Clonal expansion and activation of tissue-resident memory-like Th17 cells expressing GM-CSF in the lungs of severe COVID-19 patients

Abstract

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8⁺ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.

Fig. 1. Immune landscape of severe COVID-19 and bacterial pneumonia.

(A) Schematic representation of experimental setup. (B) Overview of baseline characteristics and clinical course of patients with COVID-19 and patients with bacterial pneumonia (LOD, limit of detection; ICU, intensive care unit). (C) Virus titers measured by quantitative polymerase chain reaction from BALF, tracheal fluid, and peripheral blood at time of sampling. (D) UMAP dimensionality reduction embedding of all cells from BALF (n = 56,735 cells, n = 8 for COVID-19, and n = 4 for bacterial pneumonia, samples of patients S6 and B1 were excluded for technical reasons) colored according to cell type assessed by gene expression and (E) epitope measurement using CITE-seq of key markers (scale bars indicate normalized expression). (F) Single-cell analysis of CD3⁺ T cells from peripheral blood of all patients (n = 77,457 cells, n = 7 for COVID-19, and n = 4 for bacterial pneumonia). (G) CITE-seq information of cluster-defining epitopes (scale bars indicate normalized expression). (H) Flow cytometry of peripheral blood and BALF of patients with COVID-19 (n = 8) and bacterial pneumonia (n = 5). Per patient, an equal number of viable CD45⁺ cells were exported for analysis and concatenated together before calculating the UMAP (total cells in peripheral blood = 129,141; in BALF = 114,927). Cell types were defined according to cell surface expression profiles by manual gating. Patient S9 was excluded from the statistical analysis due to low cell numbers. (I) Comparison of cell frequencies as measured by flow cytometry of cells from patients with COVID-19 and bacterial pneumonia (*P < 0.05). (J) Hematoxylin and eosin staining (H&E) and (K) CD3 staining of lung autopsy tissue of one representative of seven patients.

Fig. 2. T cell clonality in pulmonary inflammation.

(A) Blood-lung activation map of T cells from blood and BALF of all patients: UMAP dimensionality reduction embedding of T cells (left); clone size proportion (clone count divided by number of cells per sample) of T cells (middle), and the cytokine secretion score of T cells (right) from COVID-19 and bacterial pneumonia as indicated. (B) UMAP presentation of T cells from BALF of all patients. Clusters were annotated according to gene expression and epitopes measurement of key markers. T_CM, T central memory; T_SCM, T stem cell–like memory; lncRNA, long noncoding RNA. (C) Ratio of clonal expansion of bacterial pneumonia versus COVID-19 for the major expanded BALF T cell clusters. (D) Subclustering analysis of clonally expanded CD4⁺ T cells of all patients. Clusters were annotated according to gene expression presented in the heatmap. (E) Volcano plot showing differential gene expression between T_H17 clusters 1 and 2 of all patients. Genes were considered significant with adjust P < 0.05. Nonsignificant genes are shown in black. (F) Heatmap of selected pathogenic gene markers of T_H17 cells of all patients in comparison with other T cell clusters. (G) Clone size proportion of T cells in peripheral blood and BALF of patients with COVID-19 and presentation of high abundant clones (clone size > 5) that are shared between BALF and blood and BAL-specific clones as indicated. (H) CD4 migration and tissue residency score of T_H17 cluster1 (T_RM17) and 2 (T_EM17) from all patients. (I) Possible model of intraclonal diversification of CD4⁺ T cell subsets (left); distribution of two representative BALF clones from a patient with COVID-19 (patient S1 clone239 and clone218) on the UMAP (middle and right). (J) Bar plot of top expanded BALF clones containing T_RM17 cells from patients with COVID-19. COVID-19: n = 8 for BALF and n = 7 for blood; bacterial pneumonia: n = 4 for BALF and n = 4 for blood.

Fig. 3. Landscape of myeloid cells in the lung.

(A) UMAP dimensionality reduction embedding of myeloid cells from BALF of all patients from our study (COVID-19 n = 8 and bacterial pneumonia n = 4). (B) Heatmap of key marker gene expression of the indicated clusters. (C) UMAP plots showing expression of genes mirroring key features of macrophage polarization and function (scale bars indicate normalized expression).

Fig. 4. Interactome of T cells and myeloid cells in the lungs of patients with COVID-19.

(A) Interaction network of all BALF clusters based on the number of ligand-receptor interaction (>30 edges) based on Fruchterman-Reingold force-directed algorithm from patients with COVID-19 (n = 8). (B) Adjacency map of T cell-myeloid cell interactions. (C) Ligand and receptor interaction strength ([mean ligand expression] × [proportion of receptor expression per cluster]) of T_RM17 cells (ligands) and profibrotic macrophages (receptors). Interactions were filtered for cytokines and, for specificity, based on rank scoring. (D) Supervised interaction map of potential key players in sustaining lung inflammation in patients with COVID-19. Line width correlates with interaction strength. (E) Pathway analysis of CD40L (CD40LG), LTA, and GM-SCF (CSF2) signaling in proinflammatory and profibrotic macrophages indicating the log₂ fold change in COVID-19 versus bacterial pneumonia. (F) Cytotoxic module scores in all clusters which include CD8⁺ cells using proinflammatory and cytotoxic mediator genes in CTL from (13). (G) Module scores in the indicated clusters using the highest 50 differential expressed genes of CD8⁺ T cells receiving help or no help from CD4⁺ cells, respectively, according to (31). (H and I) Ligands and receptor interaction strength (H) between T_RM17 and CD8⁺ T_EM CTL Cluster 2 and (I) between T_RM17 and DC. Interactions were filtered according to their rank score. (J) Supervised interaction map of T_RM17, CD8⁺ T_EM CTL, and DCs with annotated ligands. Line width correlates with interaction strength.

Fig. 5. Cytokine secretion profile and cellular source of GM-CSF.

(A) GM-CSF and IL-17A protein in serum of patients with COVD-19 (n = 8) and healthy controls (n = 7) from Hamburg and of patients with moderate (n = 8) or severe COVID-19 (n = 11) from Halle as indicated. Cell map of (B) CSF2 (GM-CSF) expressing and (C) IL17A expressing cells (scale bars indicate normalized expression). Three different UMAPs with different cellular granularity showing the respective gene expression of in total cells of the BALF (left), total T cells of blood and BALF (middle), and total T cells in BALF (right) from all patients. (D) Immunofluorescence of CD4⁺ (green) CCR6⁺ (red) T_RM17 cells in the lungs of a deceased patient with COVID-19 infection [nuclear staining 4′,6-diamidino-2-phenylindol (DAPI), blue] (two additional samples are presented in fig. S10B). (E) Combined immunofluorescence (CCR6) and FISH (IL17A) of lung samples from one patient with COVID-19. (F) Concentrations of the indicated cytokines in the BALF of patients with COVID-19 and bacterial pneumonia.