Figure 1.

Optimized Protocol for Generation of Poly-functional CAR19-iNKT Cells

(A) Flow cytometric identification of iNKT cells as TCRVα24⁺Vα11⁺ pre-selection and expression of second- and third-generation CAR19 in TCRVα24⁻ T and TCRVα24⁺ iNKT cells as assessed by staining against the marker RQR8 3 days after lentiviral transduction.

(B) Expansion and absolute numbers of CAR19-T and CAR19-iNKT cells over 3 weeks using lymphapheresis (left) or peripheral blood (PB; right) (n = 3 and 4 respectively). p values are for CAR19-iNKT versus CAR19-T cells using Friedman test.

(C) Intracellular expression of cytokines in resting (n = 10) and anti-CD3/CD28-bead-activated (for 4 hr; n = 6) CD4⁻ and CD4⁺ CAR19-iNKT cells. Flow cytometric analysis was performed as shown in (D). D-B48 and δG9 monoclonal antibodies identify total and granule-associated perforin respectively. GZMB, granzyme B.

(D) Representative example of flow cytometric intracellular analysis of shown cytokines in CD4⁻ and CD4⁺ CAR19-T and CAR19-iNKT cells. In GZMB/IFNγ dot plots, intensity of perforin expression is projected as a heatmap according to the shown color scale. PFN, perforin.

(E) Proportions of cells co-expressing zero to three cytokines (mean of four independent experiments).

(F) Proportions of specific cytokines co-expressed by CD4⁻ or CD4⁺ CAR19-T and CAR19-iNKT cells.

(G) Multiple cytokine secretion after 3 and 8 hr of activation of second- and third-generation (2 and 3) CAR19-T and CAR19-iNKT cells from two healthy donors (A and B). Heatmap shows normalized CAR19-iNKT/CAR19-T cell ratios.

Error bars represent SEM.

Asterisks indicate p values as follows: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. See also Figure S1.