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. 2022 May 25;96(10):e0043222.
doi: 10.1128/jvi.00432-22. Epub 2022 Apr 27.

Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection

Takayuki Chikata  1 , Wayne Paes  2 , Nozomi Kuse  1 , Thomas Partridge  2 , Hiroyuki Gatanaga  1   3 , Yu Zhang  1 , Kimiko Kuroki  4 , Katsumi Maenaka  4 , Nicola Ternette  2 , Shinichi Oka  1   3 , Persephone Borrow  2 , Masafumi Takiguchi  1
Affiliations

Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection

Takayuki Chikata et al. J Virol. .

Abstract

There is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells. IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.

Keywords: CD8+ T cells; HIV-1; HLA-C; LC-MS/MS; epitopes; peptides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Characteristics of HLA-C*14:02 and C*14:03-bound peptides identified by LC-MS/MS. (A) Number of unique peptides identified in HIV-1 NL4-3-infected .221-C1402 and .221-C1403 cells. The frequency of p24+ cells in both NL4-3-infected .221-C1402 cells and NL4-3-infected .221-C1403 cells was 50 to 60%. The numerical overlap in peptide identification between HLA-C*14:02 and -C*14:03 is displayed in area-proportional Venn diagrams. (B) Length distribution of 8- to 13-mer peptides eluted from NL4-3-infected .221-C1402 or NL4-3-infected .221-C1403 cells. (C) Sequence logos of 9-mer peptides shared in the data sets for both alleles and 9-mer peptides detected only in eluates from either NL4-3-infected .221-C1402 or NL4-3-infected .221-C1403 cells. (D) Sequence logos of all 8- to 11-mer peptides in eluates from NL4-3-infected .221-C1402 or .221-C1403 cells.
FIG 2
FIG 2
HLA-C*14:02/14:03 binding of HIV-1 peptides eluted from NL4-3-infected .221-C1402 and .221-C1403 cells as assessed by HLA stabilization assay using RMA-S cell lines. (A) Histogram plots showing surface expression of HLA-C*14:02 or HLA-C*14:03 on RMA-S-C1402 or RMA-S-C1403 cells at 26°C and after cells cultured at 26°C had been warmed to 37°C for 3 h. MFI values of cells cultured at 26°C were 27.4 for HLA-C*14:02 and 28.1 for HLA-C*14:03. MFI values of cells cultured at 37°C for 3 h were 5.0 for HLA-C*14:02 and 6.54 for HLA-C*14:03. (B) Analysis of binding of 14 HIV-1 NL4.3 peptides identified by LC-MS/MS to HLA-C*14:02 (blue) or HLA-C*14:03 (red) as measured in HLA class I stabilization assays using RMA-S-C1402 or RMA-S-C1403 cells. (C) Analysis of binding of 9 additional variant peptides to HLA-C*14:02 (blue) or C*14:03 (red). For panels B and C, peptides were tested at a concentration of 100 μM. Results are plotted as percent surface HLA-I expression relative to that at 26°C, calculated as the ratio of the MFI of peptide-pulsed RMA-S-C1402 or RMA-S-C1403 cells to that of control (non-peptide-pulsed) cells kept at 26°C, multiplied by 100. Means and standard deviations (SDs) from triplicate assays are shown. Statistical analyses were performed by unpaired t test. The P values were corrected for multiple testing. *, P < 0.05 and q < 0.05; **, P < 0.01 and q < 0.03; ***, P < 0.001 and q < 0.005.
FIG 3
FIG 3
T cell responses to HLA-C*14:02- and/or HLA-C*14:03-binding HIV-1 peptides. (A) T cell responses to HLA-C*14:02/03-binding peptides in 20 chronically HIV-1-infected Japanese individuals with HLA-C*14:02 or HLA-C*14:03. T cell targeting of 14 putative epitopes was analyzed by testing responses to 14 NL4-3 sequence-based peptides and 9 peptides that were sequence variants thereof in ex vivo IFN-γ ELISPOT assays (peptides tested at a concentration of 1 μM). A positive response was defined as >100 spots/106 PBMCs (indicated by the horizontal dotted line). (B) Correlation between peptide binding to HLA-C*14:02 or HLA-C*14:03 and the frequency of responders to these peptides in HLA-C*14:02+ (blue) and HLA-C*14:03+ (red) individuals for NL4-3 sequence-based versions of the peptides only (left) or both NL4-3 sequence-based and variant peptides (right). Statistical analyses were performed using Pearson's correlation test.
FIG 4
FIG 4
HLA-C*14:02- and HLA-C*14:03-restricted T cell responses to 6 putative epitope peptides. (A) Histogram plots showing surface expression of HLA-C*14:02 or HLA-C*14:03 on .221-C14:02 or .221-C14:03 cells. The relative expression level of HLA-C*14:02 versus HLA-C*14:03 [(MFI of .221-C1402 stained with DT9 − MFI of .221-C1402 stained with the second antibody)/(MFI of .221-C1403 stained with DT9 − MFI of .221-C1403 stained with the second antibody)] is 1.3. (B and C) Gag LY9-, Gag AM9-, Pol KM9-, Nef YT9-, Nef IF8-, and Nef AL9-specific bulk T cell populations were expanded by stimulating responders’ PBMCs with each peptide. The ability of these peptide-specific bulk T cells to recognize peptide-pulsed .221 cells expressing HLA-C*14 molecules was then analyzed by ICS assay. (B) T cell recognition of .221 cells expressing the patient’s autologous HLA-C*14 allele and non-HLA-C*14-expressing cells pulsed with 1 μM peptide. (C) T cell recognition of HLA-C*14:02 or HLA-C*14:03-expressing cells pulsed with the indicated peptides at concentrations from 0.01 to 10 nM was analyzed by an ICS assay. The results are means and SDs from triplicate assays.
FIG 5
FIG 5
Recognition of NL4-3-infected .221-CD4 cells by peptide-specific CD8+ T cells. The response of bulk T cells specific for 6 peptides to NL4-3-infected .221-C1402 cells, NL4-3-infected .221-C1403 cells, NL4-3-infected 721.221 cells, uninfected .221-C1402 cells, and uninfected .221-C1403 cells was analyzed by ICS assay. The frequencies of p24+ cells were as follows: NL4-3-infected .221-C1402 cells, 52%; NL4-3-infected .221-C1403 cells, 54%. The results are means and SDs from triplicate assays.
FIG 6
FIG 6
Difference in frequency of responses to HLA-C*14-restricted epitopes in HLA-C*14:02+ and HLA-C*14:03+ individuals. (A) T cell responses to 6 HLA-C*14:02/C*14:03 epitopes were analyzed in 30 HLA-C*14:02+ and 30 HLA-C*14:03+ treatment-naive individuals living with HIV-1. T cell responses to each peptide tested at a concentration of 1 μM were analyzed by ex vivo IFN-γ ELISPOT assay. Mean plasma viral load (log) and CD4 count in HLA-C*14:02+ or HLA-C*14:03+ individuals were 4.74 copies/mL and 337 cells/μL and 4.58 copies/mL and 333 cells/μL, respectively. A positive response was defined as >100 spots/106 PBMCs (indicated by the horizontal dotted lines). Statistical analyses were performed by Mann-Whitney test. *, P < 0.05. (B) T cell responses to Gag, Nef, and total epitopes. Statistical analyses were performed by Mann-Whitney test. *, P < 0.05.
FIG 7
FIG 7
Peptide-induced stabilization of HLA-C*14:02 and C*14:03 molecules. (A) The relative affinity of binding of the 6 epitope peptides to HLA-C*14:02 (blue) or HLA-C*14:03 (red) was analyzed by testing HLA class I stabilization on RMA-S-C1402 or RMA-S-C1403 cells at peptide concentrations from 0.1 to 100 μM. Results are plotted as the percent increase in surface HLA-I expression relative to that at 26°C, calculated as the ratio of the MFI of peptide-pulsed RMA-S-C1402 or RMA-S-C1403 cells to that of control (non-peptide-pulsed) cells at 26°C multiplied by 100. Statistical analysis was performed by unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) The surface stability of HLA-C14-peptide complexes was determined using RMA-S-C1402 (blue) and RMA-S-C1403 (red) cells. The cells were incubated at 26°C with a 100 μM concentration of each of the 6 epitope peptides for 1 h and then warmed to 37°C for 3 h. After the excess external peptide had been washed off, cells were collected at 1 h. The relative MFI of HLA-C staining was plotted as the percentage of the maximal stabilization induced by peptide binding. Statistical analysis was performed by unpaired t test. The P values were corrected for multiple testing. *, P < 0.05 and q < 0.1; **, P < 0.01 and q < 0.05.
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