Patients and specimens
The source material from PCa patients was obtained via the leukapheresis of samples from 10 HR metastatic PCa patients obtained between November 2016 and April 2017. In the group of 10 HR metastatic PCa patients, the median age was 67.0 years (range 52–74 years), the median Gleason score was 7.5 (Gleason range 7–9), and the prostate-specific antigen (PSA) level was 35.9 ng/ml (concentration range 6.2–161.6 ng/ml). Source material was also obtained from healthy male volunteers; 2 were obtained with leukapheresis and 4 were obtained from buffy coats. The leukapheresis samples were obtained between November 2016 and April 2017 within clinical projects sponsored by SOTIO, a.s. All patients provided signed informed consent for the use of their blood-derived products for future research. The buffy coats were obtained in October 2018 from the Institute of Hematology and Blood Transfusion in Prague. In the group of healthy male volunteers, the median age was 40.0 years (age range 29–59 years). Each donor provided signed written informed consent for the use of their blood- derived products for future research.
Enrichment and expansion of antigen-specific T cells
Peripheral blood mononuclear cells (PBMCs) from leukaphereses and buffy coats were isolated as previously described [32]. The isolated PBMCs were then cryopreserved in liquid nitrogen. The cryopreserved cells were reconstituted, and a 14-day enrichment with antigen-specific T cells was performed as previously described [32]. For the enrichment of the reconstituted cells with antigen-specific T cells, a 1 mg/ml concentration of the following pooled overlapping peptide mixes spanning the indicated antigen was used: SARS-CoV-2 [22] [PepMix™ SARS-CoV-2 (Spike Glycoprotein, cat.# PM-WCPV-S-1, JPT Peptide Technologies, Berlin, Germany], and human coronavirus 229E [23] [PepMix™ HCoV-229E (Spike Glycoprotein), cat.# PM-229E-S-1, JPT]. As a positive control, pooled peptide mixes from Epstein-Barr virus (HHV-4), human cytomegalovirus (HHV-5), and influenza A [25] were used [1 mg/ml, PepMix CEF Pool (extended), cat.# PM-CEF-E, JPT].
Cell stimulation, intracellular cytokine staining, and cytokine release
The cells were processed as described previously [24,33]. Briefly, the cells were harvested, pelleted by centrifugation, and resuspended at a concentration of 1–4 × 106 cells/ml in fresh human plasma serum-containing culture medium [LM medium; RPMI 1640 medium, 5% human plasma serum (One Lambda, Canoga Park, CA), 100 U/ml penicillin-streptomycin, 2 mM Glutamax, 1 mM sodium pyruvate and nonessential amino acid mix (Thermo Scientific)]. The cell suspension (200 ml) was transferred to a 96 U- bottom well plate (Nalgene, Rochester, NY). The cells were stimulated with 50 ml of LM media containing the pertinent peptides. The final concentration of the stimulating peptides in the cell suspension was 1 mg/ml. After 1.5 h of culture (37 °C, 5% CO2), the cells were supplemented with brefeldin A (BioLegend, San Diego, CA) and then cultured for 4.5 h. Unstimulated controls (vehicle) were samples stimulated with the peptide solvent alone (20% DMSO in PBS). The cells were transferred to a V-bottom 96-well plate (Nalgene), stained with live/dead fixable stain, fixed, and permeabilized as previously described [33]. The fixed and permeabilized cells were stained with the following antibodies: CD3-PerCP-Cy5.5, CD4-PE-Cy7 (eBiosciences, San Diego, CA), CD8-Alexa Fluor 488 (Exbio, Prague, Czech Republic), TNFα-APC, and IFNγ-PE (Becton Dickinson, Franklin Lakes, NJ) for 30–60 min at 4 °C. The stained cells were washed with PBS/EDTA and analyzed by a FACSAria II (Becton Dickinson, Heidelberg, Germany). The obtained data were evaluated by FlowJo software (Tree Star, Ashland, OR). The frequency of responding T cells was determined by subtracting the frequency of the cytokine-producing T cells of the vehicle-stimulated sample from the frequency of the cytokine-producing T cells of the peptide pool-stimulated sample of the same patient or healthy volunteer. As a control (Ctrl), cell culture enriched with no peptide (vehicle) and stimulated with SARS-CoV-2 spike glycoprotein-derived peptides (peptide pools) was used. The gating strategy and determination of the cytokine-producing T cells are shown in Fig. 1A and 2A.
Statistical analysis
The means and SEM values were calculated from the indicated sample size (n) using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). Statistical significance (*P<0.05,
**P<0.01, ***P<0.001, ****P<0.0001) between two groups of differentially treated samples was determined by Wilcoxon matched-pair signed-rank tests and between three or more groups by matched-pair 1-way ANOVA with Dunn's posttest. Statistical significance (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) between two groups of subjects was determined by the Mann-Whitney U test.