Monocytes and NK Cells Activity in Covid-19 Patients

According to the medical hypothesis on which the protocol is based on, young people could benefit from a functional adaptation of innate immune cells induced through epigenetic reprogramming and, especially, a pre-existing "partially specific" immunity to the community viruses caused by "bystander effect" of preceding vaccinations. In this study, we will explore the main differences existing among patients infected by SARS-CoV-2 who experience the illness at different degree of severity. We suppose to recognize different populations of patients, each one with a specific immunological pattern. It could differ in terms of cytokines, soluble factors serum level and immune cells activity both of the innate compartment and of the acquired one.

Data will be collected using 3 approaches:

- An experimental analysis, for clusters of patients, to study the innate immune response and to identify the genetic profiles.

- An epidemiological analysis, in order to identify the patients' vaccination history;

- A clinical analysis, to detect the immunological profile;

For the specific analysis, to study the innate immune response and to identify the genetic profiles, scientists will analyze, from the peripheral blood, NK cells, monocytes, CD4 and CD8 T cells of both groups: healthy patients (tested negative for SARS-CoV-2) and sick patients of the subgroups (AS 19, PAU19, POL19, ARD19).

From different groups of patients, blood samples (10-15 mL) will be drawn into EDTA tubes, centrifuged at 360 g for 10 minutes to obtain plasma that it will be stored at -80°C for subsequent analysis for cytokines and chemokines of interest by ELISA (IL-1b, IL-6, TNF, IFN-a, IL-10, IL-12, CCL2 and CXCL10) at the end of enrolment.

The cell pellets will be brought back to the initial volume with PBS and diluted 1:1 (v/v), and then subjected to a density gradient stratification with Ficoll Histopaque-1077, at 500 g for 30 minutes. The Peripheral Blood Mononuclear Cells (PBMCs) derived from the white ring will be collected, washed twice in PBS, and then used for subsequent experiments using a flow cytometer assay (REFF). The in vitro culture using PBMCs can vary from ex vivo 1 day to a few days, and cells will be maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (P/S), at 37°C, 5% CO2.

To perform ex vivo fluorescence-activated cell sorter (FACS) phenotype analysis, 2.5x105 of fresh total PBMCs per FACS tube will be stained for 30 minutes at 4°C with monoclonal antibodies (mAbs) as follows: CD3-PerCP, CD56-APC, CD16-FITC, NKG2A-PE, NKG2C-PE, NKGD2-PE, DNAM-1-PE, CD25-PE, CD69-PE. Following Forward/Side Scatter setting, NK cells will be identified into two cell subsets, i.e. as CD3- and CD56dim CD16+ cells (CD56dim NK cells, the major subset, about 90%), and CD3- and CD56bright CD16-/low cells (CD56bright NK cells, the minor subset, about 10%). Other markers expression will be evaluated on both subsets of gated cells.

For ex vivo FACS evaluation of monocytes phenotype analysis, 2.5x105 of fresh total PBMCs per FACS tube will be stained for 30 minutes at 4°C with mAbs as follows: CD45-APC, CD14-FITC and CD16-V450, PE-CD209, PE-CD80. Following Forward/Side Scatter setting, monocytes will be identified into three subsets, i.e. as CD14+ and CD16- cells (the main subset, about 90%), CD14+ CD16+ (the minor subset, about 10%), and CD14-/low CD16+ (the other minor subset).

We will also evaluate phenotype for CD4 and CD8 T cells and CD4/CD8 T ratio using 2x105 of fresh total PBMCs, as previously described, with following mAbs: CD3-PerCP, CD4-APC, CD8-V450 as well as T regulatory (Treg) cells, as CD3-PerCP, CD4-APC, CD25-PE.

PBMCs will be also in vitro stimulated for 4 h with different types of stimuli, such as LPS (recognized by the TLR4), poly(I:C) (recognized by the TLR3), poly (I:C) plus IL-2 plus IL-12, and phorbol myristate acetate (PMA) plus ionomycin in presence of monensin and brefeldin. This procedure will allow studying specific cytokine/chemokine production from NK cells and monocytes by using a FACS intracellular assay, as described previously (REFF). For NK cells, we will measure IFN-a-PE, TNF-PE, CCL2-PE, CXCL10-PE and CD107a-PE (degranulation marker). For monocytes we will investigate: IL-6-PE, TNFa-PE, IL-12-PE, CXCL10-PE (M1-type pro-inflammatory markers) and TGFb-PE, IL-10-PE, CCL18-PE (M2-type anti-inflammatory markers) Whenever the quantity of PBMCs is sufficient, other functional in vitro tests on NK cells and monocytes will be set up. In particular, PBMCs will be studied for 4 h NK cell degranulation/cytotoxic function towards erytroleucemic K562 cell line assessing surface CD107a-PE using a flow cytofluorimetric assay (REFF).

Whenever the quantity of PBMCs is sufficient, monocytes will be purified using anti-CD14 microbeads with magnetic separator and NK cells with RosetteSep kit to obtain >90% purified cell populations.

Purified monocytes and purified NK could be maintained separated or together in culture using RPMI 1640 medium with 10%FBS, and supplemented with M-CSF and IL-2, respectively, stimulated with different stimuli (see above), and then checked for intracellular cytokines/chemokines of interest (see above). At the same time the supernatants (conditioned medium, CM) could be harvested at the end of in vitro incubation culture and assessed for cytokines/chemokines using ELISA (IL-1b, IL-6, TNFa, IFNa, IL-10, IL-12, CXCL10).

The 4-5 days in vitro culture of monocytes will be further stimulated 24 h with LPS plus IFNg (M1 stimulus) or (IL-4) (M2 stimulus) to investigate macrophage polarization studying surface M1 markers (TNF, CXCL10) or M2 markers (IL-10, CCL18) to check the prevalence of macrophage polarization in different groups of Covid-19 patients.

The epidemiological analysis will be carried out integrating both vaccination history and the data daily collected after hospital admission. ATS Insubria archives will provides missing data.

Considering the immunological profile, patients with Covid-19 will be tested for routine examinations and the following:

- lymphocyte immunophenotyping;

- determination of C3 and C4 complement fractions activity;

- immunoglobulin (IgG, IgM, IgA, IgE) serum level;

- serum protein electrophoresis;

- Angiotensin Converting Enzyme (ACE) serum level;

- CMV (Citomegalovirus) serology test;

- IL-6 serum level.