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. 2019 Aug 13;93(17):e00634-19.
doi: 10.1128/JVI.00634-19. Print 2019 Sep 1.

Identification of Immunodominant HIV-1 Epitopes Presented by HLA-C*12:02, a Protective Allele, Using an Immunopeptidomics Approach

Takayuki Chikata  1   2 , Wayne Paes  3 , Tomohiro Akahoshi  1 , Thomas Partridge  3 , Hayato Murakoshi  1   2 , Hiroyuki Gatanaga  1   4 , Nicola Ternette  5 , Shinichi Oka  1   4 , Persephone Borrow  3 , Masafumi Takiguchi  6   2
Affiliations

Identification of Immunodominant HIV-1 Epitopes Presented by HLA-C*12:02, a Protective Allele, Using an Immunopeptidomics Approach

Takayuki Chikata et al. J Virol. .

Abstract

Despite the fact that the cell surface expression level of HLA-C on both uninfected and HIV-infected cells is lower than those of HLA-A and -B, increasing evidence suggests an important role for HLA-C and HLA-C-restricted CD8+ T cell responses in determining the efficiency of viral control in HIV-1-infected individuals. Nonetheless, HLA-C-restricted T cell responses are much less well studied than HLA-A/B-restricted ones, and relatively few optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C alleles have been defined. Recent improvements in the sensitivity of mass spectrometry (MS)-based approaches for profiling the immunopeptidome present an opportunity for epitope discovery on a large scale. Here, we employed an MS-based immunopeptidomic strategy to characterize HIV-1 peptides presented by a protective allele, HLA-C*12:02. We identified a total of 10,799 unique 8- to 12-mer peptides, including 15 HIV-1 peptides. The latter included 2 previously reported immunodominant HIV-1 epitopes, and analysis of T cell responses to the other HIV-1 peptides detected revealed an additional immunodominant epitope. These findings illustrate the utility of MS-based approaches for epitope definition and emphasize the capacity of HLA-C to present immunodominant T cell epitopes in HIV-infected individuals, indicating the importance of further evaluation of HLA-C-restricted responses to identify novel targets for HIV-1 prophylactic and therapeutic strategies.IMPORTANCE Mass spectrometry (MS)-based approaches are increasingly being employed for large-scale identification of HLA-bound peptides derived from pathogens, but only very limited profiling of the HIV-1 immunopeptidome has been conducted to date. Notably, a growing body of evidence has recently begun to indicate a protective role for HLA-C in HIV-1 infection, which may suggest that despite the fact that levels of HLA-C expression on both uninfected and HIV-1-infected cells are lower than those of HLA-A/B, HLA-C still presents epitopes to CD8+ T cells effectively. To explore this, we analyzed HLA-C*12:02-restricted HIV-1 peptides presented on HIV-1-infected cells expressing only HLA-C*12:02 (a protective allele) using liquid chromatography-tandem MS (LC-MS/MS). We identified a number of novel HLA-C*12:02-bound HIV-1 peptides and showed that although the majority of them did not elicit T cell responses during natural infection in a Japanese cohort, they included three immunodominant epitopes, emphasizing the contribution of HLA-C to epitope presentation on HIV-infected cells.

Keywords: CTL; HIV-1; HLA-C; LC-MS/MS; epitope; mass spectrometry; peptide.

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Figures

FIG 1
FIG 1
Characteristics of HLA-C*12:02-bound peptides identified by LC-MS/MS. (a) Summary of the number of unique peptides identified in HIV-1 NL4-3-infected .221-C1202 cells. The numerical overlap in peptide identification between 2 technical replicates is displayed in area-proportional Venn diagrams. (b) Length distribution of 8- to 12-mer peptides eluted from NL4-3-infected .221-C1202 cells. (c) Sequence logos of 8- to 11-mer peptides eluted from NL4-3-infected .221-C1202 cells.
FIG 2
FIG 2
Confirmation of HLA-C*12:02 binding of HIV-1 peptides eluted from NL4-3-infected .221-C1202 cells. (a) Analysis of binding of 9 eluted peptides to HLA-C*12:02. Binding of peptides at concentrations from 0.1 to 100 μM (left) was measured by performing an HLA class I stabilization assay using RMA-S-C1202 cells. Results are plotted as the expression index, defined as the ratio of the mean fluorescence intensity (MFI) of peptide-pulsed RMA-S-C1202 cells to that of control (non-peptide-pulsed) cells kept at 26°C. (b) Analysis of binding of 4 eluted peptides to HLA-C*12:02. Binding of peptides at concentrations from 100 to 300 μM (right) was measured by performing an HLA class I stabilization assay using RMA-S-C1202 cells. Results are plotted as described above for panel a. (c) Refolding of the positive-control peptide Nef-MY9 (a known HLA-C*12:02-restricted epitope) with HLA-C*12:02 heavy chain and β2-microglobulin. Analysis of the products generated by fast-performance liquid chromatography shows that a complex with a predicted molecular mass coincident with the molecular mass of the complex of HLA-C*12:02, peptide, and β2m was generated. (d) Analysis of refolding of 4 eluted peptides that did not exhibit detectable binding to HLA-C*12:02 and Pol-IY11 (a known HLA-C*12:02-restricted epitope) in the stabilization assay.
FIG 3
FIG 3
Evaluation of T cell responses to the eluted HIV-1 peptides and identification of responses to the HLA-C*12:02-restricted Env-RL9 epitope. (a) Screening for T cell responses to the eluted peptides in 20 chronically HIV-1-infected HLA-C*12:02+ Japanese individuals. T cell responses to 13 eluted peptides (tested at a concentration of 1 μM) were analyzed by an ex vivo IFN-γ ELISpot assay. A positive response was defined as >100 spots/106 PBMCs. (b) Analysis of the HLA restriction of the T cell response to Env-RL9. The response of Env-RL9-expanded bulk T cells from subject KI-1407 (A*2402/–, B*5201/–, and C*1202/–) to Env-RL9 peptide-prepulsed 721.221 cell lines, each expressing a single HLA allele shared with KI-1407, was analyzed by an ICS assay. (c) Analysis of the HLA restriction of the T cell response to Vif-DY9. The response of the Vif-DY9-expanded bulk T cells from subject KI-1394 (A*0201/2402, B*3501/5201, and C*0303/1202) to Vif-DY9 peptide-prepulsed 721.221 cell lines, each expressing a single HLA allele shared with KI-1394, was analyzed by an ICS assay. (d) Recognition of NL4-3-infected cells by Env-RL9-specific CD8+ T cells. The response of Env-RL9-expanded bulk T cells to uninfected .221-C1202 cells and 721.221 cells and .221-C1202 cells infected with NL4-3 was analyzed by an ICS assay. Graphs at the right show representative fluorescence-activated cell sorter (FACS) data.
FIG 4
FIG 4
Effects of L-to-M mutation of the C-terminal residue in the Env-RL9 epitope on T cell recognition and binding affinity of mutant peptides. (a) Logo of the region of sequence containing the Env-RL9 peptide in the autologous virus of 20 chronically HIV-1-infected HLA-C*12:02+ Japanese individuals who were analyzed for T cell responses to this epitope. (b) Recognition of Env-RL9 and Env-RL9-9M peptides by CD8+ T cells. The responses of bulk T cells expanded by stimulation with Env-RL9 to .221-C1202 cells prepulsed with Env-RL9 or Env-RL9-9M peptides at concentrations from 1 to 300 nM were analyzed by an ICS assay. (c) Binding of Env-RL9 and Env-RL9-9M peptides to HLA-C*12:02. The relative binding abilities of the two peptides were measured by performing HLA class I stabilization assays with RMA-S-C1202 cells using peptide concentrations from 0.1 to 300 μM. Results are plotted as the expression index, defined as the ratio of the MFI of peptide-pulsed RMA-S-C1202 cells to that of control (non-peptide-pulsed) cells kept at 26°C. (d) T cell responses to Env-RL9 (tested at a concentration of 1 μM) were analyzed in 40 HLA-C*12:02+ individuals by an ELISpot assay. Subjects were defined as responders (res) if they exhibited Env-RL9 responses of >100 spots/106 PBMCs. Plasma viral loads (pVL) and CD4 T cell counts of responder and nonresponder (non-res) subjects are shown. The statistical significance of differences in plasma viral loads and CD4 counts between Env-RL9 nonresponders and responders was analyzed using a Mann-Whitney test.
FIG 5
FIG 5
Sequence variation in Japanese subjects in HLA-C*12:02-binding HIV-1 peptides to which no T cell responses were detected in infected individuals. (a) Logos of the autologous virus sequences of 10 peptides to which no T cell responses were observed in 20 chronically HIV-1-infected HLA-C*12:02+ Japanese individuals in whom T cell response screening was performed. (b) List of sequences of peptides chosen for synthesis based on the worldwide clade B consensus sequence in the LANL database and the consensus sequence in the 20 chronically HIV-1-infected HLA-C*12:02+ Japanese individuals evaluated here. Amino acid residues that differ in the worldwide subtype B consensus sequence and the consensus sequence in the 20 infected individuals studied here are underlined.
FIG 6
FIG 6
HLA-C*12:02 binding of four 20-patient consensus sequences and eight additional variant peptides and T cell responses to these peptides. (a) Binding of four 20-patient consensus sequences and eight additional variant peptides to HLA-C*12:02. Peptide binding was evaluated using 100 μM peptide in an HLA class I stabilization assay with RMA-S-C1202 cells. Results are plotted as the expression index, defined as the ratio of the MFI of peptide-pulsed RMA-S-C1202 cells to that of control (non-peptide-pulsed) cells kept at 26°C. (b) T cell responses to four 20-patient consensus and eight additional variant peptides in 20 chronically HIV-1-infected HLA-C*12:02+ Japanese individuals. Responses to the peptides indicated (tested at a concentration of 1 μM) were analyzed by an IFN-γ ELISpot assay. The peptides boxed in red are 3 positive-control peptides (Gag-RI8, Gag-WV8, and Env-RL9). A positive response was defined as >100 SFC/106 PBMCs.
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