Fig. 1: Generation of transgenic KSHV-specific T cell receptors from LANA-specific T cell clones.
From: LANA-specific CD4+ effector T cells accumulate at the site of KSHV infection in humanized mice
A Degranulation assay measuring the frequency of CD107a+ cells of CD4+ or CD8+ T cell clones upon co-culture with medium only (neg), PMA and Ionomycin (pos P/I), autologous unpulsed or cognate peptide pulsed LCLs or T2B35 cells. Mean ± SD of duplicates (Cl33/87/55/12/114pos) or triplicates (Cl156/114) of n = 1 experiment. Two-tailed unpaired t-test comparing unpulsed and pulsed conditions. B Schematic representation of the TCR construct used: variable α chain (Vα) followed by mouse constant α chain (mCα), P2A, variable ß chain (Vß), mouse constant ß chain (mCß), T2A, truncated mouse CD19 (mCD19). mCα and mCß are linked by an additional disulfide bond. Representative flow cytometry scatter plots of untransduced and transduced Jurkat Lucia NFAT cells showing the gating for transduced and TCR-expressing cells. C Jurkat Lucia NFAT reporter assay showing the fold change luminescent signal normalized to the background of unstimulated cells for transgenic TCR-transduced Jurkat Lucia NFAT cells stimulated with HLA-matched pulsed or unpulsed LCLs or T2B35 cells. Graph shows mean ± SD of n = 4 independent experiments. Two-tailed unpaired t-test. D Overview of the characteristics of each cloned TCR. Title contains information on the cognate antigenic peptide in brackets, the CDR3α and CDR3ß amino acid sequences, the TCR-Vß usage as well as the functional avidity EC50 as determined in Fig. 2. Plot shows the HLA-restriction assessed via Jurkat Lucia NFAT reporter assays: fold difference of normalized luminescence signals of pulsed versus unpulsed LCLs or T2B35 cells is plotted for target cells sharing the same HLA-allotype (orange, x), target cells sharing the same HLA-allele group (light blue, HLA-allele group variants) and HLA-mismatched target cells (dark blue, line). The autologous LCL0 from the CD4+ T cell donor is DQB1*06:09, DRB1*13:02 and DRB3*03:01 positive. Graph shows mean ± SD of n = 1-5 independent experiments. Exact number n for each target cell tested is indicated subscripted in the y-axis labels. (A–D) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact significant p ≥ 0.0001 from left to right (1A) p = 0.0014, p = 0.0001; (1C) p = 0.0009, p = 0.0282, p = 0.0089, p = 0.0427.
