Study subjects and sampling.
A total of 55 adult patients diagnosed with COVID-19 after testing positive for SARS-CoV-2 RNA by qPCR and 42 adult healthy volunteers without any clinical signs of COVID-19 infection were enrolled from March to July 2020 at the University Hospital Regensburg. This study was approved by the Research Ethics Committee from the University Hospital Regensburg (Study and Approval Number: 20-1785-101). Informed consent was obtained from all enrolled patients (or their legal representatives) and healthy volunteers.
The cohort of COVID-19 patients was stratified into non-ventilated (n = 25) and mechanically ventilated (n = 30) patients. Ventilated patients were further sub-grouped according to their outcome (discharged from the ICU = “survived”, or death on the ICU = “dead”) (Table 1). In most patients several consecutive blood samples were available resulting in a total of 188 samples. 14 patients were first sampled on mechanical ventilation and also after discharge from the ICU. Samples of these patients were assigned into the subgroup “ventilated” or “non-ventilated” according to the ventilation status at the time of sampling (Supplementary Table 1). None of the patients received prednisolone equivalents ≥ 40 mg/day for treatment of COVID-19.
Fresh whole blood samples for immediate immunophenotyping by flow cytometry with at least one antibody panel were available from all participants. Sufficient amounts of fresh blood to also perform whole blood stimulation were available from 38 healthy controls (38 samples), 33 non-ventilated COVID-19 patients (58 samples) and 21 mechanically ventilated COVID-19 patients (77 samples). For subgroup analysis the mechanically ventilated patients were again stratified according to their outcome into the groups “survived” (17 patients, 69 samples) and “dead” (4 patients, 8 samples). Peripheral blood mononuclear cells (PBMCs) were prepared from 25 non-ventilated patients (36 samples) and 16 ventilated COVID-19 patients (42 samples). For subgroup analysis the mechanically ventilated patients were again stratified according to their outcome into the groups “survived” (n = 14, 39 samples) and “dead” (n = 2, 3 samples). Exact numbers of patients and samples for each readout are provided in the figure legends, as for some read-outs not enough material was available.
Cell culture media, cytokines and stimulants
For PBMC cultures we used RPMI 1640 medium (Gibco, Karlsruhe, Germany) containing 1% penicillin/streptomycin (Gibco, Karlsruhe, Germany) and 1% L-glutamine (Gibco, Karlsruhe, Germany). For whole blood stimulations we used pure RPMI 1640 medium (Gibco, Karlsruhe, Germany). Anti-CD3 (clone: OKT3) was obtained from eBiosciences (San Diego, CA) and used at 5 µg/ml. The blocking anti-IL-3 antibody (clone P8C11) was generated in our lab and used at a concentration of 10 µg/ml. IL-3 was obtained from BioLegend (San Diego, USA) and used at 20 ng/ml. GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-15, IFN-γ, IFN-α, TNF-α were obtained from Peprotech (Cranbury, USA) and used at 20 ng/ml.
Whole blood stimulation
For T cell activation assays tubes (BD Bioscience) were pre-coated with 300 µl anti-CD3 antibodies (5 µg/ml in PBS) at 37°C for 4 hours and washed twice with PBS. 100 µl of heparinized blood were diluted with 200 µl RPMI 1640 medium and added to the precoated or to uncoated tubes and incubated at 37°C in 5% CO2 for 24 hours. Where indicated a blocking anti-IL-3 antibody (10 µg/ml) was added to the samples. The supernatants were recovered and released cytokines analyzed by ELISA. Cells were analyzed by multi-parametric flow cytometry. For cytokine stimulation 100 µl whole blood was diluted with 200 µl RPMI 1640 medium and added to uncoated tubes (BD Bioscience) together with 20 ng/ml of cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, IL-15, GM-CSF, IFN-γ, IFN-α and TNF-α) at 37°C in 5% CO2 for 24 hours. Cells were analyzed by flow cytometry.
Preparation and culture of PBMCs
Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Paque density gradient centrifugation from heparin-anticoagulated fresh blood samples. For cryopreservation, PBMCs were resuspended in fetal calf serum (FCS) with 10% dimethylsulfoxide (DMSO) at a concentration of 1-2 x 106 cells / ml, frozen at -80°C for 2-3 days and then transferred into liquid nitrogen. The cells were thawed in a 37°C water bath and washed 3 times with medium. The viability was controlled by Trypan-blue staining and was 90-95%. PBMCs (500.000/well) were cultured in 300 µl medium / well with or without anti-CD3 (clone: OKT3) 5 µg/ml for 24 hours by 37°C and 5% CO2. Where indicated IL-2 (20 ng/ml) was added to the samples during the incubation. Thereafter supernatants were recovered and analyzed by ELISA. Cells were analyzed by flow cytometry.
Anti-coagulated fresh blood samples (100 µl) or cells after whole blood stimulations were incubated with various panels of the following directly labeled monoclonal antibodies for 20 minutes by 4°C: anti-CD3 APC-Cy7 (clone: SK7, BioLegend), anti-CD4 V500 (clone RPA-T4, BD Bioscience), anti-CD8 APC-eFluor 780 (clone: RPA-T8, Invitrogen, Darmstadt, Germany) and anti-CD8 PE-Cy7 (clone: SK1, BioLegend), anti-CD11b PE-Cy7(clone: M1/70, BioLegend, San Diego, CA), anti-CD14 V500 (clone: MΦP9, BD Bioscience), anti-CD16 Pacific Blue (clone: 3G8, BioLegend), anti-CD19 Pacific Blue (clone: HIB19, BioLegend), anti-CD25 APC (clone: BC96, BioLegend), anti-CD116 FITC (clone: 4H1, BioLegend), anti-CD131 PE (clone: 1C1, eBioscience), anti-CD123 PE-Cy5 (BD Bioscience), anti-CD169 PE (clone: 7-239, Miltenyi Biotec, Bergisch Gladbach, Germany), CD304 APC (clone: 12C2, BioLegend), HLA-DR II APC and FITC (clone: G-46-6, BD Bioscience). Subsequently, samples were treated with FACS Lysing Solution (BD Bioscience) for 10 min, washed with 0,9% NaCl, centrifuged, resuspended in 0,9% NaCl together with FACS counting beads (Invitrogen, Darmstadt, Germany) and acquired with a BD FACSCanto II flow cytometer and analyzed with FACSDIVA 8 software (BD Biosciences). For analysis of cultured PBMCs no erythrocyte lysis was performed. The gating strategy for key cell populations is shown in Supplementary Fig. 6.
Enzyme Linked Immuno Sorbent Assay (ELISA)
Concentrations of GM-CSF and IFN-g in cell culture supernatants were measured by ELISA, according to manufacturer’s protocol (DuoSet Elisa, R&D Systems, Abington, UK). For measurement of IL-3, ELISA plates (NUNC Maxisorb, Thermofisher Scientific, Waltham, USA) were coated with a capture anti-IL-3 antibody (Clone P8C11; 5 µg/ml in PBS) in 100 µl / well at room temperature (RT) overnight. Plates were washed 3 times with PBS / 0.05 % Tween-20, blocked with PBS containing 1% bovine serum albumin (BSA) at RT for 1 hour and washed again with PBS. Samples where preincubated with 100 µg/ml mouse IgG1, kappa isotype control antibody (MOPC21, BioXCell, Lebanon, USA) for 1 hour by RT and added to the plates for 2 hours by RT (100 µl / well, diluted in PBS / 1% BSA). Recombinant human IL-3 (BioLegend) was diluted from 7.8 – 500 pg/ml in PBS / 1% BSA and served as standard. After washing plates were incubated with 400 ng/ml HRP-labelled detection anti-IL-3 antibody (Clone 13) (100 µl/well) for 1.5 hours at RT and color reaction was performed with TMB Substrate Solution (BioLegend) according to manufacturer’s protocol.
Predictive score for death in ventilated COVID-19 patients
Three parameters were used individually or in combination to predict fatal outcome in ventilated COVID-19 patients. Absolute basophil counts in fresh peripheral blood. Basophil counts < 25 / µl defined “Low Baso count”. Upregulation of CD123 on CD14+ monocytes and CD11b on neutrophils was calculated as the ratio of surface marker expression with anti-CD3 and surface marker expression without anti-CD3. Upregulation of < 130% defined “Weak upregulation”. In the combined scores a logical AND combination was used that required all parameters to be fulfilled. For all three parameters 77 data sets from 21 ventilated COVID-19 patients were available. 17 ventilated patients were discharged from the ICU (survived) (69 samples) and 4 patients died on the ICU (8 samples). The number of right and false positive samples, the test sensitivity (Sens.), specificity (Spe.) was calculated. The negative (NPV) and positive predictive value (PPV) for predicting death were calculated using a prevalence of death in ventilated COVID-19 patients of 19 % (4 dead patients / 21 ventilated patients).
Statistical analyses were performed using the GraphPad Prism 8 Software. Statistical differences between more than 2 different cell stimulations or patient cohorts were calculated using one-way ANOVA with Bonferroni multiple comparison as indicated in the figures. For two group comparisons 2-tailed unpaired t-test was used as indicated in the figures. P values less than 0.05 were considered significant and marked with one asterisk or with two asterisks if less than 0.01. P values less than 0.001 were marked with three asterisks.