HIV-1 evades a Gag mutation that abrogates killer cell immunoglobulin-like receptor binding and disinhibits natural killer cells in infected individuals with KIR2DL2+/HLA-C*03: 04+ genotype.

: HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leukocyte antigen class I (HLA-I) molecules modulating natural killer cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here, we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C03:04 and disinhibits natural killer cells in individual encoding for this genotype.

HIV-1 induced changes in HLA-C*03 :  04-presented peptide repertoires lead to reduced engagement of inhibitory natural killer cell receptors.

OBJECTIVE: Viral infections influence intracellular peptide repertoires available for presentation by HLA-I. Alterations in HLA-I/peptide complexes can modulate binding of killer immunoglobuline-like receptors (KIRs) and thereby the function of natural killer (NK) cells. Although multiple studies have provided evidence that HLA-I/KIR interactions play a role in HIV-1 disease progression, the consequence of HIV-1 infection for HLA-I/KIR interactions remain largely unknown. DESIGN: We determined changes in HLA-I presented peptides resulting from HIV-1-infection of primary human CD4 T cells and assessed the impact of changes in peptide repertoires on HLA-I/KIR interactions. METHODS: Liquid chromatography-coupled tandem mass spectrometry to identify HLA-I presented peptides, cell-based in-vitro assays to evaluate functional consequences of alterations in immunopeptidome and atomistic molecular dynamics simulations to confirm experimental data. RESULTS: A total of 583 peptides exclusively presented on HIV-1-infected cells were identified, of which only 0.2% represented HIV-1 derived peptides. Focusing on HLA-C*03 : 04/KIR2DL3 interactions, we observed that HLA-C*03 : 04-presented peptides derived from noninfected CD4 T cells mediated stronger binding of inhibitory KIR2DL3 than peptides derived from HIV-1-infected cells. Furthermore, the most abundant peptide presented by HLA-C*03 : 04 on noninfected CD4 T cells (VIYPARISL) mediated the strongest KIR2DL3-binding, while the most abundant peptide presented on HIV-1-infected cells (YAIQATETL) did not mediate KIR2DL3-binding. Molecular dynamics simulations of HLA-C*03 : 04/KIR2DL3 interactions in the context of these two peptides revealed that VIYPARISL significantly enhanced the HLA-C*03 : 04/peptide contact area to KIR2DL3 compared with YAIQATETL. CONCLUSION: These data demonstrate that HIV-1 infection-induced changes in HLA-I-presented peptides can reduce engagement of inhibitory KIRs, providing a mechanism for enhanced activation of NK cells by virus-infected cells.

Stable Frequencies of HLA-C*03:04/Peptide-Binding KIR2DL2/3+ Natural Killer Cells Following Vaccination.

Inhibitory KIRs play a central role in regulating NK cell activity. KIR2DL2/3 bind to HLA-C molecules, but the modulation of these interactions by viral infections and presentation of viral epitopes is not well-understood. We investigated whether the frequencies of KIR2DL2/3+ NK cells recognizing HLA-C*03:04/viral peptide complexes were impacted by YFV vaccination or HIV-1 and HCV infection. Ex vivo HLA class I tetramer staining of primary human NK cells derived from YFV-vaccinated individuals, or HIV-1- or HCV-infected individuals revealed that the YFV/HLA-C*03:04-NS2A4-13-tetramer bound to a larger proportion of KIR2DL2/3+ NK cells compared to HIV-1/HLA-C*03:04-Gag296-304- or HCV/HLA-C*03:04-Core136-144-tetramers. The YFV/HLA-C*03:04-NS2A4-13-tetramer also exhibited a stronger avidity to KIR2DL2/3 compared to the other tested tetramers. The proportional frequencies of KIR2DL2/3+ NK cells binding to the three tested HLA-C*03:04 tetramers were identical between YFV-vaccinated individuals or HIV-1- or HCV-infected individuals, and remained stable following YFV vaccination. These data demonstrate consistent hierarchies in the frequency of primary KIR2DL2/3+ NK cells binding HLA-C*03:04/peptide complexes that were determined by the HLA-C-presented peptide and not modulated by the underlying viral infection or vaccination.

HIV-1-Mediated Downmodulation of HLA-C Impacts Target Cell Recognition and Antiviral Activity of NK Cells.

It was widely accepted that HIV-1 downregulates HLA-A/B to avoid CTL recognition while leaving HLA-C unaltered in order to prevent NK cell activation by engaging inhibitory NK cell receptors, but it was recently observed that most primary isolates of HIV-1 can mediate HLA-C downmodulation. Now we report that HIV-1-mediated downmodulation of HLA-C was associated with reduced binding to its respective inhibitory receptors. Despite this, HLA-C-licensed NK cells displayed reduced antiviral activity compared to their unlicensed counterparts, potentially due to residual binding to the respective inhibitory receptors. Nevertheless, NK cells were able to sense alterations of HLA-C expression demonstrated by increased antiviral activity when exposed to viral strains with differential abilities to downmodulate HLA-C. These results suggest that the capability of HLA-C-licensed NK cells to control HIV-1 replication is determined by the strength of KIR/HLA-C interactions and is thus dependent on both host genetics and the extent of virus-mediated HLA-C downregulation.

Peptide-specific engagement of the activating NK cell receptor KIR2DS1.

The activating NK cell receptor KIR2DS1 has been shown to be involved in many disorders including autoimmune diseases, malignancies and pregnancy outcomes. However, the precise ligands and functions of this receptor remain unclear. We aimed to gain a better understanding of the factors involved in the binding of KIR2DS1 and its inhibitory counterpart KIR2DL1 to HLA class I molecules, and the consequences for KIR2DS1+ NK-cell function. A systematic screen that assessed binding to 97 HLA-I proteins confirmed that KIR2DS1-binding was narrowly restricted to HLA-C group 2 complexes, while KIR2DL1 showed a broader binding specificity. Using KIR2DS1ζ+ Jurkat reporter-cells and peptide-pulsed 721.221.TAP1KO-HLA-C*06:02 cells, we identified the synthetic peptide SRGPVHHLL presented by HLA-C*06:02 that strongly engaged KIR2DS1- and KIR2DL1-binding. Functional analysis showed that this HLA-C*06:02-presented peptide can furthermore activate primary KIR2DS1(+) NK cell clones. Thus, we demonstrated peptide-dependent binding of the activating NK cell receptor KIR2DS1, providing new insights into the underlying mechanisms involved in KIR2DS1-related disorders.

Sequence variations in HCV core-derived epitopes alter binding of KIR2DL3 to HLA-C∗03:04 and modulate NK cell function.

BACKGROUND & AIMS: Both natural killer (NK) cells and human leukocyte antigen (HLA)/killer cell immunoglobulin like receptor (KIR) interactions have been shown to play an important role in the control, clearance and progression of hepatitis C virus (HCV) disease. Here we aimed at elucidating the effects of viral peptides derived from HCV on HLA stabilization, changes in KIR binding and primary NK cell function. METHODS: Transporter for antigen presentation-deficient 722.221 cells stably transfected with HLA-C∗03:04 were used to screen 200 overlapping peptides, covering the non-structural protein 3 (NS3) and core protein of HCV genotype 1, for their ability to bind and stabilize HLA-C∗03:04. Binding of KIR2DL3 to the HLA-peptide complex was assessed using a KIR2DL3-IgG fusion construct. Primary NK cells were isolated from healthy donors to investigate the effects of identified peptides on KIR2DL3(+) NK cell function. RESULTS: Thirty-one peptides able to stabilize HLA-C∗03:04 were identified. One 9mer peptide, YIPLVGAPL, resulted in significantly higher KIR2DL3 binding to HLA-C∗03:04(+) 722.221 cells and suppression of primary KIR2DL3(+) NK cell function. Interestingly this sequence exhibited a high frequency of mutations in different HCV genotypes. These genotype-specific peptides showed lower HLA-C∗03:04 stabilization, decreased binding of the inhibitory KIR2DL3 and lower inhibition of NK cell function. CONCLUSIONS: Taken together we show that a viral peptide derived from the core protein of HCV genotype 1 binding to HLA-C∗03:04 results in a sequence-dependent engagement of the inhibitory NK cell receptor KIR2DL3, while the large majority of the remaining 30 HLA-C∗03:04 binding HCV core peptides did not. These data show that sequence variations within HCV can modulate NK cell function, providing potential pathways for viral escape. LAY SUMMARY: We identified a HCV peptide that dampens NK cell responses, and thereby possibly prevents killing of infected cells through this part of the innate immune system. This is facilitated via presentation of the viral peptide on HLA∗03:04 to the inhibitory KIR receptor KIR2DL3 on NK cells. Naturally occurring sequence mutations in the peptide alter these interactions making the inhibition less efficient.