Summary: This study aims to characterize pathophysiological changes in lethal COVID-19 lymph nodes. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, T-Bet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6/21 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a proinflammatory, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19.
Overall Design: Examination of autopsy lymph node tissue of lethal COVID-19 cases versus controls by gene expression profiling. Please note that the 'VLP00581_AI_Plate1_09JAN2021_Parsed-forReveal_QualityControlled.xlsx' processed data file contains 78 data columns, while 50 samples (used for the final manuscript of the study) are included in the records. The extra data columns refer to either universal RNA samples used for correlations tests by HTG Molecular, which are labelled uRNA on the second sheet ‘QC_Raw' of the count matrix excel file, -or- samples which failed the internal quality controls of HTG Molecular, such as poor quality of sample (Q0), insufficient read depth (Q1) or expression variability (Q2), marked on the third sheet ‘QC_summary’. Further samples were excluded as described in the forthcoming manuscript, such as inadequate patient data.
HTG uses an extraction free technology that involves resuspending crude sample in Lysis Buffer. The sample is then treated with Proteinase K and stored or used.
Library Construction Protocol:
HTG EdgeSeq library construction is defined in the HTG EdgeSeq System User Manual (RUO)