Fig. 6: Validation of mKRAS-specific T cell reactivity identified from peripheral blood of vaccinated patients.
From: Mutant KRAS vaccine with dual checkpoint blockade in resected pancreatic cancer: a phase I trial
a UMAP of single cell RNA/TCR sequencing performed on pre- and post-vaccine PBMCs. T cells were identified as CD4 (naïve, proliferating, central memory (TCM), and effector memory (TEM)), CD8 (naïve, proliferating, TCM, and TEM), double negative T (dnT), MAIT, and Tregs. b Phenotype of mKRAS-specific T cells identified from mKRAS expansion mapped onto single cell data by TCRβ chains (c) Specific activity of Jurkat-TCRKO-NFAT-GFP cells expressing putative mKRAS-specific TCRs and co-cultured with patient-matched LCLs pulsed with control peptide, mKRAS peptides, or KRAS WT peptide. Mean +-SD are shown. d Specific activity of Jurkat reporter line transduced with TCR03 and co-cultured with patient-matched LCLs pulsed with 10uM control, mKRAS peptide, or wild-type KRAS peptide. Mean +-SD are shown. One way ANOVA was performed followed by Tukeys multiple comparisons. e G12V reactive TCRs cocultured with HLA-null K562s transfected with patient-matched HLA class II alleles and pulsed with 2ug/mL KRAS G12V peptide. Co-cultures were assessed for GFP expression. f Specific activity of Jurkat-Public TCRβ08 expressing cells co-cultured with patient matched LCLs pulsed with control, KRAS G13D, or wild type peptide at the indicated concentrations. Specific activity of public TCRβ08 Jurkats co-cultured with LCLs from a HLA-DQB*03:01- donor (bottom left) or irrelevant HLA-DQB*03:01+ donor (bottom right) pulsed with control, KRAS G13D, or wild type KRAS peptide. g Activation (GFP expression) of KRAS TCR+ jurkat cell lines after co-culture with human CAPAN1 (KRAS G12V), SUDHL10 (KRAS WT), or MDA-MB-231 (KRAS G13D) pretreated with 100IU hIFNγ for 48 h at an effector to target ratio of 2:1 for 24 h in the presence or absence of 2ug/mL exogenous mKRAS antigen. A negative control TCR recognizing a SARS-CoV2 was used. h CD25 and CD137 expression on KRAS TCR05 knock-in healthy donor T cells 24 h after co-cultured with IFNγ pre-treated CAPAN1 or SUDHL10 cells in the absence or addition of 2ug/mL additional exogenous mKRAS antigen.
