Production of Class II MHC Proteins in Lentiviral Vector-Transduced HEK-293T Cells for Tetramer Staining Reagents

Abstract

Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC-IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable lentiviral vector-transduced derivatives of HEK-293T cells. The expression design includes allele-specific peptide ligands tethered to the amino-terminus of the MHC-II β chain via a protease-cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide exchange, enabling high-throughput production of many distinct MHC-IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label-free methods, native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp multimer reagents. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and expression line creation Support Protocol 1: Six-well assay for estimation of production cell line yield Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His-tags Basic Protocol 2: Cultures for production of Class II MHC proteins Basic Protocol 3: Purification of Class II MHC proteins by anti-leucine zipper affinity chromatography Alternate Protocol 1: IMAC purification of His-tagged Class II MHC Support Protocol 3: Protein concentration measurements and adjustments Support Protocol 4: Polishing purification by anion-exchange chromatography Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation Basic Protocol 4: Peptide exchange Basic Protocol 5: Analysis of peptide exchange by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Alternate Protocol 2: Native isoelectric focusing to validate MHC-II peptide loading Basic Protocol 6: Multimerization Basic Protocol 7: Staining cells with Class II MHC tetramers.

Keywords: Class II MHC multimers; Class II MHC tetramers; antigen-specific T cells; flow cytometry; lentiviral transduction; protein engineering; protein expression; protein purification.

Figure 1.. Design of lentiviral vectors for class II peptide/MHC protein expression.

A. The 5’ long terminal repeat (LTR), packaging signal (Ψ) rev-response element (RRE) and 3’ self-inactivating (SIN) LTR allow one round of lentivirus replication and transduction. Class II MHC ɑ or β chain expression is driven by the CMV enhancer/promotor. An internal ribosome entry site (IRES) allows coexpression of a surrogate marker (eGFP, Thy 1.1, etc.) to facilitate screening and fluorescence-activated cell sorting of transduced cell lines. The woodchuck Hepatitis virus posttranscriptional regulatory element (WPRE) enhances transgene expression. B. β chain transgene design. SS; signal sequence. 3C; HRV-3C cleavage site in a 20 aa flexible linker. C. ɑ chain transgene design. BSP85; BirA recognition site.

Figure 2.. HP-SEC analysis of peptide exchange reactions for two alleles.

3 μg each of post exchanged DRB1*0401 or DPB1*0201/DPA*0103 is analyzed on a gel filtration column (TSKgel G3000SWXL) attached to a Shimadzu HPLC at 1ml/min flow rate with PBS as the mobile phase. Each run is monitored for presence of higher order aggregates as seen with DPB1*0201.

Figure 3.. Representative chromatogram of an IMAC run and the corresponding SDS-PAGE analysis of the eluted fractions.

Supernatant from a production run of a cell line expressing Mamu-DRB*w201 was purified on 5 ml HisTrap FF Crude column and the fractions corresponding to the 280 nm absorbance peak are analyzed on SDS-PAGE. Fractions 8–13 that contain the eluted protein are then pooled and buffer exchanged.

Figure 4.. Analysis of biotinylation of Class II MHC protein by streptavidin precipitation (sap) followed by SDS-PAGE.

Purified I-A^b protein is assessed for extent of biotinylation by sap-assay followed by SDS-PAGE analysis. Either Sepharose 4b (−) or streptavidin (+) beads were added to purified I-A^b protein and incubated for one hour at room temperature with intermittent mixing. After brief centrifugation, supernatants are boiled in loading buffer and run on SDS_PAGE. RNase A protein, used as loading control is as indicated. The absence of I-A^b protein with the added streptavidin beads indicates complete biotinylation.

Figure 5.. Representative SDS-PAGE analysis of 3C cleavage.

Purified DRB1*0401 is cleaved with 3C protease for 14 hours at 25°C and the cleaved MHC is then processed on GSTrap FF column to remove the 3C protease. Protease cleavage is then monitored on SDS-PAGE by analyzing the fractions of uncleaved (U), protease cleaved prior to (Pre) or after (post) processing on GSTrap column.

Figure 6.. MALDI analyses of I-A^b loaded with peptides.

I-A^b with formerly tethered GDTAGTTHYGSLPQKSQGSGGSGGSGLEVLFQ peptide was mock exchanged (expected molecular mass of 3081) or exchanged with VRVSYYGPKTSPVQ (expected molecular mass of 1580), RSRYLTAAAVTAVLQ (expected molecular mass of 1620) or EYLSFSCYLSVT (expected molecular mass of 1411).

Figure 7.. Native IEF gel of I-A^b loaded with peptides with distinct net charges.

I-A^b with tethered TTHYGSLPQKSQ peptide (plus additional linkers) was uncleaved (U), HRV-3C-cleaved (C), or cleaved and then incubated in peptide exchange reactions (all other lanes). Peptides in exchange reactions in labeled lanes are as follows: (M) mock/no-peptide control, (+4) LPKPPKPVSKMRMATPLLMGALPM, (+3) KPVSKMRMATPLLMQAL, (+2) DITYKVHLATPINSR, (+1) DIYKGVYQFKSV, (0) PGVMYAFTTPLISFF, (−2) SQEYSGSVANEANVY. All exchange reactions contained HLA-DM, which was removed as described in Basic Protocol 4, prior to analysis.

Figure 8.. The source of streptavidin-PE conjugates can have a dramatic effect on staining.

The class II MHCp complex of I-Ab/LCMV.GP66–77. DIYKGVYQFKSV was mixed with streptavidin PE from three different suppliers and used to stain splenocytes from a C57B6 mouse 8 days after infection with LCMV Armstrong. All stains had the same concentration of I-Ab/LCMV.GP66–77. (A) FACS plots of cells gated on CD4+ positive cells inside a lymphocyte scatter gate. (B) Commercial streptavidin-conjugated PE (SA-PE) from various manufacturers (a, b, or c) were analyzed by size-exclusion chromatography on a 7.8 mm I.D. × 30 cm TSKgel G4000swxl (TOSOH Bioscience LLC) column with sodium phosphate buffer, pH 7.2, as the mobile phase at a flow rate of 1 ml/min. Relative fluorescence profiles of the conjugates are as shown. Standard proteins for calibration of the column include thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa); all were obtained from GE Healthcare (Cytvia). This figure and the associated legend are reproduced from (Altman and Davis, 2016).