Skip to main content
. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Cytotherapy. 2020 Mar 29;22(5):276–290. doi: 10.1016/j.jcyt.2020.01.011

Figure 6: CD2/CD3/CD28 stimulation significantly enhances granzyme B expression and expanded iNKT cell direct cytotoxicity.

Figure 6:

(A) mRNA expression of key cytotoxicity genes. Heat-map of log2 signal change relative to mean signal in day 0 iNKT cells, day 21 iNKT cells, and day 21 Vα24-N cells stimulated for 24 hours with media control (M), unloaded bead control (U), or anti-CD2/CD3/CD28 antibody-loaded beads (C). Red, increased relative expression; blue, decreased relative expression.

(B) Representative flow plots (top panel) showing perforin and granzyme B expression compared to isotype (black). Representative histograms and median and IQR ± range FI for granzyme B (middle panel) and perforin (lower panel) in day 21 expanded cells (N = 8) following 24-hour stimulation with media control (M, grey), unloaded bead control (U, blue), anti-CD2/CD3/CD28 antibody-loaded beads (C, red), or rhIL-2 control (IL-2, purple) compared to isotype control (pink dashed line).

(C) Representative histograms (left) and median and IQR ± range FI (right) for TRAIL (upper panels) and FasL/CD178 FI (lower panels) in day 21 expanded cells (N = 4) following a 24-hour stimulation with unloaded bead control (U, blue), anti-CD2/CD3/CD28 antibody-loaded beads (C, red) compared to isotype control (pink dashed line).

(D) Representative histograms of CD1d expression on K562 and Jurkat cells. Red, Isotype control; blue, anti-CD1d.

(E) Mean ± SEM percentage cytotoxicity in 51Cr release assay (N = 4) against K562 (upper panels) and Jurkat (lower panels) at specified effector : target (E:T) ratios for expanded and sorted day 21 iNKT cells stimulated 24 hours with media control (grey), rhIL-2 (purple) (left panels), unloaded bead control (blue), or anti-CD2/CD3/CD28 antibody-loaded beads (red) (right panels). Unloaded (open circles) or α-GalCer-loaded (triangles) targets were compared in each assay. Individual significant differences in treatment versus vehicle at the same E:T ratio are as follows: α) media + α-GalCer; β) IL-2 + α-GalCer; χ) anti-CD2/CD3/CD28; δ) unloaded beads + α-GalCer; ε) anti-CD2/CD3/CD28 + α-GalCer.