An early HIV mutation within an HLA-B*57-restricted T cell epitope abrogates binding to the killer inhibitory receptor 3DL1

Abstract

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

Fig. 1.

Viral evolution and CD8⁺ T cell recognition of the TW10 G9E variant in patient CH58. (A) Sequence analysis of the TW10 epitope in single genome amplified (SGA) viral RNA for patient CH58, illustrating the gradual decline of the wildtype (WT) TW10 epitope, coincident with the emergence of the G9E and T3N variants. Times are given in days from enrollment (E). G9E was detected as the dominant virus between days 45 and 85, and gradually declined thereafter. The T3N variant subsequently emerged as the dominant variant and was prevalent from day 154 onwards (data are from reference 11). (B) Interfeon-γ ELISPOT assays were used compare recognition of the founder viral TW10 epitope (TSTLQEQIGW) with that of the G9E and T3N variants in donor CH58. PBMCs were incubated overnight in the presence of varying amounts of each peptide (10⁻¹¹ to 10⁻⁵ M). Responses are reported as spot-forming cells (SFC) per million PBMCs.

Fig. 2.

Generation of specific HLA-B*5703 tetramers by UV-ligand exchange. Biotinylated, UV-sensitive HLA-B*5703-9MT4 complexes were photoilluminated and rescued in the presence of HLA-B*5703-restricted KAFSPEVIPMF (KF11), TSTLQEQIGW (TW10), HTQGYFPDW (HW9), and QASQEVKGW (QW9) epitopes prior to tetramer generation via conjugation to an extravidin-phycoerythrin (PE)-labeled fluorochrome. (A) The functionality and specificity of UV-exchanged B*5703-KF11 tetramers was confirmed by staining a B*5703-KF11-restricted CD8⁺ T cell clone with conventional KF11 tetramer and KF11, TW10, and QW9 tetramers produced by UV exchange. Comparable staining was observed using both KF11 tetramers (open boxes), while the irrelevant UV exchange tetramers failed to bind KF11-specifc T cells. (B) The specificities of B*5703-TW10 and HW9 tetramers were evaluated by staining HW9- and TW10-specific CD8⁺ T cell lines (day 12) derived from a single donor in vitro.

Fig. 3.

Integrity and specificity of soluble KIR3DL1-Fc. (A) The integrity of KIR3DL1-Fc was assessed by antibody binding, using the KIR3DL1-specific antibodies Z27 (which binds a linear epitope) and DX9 (conformation dependent). After the level of saturation binding of each antibody was corrected for the amount of KIR3DL1 protein immobilized in the SPR flow cell, the proportion of DX9-reactive material (correctly folded) to Z27-reactive material (total protein) was calculated as 61%. (B) The specificity of KIR3DL1 was assessed by comparing its binding to immobilized HLA-B*5703 (Bw4 motif) and B*8101 (Bw6 motif) complexes. (C) The preferential binding of KIR3DL1 to Bw4 serotypes does not reflect differences in the amounts of protein immobilized in the SPR flow cell. The data were corrected for the amount of MHC class I protein immobilized in each flow cell and are shown as both normalized binding (with the level of binding to HLA-B5703 set at 1) and fold change in binding of HLA-B*8101 compared with HLA-B*5703.

Fig. 4.

Loss of KIR3DL1 binding to the G9E TW10 variant. (A) Biotinylated B*5703-β2m complexes, irradiated and rescued in the presence of peptides corresponding to the wild-type TW10 epitope and the G9E (red) and T3N (cyan) variants, were immobilized on SA sensor chips, over which a fixed concentration of soluble KIR3DL1-Fc was injected at a constant flow rate. (B) Loss of binding of KIR3DL1 to HLA-B*5703 containing the G9E peptide did not reflect differences in the amount of material immobilized on the surface of the SPR flow cell. The data were corrected for the amount of MHC class I protein immobilized in each flow cell and are shown as both normalized binding (with the level of binding to the wild-type peptide set at 1) and fold change in binding of the G9E and T3N variants compared with the wild type.

Fig. 5.

Ligand-specific binding of KIR3DL1 to HLA-B*5703 in complex with C−1 and C−2 TW10 and IW9 variants. The sensitivity of KIR3DL1 binding in the presence of peptide epitope variants incorporating amino acid substitutions is illustrated for the entire panel of TW10 and IW9 C−1 positional variants (A and B) and a subset of C−2 TW10 and IW9 variants (C and D). The data sets were normalized relative to the total amount of MHC class I protein immobilized in the SPR flow cells and are shown as fold change in binding relative to the wild-type amino acid (G for TW10 C−1, I for TW10 C−2, A for IW9 C−1, and N for IW9 C−2) and are ranked in ascending order for the TW10 variants. The IW9 data sets are ranked in the same order as the corresponding TW10 data set. The coloring of the graphs reflects the physical properties of the individual amino acids (see Table S1 in the supplemental material).