Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100A^hi/HLA-DR^lo classical monocytes and activated LAG-3^hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8⁺ clones, unmutated IGHG⁺ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5′ single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung–blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Rationale: Fibrotic hypersensitivity pneumonitis (FHP) is a debilitating interstitial lung disease driven by incompletely understood immune mechanisms. Objectives: To elucidate immune aberrations in FHP in single-cell resolution. Methods: Single-cell 5′ RNA sequencing was conducted on peripheral blood mononuclear cells and BAL cells obtained from 45 patients with FHP, 63 patients with idiopathic pulmonary fibrosis (IPF), 4 patients with nonfibrotic hypersensitivity pneumonitis, and 36 healthy control subjects in the United States and Mexico. Analyses included differential gene expression (Seurat), TF (transcription factor) activity imputation (DoRothEA-VIPER), and trajectory analyses (Monocle3 and Velocyto-scVelo-CellRank). Measurements and Main Results: Overall, 501,534 peripheral blood mononuclear cells from 110 patients and control subjects and 88,336 BAL cells from 19 patients were profiled. Compared with control samples, FHP has elevated classical monocytes (adjusted-P = 2.5 × 10−3) and is enriched in CCL3hi/CCL4hiand S100Ahiclassical monocytes (adjusted-P < 2.2 × 10−16). Trajectory analyses demonstrate that S100Ahiclassical monocytes differentiate into SPP1hilung macrophages associated with fibrosis. Compared with both control subjects and IPF, cells from patients with FHP are significantly enriched in GZMhicytotoxic T cells. These cells exhibit TF activities indicative of TGFβ and TNFα and NFκB pathways. These results are publicly available at http://ildimmunecellatlas.com. Conclusions: Single-cell transcriptomics of patients with FHP uncovered novel immune perturbations, including previously undescribed increases in GZMhicytotoxic CD4+ and CD8+ T cells—reflecting this disease’s unique inflammatory T cell–driven nature—as well as increased S100Ahiand CCL3hi/CCL4hiclassical monocytes also observed in IPF. Both cell populations may guide the development of new biomarkers and therapeutic interventions.

Protective immunity against COVID-19 likely depends on the production of SARS-CoV-2–specific plasma cells and memory B cells postinfection or postvaccination. Previous work has found that germinal center reactions are disrupted in severe COVID-19. This may adversely affect long-term immunity against reinfection. Consistent with an extrafollicular B cell response, patients with severe COVID-19 have elevated frequencies of clonally expanded, class-switched, unmutated plasmablasts. However, it is unclear whether B cell populations in individuals with mild COVID-19 are similarly skewed. In this study, we use single-cell RNA sequencing of B cells to show that in contrast to patients with severe COVID-19, subjects with mildly symptomatic COVID-19 have B cell repertoires enriched for clonally diverse, somatically hypermutated memory B cells ∼30 d after the onset of symptoms. This provides evidence that B cell responses are less disrupted in mild COVID-19 and result in the production of memory B cells.

Background: Interstitial lung disease (ILD) evaluation often requires lung biopsy for definite diagnosis. In recent years, transbronchial cryobiopsy (TBCB) emerged as a procedure with higher diagnostic yield than transbronchial forceps biopsy (TBFB), especially for fibrotic ILDs. Nonetheless, studies comparing these modalities in non-fibrotic ILDs and for specific ILD diagnoses are scarce. Objectives: The aim of this study was to evaluate the diagnostic yield and safety of TBCB and TBFB in patients with fibrotic and non-fibrotic ILDs. Method: An observational retrospective multicenter study including patients with ILD diagnosis by multidisciplinary discussion that underwent TBCB or TBFB between 2017 and 2021. Chest CT scans were reviewed by a chest radiologist. Biopsy specimens were categorized as diagnostic (with specific histological pattern), nondiagnostic, or without lung parenchyma. Nondiagnostic samples were reassessed by a second lung pathologist. TBCB and TBFB diagnostic yields were analyzed by multivariate regression. Procedural complications were evaluated as well. Results: 276 patients were included, 116 (42%) underwent TBCB and 160 (58%) TBFB. Fibrotic ILDs were present in 148 patients (54%). TBCB diagnostic yield was 78% and TBFB 48% (adjusted odds ratio [AOR] 4.2, 95% CI: 2.4–7.6, p < 0.01). The diagnostic yield of TBCB was higher than TBFB among patients with fibrotic ILD (AOR 3.8, p < 0.01), non-fibrotic ILD (AOR 5.8, p < 0.01), and across most ILD diagnoses. TBCB was associated with higher risk for significant bleeding (10% vs. 3%, p < 0.01), but similar risk for pneumothorax. Conclusions: Diagnostic yield of TBCB was superior to that of TBFB for both fibrotic and non-fibrotic ILDs, and across most diagnoses.