Fig. 8. NKG2C⁺CD8⁺ T cells proliferate in response to HLA-E and display superior tumor killing if transduced with a CD19-CAR.

(A) Left: Representative plots showing total PBMC cells stimulated for 7 days with the indicated stimuli and pre-gated on CD3⁺CD8⁺ cells. Right: Cumulative analysis from 3 independent donors. Statistical significance was calculated by one-way ANOVA with Turkey’s multiple comparison test. (B) Total cell counts of pre-sorted NKG2C⁺CD8⁺ T cells stimulated with K562 mbIL21 HLA-E:VMAPRTLFL (ratio 1:1) for the indicated number of days. (C) Total CD8⁺ T cells were CTV labelled and exposed for 7 days to the indicated stimuli. Pre-gated NKG2C⁺ or NKG2C⁻CD8⁺ T cells were then analyzed for proliferation monitoring CTV dilutions. Statistical analysis was calculated using a 2-way ANOVA. (D) NKG2C⁻ and NKG2C⁺CD8⁺ T cells from one representative donor were FACS sorted and left untransduced or transduced with a 1928z CD19-targeting CAR construct. Cytotoxicity was assessed using a standard ⁵¹Cr assay after 24h of exposure to the CD19⁺ cell line NALM6. (E) PD-1 surface expression on NKG2C⁻CD8⁺ and NKG2C⁺CD8⁺ cells transduced with the 1928z CD19-targeting CAR construct. (F) Histogram shows HLA-E surface staining on NALM-6 cells. (G) Cumulative analysis of 3 independent donors showing degranulation (CD107a) and intracellular IFN-γ expression by pre-gated NKG2C⁻CD8⁺ T cells and NKG2C⁺CD8⁺ T cells against NALM6 in the presence of a CD94 blocking mAb (10 μg/mL). Statistical significance was calculated by one-way ANOVA with Turkey’s multiple comparison test. ****P-value ≤ 0.0001, ***P-value ≤ 0.001, **P-value ≤ 0.01, *P-value ≤ 0.05.