Figure 1. A DNA-CARζ Capable of Triggering T Cell Signaling.

(A) Schematic of the DNA-based chimeric antigen receptor system (DNA-CARζ). The SNAPf tag and His10-CLIPf were covalently labeled with complementary strands of benzyl-guanine DNA and benzyl-cytosine DNA, respectively.

(B) TIRF microscopy images of a JRT3 Jurkat cell expressing ZAP70-GFP and DNA-CARζ labeled with 16-mer ssDNA after landing on a SLB with a complementary 16-mer strand (120 molecules per μm²). Microclusters of ligand-receptor complexes formed, recruited ZAP70-GFP (inset), and then moved centripetally and coalesced near the cell center. Scale bar, 5 μm; inset scale bar, 2 μm.

(C) To measure activation of the MAP kinase pathway, cells (15 min after SLB contact) were stained for phosphoERK (red); DAPI staining of nuclei (blue); and the DNA-CARζ (green). Bar, 50 μm. Insert shows higher magnification; bar, 20 μm. Quantification (see Figure S2; STAR Methods) of the MAP kinase pathway activation by the 16-mer DNA ligand compared to PMA (10 ng/ml) is shown. Mean ± SD of 6 experiments (>2,500 cells scored per experiment).

(D) A schematic of a triggerable DNA-CARζ.

(E) Cell spreading on the SLB as a function of time after adding the DNA trigger strand. The average fold-change after addition of trigger strand reflects the mean ± SEM from three separate experiments (3–7 cells per experiment).

(F) Pseudo-color image of calcium levels. The 340 nm/380 nm fura-2 emission ratio shows the change in intracellular calcium levels from the six cells after adding the DNA trigger strand. Bar, 10 μm.

See also Figure S1.