Abstract

Many patients continue to suffer residual respiratory symptoms several months following a SARS-CoV-2 infection, known as ?pulmonary long COVID? (PLC), but the mechanisms remain unknown. We performed single-cell RNA-sequencing on bronchoalveolar lavage (BAL) fluid to characterize the immune cell population in PLC relative to healthy controls. BAL was obtained via bronchoscopy and washed twice with 0.4% bovine serum albumin in Dulbecco?s phosphate-buffered saline. Bioinformatic analysis was performed with Rsubread, SoupX, Scanpy, Scrublet, and Gseapy software. We characterized 116,856 cells from 5 PLC patients (4 females, age 43±10 years) and in two control groups: 1) 3 post-COVID patients without pulmonary symptoms (1 female, age 53 ±15 years), and 2) 3 never-infected controls (3 females, age 38±23 years). No residual SARS-CoV-2 mRNA was detected in any of the samples. Macrophages made up the majority of cells among all three groups (~70%). The proportions of other cell types were not significantly different between groups.  However, gene set enrichment analysis revealed upregulated pathways in antigen presentation and cell cycles in the dendritic cells (p<0.001) and enrichment of cytokine- and neutrophil-mediated immune pathways in T-cells (p<0.001) from PLC samples. On the other hand, T-cells, and dendritic cells in the control groups showed enrichment in protein targeting (p<0.001) and viral pathways (p<0.001). These data suggest that the airways of PLC patients are demonstrate an ongoing heightened cellular immune response, and increased antigen presentation, even in the absence of detectable viral mRNA.