Fig 6. CD4 CARs respond to Env⁺ cells and not MHC class II⁺ cells.

(A) Primary human CD8 T cells were activated with either left NTD or transduced with the indicated CD4 CARs. Two weeks post activation, the CD8 T cells were co-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562 cells expressing high levels of HLA-DR, or K562 cells expressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression is shown on the left, and intracellular IL-2 expression and CD107a surface mobilization is shown on the right. (B) A co-culture assay was designed to demonstrate that CD4 CAR⁺ CD8 T cells do not kill MHC class II-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 T cells from (A) were co-cultured with K562 cells expressing both HLA-A2 and GFP as well as K562 expressing both HLA-DR*0401 and mCherry at a 1:1:1 ratio. Flow cytometry measuring GFP and mCherry expression was performed immediately after mixing (0 hr) and after 3 days of co-culture (72 hr). C) Summary data for a single experiment performed in triplicate, measuring the ratio of HLA-A2/GFP-expressing cells to HLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours of culture. Error bars indicate SEM. Data is representative of three independent experiments.