Figure 4: Expanded iNKT cells and Vα24-N cells share gene expression profiles.

(A) Representative gates used to sort day 0 CD3⁺Vα24⁺ iNKT cells (left panel) and day 21 (right panel) CD3⁺Vα24⁺ iNKT cell and CD3⁺Vα24^neg Vα24-N cell populations.

(B) Principal component analysis (PCA) from gene expression of sorted day 0 iNKT cells (squares) (N = 3 per condition), day 21 iNKT cells (circles) (N = 4 per condition), and Vα24-N cells (triangles) (N = 4 per condition) stimulated 24 hours with media control (Stim M, yellow), unloaded bead control (Stim U, blue), or anti-CD2/CD3/CD28-loaded beads (Stim C, red).

(C) Mean fold gene upregulation (log₂) over mean background in day 21 iNKT cells vs Vα24-N cells given Stim C vs Stim U (C/U, day 21) (right panel) and Stim U vs Stim M (U/M, day 21) (left panel). Stimulations are as defined in B. Correlation coefficient (r²) is indicated above each panel.

(D) Normalized enrichment score (NES) for pathway activity identified by gene set enrichment analysis (GSEA) in sorted day 0 iNKT cells given Stim C vs Stim U (iNKT C/U, day 0) vs day21 iNKT cells given Stim C vs Stim U (iNKT C/U, day 21). Stimulations are as defined in B. Blue, pathways significantly up- or down-regulated at day 21 but not at day 0; orange, pathways significantly up- or down-regulated at day 0 but not at day 21; magenta, pathways significantly up- or down-regulated at both day 0 and day 21; grey, pathways not significantly altered at day 0 or day 21.; and green, conventional T cell activation pathways,

(E) Biocarta NKT pathway gene expression profile. Heat map of log₂ signal change in day 0 iNKT cells (N = 3), day 21 iNKT cells (N = 4), and Vα24-N cells (N = 4) stimulated under conditions identical to those in B. Red, increased relative expression; blue, decreased relative expression.