Jurkat cells

Human RPE (ARPE-19) cells were obtained from American Type Culture Collection (ATCC). HT-29 cells were purchased from Gefan Biotechnology Co., Ltd. SH-SY5Y cells were purchased from the Institute of Cells Biology, Shanghai, China. Jurkat cells were bought from Beyotime Biotechnology, Shanghai, China. ARPE 19 cells and SH-SY5Y cells were cultured in DMEM/F-12 (1:1) medium (HyClone, Utah, USA) supplemented with 2.5 mM l-glutamine, 15 mM HEPES buffer, 10% fetal bovine serum (FBS) (Gibco, NY, USA) and 1% (v/v) penicillin (100 U/ml)/streptomycin (100 μg /ml) (Hyclone, Utah, USA) at 37 °C, 5% CO₂. HT-29 cells and Jurkat cells were cultured in RPMI-1640 medium (Gibco, Beijing, China) supplemented with 10% FBS and 1% (v/v) penicillin (100 U/ml)/streptomycin (100 μg /ml) (P/S). All cell line cells used in this study were the third to seventh passage after resuscitation. Mouse primary RPE cells were isolated from the eyes of 5-day-old C57BL/6 mice and cultured as previously described^{52 (link)}.

Cas9 plasmids Px330 and Px461 were purchased from Addgene and guide sequences were annealed as described (12 (link)). Neon transfection of plasmids into cells was carried out as per manufacturer protocol for Jurkat cells (Thermo Fisher).

KIF1B_S918F-specific T cells were obtained by co-transfecting Jurkat cells (Promega) with 500 ng each of TCRα and TCRβ chain RNA together with 300 ng each of CD8α and CD8β RNA, using a Neon electroporation system (Thermo Fisher Scientific) as previously described⁴⁴ .
Antigen-specific CD8 T cell clones (i.e. obtained from isolation and expansion of primary cells) or transfected Jurkat cells (2 × 10⁵ cells) were incubated for 40 min at 4 °C with cognate NTAmers containing streptavidin-phycoerythrin and Cy5-labeled pMHC monomers in 50 µL FACS buffer (PBS supplemented with 0.5% BSA and 2 mM EDTA), as described^{15 (link)}. Irrelevant T cells were used to measure background signal and values were systematically subtracted. Specific gMFI values were plotted and analyzed using the GraphPad Prism software (v.7, or v.9, GraphPad) fitting a one phase exponential decay model.

Both strains of C57BL/6j and BALB/c mice (male, 10–12 weeks old) were received from breeding colonies in the animal facility at the Jack Bell Research Centre (Vancouver, BC, Canada), and all the experiments using these mice were carried out following an approved protocol as stated above.
A single cell suspension of splenocytes was prepared from the spleens of naïve mice as described previously [15 (link)]. Both HK-2 cells (an immortalized human kidney proximal tubular cell line) and Jurkat cells (an immortalized human T cell line) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown at 37°C in a humidified atmosphere of 5% CO₂. Mouse splenocytes and Jurkat cells were grown in RPMI 1640 complete medium (Invitrogen, Burlington, ON, Canada) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. HK-2 cells were grown in K1 complete culture medium as described previously [23 (link)]. HF was purchased from the Toronto Research Chemicals Inc. (Catalogue number: H102500, Toronto, ON, Canada). RAPA was from Caymen Chemical Company (Catalogue number: 13346, Ann Arbor, MI, USA). CsA was from Novartis (Catalogue number: 486205, Mississauga, ON, Canada). Anti-mouse anti-CD3 antibody was purified from anti-mouse CD3 hybridoma ascite [24 (link)].

Jurkat cells were purchased from ATCC (Clone E6-1). Primary T cells were purified from the spleens of 6–12-week-old B6 or DKI mice using negative-selection T-cell purification columns (CL101) from Cedarlane. Purified T cells and Jurkat cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine, 50 μM β-mercaptoethanol, and 100 μM gentamicin. Plat E and Plat GP cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 2 mM glutamine, 50 μM β-mercaptoethanol, and 100 μM gentamicin.