key: cord-1001745-5zf7o8nh
authors: Littlefield, Katherine M.; Watson, Renée O.; Schneider, Jennifer M.; Neff, Charles P.; Yamada, Eiko; Zhang, Min; Campbell, Thomas B.; Falta, Michael T.; Jolley, Sarah E.; Fontenot, Andrew P.; Palmer, Brent E.
title: SARS-CoV-2-specific T cells associate with reduced lung function and inflammation in pulmonary post-acute sequalae of SARS-CoV-2
date: 2022-02-15
journal: bioRxiv
DOI: 10.1101/2022.02.14.480317
sha: 74759b546a20b40ee436b8969a802ee5d98f2780
doc_id: 1001745
cord_uid: 5zf7o8nh

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 up to 34-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. Author Summary Long COVID-19 or post-acute sequelae of SARS-CoV-2 (PASC) impacts 20-30% of those infected with SARS-CoV-2 and is characterized by COVID-19 symptoms exceeding 4 weeks from symptom onset. While those with PASC experience a wide variety of persistent symptoms including shortness of breath, cough, chest pain, irregular heartbeat, brain fog, fatigue, and intermittent fever, lung-related conditions are the most common. Although, infection with SARS-CoV-2 is clearly the inciting factor for PASC, the mechanisms responsible for long-term lung dysfunction are unclear and current treatments are ineffective at resolving pulmonary symptoms. Generalized PASC has been associated with SARS-CoV-2-specific T cells, a component of adaptive immunity, suggesting that residual virus may persist. Here, we investigated the frequency and function of virus-specific T cells in the blood of individuals with pulmonary PASC and correlated their presence with systemic inflammation and lung function. Our findings demonstrated that T cells specific for SARS-CoV-2 are elevated in the blood of those with pulmonary PASC and are associated with increased IL-6, a cytokine strongly associated with COVID-19 severity, and decreased lung function. These findings provide mechanistic insight into the pathophysiology of pulmonary PASC needed for the development of new treatments to improve quality of life for those affected.

We measured the frequency of SARS-CoV-2-specific T cells in blood using intracellular cytokine 144 (IFN-γ, TNF-α, and IL-2) staining after stimulation with peptide pools of the SARS-CoV-2 spike 145 (S), nucleocapsid (N) or membrane (M) surface-expressed proteins. Representative density plots 146 of SARS-CoV-2-specific T cell populations are shown (Fig. 2a) . First, we analyzed the combined Δ Indicates stratification analyses were performed demonstrating these differences do not influence SARS-CoV-2specific T cell frequencies (S1 and S2 Fig.) and levels of plasma IL-6 (S4 Fig.) .

285 cells and %FEV 1 (Fig. 7c-d) . We then compared the frequency of SARS-CoV-2-specific T cells 286 with the duration of prolonged dyspnea experienced by 80% (n=16) of our pulmonary PASC 287 participants. From this analysis, we identified positive correlations between dyspnea duration and 288 frequencies of IFN-γ-producing SARS-CoV-2 total (r=0.49, P=0.02) and S-specific (r=0.55, 289 P=0.015) CD8 + T cells (Fig 7e-f ). There was also a positive correlation between TNF--producing 

SARS-CoV-2-specific T cells for all three proteins. PASC participants had 148 significantly increased frequencies of SARS-CoV-2-specific CD4 + and CD8 + T cells that produced 149 IFN-γ or TNF-α compared to the RC cohort

There was a 2.9-and 5.2-fold increased 151 frequency of CD4 + and CD8 + SARS-CoV-2-specific T cells producing IFN-γ in PASC participants 152 compared to RC participants (Fig. 2b-c)

We chose to separate pulmonary PASC participants by prior 155 hospitalization status to determine if this factor was associated with the frequency of virus-specific

The same significant differences were observed when comparing only PASC-NH and RC 157 participants and no significant differences were observed between PASC-NH and PASC-H groups 158 (Fig. 2b-g). Stratification analyses for age, comorbidities and time of sample collection showed no 159 significant differences within the RC or pulmonary PASC cohorts

Similar 167 findings were seen for IFN-γ expression in CD8 + T cells between PASC and RC cohorts and again 168 no difference was observed comparing the PASC-NH and PASC-H groups (Fig. 3b). Again, there 169 were no significant differences based on age

respectively) (Fig. 3c) and significantly 174 increased frequencies of TNF-α-producing CD8 + T cells in response to all three SARS-CoV-2 175 proteins (Fig. 3d) compared to RC participants. Approximately 50% of CD4 + and CD8 + T cells 176 from PASC participants produced TNF-α in response to all 3 SARS-CoV-2 proteins, whereas these 177 percentages were 33% and 13%, respectively in RC participants. Only one PASC participant had 178 no detectable CD4 + T cell cytokine response to any SARS-CoV-2 protein -this individual did 179 have CD8 + T cell cytokine responses -while 5 RC participants had no detectable responses in 180 either CD4 + or CD8 + T cells. Interestingly, female PASC participants had significantly higher total 181 (P=0.0015) and S-specific

SARS-CoV-2-specific T cells in PASC are less polyfunctional than in RC and exhibit recent 185 proliferation

Next, we compared the expression of cytokines and phenotypic markers on SARS-CoV-2-specific

As confirmation of 189 waning immunity, we examined SARS-CoV-2-specific T cell frequencies in 2 RC participants 190 who provided blood at 2-and 30-weeks post resolution of symptoms. As expected, their T cell 191 responses decreased over time (S2 Fig). Based on these data, we collected blood from RC 192 participants at early time points after resolution of infection to ensure that detectable frequencies 193 of SARS-CoV-2-specific T cells were present. Thus

The SPICE analysis revealed the 198 majority of SARS-CoV-2-specific T cells in individuals with pulmonary PASC only produce one 199 of the three cytokines tested with TNF- dominating the virus-specific CD4 + T cell response and 200 IFN- dominating the CD8 + T cell response, while RC participants tended to produce multiple 201 cytokines, indicating the T cell cytokine response is more restricted PASC compared to RC. A 202 deeper analysis revealed significant differences in the distribution of CD4 + T cell cytokine 203 expression in response to N and M proteins when comparing PASC and RC participants (P=0.012 204 and P=0.046, respectively) (Fig. 4). The proportion of N-specific CD4 + T cells producing both 205 IFN- and IL-2 was significantly higher in RC participants compared to PASC participants 206 (P=0.017) while the proportion of TNF--and IL-2-producing N-specific CD4 + T cells was higher 207 in pulmonary PASC participants (P=0.024). For CD8 + T cells, the overall proportions of cytokine 208 co-expression were also significantly different between PASC and RC cohorts for S-and N-209 specific T cells (P=0.012 and P=0.008, respectively) and although at an overall low frequency, the 210 proportion of TNF--and IL-2-producing N-specific CD8 + T cells was also

S-specific CD8 + T cells was significantly greater in RC compared to PASC (P=0.026), while the 213 proportion of CD8 + T cells secreting this combination of cytokines trended higher in PASC in 214 response to N-and M-peptide pools

Ki-67) on total (S3 Fig) and virus-specific (Fig. 5) T cells from PASC and 218 RC participants. No differences were seen in the frequency of naïve, effector memory or terminally 219 differentiated effector memory for total CD4 + or CD8 + T cells between the two groups; however, 220 there was an increased frequency of central memory (CD27 + CD45RA -) CD4 + T cells in the blood 221 of PASC participants

Ki-67 expression in 225 SARS-CoV-2-specific TNF-α-producing CD4 + and CD8 + T cells was significantly higher than in 226 cells expressing either IFN-γ or IL-2 (Fig. 5). For example, in response to M protein, the number 227 of TNF-α-producing cells expressing Ki-67 was 2.6-fold higher for CD4 + T cells (P<0.0001) and 228 3.2-fold higher for CD8 + T cells (P=0.0059) compared to IFN-γ-producing T cells (Fig. 5). TNF-229 α-producing T cells exhibited significantly higher frequencies of Ki-67 than IFN-γ-producing T 230 cells for both CD4 + and CD8 + T cell subsets, and Ki-67 was higher on TNF-α-producing T cells 231 for all conditions

For S-specific CD8 + and N-specific CD4 + T cells the frequency 233 of Ki-67 on IL-2-producing T cells was somewhat higher than that of IFN-γ-producing T cells 234

Plasma IL-6 levels in pulmonary PASC correlated with the frequency of SARS-CoV-2

Assessed independently of hospitalization status, both IL-6 and CRP were significantly elevated 241 compared to the RC cohort: IL-6 (PASC median=2.9 pg/mL, RC median=1.7 pg/mL

There was also no difference in IL-6 comparing female and 247 male PASC participants (Fig. 6b). Assessing CRP in PASC-NH participants, this group trended 248 higher than RC participants (PASC-NH median=3.10 mg/L, RC median=1

This 253 observation suggests that elevated plasma CRP in pulmonary PASC is likely related to initial 254 disease severity, known to be associated with male sex(26), while IL-6 elevation is specific to 255 pulmonary PASC regardless of disease severity or gender. Stratification analyses show no 256 significant differences within the PASC or RC cohorts based on age or pre-existing conditions, 257 although CRP did trend higher in those with pre-existing conditions (S4 Fig). No correlations 258 between duration of symptoms, time from onset to sample collection or age with IL-6 or CRP were 259 found (data not shown). We also compared IgG and IgA antibody levels to the S1 region of the 260 spike protein and found no differences when comparing all PASC or PASC-NH

IgG and IgA antibody levels than PASC-NH participants

We next explored the relationship between the frequencies of SARS-CoV-2-specific CD4 + and

+ T cells with IL-6 and CRP. We identified significant positive correlations between total, S-266 specific, and N-specific frequencies of TNF-α-producing SARS-CoV-2-specific CD8 + (r=0

No 269 significant correlations between plasma CRP and the frequency of SARS-CoV-2-specific T cells 270 in either PASC or

We 277 correlated the frequencies of SARS-CoV-2-specific T cells with the following variables: percent 278 predicted forced vital capacity (%FVC), absolute and percent predicted forced expiratory volume 279 during the 1 st second (FEV 1 , %FEV 1 respectively), FEV 1 /FVC, total lung capacity percent 280 predicted (%TLC), single-breath diffusing capacity of the lung for CO percent predicted 281 (%DLCO_SB), and diffusing capacity of the lung per alveolar volume percent predicted 282 (%DLCO/VA). As shown in Fig. 7a and 7b, the total frequencies of IFN-γ-producing SARS-CoV-283 2-specific CD4 + and CD8 + T cells negatively correlated with %FEV 1 (r=-0.81, P=0.011; r=-0.9, 340 inflammatory conditions and decrease mortality in severe COVID-19 cases(38-41). Interestingly, 341 IL-6 levels strongly associated with the frequencies of SARS-CoV-2-specific CD8 + T cells which 342 have been shown in other diseases to directly impact tissue-specific monocyte and macrophage 343 production of IL-6 and TNF-α

In severe COVID-349 19, decreased pulmonary function is connected to elevated levels of systemic IFN-γ and TNF-α, 350 and analysis of immune cells isolated from bronchoalveolar lavage fluid suggests T cell 351 dysfunction potentially exacerbates tissue damage in severe cases

CoV-2 infections. However, this association had not been examined in pulmonary PASC. Here, 354 we found that elevated frequencies of IFN-γ-and TNF-α-producing SARS-CoV-2-specific T cells 355 were positively associated with decreased lung function in pulmonary PASC. We also found the 356 duration of dyspnea correlated with increased frequencies of CD8 +

SARS-CoV-2-specific T cells and decreased levels of CD4 + IL-2 producing T cells. Similar to the 358 effects of systemic cytokines and T cell expression of inflammatory cytokines in other pulmonary 359 conditions

396 biotinylated detection antibody, HRP conjugate, substrate reagent, and stop solution, the plates

The frequency of antigen-specific, cytokine-secreting T cells in blood was determined by 401 intracellular cytokine staining

PBMCs (2-4 and anti-CD28 and anti-CD49d mAbs (each at 1 μg/ml

Cells were stimulated under the following conditions: peptide arrays of SARS-CoV-2 spike (S)

µg/ml final concentration of 406 each peptide; BEI Resources from USA-WA1/2020 strain, NR-52402, NR-52404, NR-52403), 407 combined phorbol 12-myristate 13-acetate (PMA) and ionomycin (25 µg/ml and 32.5 µg/ml, 408 respectively; Sigma) or medium alone. S and N arrays were 17-or 13-mer peptides with 10 amino 409 acid overlap, and the M array consisted of 17-or 12-mer peptides

Invitrogen L34957) was used per the manufacturer's protocol after washing. Cells were surfaced 413 stained with the following mAbs: anti-CD3, anti-CD4, anti-CD8, anti-CD27, anti-CD45RA, and 414 anti-PD-1 for 30 min at 4°C. Cells were washed and stored in a fix permeabilization buffer 415 (eBioscience, 421403) overnight at 4°C. Cells were washed in permeabilization buffer and stained 416 with anti-IFN-, anti-IL-2, anti-Ki-67, and anti-TNF- mAbs for 120 min at 4°C, washed, and 417 fixed with 1% formaldehyde. Fluorescence −1 (FMO) controls were used in anti-PD-1, anti-CD27 418 and anti-CD45RA staining. Full information on staining fluorophores are provided in S2 Table. events were collected for each tested condition. Antibody capture beads (BD Biosciences) by their forward and side scatter profile. Live and CD3 + cells were selected, and expression 425 of CD4 was analyzed in a bivariate dot

Ki-67 was examined on cytokine-producing cells with at least 100 events to ensure an adequate 428 number of events for analysis(52, 53). FMO controls were used to set gates for determining the 429 percentage of PD-1-expressing T cells. To ensure accuracy and precision of the measurements 430 taken from day-to-day

This program uses standardized 432 CS&T beads (BD Biosciences) to determine voltages, laser delays, and area scaling to track these 433 settings over time. A manual quality control (QC) using rainbow beads was also performed daily using GraphPad-Prism (Graphpad

U test or Wilcoxon's matched pairs test were utilized to determine significance of 439 differences between groups. Correlations were calculated using the nonparametric Spearman test

To visualize and evaluate differences 441 in expression of multiple cytokines between the PASC and RC cohorts, simplified presentation of 23 442 incredibly complex evaluations (SPICE) analysis was utilized as well as permutation tests with 443 10,000 iterations and student T tests for statistical significance where P<0.05 were considered 444 statistically significant

This study was approved by the Colorado Multiple Institutional Review Board (COMIRB# 20-449 1219) at the University of Colorado Anschutz Medical Campus

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Fig 1. Symptom characteristics of PASC and RC participants

685 (b) Number of symptoms reported <4 weeks or >4 weeks from symptom onset for PASC and RC 686 participants. For each graph, the horizontal bars represent the median of each cohort and the error 687 bars represent the 95% confidence interval. Blue represents PASC participants not hospitalized 688 (PASC-NH, n=12), red represents PASC-hospitalized (PASC-H, n=8) and orange represents RC 689 participants (n=15)

Representative flow cytometry density plots of SARS-CoV-2-specific T cells stimulated with

Samples 707 were gated through lymphocytes, live, CD3+, separated by CD4+/CD8+ and then frequencies of 708 cytokines were assessed. Percent of total CD4 + producing (b) IFN- (c) TNF- (d) IL-2 or CD8 + 709 producing (e) IFN- (f) TNF- or (g) IL-2 T cells in response to S, N and M peptide pools. Each 710 point represents the sum of the combined frequencies of virus-specific T cells to the peptide pools 711 from each participant. The horizontal bars depict the median values for each cohort

Percent of CD4 + T cells producing (a) IFN- or (b) TNF- and CD8 + T cells producing (c) IFN- 725 or (d) TNF- in response to S, N and M peptide pools separately. (e) Frequency of total (S, N, and 726 M) CD8 + T cells or (f) S-specific CD8 + T cells producing TNF- for PASC participants compared 727 by sex. Blue represents PASC-NH

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

Each pie chart represents the proportions of 735 combinations of IFN-, TNF- and IL-2 producing T cells in response to one SARS-CoV-2 736 protein. Arcs surrounding each pie chart depict the proportion of cells secreting each individual 737 cytokine. Colors for pie charts and arcs represent different cytokines or combinations of cytokines 738 and are listed in their corresponding legend. Stars denote significant differences determined by 739 student t test between PASC and RC cohorts for a particular combination of co-expressed 740 cytokines matching as indicated by the color corresponding to the pie legend

TNF--producing T cells have the highest proportion of Ki-67 expression

Shown are the percentages of CD4 T cells (left panels) and CD8 T cells (right panels) obtained 746 from the blood of PASC participants that are positive for Ki-67 expression. T cell populations 747 are further grouped by their production of cytokines and responses to peptide pools of SARS

CoV-2 structural proteins (S, top panels

Note, data points 749 from individual PASC participants were obtained for 1 or more of the cytokines assessed; 750 however, in no instances are there multiple values obtained from the same participant for a 751 particular cytokine. Blue represents IFN- + T cells, brown represents TNF- + T cells and green 752 represents IL-2 + T cells. Mann-Whitney tests were used to determine statistical significance

PASC 762 plasma IL-6 (pg/mL) levels and (d) PASC plasma CRP levels (mg/L) in female versus male. € 763 Correlations between plasma IL-6 and frequency of TNF--producing CD8 + T cells in PASC 764 participants. (f) Correlations between plasma IL-6 and frequency of TNF--producing CD8 + T 765 cells in RC participants. For a-d, bar represents median of cohort and error bar is 95% confidence 766 index. Each point represents data from one participant. Blue: PASC-NH (not hospitalized), red: 767 PASC-Hospitalized and orange: RC participants. For a-d Mann-Whitney tests were used to 768 determine statistical significance

Fig 7. Correlations between SARS-CoV-2 specific T cells and FEV 1 and symptoms in PASC

CD8 + SARS-CoV-2-775 specific T cells, TNF--producing (c) CD4 + , (d) CD8 + SARS-CoV-2-specific T cells

IL-2-producing CD4 + T cells and (h) S-specific TNF--producing CD8 + T 778 cells with duration of prolonged dyspnea in days. Each point represents data from one PASC 779 participant. Blue and red symbols represent PASC-NH (not hospitalized) and PASC-Hospitalized 780 participants, respectively