Fig. 3: Cytokine therapy promotes the maturation of NKG2a/c^lowCD16⁺ NK cells with enhanced ex vivo innate activity, which correlates with the content of lymphoid replication competent virus.

a The frequency of NK cells (CD45⁺CD20⁻CD3⁻NKG2a/c⁺) of PBMC CD45⁺ lymphocytes was longitudinally measured by flow cytometry, b as was their CXCR5 expression. The frequency of each differentiation stage of NK cells was determined based on the following definition: c Stage 0 (red, NKG2a/c^lowCD16⁻), d Stage 1 (blue, NKG2a/c^highCD16⁻), e Stage 2 (orange, NKG2a/c^highCD16⁺), and f Stage 3 (purple, NKG2a/c^lowCD16⁺). g The mean frequency of each NK cell differentiation stage from above was also re-visualized as a color-coded (as annotated below), parts-of-whole stacked bar plot for the cytokine-treated (n = 8; at left) and control RMs (n = 5; at right) over time (indicated at left). Flow cytometry was used to quantify the ex vivo innate frequency of h IFN-γ⁺ (intracellular) NK cells and i CD107a⁺ (surface) stage 3 (NKG2a/c^lowCD16⁺) NK cells. a–i Data from individual RMs (staggered open circles) are overlaid against the mean (solid line) ± SEM (shaded region within the dashed lines): control (ART-only, black; n = 5) and cytokine-treated (ART + IL-21 + IFNα, blue; n = 8). Treatment phases are indicated with the following background shading: IL-21 (orange), rIFNα (red), and ART (gray). Data were analyzed with two-sided (95% CI), two-way ANOVA with Bonferroni’s correction with cross-sectional comparisons relative to controls. j The frequencies of each differentiation stage of NK cells in PBMCs (as indicated below) were correlated against levels of cell-associated and replication competent SIV content in tissue, and the ex vivo innate and Env-specific NK cell activities in PBMCs (as indicated at left) in all RMs (n = 13; days 35, 77, 217, and 374 p.i. as matched data are available). Per each correlation the two-tailed (95% CI) Spearman’s rank correlation coefficient (rho) is represented as a double-gradient heatmap and the size of each data point corresponds inversely to the log₁₀-transformed Spearman’s p value. The false discovery rates (FDR) were calculated using SAS and significant values (Q < 0.05) are represented by a black border.