Patients and Controls
Forty patients admitted from March 2020 to June 2020 to the Fondazione Don Carlo Gnocchi Onlus inpatient rehabilitation program (Milan – IRCCS Santa Maria Nascente and Rovato – Centro Spalenza) were enrolled in the study. All patients were undergoing acute rehabilitation due to the disability caused by a neurological disease, and all of them received at least one SARS-CoV-2 RNA test on their nasopharyngeal swab (NPS) or bronchial aspirate. Thirty patients contracted SARS-CoV-2 infection, while ten other patients were repeatedly negative on their NPSs (control group).
Patients were divided into three diagnostic groups: brain injury causing a disorder of consciousness (DOC), injury of the central nervous system (CNS) without DOC history and disease of the peripheral nervous system (PNS).
It is well known that COVID severity can be highly variable, with the disease ranging from mild flu-like symptoms to severe interstitial pneumonia. In line with Chen and colleagues , COVID severity was graded on three levels:
- mild: asymptomatic or flu-like symptoms without pneumonia manifestation;
- moderate: pneumonia manifestation in imaging without respiratory distress;
- severe: pneumonia with respiratory distress treated with respiratory support (from continuous positive airway pressure up to oro-tracheal intubation or tracheostomy).
The study was approved by the local ethics committee (Comitato Etico – IRCCS Fondazione Don
Gnocchi, Milano) and patients or guardians provided written informed consent.
Blood sample collection
Whole blood was collected in vacutainer tubes containing ethylenediamine tetra-acetic acid (EDTA) (Becton Dickinson & Co., Rutherford, NJ). PBMCs were isolated by density gradient centrifugation on Ficoll (Cedarlane Laboratories Limited, Hornby, Ontario, Canada) and counted with the automated cell counter ADAM-MC (Digital Bio, NanoEnTek Inc., Korea), which allows for discrimination of viable from non-viable cells.
Immunofluorescent staining and analysis by flow-cytometry
Immunophenotypic analysis of NK subsets and KIR expression were performed on 100ml of EDTA peripheral blood incubated for 30 minutes at 4° C with the fluorochrome-labeled monoclonal antibodies. Erythrocyte lysis was obtained with the Immuno-Prep Epics Kit and Q-Prep Work Station (Beckman-Coulter Brea, CA, USA). The analyses were performed using a Beckman-Coulter GALLIOS flow cytometer equipped with a 22 mW Blue Solid State Diode laser operating at 488 nm and with a 25 mW Red Solid State Diode laser operating at 638 nm, and interfaced with Kaluza analysis software. For each analysis, 20.000 events were acquired and gated on Forward and Side scatter properties for lymphocyte and on the CD3-CD19-CD14- and Side scatter properties to exclude T, B and monocyte cells; the remaining triple-negative cells were analyzed in a CD56 versus CD16 dot plot to identify the natural killer (NK) cell subsets, CD56brightCD16−, CD56brightCD16dim, CD56dimCD16bright, CD56dimCD16dim, CD56dimCD16− and CD56−CD16bright, considering isotype background (Figure 1). The expression of KIR receptors was performed on NK subsets. Sample were first run using single fluorocrome-stained preparation for colour compensation.
Monoclonal antibodies (mAbs)
The following mAbs were used: anti- CD3 phycoerythrin-cyanine 7 (PE -Cy7) (Mouse IgG1, Clone: UCHT1) (Beckman-Coulter); anti- CD19 PC-7(Mouse IgG1, Clone: J3-119) (Beckman-Coulter); anti- CD14 PC- 7 (IgG2a Mouse, clone: RMO52)( Beckman-Coulter); anti-CD16 Fluorescein (FITC) or phycoerythrin-cyanine 5 (PE-Cy5) (Mouse IgG1, Clone: 3G8) (Beckman Coulter); anti-CD56 phycoerythrin (PE) (Mouse IgG1, Clone: N901 (NKH-1) (Beckman Coulter);anti-Human KIR2DL1/CD158a FITC (Mouse IgG1, Clone: 143211), (R&D Systems, Minneapolis, MN, USA), anti-Human KIR2DS4/CD158i Allophycocyanin (APC) (Mouse IgG2a, Clone: 179315) (R&D Systems); anti-Human ILT2/CD85j APC (Mouse IgG1, Clone: 292305) (R&D Systems); anti-Human KIR2DS1/CD158h Alexa Fluor 700 (Rabbit IgG, Clone: 1127B) (R&D Systems); anti-Anti-KIR2DS2 / CD158b Polyclonal Antibody FITC (Rabbit IgG aa39-65) (LSBio,Seattle WA USA).
KIR and HLA ligand genotyping
Genomic DNA was isolated from peripheral blood by phenol-chloroform extraction using standard procedures. Molecular genotyping of KIRs and HLA-Bw4+/Bw4- C1/C2 Kir ligands was performed by PCR on genomic DNA using sequence specific primers (SSP) according to the manufacturer’s instructions (BAG- Lich, Germany, Astra Formedic, Milan Italy). Allele detection was done after amplification in a GeneAmp PCR 9700 thermocycler (Applied Biosystem, Foster City, CA, USA) by gel electrophoresis on 2% agarose gel.
KIR-HLA complexes were defined as follows:
- KIRs 2DL1 and 2DS1 ligate the C2 epitope (Asp at position 77, Lys at position 80).
- KIRs 2DL2, 2DL3 and 2DS2 ligate the C1 epitope (Ser at position 77, Asp at position 80).
- HLA-Bw4*80I was considered the ligand for 3DL1 and 3DS1 [16,17].
The normality of distribution of continuous variables was evaluated using the Kolmogorov–Smirnov test. Quantitative data were defined normally or not normally distributed (Shapiro-Wilk test) and are therefore summarized as mean and standard deviation or median and interquartile range (IQR; 25th and 75th percentiles) respectively. Comparisons between groups were performed using a T-Test or two-tailed Mann-Whitney test for independent samples. Kruskal-Wallis analysis of variance was utilized for each variable. Chi-square analysis was used to evaluate KIR and HLA genetic distribution between groups. Data analysis was performed using the MEDCALC statistical package (MedCalc Software bvba, Mariakerke Belgium).