Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection

Abstract

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

Keywords: Autopsia; Broncho-alveolar lavage (BAL); COVID-19; T cell responses; Vasculitis.

Figure 1

SARS-CoV-2 antibody response in ICU COVID-19 patients. Number of patients tested during the first and second wave (A). (B–I) Antibodies against the S1 domain of the spike protein were measured in ELISA assay in first-wave survivors (closed symbols) or deceased (open symbols) patients at different time points from first COVID symptoms. (B, C) Serum IgA (B) and IgG responses (C) are depicted during ICU stay. (D, E) First time point IgA (D) and Ig IgG (E) titers in survivors and deceased patients. In BAL, the IgA (F, H) and IgG (G, I) antibody response was weak in deceased patients (open symbols) when compared to survivors (closed symbols). Chi-square test was used for serology comparison, while Mann–Whitney test was used for comparing BAL antibody levels.

Figure 2

SARS-CoV-2-specific T cells trafficking to the lungs in severe COVID-19 infection. (A, B) PBMC ELISpot IFN-γ assays against spike protein (S, 2 peptide pools) and nucleocapsid (NC, 4 peptide pools) were assessed in 36 first-wave ICU patients at first time point and in 24 second-wave ICU patients (A, B). Results are expressed as SFC units (A) or as a number of recognized peptide pools (B). (C) Percentage of the total SFC response against SARS-CoV-2 peptide pool in each group. (D) Proportion of lymphocyte (Ly) on first BAL elements in first-wave patients (round symbols) and second-wave patients (triangles). (E, F) %CD3+CD4+ and CD3+CD8+ T cell in BAL lymphocytes in all BAL samples from first wave (E) and second wave (F), and comparison of the CD4/CD8 BAL ratio during the first and second waves (G). Open symbols are deceased patients. (H, I) Comparison of BAL lymphocytes and PBMC SARS-CoV-2 ELISpot IFN-γ assays in two representative patients (H) and in all patients (I). *p<0.05 ***p<0.001; NS, not significant.

Figure 3

Serum and bronchoalveolar supernatant cytokine levels in severe COVID-19 infection. First-wave serum (A) and BAL (B) cytokine levels in survivors (n=8, black symbols), and deceased patients (n=4, open symbols). The BAL/serum ratio is shown in (C). Heatmaps represent individual data of survivors and deceased patients and cytokine orders in whole Figure 3 follow the hierarchical clustering of the corresponding heatmap. Cytokine serum (D), BAL (E), and BAL/serum ratio (F) levels for first- wave patients (rounded symbols) and second-wave patients (triangles).

Figure 4

Lung histopathology of deceased COVID-19 cases. Lung sections of three COVID-19 cases were studied with hematoxylin–eosin staining (HES) and immunostaining. (A) HES staining showed sparse (case #1) or abundant (cases #2 and #3) lymphocytic vasculitis of middle-sized pulmonary vessels. (B) The lymphocytic infiltration was mainly CD3 positive (C) and also CD45RO+CD4+ positive in imaging mass cytometry (C). (D) Immunostaining showed SARS-CoV-2-positive cells around middle-sized vessels (arrows). (E) Some vascular formations were surrounded (F) by CD20+ lymphocytes accumulating in what could be a tertiary lymphoid structure. This perivascular inflammation was not observed in an influenza H1N1 case (bottom panel).