Fig. 5. Treatment of CART19-28ζ cells with hrIL-4 leads to phenotypical and functional signs of exhaustion in a CD4-independent manner.

a Percent killing as measured with bioluminescent imaging after Day 8 CD3⁺ or CD8⁺ CART19-28ζ cells were co-cultured with luciferase⁺ JeKo-1 cells at various E:T cell ratios for 48 hours in the presence of either 20ng/mL human recombinant IL-4 (hrIL-4) or diluent (Two-way ANOVA, average of two technical replicates for three biological replicates, mean ± SD). b, f CD3⁺ and CD8⁺ CART19-28ζ cells were kept in media supplemented with 100 IU/mL hrIL-2 and chronically stimulated from Day 8 to Day 15 of the in vitro model for exhaustion in the presence of either 20ng/mL hrIL-4 or diluent control. b Absolute CD3⁺ cell count as measured with flow cytometry after Day 15 CART19-28ζ cells were co-cultured with JeKo-1 cells at a 1:1 E:T cell ratio for five days. (Two-way ANOVA, average of two technical replicates for three biological replicates). c, d The percent of CD3⁺ cells producing IL-2 and IFN-γ as determined with intracellular staining and flow cytometry after Day 15 CART19-28ζ cells were co-cultured with JeKo-1 cells at a 1:5 E:T cell ratio for four hours (Two-way ANOVA, average of two technical replicates for three biological replicates). e The percent of CART19-28ζ cells co-expressing multiple inhibitory receptors (0—black, 1—pink, 2—green, 3—dark purple, 4—light purple) on Day 15 as determined by flow cytometric detection of PD-1, CTLA-4, TIM-3, and LAG-3 on CD3⁺ cells (Circle plots from one representative biological replicate). f The change in the transcription of EOMES as determined with RT-qPCR of Day 15 CD3⁺ or Day 15 CD8⁺ CART19-28ζ cells (Paired two-sided t-tests average of two technical replicates for three biological replicates, mean ± SD). Source data are provided as a Source Data file.