Journal of Immunological Methods 464 (2019) 22–30 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim Expression and characterization of soluble epitope-defined major histocompatibility complex (MHC) from stable eukaryotic cell lines T ⁎ Amanda L. Wooster, Trevor S. Anderson, Devin B. Lowe Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center, Abilene, TX 79601, United States ARTICLE INFO ABSTRACT Keywords: MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) have greatly advanced the Major histocompatibility complex (MHC) class understanding of CD8+ T cells under normal and diseased states. However, recombinant MHC class I compo- I nents (comprising MHC class I heavy chain and β2 microglobulin) are usually produced in bacteria following a Single-chain trimer lengthy purification protocol that requires additional non-covalent folding steps with exogenous peptide for Tetramer complete molecular assembly. We have provided an alternative and rapid approach to generating soluble and Dextramer fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using a Sleeping Beauty trans- Sleeping beauty transposon system Stable cell line generation poson system. Importantly, this method culminates in generating stable cell lines that reliably secrete epitope- defined MHC class I molecules into the tissue media for convenient purification and eventual biotinylation/ multimerization. Additionally, MHC class I components are covalently linked, providing the opportunity to produce a diverse set of CD8+ T cell-specific reagents bearing peptides with various affinities to MHC class I. 1. Introduction inclusion bodies through a laborious lysis/solubilization process. A defined MHC class I peptide is then added alongside β2 microglobulin The major histocompatibility complex (MHC) class I and II mole- and heavy chain in a precise folding reaction mixture that requires cules play an integral role in T cell development and peripheral effector several days to complete prior to affinity chromatography (AC) pur- responses (Alcover et al., 2018). MHC class I is retained on the plasma ification of properly folded peptide/MHC and later biotinylation steps. membrane of nucleated cells and consists of a multi-unit heavy chain Although this standard production process works to eventually yield whose tertiary structure is stabilized by β2 microglobulin through non- excellent reagents for immunologic assays, there exist a number of covalent forces (Wieczorek et al., 2017). To provide specific binding to major disadvantages. Namely, the standard method [i] is time con- antigen specific CD8+ T cells, MHC class I usually retains a short 8–10 suming, [ii] requires substantial levels of raw ingredients (particularly amino acid peptide within the MHC peptide binding groove that is purified MHC class I peptide), and [iii] cannot guarantee large-scale derived from degraded intracellular proteins (hereafter referred to as production of properly folded peptide/MHC molecules based on pre- peptide/MHC). dicted peptide binders. For example, it is extremely difficult to stably Our understanding of basic T cell properties and dynamics under a produce MHC molecules bearing peptides with low-to-moderate affinity variety of normal and diseased settings has been greatly advanced by to the MHC peptide binding groove. the ability to produce and purify peptide/MHC for use in assays to To circumvent these perceived drawbacks (particularly in stabi- specifically engage the T cell receptor (TCR). Arguably the most lizing peptide binding to the MHC peptide binding groove), previous widespread approach incorporates fluorochrome-conjugated peptide/ efforts have revealed the ability to engineer and produce peptide/MHC MHC multimers (e.g., tetramers) for analyzing or isolating antigen- molecules in bacteria by covalently joining the MHC class I peptide, β2 specific CD8+ T cells from biological samples (Khairnar et al., 2018; microglobulin, and heavy chain with discrete amino acid linkers (de- Soen et al., 2003). Peptide/MHC generation has continued similarly to signated single-chain trimers [SCTs]) (Yu et al., 2002). For most SCTs the process outlined in the landmark work by Altman et al. (1996). reported, these engineered proteins fold correctly and specifically en- Briefly, β2 microglobulin and MHC class I heavy chain (containing a gage CD8+ T cells as tetramers (Mitaksov et al., 2007), irrespective of BirA tail) are individually expressed in E. coli and later purified from the arti ficial linker design (Hansen et al., 2009). However, this Abbreviations: MHC, Major histocompatibility complex; TCR, T cell receptor; SCT, single-chain trimer; CHO, Chinese hamster ovary; SB, sleeping beauty; sHeavy, secretory MHC class I heavy chain; AC, affinity chromatography; Ab, antibody ⁎ Corresponding author: 1718 Pine Street, Office 1306, Abilene, TX 79601, United States. E-mail address: [email protected] (D.B. Lowe). https://doi.org/10.1016/j.jim.2018.10.006 Received 15 September 2018; Received in revised form 16 October 2018; Accepted 16 October 2018 Available online 19 October 2018 0022-1759/ © 2018 Elsevier B.V. All rights reserved. A.L. Wooster et al. Journal of Immunological Methods 464 (2019) 22–30 particular SCT method still utilizes a bacterial expression system and then amplified and cloned into the pSBbi-pur transposon vector (kindly requires substantial purification and refolding efforts. provided by Dr. Eric Kowarz [Addgene plasmid # 60523]) using the SfiI We, therefore, sought an alternative method to potentially improve restriction enzyme sites (Kowarz et al., 2015). Plasmid transformations the production of peptide/MHC based on the SCT approach. Our cur- were carried out in chemically-competent NEB-5 alpha E. coli (NEB) rent work highlights the ability to rapidly generate eukaryotic cell lines using ampicillin selection. All vector constructs were confirmed by re- that stably express and secrete peptide/MHC into the tissue media for striction enzyme digestion and DNA sequencing. purification and biotinylation. This modified protocol could potentially provide a much faster/convenient route to generating properly folded 2.4. Sleeping Beauty (SB) transposon system peptide/MHC with minimal user intervention, especially for MHC class I targets with high demand (such as the model OVA epitope SIINFEKL). Parental cell lines were transiently transfected with transposon-re- Since we have adopted the SCT strategy, MHC molecules presenting a lated vectors using Lipofectamine reagent (Thermo Fisher Scientific) as range of class I peptides (i.e., low-to-high binding affinity) can also be directed by the manufacturer. A plasmid encoding the SB 100× reliably generated. Additionally, it remains possible that these eu- transposase (pCMV[CAT]T7-SB100; designated Vector 1), a gift from karyotic-derived peptide/MHC molecules more accurately recapitulate Dr. Zsuzsanna Izsvak (Addgene plasmid # 34879), and a transposon binding dynamics with TCRs in downstream assays (Schmidt and Lill, plasmid containing the necessary inverted terminal repeats (pSBbi-pur; 2018). designated Vector 2) were used. Both vectors were provided con- currently to stably integrate peptide/MHC transgenes into cells. Briefly, 2. Materials and methods 1×105 cells were plated in 24-well plates (Corning, Corning NY) and exposed to Vector 1 (12.5 ng)/Vector 2 (488 ng) in a total volume of 2.1. Mice 500 μl media as similarly described (Kowarz et al., 2015). Culture media was replenished with 2 ml fresh media after 24 h and 48 h, Female 6–8-week-old C57BL/6J (stock #000664) and OT-1 (stock whereupon cells were exposed to lethal doses of puromycin (CHO-S - #003831) mice were purchased from The Jackson Laboratory (Bar 10 μg/ml; 4 T1–5 μg/ml) (Invivogen, San Diego, CA). Fresh media Harbor, Maine, USA) and maintained in micro-isolator cages under containing puromycin was provided every 2–3 days as needed. Gen- sterile conditions. Animals were humanely euthanized and spleens/ erally, actively dividing cells were ready for expansion by 2 weeks, with lymph nodes harvested and combined for Ficoll gradient centrifugation virtually all cell clones expressing peptide/MHC molecules. (GE HealthCare, Piscataway, NJ). The lymphocyte interphase was then subjected to ACK lysis and eventual CD8+ T cell purification using 2.5. SDS-PAGE and western blot MACS bead positive selection as instructed by the manufacturer (Miltenyi Biotec, Cambridge, MA). Purified CD8+ T cells were ali- To remove extraneous tissue media components such as surfactants, quoted in 90% FBS/10% DMSO and stored in liquid nitrogen until use. CHO cell supernatants were passed onto PD10 columns (GE Healthcare) All mouse procedures were followed in accordance with TTUHSC and concentrated/washed in PBS using a 30 MWCO Amicon centrifugal IACUC-approved protocols. unit (MilliporeSigma, Burlington, MA). Protein concentration was de- termined using a BCA protein assay kit (Thermo Scientific Fisher) and 2.2. Cell lines and culture sample aliquots stored at −80 °C until use. Protein purity was assessed with SDS-PAGE and coomassie blue staining while protein identity was FreeStyle™ Chinese Hamster Ovary (CHO-S) (Thermo Fisher confirmed through western blotting. Briefly, proteins were resolved on Scientific, Waltham, MA) and 4T1 (ATCC, Manassas, VA) cells were a 4%/12% polyacrylamide gel, transferred to a PVDF membrane utilized for in vitro studies. CHO-S