universal primer mix smarter race cdna amplification kit clontech catalog 634858  (TaKaRa)

Journal: Journal for Immunotherapy of Cancer

Article Title: Generation of TGFβR2(-1) neoantigen-specific HLA-DR4-restricted T cell receptors for cancer therapy

doi: 10.1136/jitc-2022-006001

Figure Lengend Snippet: Identification of TGFβR2(-1)-specific TCR-α and TCR-β chain combinations. (A) Frequencies of CDR3 regions and V/J gene segments of TCR α-chains and β-chains. After RACE-PCR from responding CD4 + T cells, the amplified PCR product of each chain was separately cloned and around 50 clones for each TCRα and TCRβ were sequenced and their frequencies are presented in percentages. (B) Combinatorial expression of TCRα and TCRβ genes in human CD4 + T cells with given transduction efficiency in percentages, stained with antibodies specific for mouse TCRβ constant region (mTCRβ) and human CD4. Cells are gated on CD3 + lymphocytes. (C) Identification of functional pairs TCR 1A/1B and TCR 2A/3B. Human CD4 + T cells transduced with 15 ABabDR4-derived TCR combinations were co-cultured with HLA-DR4 + BM14 cells loaded with 10 -6 M TGFβR2(-1) peptide (+pep). After 16 hours, IFNy was measured in the supernatant. As negative control, T cells alone were used and as positive control, T cells stimulated with PMA/Ionomycin (P/I). Intra-assay duplicates with SD are displayed. Similar data were obtained with HLA-DR4 + BSM and K562-DR4 cells (data not shown). PMA, phorbol 12-myristate 13-acetate; TCR, T cell receptor.

Article Snippet: This was followed by synthesis of cDNA and 5’ RACE PCR (SMARTer RACE cDNA Amplification Kit, Clontech) with 0.5 µM primers specific for the constant region of either TCRα (5′-CGGCCACTTTCAGGAGGAGGATTCGGAAC-3′) or TCRβ (5′-CCGTAGAACTGGACTTGACAGCGGAAGTGG-3′) chain.

Techniques: Amplification, Clone Assay, Expressing, Transduction, Staining, Functional Assay, Derivative Assay, Cell Culture, Negative Control, Positive Control, Intra Assay