Optimized CAR T cells induce target-specific, dose-dependent cytotoxicity and proliferation. (A) Real-time cell analysis (RTCA) assay quantifying CAR T-cell cytotoxicity, using cell products from Figure 1. Target cells were the unmodified rhesus macaque kidney epithelial cell line LLC-MK2 (“Mock”) or LLC-MK2 that underwent electroporation with messenger RNA expressing the HIV-1 clade C Env from SHIV-1157ipd3N4 (“ENV”). Replicate wells containing 10:1 (red), 5:1 (orange), and 2.5:1 (yellow) ratios of CD4CAR T cells:Env targets were compared with CD4CAR T cells plated at a 10:1 ratio with Mock targets (gray) as a control. “Normalized Cell Index” is a real-time surrogate of LLCMK-2 viability, quantified via adherence to substrate (details are given in "Methods"). Target cell adherence increases until addition of CD4CAR T cells (dotted line). (B) CAR T cells were labeled with proliferation dyes during RTCA to quantify proliferation in response to mock (gray) or Env-expressing targets (red). Details of the RTCA assay are included in the supplemental Methods.