions, Inc

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

Prediction Of T-cell Epitopes for Cancer Therapy

The human immune system can identify malfunctioning, damaged, or infected cells within the body. The integrity of nucleated cells is communicated to the immune system through short peptides which they display on their exterior using MHC proteins. Mutated peptides produced in cancer cells due to single nucleotide events, short insertion or deletion events, or due to large chromosomal rearrangements can help the immune system identify tumor cells as threats. Adoptive T-cell and Vaccine-based therapies that stimulate autologous T-cells to treat a patient's disease are more specific than standard chemo- and radiotherapies because they directly target tumor-specific mutations. They also potentially protect the body from recurrence of the tumor due to their unique memory potential. However, T-cells are often perceptive to only those cells originating from the patient's body, making this a personalized treatment for the disease. Rapid identification of tumor neoepitopes in a patient diagnosed with cancer will greatly reduce the time taken to develop a personalized therapy.This thesis primarily covers ProTECT, a computational workflow for the Prediction of Epitopes for Cancer Therapy. ProTECT is an automated, scalable, and reproducible end-to-end pipeline that processes patient sequencing data to produce a ranked list of neoepitopes of therapeutic significance. ProTECT also attempts to predict the state of the MHC presentation pathway in the tumor, and the tumor response to immunotherapies based on previously published immune signatures. I describe ProTECT and demonstrate its features using a cohort of 326 Prostate Adenocarcinoma patients. I then demonstrate its clinical utility through the identification of a neoepitope arising from a hotspot mutant in pediatric Neuroblastoma.To enable ProTECT, I worked on two auxiliary projects, Toil and TransGene. Toil is a workflow manager developed at the Genomics Institute at UCSC, and is the backbone of the ProTECT workflow. I spent some of my time at UCSC working on enhancing the efficiency of Toil workflows. TransGene is a tool I wrote that translates genomic variants (single nucleotide, short insertions and deletions, and fusion genes) into peptides that are compatible with existing peptide:MHC binding affinity predictors. These methods are also included in this thesis

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer

International audienceTissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophages (TAMs) identity and functions. Combining scRNAseq to spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in spontaneous tumor model and track the different stroma territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ T cells and to phagocyte tumor cells supporting that specific tumor regions rather than defined activation states are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design new cancer treatment targeting specific TAM niches

Sex-Stratified Single-Cell RNA-Seq Analysis Identifies Sex-Specific and Cell Type-Specific Transcriptional Responses in Alzheimer's Disease Across Two Brain Regions.

Alzheimer's disease (AD) is a pervasive neurodegenerative disorder that disproportionately affects women. Since neural anatomy and disease pathophysiology differ by sex, investigating sex-specific mechanisms in AD pathophysiology can inform new therapeutic approaches for both sexes. Previous bulk human brain RNA sequencing studies have revealed sex differences in dysregulated molecular pathways related to energy production, neuronal function, and immune response; however, the sex differences in disease mechanisms are yet to be examined comprehensively on a single-cell level. We leveraged nearly 74,000 cells from human prefrontal and entorhinal cortex samples from the first two publicly available single-cell RNA sequencing AD datasets to perform a case versus control sex-stratified differential gene expression analysis and pathway network enrichment in a cell type-specific manner for each brain region. Our examination at the single-cell level revealed sex differences in AD prominently in glial cells of the prefrontal cortex. In the entorhinal cortex, we observed the same genes and networks to be perturbed in opposing directions between sexes in AD relative to healthy state. Our findings contribute to growing evidence of sex differences in AD-related transcriptomic changes, which can fuel the development of therapies that may prove more effective at reversing AD pathophysiology

Bioinformatics Analysis of Publicly Available Single-Nuclei Transcriptomics Alzheimer's Disease Datasets Reveals APOE Genotype-Specific Changes Across Cell Types in Two Brain Regions.

Alzheimer's Disease (AD) is a complex neurodegenerative disease that gravely affects patients and imposes an immense burden on caregivers. Apolipoprotein E4 (APOE4) has been identified as the most common genetic risk factor for AD, yet the molecular mechanisms connecting APOE4 to AD are not well understood. Past transcriptomic analyses in AD have revealed APOE genotype-specific transcriptomic differences; however, these differences have not been explored at a single-cell level. To elucidate more complex APOE genotype-specific disease-relevant changes masked by the bulk analysis, we leverage the first two single-nucleus RNA sequencing AD datasets from human brain samples, including nearly 55,000 cells from the prefrontal and entorhinal cortices. In each brain region, we performed a case versus control APOE genotype-stratified differential gene expression analysis and pathway network enrichment in astrocytes, microglia, neurons, oligodendrocytes, and oligodendrocyte progenitor cells. We observed more global transcriptomic changes in APOE4 positive AD cells and identified differences across APOE genotypes primarily in glial cell types. Our findings highlight the differential transcriptomic perturbations of APOE isoforms at a single-cell level in AD pathogenesis and have implications for precision medicine development in the diagnosis and treatment of AD