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. 2024 Mar 21;5(3):101469. doi: 10.1016/j.xcrm.2024.101469

Figure 5.

Figure 5

SJ1-4 and SJ1-12 TCRs are fusion specific, functional, and able to kill fusion-positive target cells in vitro

(A) A∗68:02-EIFDRYGEEV tetramer staining of SJ1-4 and SJ1-12 TCRs reconstructed and expressed in TCR-null Jurkat cells.

(B) Normalized frequency of IFNγ+TNFα+ cells among SJ1-4- or SJ1-12-transduced primary human T cells (donor Aph34) after stimulation with increasing doses of fusion (EIFDRYGEEV) or WT peptide (EIFDRYGEEG) (see STAR Methods for calculation). Data for each TCR were collected in separate single experiments, each using 3 PBMC donors. See also Figure S6D.

(C) Frequency of IFNγ+TNFα+ cells among SJ1-4 or SJ1-12 primary human T cells after stimulation with aAPCs expressing DNAJB1-PRKACA fusion or WT transgenes. All data were collected in the same single experiment using 3 PBMC donors. See also Figure S6F.

(D) xCelligence assay measuring SJ1-4 or SJ1-12 primary human T cell killing of fusion- or WT-expressing target cells. Target cells adhered for 24 h before addition of T cells at effector:target ratios of 20:1, 5:1, and 1.25:1 (mean ± SD across technical triplicates). Cell index was normalized to 1 at the time of T cell addition. Killing is indicated by a decrease in cell index as target cells die and lift from the plate. SJ1-12 and mock-transduced control data were collected in the same plate; SJ1-4 data were collected in a separate plate with an additional set of mock-transduced controls (comparable with those shown). A t test on normalized cell index values was performed at the final time point. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, p > 0.05. See also Figure S6G.

(E) Frequency of cleaved caspase-3+ wells in the Berkeley Lights Lightning assay co-culturing a single SJ1-4 or SJ1-12 primary human T cell with a single fusion- or WT-expressing aAPC for 24 h. Data for SJ1-12 and mock-transduced controls were collected in the same experiment; data for SJ1-4 were collected in a separate experiment with an additional set of mock-transduced controls (comparable with those shown). Fisher’s exact test was performed on the proportion of caspase+ cells. ∗∗p < 0.01; ns, p >0 .0.05. See also Figure S6H.

(F) Frequency of IFNγ, IL-2, and TNFα production during same Berkeley Lights Lightning assay. Fisher’s exact test was performed on proportions of cytokine+ cells (i.e., single, double, or triple cytokine+). ∗∗p < 0.01; ns, p >0 .0.05.