American Society of Hematology

Figure 2.

$NK cells with adaptive phenotype are largely GPI positive. PMBCs from 15 PNH patients were analyzed by flow cytometry. (A-D) Plots depict Barnes-Hut t-SNE analysis of 9-parametric data performed on gated CD56+ NK cells from all PNH patients. FLAER expression was not included as a parameter in the t-SNE analyses. (A) Distribution in the t-SNE field of GPIpos (FLAER+, top plot) and GPIneg (FLAER−, bottom plot) CD56+ NK cells from all patients. (B) Cumulative enrichment of GPIpos (FLAER+) and GPIneg (FLAER−) CD56+ NK cells from all patients according to the t-SNE field, as indicated. (C) Protein expression levels for single parameters in t-SNE field, as indicated. (D) Cell density in the t-SNE field for selected, individual patients, as indicated, with black and red dots indicating GPIpos (FLAER+) and GPIneg (FLAER−) CD56+ NK cells, respectively. Supplemental Figure 3 shows the individual t-SNE fields for all patients. (E-F) Flow plots depict (E) PLZF or (F) NKG2C vs FLAER expression on gated CD56dim NK cells in individual patients, as indicated. (G-H) Graphs indicate the frequency of (G) PLZF− and (H) NKG2C+ cells, also designating the proportion of which expressed FLAER or not, among CD56dim NK cells in individual patients, as indicated. (I) Venn diagrams depict the relative abundance of canonical and different adaptive CD56dim NK-cell subsets, as defined by lack of PLZF, FcεRγ , and SYK expression. Only samples with GPIneg neutrophils >75% are included (n = 9). (J) Analysis of the fractions of GPIneg cells in different NK cells subsets, neutrophils, and CD3+ T cells of 15 PNH patients. GPI expression was quantified in CD56dim NK cells either coexpressing NKG2A, NKG2C, and CD57 or lacking expression of PLZF or FcεRγ. The gating strategy defining positive and negative cells for each NK-cell marker, and expression profile for each patient, can be found in supplemental Figure 3. Each color depicts an individual patient. Full circles indicate CMV seropositive, whereas open circles indicate CMV seronegative. n.s., not significant, 1-way analysis of variance followed by multiple comparisons (Holm-Šídák).$

NK cells with adaptive phenotype are largely GPI positive. PMBCs from 15 PNH patients were analyzed by flow cytometry. (A-D) Plots depict Barnes-Hut t-SNE analysis of 9-parametric data performed on gated CD56⁺ NK cells from all PNH patients. FLAER expression was not included as a parameter in the t-SNE analyses. (A) Distribution in the t-SNE field of GPI^pos (FLAER⁺, top plot) and GPI^neg (FLAER⁻, bottom plot) CD56⁺ NK cells from all patients. (B) Cumulative enrichment of GPI^pos (FLAER⁺) and GPI^neg (FLAER⁻) CD56⁺ NK cells from all patients according to the t-SNE field, as indicated. (C) Protein expression levels for single parameters in t-SNE field, as indicated. (D) Cell density in the t-SNE field for selected, individual patients, as indicated, with black and red dots indicating GPI^pos (FLAER⁺) and GPI^neg (FLAER⁻) CD56⁺ NK cells, respectively. Supplemental Figure 3 shows the individual t-SNE fields for all patients. (E-F) Flow plots depict (E) PLZF or (F) NKG2C vs FLAER expression on gated CD56^dim NK cells in individual patients, as indicated. (G-H) Graphs indicate the frequency of (G) PLZF⁻ and (H) NKG2C⁺ cells, also designating the proportion of which expressed FLAER or not, among CD56^dim NK cells in individual patients, as indicated. (I) Venn diagrams depict the relative abundance of canonical and different adaptive CD56^dim NK-cell subsets, as defined by lack of PLZF, FcεRγ , and SYK expression. Only samples with GPI^neg neutrophils >75% are included (n = 9). (J) Analysis of the fractions of GPI^neg cells in different NK cells subsets, neutrophils, and CD3⁺ T cells of 15 PNH patients. GPI expression was quantified in CD56^dim NK cells either coexpressing NKG2A, NKG2C, and CD57 or lacking expression of PLZF or FcεRγ. The gating strategy defining positive and negative cells for each NK-cell marker, and expression profile for each patient, can be found in supplemental Figure 3. Each color depicts an individual patient. Full circles indicate CMV seropositive, whereas open circles indicate CMV seronegative. n.s., not significant, 1-way analysis of variance followed by multiple comparisons (Holm-Šídák).

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