Anti phycoerythrin pe magnetic beads

The STEAP1¹³⁰/HLA-A*02:01-specific TCR transgene was introduced into T cells using the retroviral vector pMP-71 [16 (link)]. Purified CD4⁺ and CD8⁺ T cells were activated with anti-CD3/CD28 Dynabeads™ (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) according to manufacturer’s recommendation and 100 U/mL recombinant human (rh) IL-2 (Novartis) and 2 ng/mL rh IL-15 (Bio-Techne, Minneapolis, MI, USA) for CD8⁺ T cells or 50 U/mL rh IL-2 and 5 ng/mL rh IL-7 (Bio-Techne, Minneapolis, MI, USA) for CD4⁺ T cells was added. Two days later, spin infection with virus supernatant from the packaging cell line RD114 was performed, as previously published [28 (link)]. On the next day, T cells were re-infected with the same approach. After verifying successful transduction via FACS staining, T cell subsets were purified with anti-phycoerythrin (PE) magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) coupled to STEAP1¹³⁰/HLA-A*02:01-specific PE labeled multimers (in-house production), as described previously [29 (link)]. Detailed assessment of STEAP1¹³⁰/HLA-A*02:01-specific TCR functionality and potential cross reactivity was published previously [30 ].

The CHM1³¹⁹/HLA-A*02:01-specific TCR transgene was introduced into T cells using the retroviral vector pMP-71 [12 (link),13 ]. Purified CD8⁺ T cells were activated with 3 µg/mL OKT3 according to manufacturer’s recommendation and 100 U/mL IL-2 (Novartis) was added for T cell proliferation. Two days later, spin infection with virus supernatant from the packaging cell line RD114 was performed. On the next day, T cells were re-infected with the same approach. After verifying successful transduction via FACS staining, T cell subsets were purified with anti-phycoerythrin (PE) magnetic beads (Miltenyi Biotec) coupled to CHM1³¹⁹/HLA-A*02:01-specific PE labeled multimers as previously published [6 (link)]. Upon generation, T cells were frozen and stored in liquid nitrogen for further use.