Patients and blood samples
Peripheral blood mononuclear cells (PBMCs) asserted from blood donations of cancer patients were collected prior to the SARS-CoV-2 pandemic (04/2009 – 11/2019) at three centers (University Hospital Tübingen, Germany; University Hospital Bonn, Germany; University Hospital St. Gallen, Switzerland) to assess the prevalence of pre-existing cross-reactive SARS-CoV-2 T cell responses (PRE group, n = 199).
Blood and serum samples from cancer patients after SARS-CoV-2 infection (SARS group, n = 17) were collected at the University Hospital Tübingen, Germany from 4/2020 – 12/2020. SARS-CoV-2 infection was confirmed by PCR after nasopharyngeal swab. Sample collection for COVID-19 cancer patients was performed between 14 – 263 days (median 47 days) after positive PCR. In non-hospitalized patients, donor characteristics and COVID-19 symptoms were assessed by questionnaire. For hospitalized patients, data was obtained from clinical data records.
Informed consent was obtained in accordance with the Declaration of Helsinki protocol. The study was approved by and performed according to the guidelines of the local ethics committees (University of Tübingen: 454/2016/BO2, 406/2019/BO2, 179/2020/BO2; University of Bonn: 266/08; Kantonsspital St. Gallen: Ethikkommission Ostschweiz [EKOS] 16/079).
PBMCs were isolated by density gradient centrifugation. Serum was separated by centrifugation for 10 min and the supernatant was stored at -80°C. Detailed cancer patient characteristics are provided in Tables 1 and 2 and Supplementary Tables S1 and S2.
To delineate differences in SARS-CoV-2 immune responses in cancer patients, a reference group of SARS-CoV-2 convalescent and unexposed healthy individuals, described in a previous work, was applied (25). PBMCs of unexposed HV (HV-PRE, n = 94) were collected prior to the SARS-CoV-2 pandemic (06/2007 – 11/2019). Sample collection for COVID-19 convalescent HV (HV-SARS, n = 193) was performed between 16 – 59 days (median 41 days) after positive PCR.
Synthetic peptides were provided by EMC Microcollections GmbH and INTAVIS Peptide Services GmbH & Co. KG. The SARS-CoV-2 HLA class I and HLA-DR T cell epitopes as well as the applied epitope compositions (EC) were characterized in detail in a previous work (25) analyzing T cell responses in convalescents after COVID-19 and in healthy donors never exposed to the virus. To standardize analyses of SARS-CoV-2 T cell responses, broadly applicable HLA class I and HLA‑DR SARS-CoV-2-specific EC (16 and 5 HLA class I and HLA-DR peptides, respectively) recognized exclusively in COVID-19 convalescents or cross-reactive EC (9 and 10 HLA class I and HLA-DR peptides, respectively) recognized by both, convalescents and individuals never exposed to SARS-CoV-2 (Supplementary Table S3) were used. For the analyses of T cell response diversity, which requires the analysis of multiple peptides, promiscuous SARS-CoV-2 HLA-DR T cell epitopes (20 peptides with multiple HLA-DR restrictions) were used.
HLA class I and HLA-DR viral peptide panels comprising peptides derived from EBV, CMV, and ADV (Supplementary Table S3) were used to assess the general T cell functionality in cancer patients.
IFN-g ELISPOT assay ex vivo or following 12-day in vitro expansion
For 12-day in vitro expansion, PBMCs were pulsed with HLA class I or HLA‑DR peptide pools (1 mg/mL per peptide for HLA class I or 5 mg/mL for HLA-DR) and cultured for 12 days adding 20 U/mL IL-2 (Novartis) on days 3, 5, and 7. Peptide-stimulated (in vitro expanded) or freshly thawed (ex vivo) PBMCs were analyzed by IFN-g ELISPOT assay as described previously (25). In brief, 2‑8 × 105 cells per well were incubated with 1 mg/mL (HLA class I) or 2.5 mg/mL (HLA‑DR) of EC or single peptides in 96-well ELISPOT plates coated with anti-IFN-g antibody (clone 1-D1K, 2 mg/mL, MabTech, Cat# 3420-3-250, RRID: AB_907283). PHA (Sigma-Aldrich) served as positive control. An irrelevant HLA-matched control peptide (HLA-DR, ETVITVDTKAAGKGK, FLNA_HUMAN1669−1683) or 10% dimethyl sulfoxide (DMSO) in double-distilled water (ddH2O) for HLA class I served as negative control. After 24 h of incubation, spots were revealed with anti-IFN-g biotinylated detection antibody (clone 7-B6-1, 0.3 mg/mL, MabTech, Cat# 3420-6-250, RRID: AB_907273), ExtrAvidin-Alkaline Phosphatase (1:1,000 dilution, Sigma-Aldrich), and BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro-blue tetrazolium chloride, Sigma-Aldrich). Spots were counted using an ImmunoSpot S5 analyzer (CTL) and T cell responses were considered positive when the mean spot count was ≥ 3-fold higher than the mean spot count of the negative control. The intensity of T cell responses is depicted as calculated spot counts, which represent the mean spot count of duplicates normalized to 5 x 105 cells minus the normalized mean spot count of the respective negative control. The recognition frequency of T cell responses within groups indicates the percentage of donors recognizing the respective EC or peptide. The diversity of T cell responses for single donors represents the number of recognized SARS-CoV-2-derived peptides (positive peptides/tested peptides).
Intracellular cytokine and cell surface marker staining
Peptide-specific T cells were characterized by intracellular cytokine and cell surface marker staining as previously described (25). In brief, PBMCs were incubated with SARS-CoV-2 peptide/EC or negative control peptide, Brefeldin A (Sigma-Aldrich), and GolgiStop (BD Biosciences). Staining was performed using Cytofix/Cytoperm solution (BD), Aqua live/dead (1:400 dilution, Invitrogen), APC/Cy7 anti-human CD4 (1:100 dilution, BioLegend, Cat# 300518, RRID: AB_314086), PE/Cy7 anti-human CD8 (1:400 dilution, Beckman Coulter, Cat# 737661, RRID: AB_1575980), Pacific Blue anti-human TNF (1:120 dilution, BioLegend, Cat# 502920, RRID: AB_528965), FITC anti-human CD107a (1:100 dilution, BioLegend, Cat# 328606, RRID: AB_1186036), and PE anti-human IFN-g monoclonal antibodies (1:200 dilution, BioLegend, Cat# 506507, RRID: AB_315440). PMA and ionomycin (Sigma-Aldrich) served as positive control. All samples were analyzed on a FACS Canto II cytometer (BD).
Flow cytometry-based analysis of T cell exhaustion marker expression
T cell exhaustion was assessed based on cell surface expression of CD279 (PD-1) and CD366 (TIM-3) as well as intracellular expression of CD152 (CTLA-4) and CD223 (LAG-3). Staining was performed using Cytofix/Cytoperm solution (BD), Pacific Blue anti-human CD4 (1:100 dilution, BioLegend, Cat# 300524, RRID: AB_493099), FITC anti-human CD8 (1:100 dilution, BioLegend, Cat# 300905, RRID: AB_314908), PE anti-human CD152 (1:50 dilution, BioLegend, Cat# 349905, RRID: AB_10645522), PE-Cy7 anti-human CD223 (1:100 dilution, BioLegend, Cat# 369309, RRID: AB_2629752), APC anti-human CD279 (1:100 dilution, BioLegend, Cat#621609, RRID: AB_2832829), APC/Cy7 anti-human CD366 (1:100 dilution, BioLegend, Cat# 345025, RRID: AB_2565716). Viable cells were determined using Aqua live/dead (1:400 dilution, Invitrogen). All samples were analyzed on a FACS Canto II cytometer (BD).
SARS-CoV-2 IgG and IgA ELISA (EUROIMMUN)
SARS-CoV-2 IgG and IgA ELISA (EUROIMMUN) assays were performed as previously described (25) on an automated BEP 2000 Advance® system (Siemens Healthcare Diagnostics GmbH) according to the manufacturer’s instructions. The assay detects anti-SARS-CoV-2 IgG and IgA directed against the S1 domain of the viral spike protein (including the immunologically relevant receptor binding domain) and relies on an assay-specific calibrator to report a ratio of specimen absorbance to calibrator absorbance. The final interpretation of positivity is determined by the ratio above a threshold value given by the manufacturer: positive (ratio ≥ 1.1), borderline (ratio 0.8 - 1.0), or negative (ratio < 0.8). Quality control was performed following the manufacturer’s instructions on each day of testing.
Elecsys® anti-SARS-CoV-2 immunoassay (Roche Diagnostics GmbH)
The Elecsys® anti-SARS-CoV-2 ECLIA (electrogenerated chemiluminescence immunoassay) assay was performed as previously described (25). The assay detects high-affinity antibodies (including IgG) directed against the nucleocapsid protein of SARS-CoV-2 in human serum. Readout was performed on a Cobas e411 analyzer. Negative results were defined by a cut-off index of < 1.0. Quality control was performed following the manufacturer’s instructions on each day of testing.
Software and statistical analysis
Data are displayed as mean with standard deviation (for n > 3), scatter dot plot with mean, box plot as median with 25th or 75th percentiles and min/max whiskers. Description of the applied tests used for statistical analysis are provided within the respective figure legends. Continuous data were tested for distribution (Shapiro-Wilk test) and individual groups were tested by use of Wilcoxon, Mann-Whitney U, Kruskal-Wallis test, or Kruskal-Wallis with Dunn’s multiple comparisons test, where appropriate. Categorial data were tested by use of Fisher’s exact test of Pearson Chi Square test. Univariate logistic regression analysis was performed to assess the predictive value of patient demographics and clinical parameters for SARS-CoV-2 cross-reactive EC recognition. Flow cytometric data was analyzed using FlowJo 10.0.8 (BD). Graphs were plotted using RStudio and GraphPad Prism 9.0.0. Statistical analyses were conducted using GraphPad Prism 9.0.0 and SPSS 26 (IBM) software. P-values of < 0.05 were considered statistically significant.
Conceptualization: T.B., M.R., J.S.W.; Data curation: T.B., M.R., J.S.H.; Formal analysis: T.B., M.R.; J.S.H.; Funding acquisition: J.S.W.; Investigation: T.B., M.R., Y.M., A.N., J.S.H., A.P., S.H., J.B., J.R., M.W., F.B., L.F., S.H., P.B.; Methodology: T.B., M.R., A.N., J.S.H., H.-G.R., J.S.W.; Project administration: J.S.W.; Resources: A.P., S.H, L.F., H.-G.R., H.R.S., J.S.W.; Supervision: H.-G.R., H.R.S., J.S.W.; Visualization: T.B., M.R., M.L.D.; Writing – original draft preparation: T.B., M.R., J.S.W.; Writing – review and editing: T.B., M.R., Y.M., A.N., J.S.H., M.L.D., A.P., S.H., J.B., J.R., M.W., F.B., L.F., S.H., P.B., M.M., H.-G.R., H.R.S., J.S.W.