Cell Collection and Isolation

Bone marrow (BM) samples were collected by aspiration from the posterior iliac crests of healthy donors undergoing stem cell harvesting, at the Unità Operativa Ematologia, Ospedale Policlinico San Martino -IRCCS, Genova, Italy. Mononuclear cells were isolated from BM samples by Ficoll-gradient centrifugation (Bignold and Ferrante 1987), cryopreserved, and stored under liquid N2.

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of healthy donors by Ficoll-gradient centrifugation.

Flow Cytometry

Cryopreserved BM mononuclear cells were thawed and potential dead cells removed through Ficoll-gradient centrifugation. Common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) were identified as Lineage (Lin)- CD34 + CD38 + CD10 + and Lin- CD34 + CD38 + CD123Low CD45RA-, respectively, using fluorescent labelled monoclonal antibodies (mAb) (Table 1). Immune cell subsets relevant to MS pathogenesis were assessed in PBMCs as previously described (Cellerino et al. 2020), using Lyotubes (BD Biosciences, Italy, Cat. No. 625,148) optimised to monitor broad subpopulations of effector CD4 + T cells (Th1, CD3 + CD4 + CXCR3 + CCR6-CD161-; Th17, CD3 + CD4 + CXCR3-CCR6 + CD161 + CCR4+; Th1/17, CD3 + CD4 + CCR6 + CD161 + CXCR3hiCCR4low), regulatory CD3 + CD4 + T cells (Total Treg, CD25 + CD127-; T naïve CD45RA + CD25low), regulatory CD3 + CD8 + T cells (Treg, CD28 Treg CD28- CD127-), effector B cells (B memory CD19 + CD14-CD24highCD38-; B mature CD19 + CD14-CD24lowCD38low), regulatory B cells (CD19 + CD14-CD24highCD38high), effector NK cells (CD3-CD16 + CD56dim), and regulatory NK cells (CD3-CD16 + CD56bright) (Cellerino et al. 2020). Progenitor cells and mature cell subsets were first stained for surface markers; they were then permeabilized with Cytofix/Cytoperm™ kit (BD Bioscence, Italy) and stained for the intracellular enzymes, using the primary mouse mAb anti-dCK (clone OTI3F5, MA5-25500 Thermo Fisher Scientific, Italy) and rabbit anti-NT5C2 polyclonal (p)Ab (ab96084, Abcam, UK) antibodies, followed by secondary Alexa Fluor® 488 anti-mouse IgG2a (Biolegend, CA) and Alexa Fluor® 488 anti-rabbit IgG (Biolegend, CA) antibodies. Fixable Viability Stain (FVS)-780 was used to exclude dead cells from the analysis. The gating strategies for CLP/CMP and the immune cells subsets are shown in Supplementary Figures 1 and 2.

Table 1 Antibody and fluorochrome panels to identify CLP and CMP from bone-marrow mononuclear cells In-vitro CD4 + T- and CD19 + B-cell Activation: Assessment of Cell Viability And/or Proliferation

CD4 + T cells and CD19 + B cells were isolated by negative selection from freshly isolated PBMC using Human CD4 + T- and CD19 + B-cell Isolation Kits (Milteny, Germany) according to manufacturer’s instructions. The purity of CD4 + T cells and CD19 + B cells after isolation was 97.8 (SD = 1.8) and 98.5 % (SD = 2.1), respectively, as verified by flow cytometry using anti-CD4 + mAb (clone SK7, V500-C, BD Bioscence, Italy) and anti-CD19 + mAb (SJ25CL, PE-Cy7, BD Bioscence, Italy).

Freshly isolated CD4 + T cells or CD19 + B cells were seeded in 24-well flat-bottom plates (500,000 cells/well) and activated with Dynabeads™ Human T-Activator CD3/CD28 kit (25 µl/106 cells; Thermo Fisher Scientific, Italy) or with 10 ng/ml IL-15 (VWR, Italy) plus 5 µM CpG (InvivoGen, France) (Gupta et al. 2018), respectively, for 48 and 72 h in RPMI medium containing 10 % foetal bovine serum and 1 % Penicillin-Streptomycin (Thermo Fisher Scientific, Italy). 2CdA was added (Merck KGaA, Germany) at different concentrations (see Results) at the same time as the T- or B-cell activators, for the duration of the culture. Viability of CD4 + T cells and CD19 + B cells was assessed by flow cytometry with Annexin V (Biolegend, CA) and propidium iodide (Merck KGaA, Germany).

Real‐time PCR

RNA extracted from CD4 + T cells and CD19 + B cells using QIAzol Lysis Reagent (Qiagen, France) was reverse-transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen, France) according to manufacturer’s protocol. Real-time PCR was performed using LightCycler 480 (Roche Applied Science, Germany) in a final reaction volume of 20 µl containing 10 ng cDNA, 2 µl of primers/probe mix (0.5 µM and 0.02 µM final concentration for primers and probes, respectively), and 10 µl FastStart Essential DNA Probes Master ready-to-use reaction mix (Roche Applied Science, Germany). Measurement of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used for normalization of expression data. Primers and probe for DCK were designed from the mRNA reference sequence (NM_000788.2) [DCK : Forward primer (Fw) 5’-CCACCCCGCCCAAGAGA-3’; Reverse primer (Rw) 5’-CTTCCCTGCAGCGATGTTCCC-3’; Probe (Pb) FAM-TGCCCGTCTTTCTCAGCCAGCTCT-BBQ]. Since both NT5C2 and GAPDH possess multiple mRNA isoforms, sequences of primers and probes were designed to target all the different transcript variants listed in the National Center for Biotechnology Information database (Sayers et al. 2020) [NT5C2 : Fw 5’-GGCAAGCTGAAAATTGGTACCT-3’; Rw 5’-TCGTATCAGAAGAACCTCCTGAGTAG-3’; Pb FAM-ACAGGGCCCCTACAGCATGGTATCG-BBQ; GAPDH: Fw 5’-TCACCACCATGGAGAAGGC-3’; Rw 5’-GCTAAGCAGTTGGTGGTGCA-3’; Pb FAM-ATGCCCCCATGTTCGTCATGGGTGT-BBQ]. Quantification was carried out using the relative standard curve method as previously described (Carlini et al. 2017). An efficiency equal to two was assumed (Bustin et al. 2009).

Western Blotting

CD4 + T cells were lysed using RIPA buffer containing protease inhibitors (Roche Applied Science, Germany). Protein sample load was based on the number of cells (3 × 105 cells per lane) to quantify the cellular changes in expression of the protein tested, under different experimental conditions. Electrophoresis was performed on a 4–15 % Mini-PROTEAN® TGX™ Precast Gels (BioRad, CA), using Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (BioRad, CA) and transferred to nitrocellulose membrane (BioRad, CA) using XCell II™ Blot Module (Thermo Fisher Scientific, Italy). Membranes were blocked for 2 h with 5 % BSA in PBS/0.1 % Tween 20 and incubated overnight with primary antibodies, mAb anti-dCK (clone OTI3F5, 1:10,000; Thermo Fisher Scientific, Italy), pAb NT5C2 (ab96084, 1:5000; Abcam, UK), mAb anti-vinculin (VCN) (clone hVIN-1, 1:20,000; Merck KGaA, Germany). Membranes were washed with PBS/Tween 20 and incubated for 1 h with secondary horseradish peroxidase-conjugated antibodies (anti-mouse IgG 1:10,000, NA931; anti-rabbit IgG, 1:10,000, AP307P; Merck KGaA, Germany). Reactive bands were visualized using ECL Plus (Thermo Fisher Scientific, Italy). Densitometric analysis for relative protein quantification was performed with ImageJ software (NIH) and normalized to loading control protein, VCN.

Assessment of dCK Activity

Cells (5 × 106 collected from 10 wells of 24-well plates) were lysed using CytoBuster™ Protein Extraction Reagent (Merck KGaA, Germany) following manufacturer’s protocol. Enrichment of dCK from cell lysates was performed essentially as described (Hao et al. 2014) with some modifications. Briefly, 0.5 ml cell lysate were incubated in a 1.5 ml vial for 5 min with Q Sepharose ® anionic exchange beads (1:3 volume of lysate; Merck KGaA, Germany) pre-equilibrated with 0.01 M Tris-HCl buffer (pH 7.0). The beads were washed three times with 0.01 M Tris-HCl buffer (pH 7.0) containing 5 mM β-mercaptoethanol. dCK elution was repeated three times using the same Tris-HCl buffer containing 0.5 M NaCl (1:1 initial volume of lysate). dCK-enriched extracts were concentrated on Amicon® Ultra 0.5 ml Centrifugal Filter devices (Merck KGaA, Germany) with 10-kDa cut-off. The whole procedure was conducted at 4° C (Fig. 1a).

Fig. 1

Schematic representation of dCK enrichment procedure and assay of dCK activity. (a) The procedure involves the use of Q Sepharose ® anionic exchange beads to enrich dCK from cell lysates followed by concentration of the eluted fraction using Amicon® Ultra 0.5 ml Centrifugal Filters. Assessment of dCK activity is performed using the Kinase Glo ™ kit which measures the kinase activity indirectly by converting ATP left in the reaction mixture to light (luminescence) through luciferin/luciferase-dependent reaction. (b) Standard curve reflecting luciferase conversion of ATP. Different amount (µM) of ATP are converted into a sustained light output (λ = 570 nm), expressed as relative light units (RLUs), through a luciferin/luciferase-dependent reaction. dCK activity is inversely proportional to residual ATP concentration in the reaction medium

Quantification of the enzymatic activity was conducted on dCK-enriched extracts using Kinase-Glo ™ reagents (Promega, Wisconsin, USA) according to manufacturer’s protocol. Briefly, dCK-enriched extracts (1 µg) in Kinase Reaction Buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml bovine serum albumin) were incubated in 0.5 ml Eppendorf tubes at 37 °C in the presence of 10 µM ATP and 30 µM 2CdA (final volume 5 µl) for 0, 5, and 10 min. The reaction mixtures were moved to White Bio-One 384-well plates (Greiner Bio-One, Italy) and incubated for 10 min at room temperature in the presence of 10 µl Kinase-Glo® Reagent. The luminescence emitted by the samples was read on a Spark® Multimode Microplate Reader (Tecan, Switzerland). dCK activity was calculated as luminescence/min after subtracting the blank sample value (Kinase Reaction Buffer alone) (Fig. 1b). dCK-enriched extract without 2CdA was used to control that the activity observed was not due to other kinase contaminants potentially present after dCK enrichment procedure.