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Transient and durable T cell reactivity after COVID-19

This study analyzed whole blood samples (n = 56) retrieved from 30 patients at 1 to 21 (median 9) mo after verified COVID-19 to determine the polarity and duration of antigen-specific T cell reactivity against severe acute respiratory syndrome coronavirus 2–derived antigens. Multimeric peptides spanning the entire nucleocapsid protein triggered strikingly synchronous formation of interleukin (IL)-4, IL-12, IL-13, and IL-17 ex vivo until ∼70 d after confirmed infection, whereafter this reactivity was no longer inducible. In contrast, levels of nucleocapsid-induced IL-2 and interferon-γ remained stable and highly correlated at 3 to 21 mo after infection. Similar cytokine dynamics were observed in unvaccinated, convalescent patients using whole-blood samples stimulated with peptides spanning the N-terminal portion of the spike 1 protein. These results unravel two phases of T cell reactivity following natural COVID-19: an early, synchronous response indicating transient presence of multipolar, antigen-specific T helper (TH) cells followed by an equally synchronous and durable TH1-like reactivity reflecting long-lasting T cell memory.

Ethical Review Authority (permits no. 2020-03276, 2021-00374, and 2021-00539) and by the Swedish Medical Products Agency (EudraCT no. 2021-000349-42). Peripheral blood (8-24mL) was collected in serum tubes and lithium-heparin tubes (Becton Dickinson, Stockholm, Sweden). Thirty patients previously diagnosed with COVID-19, confirmed by SARS-CoV-2 RNA RT-qPCR, contributed 56 whole blood samples. One patient had severe COVID-19 requiring hospitalization; the remaining 29 patients had mild infection. Median age was 38 (range 25-66) years and 77% were female. Fourteen participants contributed with 1 sample, 9 with 2 samples, 4 with 3 samples and 3 with 4 samples at different timepoints after infection that were analyzed for presence of nucleocapsid specific T cells. Twenty-three previously infected patients contributed one sample prior to vaccination that was analyzed for presence of spike 1-specific T cells. Three participants were infected during the study and were thereafter analyzed together with infected patients. Median days from verified infection to sampling was 267 (range 25-629) days. Uninfected control subjects (n=17) were defined by lack of PCRverified COVID-19 and seronegativity for IgG against the receptor binding domain of spike 1 at baseline sampling prior to vaccination. Ten controls contributed with 1 sample, 6 with 2 samples and 1 with 3 samples for analysis of nucleocapside specific T cells. Eleven controls contributed one sample each prior to vaccination for analysis of spike-1-specific T cells.

Serum IgG against the receptor-binding domain spike 1
To exclude prior COVID-19 among control subjects, serum samples were analyzed for antibodies against the receptor-binding domain of SARS-CoV-2 spike 1 using the chemiluminescent microparticle immunoassay Abbott Alinity SARS-CoV-2 IgG II Quant (Abbott, Abbott Park, IL, USA).

Assay of antigen-specific cytokine release in whole blood samples
One ml of peripheral venous lithium-heparinized blood was incubated with nucleocapsid or spike 1 protein peptides at 1µg/ml/peptide in conical 10 ml tubes (Sarstedt AB, Helsingborg, Sweden. Each sample was stimulated with 15-mer peptides with 11-amino acid overlap spanning the complete sequence of the SARS-CoV-2 nucleocapsid phosphoprotein (aa 1-419, 102 peptides) (NC;Miltenyi Biotec,Lund,Sweden), the N-terminal S1 domain (aa 1-692, 170 peptides) of the SARS-CoV-2 spike 1 surface glycoprotein (S1; 130-127-041, Miltenyi Biotec) or was left unstimulated. After 48 hours of incubation at 37 o and 5% CO2 samples were centrifuged at 1500 rpm for 5 minutes. Plasma supernatants were collected and stored at -80 o C until analysis for cytokine content. Only unvaccinated participants were included in analysis of spike 1 peptide-induced cytokines.
Cytokine levels in stimulated samples are presented with levels in unstimulated samples subtracted, thus reflecting cytokines induced by SARS-CoV-2-peptides.
The levels of IL-21 in plasma supernatant from unstimulated, NC-and S1-stimulated whole blood were determined by IL-21 ELISA (DY8879-05, R&D Systems) according to manufacturer's instructions. Plasma was diluted 1:2 in PBS containing 1%BSA and 10% rat serum (Stemcell Technologies) to minimize unspecific binding. Optical density was measured at 450 nm and 570 nm using a FLUOstar Omega plate reader (BMG, Ortenberg, Germany).
Results are presented as peptide-induced IL-21 obtained by subtracting levels of IL-21 in unstimulated samples from levels in peptide-stimulated samples.

Data analysis and statistics
Mean differences between SARS-CoV-2 peptide-induced cytokine production in uninfected control subjects, patients with samples available <70 days after infection and patients with samples available 70-629 days after infection were compared using permutation tests. This test is similar to the Fisher-Pitman test but in addition accounts for repeated measures. Correlations between SARS-CoV-2-induced cytokine levels were calculated using mean concentrations per time phase when repeated measures were available. The impact of time after infection on SARS-CoV-2 peptide-induced cytokines was assessed using linear regression or linear mixedeffects models, if repeated measures were present. Model assumptions were examined with residual plots. P-values are two-sided throughout. Statistical analyses were performed using IBM SPSS 28.0 and R 4.0.5 and figures were prepared using GraphPad Prism version 9.3.1.

Data availability
The Supporting information contains a complete set of deidentified primary data showing donor characteristics, SARS-CoV-2-specific serology, SARS-CoV-2 peptide-induced cytokines and background cytokine levels in unstimulated samples at different time points after infection.