Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8
+ T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I-bound peptides on HIV-infected cells. We demonstrate that HIV-1-derived spliced peptides comprise a relatively minor component of the HLA-I-bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8 + T cell responses relatively infrequently during infection, CD8 + T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection.
T cell epitope; human immunodeficiency virus; immunopeptidome; peptide splicing; proteasome.
Copyright © 2019 the Author(s). Published by PNAS.
Conflict of interest statement
The authors declare no competing interest.
Workflow for identification of HLA-I–bound spliced peptides by de novo sequencing. HLA class Ia (HLA-Ia)-deficient CD4.221 cells individually transfected with a panel of HLA-I alleles, or the C8166 cell line (expressing 5 distinct HLA-Ia alleles), were lysed and HLA-I–peptide complexes isolated using the pan anti–HLA-I antibody W6/32. Peptides were eluted and then separated by high-performance liquid chromatography fractionation prior to LC-MS/MS sequencing. Following spectral assignment to contiguous peptide sequences in the UniProt database, sequence interpretations (including all L/I permutations) for each DNUP residing within the top 5 ALC scores in each scan were considered. Scans containing single amino acid variants of contiguous sequences within the canonical proteome were removed and the remainder fragmented in silico into 2 splice partners and matched to the annotated UniProt proteome to create a list of sDNUPs. Post hoc database matching with artificial spliced proteins was not implemented (see also
Materials and Methods and ). SI Appendix, Fig. S1
Proportion and redundancy of identified sDNUPs. (
A) The number of unique, L/I redundant, and ALC redundant sDNUPs in datasets from the indicated cell lines are shown at each ALC cutoff. ( B) The number of nonspliced, sDNUP, and other/noncanonical (non- cis-spliced) peptides contributing to the MS-detectable immunopeptidome of the indicated cell lines at each ALC cutoff. ( C) The proportion of assigned sDNUPs (expressed as a percentage of all unique MS spectra) at each ALC threshold.
Characteristics of sDNUPs and nonspliced peptides. (
A) Peptide length distributions of nonspliced peptides and sDNUPs at the indicated ALC thresholds, expressed as a percentage of all unique 8- to 12-mers. ( B) Sequence motifs of nonspliced and sDNUP 9-mers identified at each ALC threshold as generated by Seq2logo. The number of peptides (n) incorporated at each ALC cutoff is indicated below each panel. See also . ( SI Appendix, Fig. S2 C) Percentage of nonspliced peptides and sDNUPs at the indicated ALC cutoffs predicted to bind to the relevant HLA-I allele by NetMHCpan4.0. ( D) Percentage of the nonspliced peptides and sDNUPs tested of those with ALC scores in the indicated ranges that exhibited spectral matches to corresponding synthetic peptide standards. See also Dataset S3.
HIV-1 spliced peptides presented on HLA-I are generated by (immuno)proteasomal splicing. (
A) Spectral matching of HLA-I–bound HIV-1–derived sDNUPs with synthetic peptide standards. ( B) Schematic illustrating generation of the spliced peptide products from HIV-1 precursor peptides following (immuno)proteasomal digestion. See also . ( SI Appendix, Fig. S3 C) RMA-S HLA-I stabilization assays confirming binding of HIV-1–derived spliced peptides to relevant HLA-I alleles. The relative fluorescence intensities of peptide-pulsed to non-peptide-pulsed cells are depicted at log-fold titrations of peptide. Data are expressed as the mean relative fluorescence values ± the SD from triplicate experiments.
HIV-1 spliced peptides are cross-recognized by CD8
+ T cell responses primed to overlapping contiguous epitopes in infected individuals. ( A) Responses detected in IFN-γ ELISpot assays ex vivo ( Top) or following T cell expansion by in vitro culture with the contiguous epitope LADQLIHLY ( Bottom) to the HIV-1 IIIB contiguous LADQLIHLY (LY9) and spliced FSD-QLIHLY (FD9) epitopes and the predominant subtype B consensus sequence (see also ) version of the spliced epitope FSE-QLIHLY (FE9) in 4 A*01:01 SI Appendix, Fig. S4 + HIV-infected individuals (700010390, 701010114, 701010529, and 700010945). Cells stimulated with PHA and medium only (unstim) served as positive and negative controls, respectively. Data are expressed as SFU per million PBMC, and the mean ± the SD of duplicate wells are shown. ( B) Sequence alignments of HIV-IIIB and predominant autologous viral sequences present in the 4 HIV-1 + A*01:01 + individuals at the indicated sampling time points during acute and/or chronic infection. Amino acid residues that differ from the IIIB sequence are shown in red. See also . ( SI Appendix, Fig. S4 C) Schematic illustrating HIV-1 precursor peptides subjected to (immune)proteasomal digestion and the spliced and optimal length contiguous epitope products detected by MS. ( D) RMA-S HLA-I stabilization assays comparing the relative binding affinities of contiguous (LADQLIHLY) and spliced viral epitopes (FSD-QLIHLY, FSE-QLIHLY, and FTE-QLIHLY) to HLA-A*01:01. Data are expressed as the mean relative fluorescence values ± SD from triplicate experiments. ( E) Responses detected in an IFN-γ ELISpot assay following T cell expansion by in vitro culture with the contiguous epitope LADQLIHLY to the former peptide and to the IIIB FSD-QLIHLY (FD9) and autologous virus FTE-QLIHLY (FT9) versions of the spliced epitope peptide. Data are expressed as in A. ( F) Abundance values of unique contiguous and spliced peptides originating from immunoproteasomal digests of HIV polypeptides described in Figs. 4 B and C. Group medians are indicated; ** P<0.01, two-tailed unpaired Mann-Whitney t test.
Mutations abrogate CD8
+ T cell recognition of both contiguous and spliced epitopes. ( A) Responses detected to mutant contiguous LADQLIHMY (L8M), LADQLIHLF (LF9), and LADQLIHLH (LH9) and mutant spliced FSE-QLIHMY (F8M), FSE-QLIHLH (FH9), and FSE-QLIHLF (FF9) peptides in cultured IFN-γ ELISpot assays following T cell expansion by in vitro culture with autologous contiguous viral epitopes LADQLIHLY (LY9) or TADQLIHLY (TY9). Data are expressed as SFU per million PBMC, and the mean ± SD of duplicate wells are shown. ( B) RMA-S HLA-I stabilization assays comparing the relative binding affinities of dominant mutant versions of the nonspliced (LADQLIHLF and TADQLIHMY) and spliced (FSE-QLIHLF and FSE-QLIHMY) viral epitopes to HLA-A*01:01. Data are expressed as the mean relative fluorescence values ± SD from triplicate experiments.
CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.
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