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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Cancer Immunol Res. 2015 Dec 23;4(3):204–214. doi: 10.1158/2326-6066.CIR-15-0188

Figure 4.

Figure 4

Characteristics of HLA-A*11:01–restricted KRAS G12D-reactive murine TCR, TRAV4-4*01/BV12-2*01. (A) Affinity of the KRAS G12D-reactive TCR. Anti-CD3 stimulated human allogeneic PBL were transduced with retrovirus encoding TRAV4-4*01/BV12-2*01. Three days after transduction, TCR-transduced cells were co-cultured with COS7/A11 pulsed with 1:10 serial diluted peptides. (B) TRAV4-4*01/BV12-2*01 was HLA-A*11:01–restricted. TCR-transduced T cells were cocultured with KRAS G12D-positive pancreatic tumor lines with or without HLA-A*11:01 expression. (C) TRAV4-4*01/BV12-2*01 was KRAS G12D specific. TCR-transduced T cells were cocultured with a panel of HLA-A*11:01 expressing pancreatic tumor lines with or without KRAS G12D mutation. (D) Reactivity of KRAS G12D-specific TCR against PANC-1. TCR-transduced T cells were cocultured with PANC-1, PANC-1 pulsed with 10-mer peptides, or PANC-1 transduced to overexpress HLA-A*11:01. (E) Reactivity of KRAS G12D-specific TCR against IFNγ treated pancreatic tumor lines. Pancreatic tumor lines were pre-treated with IFNγ (10ng/ml) for 48hrs, and then cocultured with TCR-transduced T cells. From (A) to (E), supernatant of cocultures were harvested and IFNγ production was assessed. (F) T cells transduced with TRAV4-4*01/BV12-2*01 proliferated upon antigen-specific stimulation. T cells transduced with TRAV4-4*01/BV12-2*01 were labeled with CFSE, cocultured with various targets. Three days after coculture, T cells were labeled with antibodies to human CD3 and to murine TCRβ, and then analyzed on a FACS Canto II. Data was gated on the live CD3+ population. (G) Antigen-specific degranulation of TRAV4-4*01/BV12-2*01–transduced T cells. T cells transduced with TRAV4-4*01/BV12-2*01 were cocultured with various targets in the presence of anti-CD107a-FITC for 4 hr, labeled with antibodies to human CD3 and to murine TCRβ, and then analyzed on FACS Canto II. Data was gated on live CD3+CD8+ populations.