Journal: Cancer discovery
Article Title: Recurrent immunogenic neoantigens and their cognate T-cell receptors in treatment-resistant metastatic prostate cancer
doi: 10.1158/2159-8290.CD-24-1213
Figure Lengend Snippet: A, Healthy donor PBMCs were transduced with TCR-MSGV1 (T157.1, T157.3, and T112.1) plasmids using retroviruses. One week after transduction, the cells were co-cultured with mono-allelic B cells (HLA-A*01:01 or HLA-B*15:01) pulsed with 1 μg/ml AR H875Y peptides (VQPIAREL Y , SVQPIAREL Y , or LLDSVQPIAREL Y ) or AR WT peptides (VQPIARELH, SVQPIARELH, or LLDSVQPIARELH) at 1.5:1 E:T ratio. Flow cytometry analysis was used to evaluate the activation of T-cells based on IFN-γ secretion, TNF-α secretion, and 4–1BB expression. B, The LNCaP cancer cell line harboring the HLA-A*01:01 allele was stably transduced with the AR H875Y MNG and HLA-B*15:01 alleles (LNCaP AR H875Y , LNCaP AR H875Y/B*15:01 ). The cells were co-cultured at a 1:1 E:T ratio with T-cells stably expressing the AR H875Y-specific TCRs, followed by flow cytometry analysis of activation markers, including IFN-γ secretion, TNF-α secretion, and 4–1BB expression. C, LNCaP cells were stably transduced with AR H875Y full-length cDNA and the HLA-B*15:01 allele (LNCaP AR H875Y , LNCaP AR H875Y/B*15:01 ). The cells were co-cultured at a 1:1 E:T ratio with T-cells stably expressing the AR H875Y-specific TCRs, followed by flow cytometry analysis of 4–1BB expression. D, Diagram illustrating the CRISPR knock-in. LNCaP cells were co-transfected with a plasmid encoding Cas9-GFP, sgRNA targeting the AR gene, and oligonucleotide repair template for homology-directed repair. After 48-hour, GFP-expressing cells were isolated as single cells in individual wells of a 96-well plate using FACS. Single cells were expanded into clonal populations. Genomic DNA was extracted from each clone and Sanger sequencing was performed to verify the incorporation of the intended knock-in mutation. A schematic was created using BioRender.com . E, CRISPR KI AR H875Y LNCaP cells (SCC18) and control cells (SCC8) were stably transduced with the HLA-B*15:01 allele. The cells were co-cultured at a 1:1 E:T ratio with T-cells stably expressing the AR H875Y-specific TCRs, followed by flow cytometry analysis of 4–1BB expression. F, C4–2 cells were stably transduced with AR H875Y full-length cDNA and the HLA-B*15:01 allele (C4–2 AR H875Y , C4–2 AR H875Y/B*15:01 ). The cells were co-cultured at a 1:1 E:T ratio with T-cells stably expressing the AR H875Y-specific TCRs, followed by flow cytometry analysis of 4–1BB expression. G, The 22Rv1 cancer cell line harboring the AR H875Y mutation was stably transduced with HLA-B*15:01 allele. The cells were co-cultured at a 1:1 E:T ratio with T-cells stably expressing the AR H875Y-specific TCRs, followed by flow cytometry analysis of activation markers, including IFN-γ secretion, TNF-α secretion, and 4–1BB expression. The plots represent ≥2 biological replicates. The normality of the data distribution was confirmed using the Shapiro-Wilk test (p > 0.05). Levene's Test for Homogeneity of Variance was not violated (p > 0.05) and statistical significance was determined using an unpaired two-tailed Studenťs t-test. Specific p-values are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Article Snippet: The constructs were cloned into the MSGV1 retroviral vector (RRID:Addgene_11174) in a TCRβ-TCRα orientation.
Techniques: Transduction, Cell Culture, Flow Cytometry, Activation Assay, Expressing, Stable Transfection, CRISPR, Knock-In, Transfection, Plasmid Preparation, Isolation, Sequencing, Mutagenesis, Control, Two Tailed Test
