CRISPR-Cas9 screening reveals a distinct class of MHC-I binders with precise HLA-peptide recognition

Abstract

Human leukocyte antigen (HLA) class-I molecules present fragments of the cellular proteome to the T cell receptor (TCR) of cytotoxic T cells to control infectious diseases and cancer. The large number of combinations of HLA class-I allotypes and peptides allows for highly specific and dedicated low-affinity interactions to a diverse array of TCRs and natural killer (NK) cell receptors. Whether the divergent HLA class-I peptide complex is exclusive for interactions with these proteins is unknown. Using genome-wide CRISPR-Cas9 activation and knockout screens, we identified peptide-specific HLA-C∗07 combinations that can interact with the surface molecules CD55 and heparan sulfate. These interactions closely resemble the HLA class-I interaction with the TCR regarding both the affinity range and the specificity of the peptide and HLA allele. These findings indicate that various proteins can specifically bind HLA class-I peptide complexes due to their polymorphic nature, which suggests there are more interactions like the ones we describe here.

Keywords: Biochemistry; Bioinformatics; Biological sciences; Immunology; Natural sciences.

Graphical abstract

Figure 1

Receptor-ligand CRISPR-Cas9 activation screen reveals that CD55 interacts with HLA-C∗07:01-VRIG tetramers (A) Schematic of the receptor ligand CRISPR-Cas9 activation screen. K562 cells transduced with a genome-wide activation library were stained with a pool of three HLA tetramers (HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, and HLA-C∗07:01-VRIG), and enriched gRNAs in stained cells were identified using NGS. (B) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. (C) K562 cells stably expressing dCas9 and transduced with a gRNA upregulating CD55 or a control guide were stained with the HLA-A, -B, -C, or tetramers as in (A) or with HLA-E∗01:01-VMAP tetramers and analyzed by flow cytometry. (D) In vitro co-immunoprecipitation of recombinant CD55-Fc with HLA-A∗02:01-NLVP, HLA-B∗07:02-TPRV, HLA-C∗07:01-VRIG, or HLA-E∗01:01-VMAP tetramers. (E) Three different cell lines (HeLa, PC-3M, or SiHa) that express CD55 endogenously were stained for CD55 (top) or with HLA-C∗07:01-VRIG tetramers (bottom) and analyzed by flow cytometry. (F) HeLa wild-type or HeLa CD55 KO cells were stained with αCD55 or HLA-C∗07:01-VRIG tetramers and analyzed by flow cytometry. All data except (B) represent at least three independent experiments. CRISPRa, CRISPR activation screen; TMs, tetramers; WT, wild-type; KO, knockout. Related to Figure S1 and Table S1.

Figure 2

Interaction of CD55 with HLA-C∗07:01-VRIG tetramers is allotype and peptide specific (A) HEK293T cells were transfected with a plasmid containing GFP and a truncation mutant of CD55 and analyzed by flow cytometry. GFP+ positive cells were analyzed for staining with HLA-C∗07:01-VRIG. Each mutant removes an additional SCR domain from CD55. Data are represented as mean ± SD. (B) HeLa cells were stained with HLA-C∗07:01-VRIG tetramers after pre-incubation with CD55 blocking antibodies targeting different SCR domains on CD55 and analyzed by flow cytometry. (C) HeLa cells were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with the VRIG peptide and analyzed by flow cytometry. (D) HeLa cells were stained with HLA-C∗07:01 tetramers loaded with different alanine mutants of the VRIGHLYIL peptide and analyzed by flow cytometry. (E) CD55-Fc was immobilized on a Prot-G chip for SPR data using HLA-C∗07:01-VRIG tetramers as analyte to determine interaction on and off rates and K_D. Response units were measured with increasing concentrations of HLA-C∗07:01-VRIG tetramers. All data represent at least three independent experiments, except (E), which represents a biological duplicate. FL, full length. Related to Figure S2, Tables S2 and S3.

Figure 3

CRISPR-Cas9 activation and KO screens identify an interaction between HLA-C∗07:02-YRFR and heparan sulfate chains (A) HeLa cells were stained with a variety of HLA-C∗07:02 tetramers loaded with different peptides and analyzed by flow cytometry. (B) The indicated cell lines were stained with HLA-C∗07:02-YRFR tetramers and analyzed by flow cytometry. Staining is normalized to unstained levels of that cell line. (C) Schematic of CRISPR-Cas9 KO screen in MelJuSo cells. Cells were stained with HLA-C∗07:02-YRFR, and enriched gRNAs in non-binding cells were identified using NGS. (D) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and genes involved in the heparan sulfate biosynthesis pathway are depicted in purple. Hits used for further validation are annotated. (E) Schematic of the CRISPR-Cas9 activation screen. K562 cells stably expressing dCas9 transduced with a genome-wide activation library were stained with HLA-C YRFR∗07:02 tetramers, and enriched gRNAs in positive cells were identified. (F) SigmaFC scores of genes from two replicate screens. SigmaFC scores were calculated using PinAplPy, and top hits are annotated. Proteoglycans of interest are depicted in purple. CRISPRa, Crispr activation screen. Related to Figure S3, Table S1 and S4.

Figure 4

Interaction between HLA-C∗07:02-YRFR and heparan sulfate chain is allotype and peptide specific (A) K562 cells stably expressing dCas9 were transduced with a gRNA activating SDC1, SDC2, or SDC4, or a control guide and were stained with either HLA-C∗07:02-YRFR tetramers or an antibody targeting heparan sulfate chains prior to analysis using flow cytometry. (B) MelJuSo cells were transduced with a gRNA knocking out GOLPH3, EXT1, EXT2, or UGDH, stained with HLA-C∗07:02 tetramers, and analyzed by flow cytometry. Data are represented as MFI relative to the C∗07:02-YRFR tetramer staining on WT MelJuso cells. (C) MelJuSo cells were treated with heparinase II and subsequently stained with either HLA-C∗07:02-YRFR tetramers (left) or with an antibody targeting heparan sulfate chains (right), followed by analysis by flow cytometry. Data are relative staining to unstained levels. (D) MelJuSo cells were stained with HLA-C∗07:02-YRFR tetramers pre-incubated with recombinant heparan sulfate chains when indicated and analyzed by flow cytometry. (E) MelJuSo were stained with either HLA-C∗07:01 or HLA-C∗07:02 tetramers loaded with YRFR peptide and analyzed by flow cytometry. (F) MelJuSo cells were stained with HLA-C∗07:02 tetramers loaded with iterative alanine mutants of the YRFRFRSVY peptide and analyzed by flow cytometry. (G) Space-filling model of HLA-C∗07:01-VRIG, HLA-C∗07:02-YRFR, and the camelid antibody cAb-005. Shown is a top view of the different proteins. Residues identified to contact the binding partner of each molecule are depicted in red, and the calculated surface binding area is noted below the figure. Figures created using PyMol. All data represent at least three independent experiments, and bar graphs represent mean ± SD. gCtrl, guide control; SDC, syndecan; OE, overexpression; KO, knockout. Related to Figure S4 and Table S3.