FIG 6.

HIV-naive CD8⁺ T cells can be transduced and engineered to express HIV-specific TCRs that bind to KK10-specific dextramer. (A) The TCR α and β genes of effective (EC1) and ineffective (IC1 and IC5) CD8⁺ T-cell clones were cloned into a lentiviral transfer vector driven by a spleen focus-forming virus (SFFV) viral promoter. TCR α and β genes were linked by a self-cleaving P2A peptide followed by a T2A self-cleaving peptide-linked GFP reporter cassette to allow visualization of the transduced cells by flow cytometry. (B) Jurkat/MA cells were transduced with lentiviral vectors expressing EC1-TCR, ICI-TCR, IC5-TCR, or a control glutamic acid decarboxylase peptide (residues 555 to 567)-specific TCR linked with a T2A sequence to a GFP reporter gene or a control lentiviral vector expressing only the GFP reporter gene. Expression of a KK10-specific TCR was determined by flow-cytometric analysis 48 h after transduction after staining with HLA-B*2705-KK10 dextramer or anti-human TCR αβ antibody. (C) Primary human CD8⁺ T cells, isolated from PBMC of HIV-naive donors, were activated and transduced with lentiviral vectors expressing EC1-TCR, IC1-TCR, or IC5-TCR linked with a T2A sequence to a GFP reporter gene. After 8 days of culture, the CD8⁺ T cells were evaluated by flow cytometry for transduction by quantifying the fraction of GFP⁺ cells. After gating on the GFP⁺ cells, their expression of the KK10-specific TCR was determined by measuring their binding to HLA-B*2705-KK10 dextramer. Results shown are representative of 3 experiments with 2 different donors. SSC, side scatter. (D) Primary human CD8⁺ T cells were activated and transduced with the lentiviral vectors expressing EC1-TCR or IC1-TCR linked to a GFP reporter gene by a T2A peptide or expressed by an IRES-driven GFP reporter gene. After 4 days of culture, the CD8⁺ T cells were evaluated by flow cytometry for transduction by quantifying the fraction of GFP⁺ cells. The values presented are the means ± SEM of the percentage of GFP-positive CD8⁺ T cells from 9 different experiments using primary CD8⁺ T cells from 3 different HIV-naive donors. (E) After gating, the GFP⁺ cells were evaluated for their expression of the KK10-specific TCR by measuring their binding to HLA-B*2705-KK10 dextramer. The values presented are the means ± SEM of the percentage of HLA-B*2705-KK10 dextramer binding-CD8⁺ T cells from 3 different experiments using primary CD8⁺ T cells from 2 different HIV-naive donors. *, P < 0.05; **, P < 0.01.