SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2

Abstract

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV1), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.

Fig 1. Cumulative and S-, N- and M- specific SARS-CoV-2 specific T cell frequencies are elevated in PASC.

(A) Representative flow cytometry density plots showing the frequency of SARS-CoV-2 (S, N and M) IFN-γ-, TNF-α- and IL-2-specific T cells from a participant with PASC. The cummulative frequency of SARS-CoV-2 S, N and M IFN-γ-producing (B) CD4⁺ and (C) CD8⁺, TNF-α-producing (D) CD4⁺ and (E) CD8⁺ and IL-2-produing (F) CD4⁺ and (G) CD8⁺ T cells are shown. For B-G each point represents the sum of the combined frequencies of SARS-CoV-2 S, N and M-specific T cells of each participant. The frequency of individual SARS-CoV-2 S-, N- or M-specific IFN-γ-producing (H) CD4⁺ and (I) CD8⁺ and TNF-α-producing (J) CD4⁺ and (K) CD8⁺ T cells are shown. The horizontal bars depict median values for each cohort. Blue: PASC-NH (not hospitalized), red: PASC-Hospitalized and orange: RC participants. Mann-Whitney tests were used to determine statistical significance.

Fig 2. Cytokine co-expression of SARS-CoV-2 specific T cells differs between PASC and RC.

Cytokine co-expression on SARS-CoV-2 specific T cells visualized using simplified presentation of incredibly complex evaluations (SPICE) analysis. Each pie chart represents the proportions of combinations of IFN-γ, TNF-α and IL-2 producing T cells in response to one SARS-CoV-2 protein. Arcs surrounding each pie chart depict the proportion of cells secreting each individual cytokine. Colors of pie charts and arcs represent different cytokines or combinations of cytokines and are listed in the corresponding legend. Stars denote significant differences determined by student t test corrected for multiple comparsons between PASC and RC cohorts for a particular combination of co-expressed cytokines matching as indicated by the color corresponding to the pie legend. Stars are positioned next to the cohort with the higher proportion. P values positioned between PASC and RC pie charts denote statistical significance of overall composition by permutation test with 10,000 iterations corrected for multiple comparsons.

Fig 3. TNF-α-producing T cells have the highest proportion of Ki-67 expression.

The percentages of Ki67 expressing SARS-CoV-2-specific CD4⁺ (left panels) and CD8⁺ (right panels) T cells obtained from the blood of PASC participants are shown. SARS-CoV-2 S- (top panel), N- (middle panel) and M- (bottom panel) specific T cells are grouped by production of IFN-γ, TNF-α and IL-2. Note, data points from individual PASC participants were obtained for 1 or more of the cytokines assessed; however, in no instances are there multiple values obtained from the same participant for a particular cytokine. Blue represents IFN-γ⁺ T cells, brown represents TNF-α⁺ T cells and green represents IL-2⁺ T cells. Kruskal-Wallis tests with corrections for multiple comparisons were used to determine statistical significance.

Fig 4. Plasma IL-6 in PASC is higher than RC and correlates with frequency of SARS-CoV-2-specific TNF-α producing CD8⁺ T cells.

(A) Plasma IL-6 levels (pg/mL) and (B) plasma CRP levels (mg/L) in PASC and RC. (C) Correlations between plasma IL-6 and frequency of SARS-CoV-2-specific TNF-α-producing CD8⁺ T cells in PASC participants. (D) Correlations between plasma IL-6 and frequency of SARS-CoV-2-specific TNF-α-producing CD8⁺ T cells in RC participants. For A-B, the horizontal bar represents median of cohort and error bars are 95% confidence index. Each point represents data from one participant. Blue: PASC-NH (not hospitalized), red: PASC-Hospitalized and orange: RC participants. For A-B Mann-Whitney tests were used to determine statistical significance. For C-D Spearman correlations were used to determine statistical significance.

Fig 5. Correlations between SARS-CoV-2 specific T cells and FEV₁ and symptoms in PASC.

Correlations between the percent predicted FEV₁ and the combined SARS-CoV-2 S-, N- and M-specific frequencies of IFN-γ-producing (A) CD4⁺ and (B) CD8⁺ T cells and TNF-α-producing (C) CD4⁺ and (D) CD8⁺ T cells are shown. Correlations of between the duration of dyspnea in days and the frequencies of (E) combined SARS-CoV-2 S-, N- and M-specific CD8⁺ T cells, (F) S-specific IFN-γ-producing CD8⁺ T cells, (G) combined S-, N- and M-specific IL-2-producing CD4⁺ T cells and (H) S-specific TNF-α-producing CD8⁺ T cells are shown. Each point represents data from one PASC participant. Blue and red symbols represent PASC-NH (not hospitalized) and PASC-Hospitalized participants, respectively. Spearman correlations were used to determine statistical significance.