Mono-allelic expression of HLA-I alleles for HLA-peptidomics analysis

Adi Nagler¹*, Shelly Kalaora²*, Chaya Barbolin³, Michal Alon³, Polina Greenberg³, Gal Yagel³, Aviyah Peri³, Yishai Levin⁴, Yardena Samuels^3**

¹ Dana-Farber Cancer Institute; Harvard Medical School, Boston, Massachusetts, United States

² Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States

³ Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

⁴ The de Botton Institute for Protein Profiling, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel

* Contributed equally to this work

** Corresponding author: yardena.samuels@weizmann.ac.il

Lead Contact: Prof. Yardena Samuels

Abstract 

The identification of human leukocyte antigen (HLA)-bound peptides using HLA-peptidomics (also called “Immunopeptidomics”) analysis has provided an in-depth understanding of antigen presentation. A possible obstacle to such an analysis is the co-expression of multiple HLA alleles, thus reducing the specificity of this assay. Here we present a mono-allelic strategy for HLA class-I (HLA-I) expression, which can be further utilized for HLA peptidome profiling by liquid chromatography-tandem mass spectrometry (LC/MS-MS). Using this application combined with the overexpression of genes of interest has been demonstrated to enable the identification of SARS-CoV-2-derived HLA-I peptides. We thus demonstrate a strategy for systematic HLA- allele specific analysis of antigen presentation.

Keywords: HLA-I, mono-allele, lentivirus, peptides

Graphical Abstract

Background 

The HLA-I molecules (HLA-A/B/C) are heterodimeric proteins presented on the surface of human nucleated cells, encoded by genes displaying a large degree of polymorphism with hundreds of allelic variations (Jin and Wang, 2003; Robinson et al., 2015). HLA-I bound peptides arise from intracellular proteins that are cleaved by the proteasome and displayed by the surface HLA-I proteins serving as an immunological signature for the detection by CD8+ T cells via their T cell receptor.

Characterization of the HLA-bound peptides by immunopurification and LC-MS/MS has revealed rules for peptide binding to the HLA molecules and has been performed on various HLA alleles.(Abelin et al., 2017; Bassani-Sternberg et al., 2015; Hickman et al., 2004; Rammensee et al., 1995; Vita et al., 2019) This unbiased powerful approach can elucidate novel peptides presented by the HLA-I alleles informing the rational design of CD8+ T cell inducing vaccines to control pathogen infections or to combat tumors. 

Here we provide a mono-allelic approach that has been used to detect the presentation of SARS-CoV-2 viral peptides (Nagler et al., 2021), bacterial peptides (Kalaora et al., 2021) and has also been demonstrated efficient in the detection of neoantigens recognized by CD8+ T cells (Bear et al., 2021; Peri et al., 2021). Briefly, the protocol can be broadly outlined as a three-step process. In the first step, DNA sequences coding for the HLA-I alleles are cloned into a lentivirus vector with neomycin selection and lentiviral particles are produced. In the second step, human B-LCL 721.221 (HLA-I null) cells are infected and selected with antibiotics. Productive over-expression of HLA-I is validated approximately two weeks post-infection via flow cytometry. In the third step, the 721.221 HLA-I cells are re-infected with lentivirus containing a gene of interest. This HLA-I monoallelic expression protocol can be further implemented for HLA-I peptidome analysis in which the HLA-I peptide complexes are purified and the HLA-I presented peptides are eluted off the complexes. These peptides are sequenced with LC-MS/MS and identified by a database search.

Materials and reagents

1. dsDNA sequences coding for HLA-I alleles (IPD-IMGT/HLA, twist bioscience)
2. pCDH-CMV-MCS-EF1α-Neo vector (SBI, #CD514B-1)
3. PMD2.G (#12259, Addgene)
4. psPAX2 (#12260, Addgene)
5. HEK293T cells (ATCC)
6. Human B-LCL 721.221 (ATCC)
7. XbaI (NEB, #R0145L)
8. NotI (NEB, #R0189L)
9. T4 DNA ligase (NEB, #M0202L)
10. Lipofectamine 2000 (Invitrogen, #11668027)
11. Mouse monoclonal anti-pan HLA-I (clone W6/32, Purified from HB95 hybridoma cells)
12. RPMI 1640 Medium(BI, 01-100-1A)
13. Opti-MEM™ I Reduced Serum Medium (Gibco, 11058021)
14. Steriflip-GP Sterile Centrifuge Tube Top Filter Unit (Millipore, SE1M179M6)
15. Geneticin™ Selective Antibiotic (G418 Sulfate, ThermoFisher, 10131035)
16. 50ml conical tubes
17. 10 cm² dishes
18. 6 Well Cell Culture Plate

Equipment

1. Tissue culture hood
2. Incubator 37°C
3. BD LSR II (BD Biosciences)

Software

1. FACSDiva (BD, biosciences, https://www.bdbiosciences.com/en-us/products/software/instrument-software/bd-facsdiva-software)
2.  FloJo (FlowJo, LLC, https://www.flowjo.com)

Procedure

1. DNA sequences coding for HLA-I alleles were taken from IPD-IMGT/HLA database (https://www.ebi.ac.uk/ipd/imgt/hla/allele.html).
2. Synthetic dsDNA of HLA-I alleles were purchase from Twist bioscience with flanking XbaI and NotI restriction sites.
3. Coding sequence was cloned into pCDH-CMV-MCS-EF1α-Neo vector.
4. Lentivirus production was performed by co-transfection of 4.4 µg of the HLA-I plasmid, with 2.2 µg of PMD2.G and 3.4 µg of psPAX2 into 3X10⁶ HEK293T seeded on a 10 cm² dish in RPMI 1640 medium, using Lipofectamine 2000, as described by the manufacturer.
5. After 24 hours post-transfection, media was changed to Opti-MEM™ Reduced Serum Medium. 72 hours post-transfection, media was collected, filtered using Steriflip-GP Sterile Centrifuge Tube, aliquoted and stored at −80°C.
6. 2 X10⁶ Human B-LCL 721.221 were seeded in a 6 well cell culture plate, infected with 1 ml of HLA-I lentivirus and after 72 hours were selected with 800 µg/ml neomycin (G418) for 2 weeks.
7. HLA-I expression was confirmed via flow cytometry, using the monoclonal antibody W6/32.
8. The B-LCL 721.221 HLA-I expressing cells could be re-infected with a vector containing a gene of interest and an alternative antibiotic selection such as Puromycin.
9. B-LCL 721.221 HLA-I expressing cells could be further expanded and used for HLA-peptidomics, which is based on the immunoaffinity purification of the HLA complexes followed by the extraction of the HLA-bound peptides and their further analysis by mass spectrometry (Kalaora et al., 2016; Kalaora and Samuels, 2019; Kalaora et al., 2018) 

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