Expi293 expression media

HEK293 cells were cultured in EXPI293 expression media (Life Technologies), and cell concentration and viability were determined using trypan blue (Gibco, Life Technologies). Cells were routinely cultured at 37°C, in 8% CO₂, in vented Erlenmeyer flasks (Corning, Surrey, UK), shaking at 180 rpm and sub-cultured every 3–4 days, at a seeding density of 0.5 × 10⁶ cells/mL.
Cells were transfected with DNA-lipid complexes comprising DNA and Expifectamine293 (Life Technologies) at a 1 µg DNA:1 or 3 × 10⁶/mL cell ratio for target and antibodies, respectively, and prepared according to manufacturer’s protocol. Transfected cells were incubated for 24 h, or up to 7 days, respectively, prior to use.

VLPs were produced by transiently transfecting Expi293 cells (Life Technologies) grown in Expi293 expression media (Life Technologies) on an orbital shaker at 37 °C and 8% CO₂. Cells were transfected with a plasmid vector expressing Rev-independent HIV-1 Gag-Pol (pHDM-Hgpm2 plasmid; PlasmID Repository, Harvard Medical School) or a Gag-EGFP fusion protein (HIV-1 HXB2 Gag-EGFP expression vector; NIH AIDS Reagent Program). To generate CD4-VLPs and CD4-CCR5-VLPs, cells were cotransfected with a second plasmid (cDNA sequences of CD4 and CCR5 subcloned into the pHAGE-CMV-IRES-ZsGreen plasmid; PlasmID Repository, Harvard Medical School) encoding CD4 alone or CD4 and CCR5 at a DNA ratio of 4:1 Gag-Pol:CD4-(CCR5). Control VLPs were generated by expression of HIV-1 Gag-Pol alone. Expi293 cells were also transfected with CD4 in the absence of Gag-Pol to make CD4⁺ extracellular vesicles. At 48 to 72 h posttransfection, cells were centrifuged at 350 × g for 8 min, and supernatants were collected and passed through a 0.45-μm syringe filter.

Expi293F (Thermo Fisher Scientific) cells were cultured in Expi293 Expression media (Thermo Fisher Scientific) at 37°C, 125 rpm, and 8% CO₂. The generation of tapasin-KO cells is previously described (50 (link)). HLA-A*01:01-knockin/tapasin-KO cells were generated by first transfecting Expi293F cells with EcoRV-linearized pCEP4-HLA-A*01:01 (untagged). The cells were selected with hygromycin B (100 μg/ml) and FACS-sorted for HLA-A*01:01–positive cells after staining with anti-HLA-A*01:01 clone 8.L.101 (USBio, H6098-05). Following sorting, the HLA-A*01:01–positive cells were transfected with plasmids encoding human codon–optimized Cas9 and tapasin guide RNA, as previously described (50 (link)) and then FACS-sorted for cells that no longer processed HLA-A*01:01 to the cell surface.
TAPBPR-TM was cloned into pCEP4 (Invitrogen) with a canonical N-terminal MHC-I signal peptide, FLAG tag, linker, luminal domain of hTAPBPR, linker, and TM domain of HLA-G (Addgene, no. 135500). Mouse and chTAPBPR were cloned using the same design and included C98A and C94A, respectively. HLA-A*01:01 was subcloned by removing the myc tag from pCEP4-myc- HLA-A*01:01, as previously described (27 (link)). Site-directed mutagenesis of TAPBPR-TM mutants was achieved using overlap extension PCR. The inserts of all plasmids were confirmed by Sanger sequencing.

Suspension Expi293HEK cells (Thermo Fisher Scientific) were cultured in Expi293 expression media (Thermo Fisher) at 110–130 rpm in a humidified Multitron cell incubator (Infors HT) at 37 °C with 7% CO₂. Cells were transfected transiently using ExpiFectamine 293 transfection reagent (Thermo Fisher Scientific) with either pENTR4-LPTOS-CyRPA-SnoopTagJr or pENTR4-LPTOS-CyRPA. Transfection enhancers (Thermo Fisher) were added 16–18 h after transfection. Cell supernatants were harvested 4 days post transfection, filtered through 0.45 μm syringe filters and the proteins were purified using Capture Select C-tag Affinity Matrix (Thermo Fisher Scientific)⁵⁵ followed by size exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (GE Healthcare). The proteins were eluted with 50 mM Tris•borate pH 7.25.