Journal: Frontiers in Immunology

Article Title: CD94-driven in vitro expansion of highly functional adaptive NKG2C + NKG2A - CD57 + NK cells from CMV + healthy donors

doi: 10.3389/fimmu.2025.1481745

Figure Lengend Snippet: CD94 mAb-induced growth of NKG2C + NK cells by efficient proliferation of NKG2C/CD57 NK cell subsets. (A) The frequency of the different NKG2C/CD57 NK cell populations was evaluated by flow-cytometry comparing day 0 (circle) with day 14 of culture with IL-2 (empty square), IL-2+mAb (square), IL-15 (empty triangle) or IL-15+mAb (triangle). The frequency of total NKG2C + (left), NKG2C + CD57 + (central) or NKG2C + CD57 – NK cells (right) are shown. Bars indicate the mean ± SD. Differences in NK cell subsets abundance at day 0 with respect to day 14 in IL-2 or IL-15 conditions were evaluated using Mann-Whitney test and statistical significances are reported (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (B) T-depleted PBMCs labeled with CFSE were cultured according to the protocol in Figure 1B in parallel cultures. The gradual CFSE dilution (left) and the progressive increase of NKG2C + CD57 + NK cell frequencies (right) are shown at different time points for both IL-2+mAb and IL-15+mAb culture conditions. Percentages of highly proliferating, CFSE negative NKG2C + NK cells (identified by a dotted line) and NKG2C/CD57 NK cell subsets frequencies are reported in the corresponding quadrants. A representative experiment out of 13 is shown. (C) The number of NKG2C + NK lymphocytes was evaluated at various culture times starting from day 0, when 10 5 T-depleted PBMC per well were seeded. Growth curves of cell cultures carried out with (squares) or without (circles) the addition of the anti-CD94 mAb, in the presence of IL-2 (left) or IL-15 (right) are shown. The number of NKG2C + NK lymphocytes was calculated for each experiment (n=13) based on the composition of the cell culture as indicated in Figure 1C (gating strategy for cultured NKG2C + NK cells in Supplementary Figure S1B ). Bars indicate the mean ± SD. Differences in NK cell numbers comparing cultures in the presence and in the absence of the mAb were evaluated for each time point (7, 10, 14 days) using the Mann-Whitney test. Statistical significance is reported at each point (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (D) Fold expansion in cell numbers of NKG2C + NK cells in the different culture conditions on day 14 (n=13). Statistical significance is reported (**p<0.01; ***p<0.001).

Article Snippet: In each experiment a fraction of T cell-depleted PBMCs was labeled with the intracellular fluorescent dye 5 ( )-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Life Technologies, Thermo Fisher Scientific, California, USA), as previously described , and cultured according to the protocol shown in .

Techniques: Flow Cytometry, MANN-WHITNEY, Labeling, Cell Culture