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. Author manuscript; available in PMC: 2024 Nov 2.
Published in final edited form as: Cancer Immunol Res. 2024 May 2;12(5):530–543. doi: 10.1158/2326-6066.CIR-23-0467

Fig. 4. Tsyn reporter system for detecting bidirectional signaling.

Fig. 4.

(A) Schematic of Tsyn-seq dual-reporter system. Simultaneous activation of Fas-iNFκB reporter in FIR-APCs and NFAT-mCherry reporter in JR-T cells through FasL/FasR and TCR/pMHC interactions, respectively. (B) Frequency of Fas-iNFκB reporter positive populations in NFAT-mCherry reporter positive or negative aggregates. E7-peptide-pulsed FIR-APCs are cocultured with αE7-JR-T cells (red line), while NLV-peptide-pulsed FIR-APCs are cocultured with αE7-JR-T cells as control (grey line). Gates demarcating mCherry positive (orange arrow) and negative (green arrow) populations among the CTV/CTFR double-positive aggregates are shown. (C) Coculture of JR-T cells and FIR-APCs pulsed with cognate and non-cognate peptides. Statistical significance is determined by the Mann-Whitney U test. Error bars indicate SD across 5 replicates. (D) ROC curve analysis of dual-reporter signaling in the coculture of (B). Green ROC curve represents GFP fluorescence intensity of aggregates population (left panel). Orange ROC curve represents mCherry fluorescence intensity of GFP positive population in aggregates population (right panel).