CD8+ T cells were negatively selected using RosetteSep Human CD8+ T Cell Enrichment Cocktail (Stemcell). Afterward, naive CD8+ T cells (CD8+CD45RA+CD45RO−) were isolated by FACS to 91.0 ± 2.5% purity (Aria III; BD Biosciences). Lentiviral transduction with vectors containing TCR expression constructs pCCL.IGRPopt.eGFP recognizing the autoantigen IGRP (29 (link),34 (link)) or LV.Mart1.TCR.RK recognizing the non–β-cell antigen MART1 (27 (link),35 ) and subsequent T-cell expansion were carried out as outlined in Supplementary Fig. 1 . CTL avatars were cryopreserved after 9 days of expansion.
Protocol detail
Generation of Antigen-Specific CTL Avatars
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Antigen
Aria
Autoantigen
Cd45ro
Cd8 t cells
Human
Mart1
Stemcell
T cell
Vectors
β cell
Corresponding Organization : University of Florida
Other organizations : Florida College
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Variable analysis
independent variables
- Lentiviral transduction with vectors containing TCR expression constructs pCCL.IGRPopt.eGFP recognizing the autoantigen IGRP
- Lentiviral transduction with vectors containing TCR expression constructs LV.Mart1.TCR.RK recognizing the non–β-cell antigen MART1
- Purity of naive CD8+ T cells (CD8+CD45RA+CD45RO−) after FACS isolation (91.0 ± 2.5%)
- CD8+ T cells were negatively selected using RosetteSep Human CD8+ T Cell Enrichment Cocktail (Stemcell)
- T-cell expansion was carried out as outlined in Supplementary Fig. 1
- Positive control: None specified
- Negative control: None specified
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