Figure 3. CD3ζ sharing in the dual CAR relies on CD8α mediated dimerization.
(a, b) T cells co-expressing B7-H3.BB and dNGFR.28ζ or 28ζ were stimulated with the B7-H3-Fc protein followed by incubation with an anti-Fc secondary Ab for 20 minutes at 37°C. Cells were then lysed in Laemmli buffer in non-reducing (without β-mercaptoethanol) (a) or reducing (with β-mercaptoethanol) (b) conditions for 10 minutes at 100°C, and separated on non-reducing gel or reducing gels. Membranes were stained with the anti-CD3ζ antibody. Data are representative of two independent experiments in (a, b). (c) Schematic representation of the retroviral vectors encoding dNGFR.28ζ/B7-H3.BB(CD8m) and 28ζ/B7-H3.BB(CD8m). CD8m, the stalk and transmembrane region of human CD8α that carrying the C164S and C181S mutations. (d-f) Summary of residual tumor cells (d), IFN-γ (e) and IL-2 (f) in the co-culture experiments of CAR-T cells with CHLA-255 at T cell to tumor cell ratio of 1 to 5. Data are shown as individual values and the mean + SD, n = 4 independent co-culture with CAR-T cells generated from 4 different donors; ****p <0.0001 by one-way ANOVA with Tukey’s multiple comparison test adjusted p value. (g) Representative confocal microscopy imaging showing CARs clustering in T cells expressing GFP-tagged GD2.28ζ (green) and B7-H3.BB (red) with and without CAR engagement using either the anti-14g2a idiotype antibody (1A7) or the B7-H3-Fc protein. Blue staining indicates the DAPI. Shown are representative cells. Data are representative of three independent validations. Shown in white are the scale bars that correspond to 5 μm.