Fig 1.
(A) Schema of T cell transduction and culture as described in Materials and Methods. (B) Diagram of QuIK assay set up: tumor cells loaded with target peptide at different concentrations were cocultured in 384-well plates, and incubated in an IncuCyte® to monitor morphological changes. (C) Examples of readout: Target cell confluency was determined by phase contrast. Calculated confluent area is marked in orange. T cells are loaded with CellTracker™ Green, and live T cells (green-positive and red-negative) are marked in magenta. Dead/dying cells that incorporated Annexin V/PI are red. Dead/dying target cells were separated from T cells by size and shape and are marked in blue.
Table 1.
Receptor constructs used in this study.
Fig 2.
Summary of T cell proliferation and specific killing.
(A) Time course for 3:1 E:T ratio of target cell confluency, T cell counts, relative T cell proliferation, percentage of target cells killed, and specific target cell killing per T cell, summarized for T cells that are either untransduced or transduced with one of the CAR/TCR constructs in target cells loaded with target peptide at 100 μM (green), 0 μM (red), or a concentration similar to their EC50 (blue) determined. Similar data for 1:1 and 1:3 in S2 and S3 Figs. (B) Dose-response of relative T cell proliferation; and, (C) specific killing. Measurements are made for both Fig 2A and 2B for all constructs at 3:1, 1:1, and 1:3 E:T ratios, at the time point when the peak value is reached: 72 hours for proliferation and 48 hours for killing.
Table 2.
Summary of parameters measured for constructs used in this study.
Fig 3.
Summary of sensitivity (dose-response) of T cell proliferation and kill rates.
Time point for dose-response chosen to be maximum rate for each peptide concentration between t = 30–72 hours; maximum rate is plotted vs. peptide dose. (A) Normalized proliferation rate of each construct. (B) Specific kill rate.
Fig 4.
Summary of kinetic profile of T cell proliferation and specific kill rates.
Time course plots were divided into three groups: 1) High: [peptide] >100x EC50; 2) Mid: 100x EC50 > [peptide] >1x EC50; 3) Low: [peptide] < 1x EC50. When proliferation time courses (A) are plotted, each group shows a different time dependent profile from the others. The highest proliferation is observed in the 2nd group. In the specific killing time course (B), all groups follow similar time course patterns. Specific killing peaks occur sooner than T cell proliferation peaks.
Fig 5.
Example of non-specific activity determined by QuIK assay.
(A) Jurkat/T2 assay. (B) E:T ratio assays with primary T cells and either A375 (HPV-negative) or CaSki (HPV-positive) as target cells; UTD, untransduced. (C) Peptide-loading, luciferase viability assay. (D) IFNγ as a readout for T cell activation. The signal was measured with AlphaLISA as described in Materials and Methods. T cells-only and T cells co-cultured with A375 without loaded peptide were used to determine target-independent activation. (E) Total target cell death and specific kill/cell as described in Results and Materials and Methods. (F) QuIK assay showing on-target cell killing and T cell proliferation of HPV E7 TCR but not HPV E7 CAR.