Generation of TCR-modified T Cells

Jurkat 76 was transduced with CD8α and CD8β cDNAs to generate Jurkat 76/CD8 as reported previously18 (link). Jurkat 76 or Jurkat 76/CD8 transfectants were further transduced with individual TCRβ genes along with SIG35α, and the transfectants were purified using anti-CD3 Microbeads (Miltenyi Biotec). K562-based artificial antigen-presenting cells (aAPC) stably express mutated HLA–A2 in conjunction with CD80 and CD8323 (link)26 (link). The mutated HLA–A2 molecules bear two amino acid substitutions at positions 227 and 228 that abrogate the interaction with CD857 (link). aAPC was engineered to constitutively secrete IL-21 to enable T cell expansion26 (link)58 (link). PG13-based packaging cells were generated by first transfecting Phoenix Eco cells with 20 μg of DNA for each construct using the TransIT-293 reagent (Mirus Bio, Madison, WI). PG13 cells were then transduced with supernatant from Phoenix Eco cells. PG13-derived retrovirus supernatant was utilized to transduce TCR genes into Jurkat 76, Jurkat 76/CD8, and human primary T cells as reported previously18 (link). A retroviral vector encoding ΔNGFR alone was employed as a control vector.

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Nakatsugawa M., Rahman M.A., Yamashita Y., Ochi T., Wnuk P., Tanaka S., Chamoto K., Kagoya Y., Saso K., Guo T., Anczurowski M., Butler M.O, & Hirano N. (2016). CD4+ and CD8+ TCRβ repertoires possess different potentials to generate extraordinarily high-avidity T cells. Scientific Reports, 6, 23821.

Publication 2016

anti-CD3 Microbeads TransIT-293 reagent

Amino acid substitutions Anti cd3 Antigen presenting cells Bear Cdnas Cells First cells Hla a2 Human Il 21 Microbeads Retroviral T cells Tcr genes Tcrβ genes Vector

Corresponding Organization :

Other organizations : Princess Margaret Cancer Centre, University Health Network, University of Toronto

Top 5 similar protocols

Variable analysis

independent variables

Transduction of Jurkat 76 cells with CD8α and CD8β cDNAs to generate Jurkat 76/CD8 cells
Transduction of Jurkat 76 or Jurkat 76/CD8 cells with individual TCRβ genes along with SIG35α
Generation of K562-based artificial antigen-presenting cells (aAPC) that stably express mutated HLA–A2 in conjunction with CD80 and CD83
Engineering of aAPC to constitutively secrete IL-21

dependent variables

Purification of transfectants using anti-CD3 Microbeads (Miltenyi Biotec)

control variables

Mutated HLA–A2 molecules with two amino acid substitutions at positions 227 and 228 that abrogate the interaction with CD8

positive controls

PG13-based packaging cells generated by first transfecting Phoenix Eco cells with DNA constructs using the TransIT-293 reagent, followed by transduction of PG13 cells with the supernatant from Phoenix Eco cells
PG13-derived retrovirus supernatant used to transduce TCR genes into Jurkat 76, Jurkat 76/CD8, and human primary T cells

negative controls

A retroviral vector encoding ΔNGFR alone was employed as a control vector

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