HLA-C-restricted viral epitopes are associated with an escape mechanism from KIR2DL2+ NK cells in Lassa virus infection

Abstract

Background: Lassa virus (LASV) is the etiologic agent of an acute hemorrhagic fever endemic in West Africa. Natural killer (NK) cells control viral infections in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their ligands. LASV infection is associated with defective immune responses, including inhibition of NK cell activity in the presence of MHC-class 1⁺-infected target cells.

Methods: We compared individual KIR and HLA-class 1 genotypes of 68 healthy volunteers to 51 patients infected with LASV in Sierra Leone, including 37 survivors and 14 fatalities. Next, potential HLA-C1, HLA-C2, and HLA-Bw4 binding epitopes were in silico screened among LASV nucleoprotein (NP) and envelope glycoprotein (GP). Selected 10-mer peptides were then tested in peptide-HLA stabilization, KIR binding and polyfunction assays.

Findings: LASV-infected patients were similar to healthy controls, except for the inhibitory KIR2DL2 gene. We found a specific increase in the HLA-C1:KIR2DL2 interaction in fatalities (10/11) as compared to survivors (12/19) and controls (19/29). We also identified that strong of NP and GP viral epitopes was only observed with HLA-C molecules, and associated with strong inhibition of degranulation in the presence of KIR2DL⁺ NK cells. This inhibitory effect significantly increased in the presence of the vGP₄₂₀ variant, detected in 28.1% of LASV sequences.

Interpretation: Our finding suggests that presentation of specific LASV epitopes by HLA-C alleles to the inhibitory KIR2DL2 receptor on NK cells could potentially prevent the killing of infected cells and provides insights into the mechanisms by which LASV can escape NK-cell-mediated immune pressure.

Keywords: HLA-C; KIR-L; Lassa virus; NK cells; Viral escape.

Fig. 1

Stabilization of HLA Class-1-Transfected 721.221-ICP47 Cells Pulsed with NP and GP LASV Peptides. (a) Representative histograms of HLA-B*27:07 stabilization without peptide (None), with negative NP₂₃₀ control peptide, or with GP₁₉₂ peptide. Peptides were added at a final concentration of 100 μM. Surface level expression of HLA class-1 molecules was determined by flow cytometry using the anti-pan HLA class-1 W6/32 mAb. (b) Quantification of different HLA class-1 stabilization (HLA-C*07:01, C*06:03, and B*27:05) (N = 5) in the absence (None) or in the presence of negative NP₂₃₀ control peptide, or of selected peptides (NP₂₁₈, GP₁₉₂, and GP₂₅₆). Data represents the mean of 5 experiments and error bars indicate standard deviation. (c) Representative histograms of HLA-B*53:03 stabilization without peptide (None) or with wild-type NP₃₅₈ and vNP₃₅₈ variant peptides. (d-e) Representative histograms and quantification of 5 HLA-C*08:01 stabilizations without peptide (None) or with wildtype GP₄₂₀ and vGP₄₂₀ variant peptides. Cells were then stained with isotype control (single line) or with W6/32 monoclonal antibody (solid grey line). Data are expressed as the geometric mean of fluorescence (gMFI).

Fig. 2

Binding of KIR-Fc by HLA Class-1-Transfected 721.221-ICP47 Cells Pulsed with NP and GP LASV Peptides. (a) Representative dot plots of KIR-Fc staining of HLA class-1-transfected 721.221-ICP47 cells pulsed without peptide (None), with negative NP₂₃₀ control peptide, or GP₁₉₂ peptide. Data are shown with HLA class-1-transfected 721.221-ICP47 cells transfected with HLA-C*06:02, C*07:01, and B*27:01, and stained with KIR2DL1-Fc, KIR2DL2-Fc, or KIR3DL1-Fc, respectively. (b-c) Representative dot plots and quantification of KIR2DL2-Fc and KIR2DL1-Fc staining (N = 5) from 721.221-ICP47 cells transfected HLA-C*08:03 or C*05:01, respectively, and pulsed without peptide (None) or with wild-type GP₄₂₀ and vGP₄₂₀ variant peptides.

Fig. 3

Functional NK cell activity in the presence of HLA class-1-transfected 721.221-ICP47 cells pulsed with NP and GP LASV peptides. (a) Representative polyfunctional assay (degranulation and cytokine production) of KIR2DL2⁺ NK-NAM cell line, in the presence of HLA class-1-transfected 721.221-ICP47 cells pulsed without peptide (None), with negative NP₂₃₀ control peptide, or with GP₁₉₂ peptide. Polyfunctionality was determined using SPICE and PESTLE software (NIH). Single, double or triple cytokine producing cells are color coded in pies as indicated in the Pie slice legend. Pie chart arcs represent overlapping production of cytokines and are color coded as indicated in the corresponding legend. (b) Quantification of CD107a frequency (n = 5) in selected KIR⁺ NK cell lines in the presence of HLA-C-transfected 721.221-ICP47 cells pulsed without peptide (None), with negative NP₂₃₀ control peptide, or with GP₁₉₂ peptide. (c) Representative polyfunctional assay of KIR2DL1⁺ NK-RIM cell line in the presence of HLA-C*05:01-transfected 721.221-ICP47 cells pulsed without peptide (None) or with wild-type GP₄₂₀ and vGP₄₂₀ variant peptides. (d) Quantification of CD107a frequency (n = 5) in select KIR⁺ NK cell lines in the presence of HLA-C transfected-721.221-ICP47 cells pulsed without peptide (None) or with wild-type GP₄₂₀ and vGP₄₂₀ variant peptides.