Fig 2: B- and T-cell activation increases dCK activity but exposure to 2CdA abrogates such activity in stimulated B cells. (a) Validation of dCK activity assay on different amounts of dCK in enriched preparation from PBMC. dCK activity is presented as RLUs), corresponding to the residual ATP from kinase reaction. Data are presented for PBMC isolated from 3 healthy donors assayed in independent experiments; (b) dCK activity in CD4 + T cells stimulated with anti-CD3/CD28 beads for 72 h in the presence or absence of 2CdA (500 nM). Data are presented for T cells isolated from 6 healthy donors assayed in independent experiments. Unstim. = unstimulated CD4 + T cells; Unstim. + 2CdA = unstimulated CD4 + T cells treated with 2CdA (500 nM); Stim. = CD4 + T cells stimulated with anti-CD3/CD28 beads; Stim. + 2CdA = CD4 + T cells stimulated with anti-CD3/CD28 beads and treated 2CdA (500 nM). (c) dCK enzymatic activity in CD19 + B cell stimulated with anti-IL-15/CpG for 72 h in the presence or absence of 2CdA (500 nM). Data are presented for B cells isolated from 6 healthy donors assayed in independent experiments. Unstim. = unstimulated CD19 + B cells; Unstim. + 2CdA = unstimulated CD19 + B cells treated with 2CdA (500 nM); Stim. = CD19 + B cells activated with IL-15/CpG; Stim.+ 2CdA = CD19 + B cells activated with IL-15/CpG treated with 2CdA (500 nM). * P < 0.05, ** P < 0.01, **** P < 0.0001

Fig 3: The ratios of dCK and NT5C2 expression (MFI) do not differ between subsets within the T, B, and NK cell populations. Quantification of the MFI dCK/NT5C2 ratio from flow cytometry analysis of Teff-cell (a), Treg-cell (b), B-cell (c), and NK-cell (d) subsets, in PBMC. Data are presented for PBMC isolated from 8 healthy donors assayed in independent experiments

Fig 4: DCK and NT5C2 mRNA and/or protein expression are upregulated by 2CdA in T and B cells. (a) DCK and (b) NT5C2 mRNA expression measured by Real-time PCR in CD4 + T cells stimulated with anti-CD3/CD28 beads for 48 and 72 h in the presence or absence of 2CdA (500 nM). The mean ?CT + SD baseline levels of expression of DCK and NT5C2 mRNA in unstimulated cells were 9.98 + 0.41 and 8.5 + 0.13, respectively. Data are presented for T cells isolated from 8 healthy donors assayed in independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001. (c) Representative Western blot (left panel) and quantification (right panel) of dCK in stimulated CD4 + T cells exposed or not to 2CdA (500 nM) for 72 h. Data were normalized to loading control protein, VCN. Fold changes were calculated using Unstim. controls as reference. Data are presented for T cells isolated from 5 healthy donors assayed in independent experiments; * P < 0.05, ** P < 0.01. Unstim. = unstimulated cells;. Stim. = CD4 + T cells stimulated with anti-CD3/CD28 beads; Stim. + 2CdA = CD4 + T cells stimulated with anti-CD3/CD28 beads and treated with 2CdA (500 nM). (d) DCK and NT5C2 mRNA expression measured by Real-time PCR in CD19 + B cells stimulated anti-IL-15/CpG for 72 h in the presence or absence of 2CdA. The mean ?CT + SD baseline levels of expression of DCK and NT5C2 mRNA in unstimulated cells were 10.5 + 0.40 and 7.475 + 0.12, respectively. Data are presented for B cells isolated from 5 healthy donors assayed in independent experiments; * P < 0.05. Stim. = CD19 + B cells activated with IL-15/CpG; Stim. + 2CdA = CD19 + B cells stimulated with IL-15/CpG treated with 2CdA (500 nM). GAPDH mRNA was used as endogenous control to normalize the expression data in the PCR analyses

Fig 5: 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 (? = 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