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.


INTRODUCTION
Natural killer (NK) cells are an important component of the innate immune system and play a critical role in the early control of infections and malignancies. NK cells can recognize virusinfected or malignantly transformed cells through sensing the downregulation of human leukocyte class I (HLA-I) molecules on the cell surface, changes in the HLA class I-presented peptide repertoire, and upregulation of ligands for activating NK cell receptors (1). Function of NK cells is regulated through a balanced interplay between activating and inhibitory receptors.
One of the major receptor families modulating NK cell function is the group of killer-cell immunoglobulin-like receptors (KIRs), which interact with HLA-I molecules expressed on nucleated cells (2). KIRs contain either two or three immunoglobulin-like domains in the extracellular region, and are named accordingly as KIR2D or KIR3D receptors. KIRs exhibiting a short cytoplasmic tail deliver an activating signal upon stimulation through interaction with the adaptor molecule DAP-12. Inhibitory KIRs possess a long cytoplasmic tail containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and prevent NK cell activation after ligand binding (3). Several epidemiological studies have shown that expression of specific KIRs can be beneficial in the context of viral infections, especially when expressed in combination with their respective HLA-I ligands. As an example, the combination of KIR2DL3 and HLA-C alleles of the HLA-C group 1 (HLA-C1) was associated with spontaneous clearance of HCV infection (4,5).
Multiple studies have shown that interactions between KIRs and HLA-I are not only determined by the HLA-I ligand but also modulated by the peptide presented by the respective HLA-I molecule (6)(7)(8)(9). The resolution of crystal structures of inhibitory KIRs in complex with HLA-I/peptide complexes has further unraveled the importance of the sequence of HLA-I presented peptides, showing that KIR-binding is highly influenced by carboxyl-terminal residues of the HLA-I-presented peptides (10,11). Several studies furthermore demonstrated that naturally occurring sequence mutations in virus-derived peptides are able to restore binding of HLA-I molecules to inhibitory KIRs, thereby preventing NK cell activation, and this has been suggested as a potential mechanism of viral escape from innate immune pressure (6)(7)(8)12). NK cell receptor repertoires can also be shaped by viral infections, and expansion of NK cells expressing specific receptors has been observed in HCV, HCMV and HIV-1 infections, as well as in MCMV-infected mice (13)(14)(15)(16). However, it remains unclear whether exposure to virus-derived HLA class I-presented peptides during infections or following vaccination has an impact on frequencies of KIR + NK cells in vivo. To gain a better understanding of how viral infections and HLA-I-presented viral epitopes might influence frequencies of KIR + NK cell subsets, we investigated the impact of YFV vaccination or HIV-1 and HCV infection on the frequencies of KIR2DL2/3 + NK cells binding to HLA-C * 03:04/viral peptide complexes.

Ethics Statement
All study subjects provided written informed consent for participation under protocols approved by the Ärztekammer Hamburg.

Study Population
The demographics and clinical characteristics of study subjects are summarized in Table 1. Study subjects included: 5 individuals enrolled in a Yellow Fever Virus (YFV) vaccine study at the Bernhard Nocht Institute for Tropical Medicine. Peripheral blood samples were collected one day prior to vaccination and at day 1, day 3, day 5, and day 28 post vaccination.

Data Analysis
Data analysis was performed using BD FACS Diva Software, FlowJo V10, Microsoft Excel 2017, GraphPad Prism 7 and Adobe Illustrator CC 2015.

Data Availability
Data used in this study have been collected in a clinical study and are subject to the regulation of the Ethics Committee of the Ärztekammer Hamburg that approved these studies. Participant's written consent has been provided to data generation and handling according to the approved protocols. Data storage is performed by the HPI. Data are available upon request and can be shared after confirming that data will be used within the scope of the originally provided informed consent.
Binding of KIR2DL3-Fc construct to 221-TAPko-HLA-C * 03:04 pulsed with YFV/NSA2A 4−13 , HIV/Gag 296−304 , HCV/Core 136−144 peptide, respectively, showed similar hierarchies (Supplementary Figure 1). The percentage of YFV/HLA-C * 03:04-NS2A 4−13 -tetramer-binding KIR2DL2/3 + NK cells did furthermore not differ between individuals encoding for HLA-C * 03 and HLA-C * 03-negative individuals (in the 10 individuals from the YFV vaccine and healthy cohorts for which HLA class I typing was available, median of 74.2 vs. 78.8%, p > 0.9, Supplementary Figure 2). Taken together, these data show that KIR2DL2/3 + NK cells follow a consistent peptidedependent hierarchy in their binding to HLA-C * 03:04 tetramers, which is not influenced by whether a study subject encodes for HLA-C * 03 and is furthermore independent of the underlying viral setting, suggesting a lack of antigen-dependent expansion of these NK cell populations. HLA-C group 1 tetramers, such as the HLA-C * 03:04 tetramers used here, can therefore serve as a reagent to monitor the frequencies of KIR2DL2 + or KIR2DL3 + NK cells.

DISCUSSION
Inhibitory KIRs expressed on NK cells bind to HLA class I molecules, and this interaction is modulated by the HLA class I-presented peptide. However, it is not known whether peptide-dependent KIR/HLA interactions provide a peptidespecific signal to NK cells and modulate frequencies of KIR + NK cells. We investigated the impact of viral infections and vaccination on frequencies of HLA-C * 03:04/peptide-binding KIR2DL2/3 + NK cell populations and their function. Crosssectional studies of HCV-and HIV-1-infected individuals and longitudinal studies in YFV vaccine recipients showed that frequencies of HLA-C * 03:04/peptide-binding KIR2DL2/3 + NK cells in peripheral blood were not influenced by whether a study subject encoded for HLA-C * 03, and were furthermore independent of the underlying viral infection and did not change following YFV vaccination. Rather, hierarchies of YFV/HLA-C * 03:04-NS2A 4−13 -, HIV/HLA-C * 03:04-Gag 296−304and HCV/HLA-C * 03:04-Core 136−144 -tetramers + KIR2DL2/3 + NK cells in these different infections and following vaccination were determined by the affinity of KIR2DL2/3 to the respective HLA-C * 03:04/peptide complex. HLA-C group 1 tetramers, such as the HLA-C * 03:04 tetramers used here, can therefore serve as a reagent to monitor the frequencies of KIR2DL2 + or KIR2DL3 + NK cells over time.
Several studies demonstrated that interactions between HLA class I molecules and their KIR ligands are influenced by the sequence of the peptide that is presented by the respective HLA-I molecule. Crystal structures of KIR2DL2 and KIR3DL1 in complex with HLA-I/peptide complex provided structural evidence that KIR/HLA-I interactions are highly susceptible to changes in the C-terminal sequence of HLA-I-presented peptides (10,11). While the peptide-dependence of these interactions is well established, it remains unknown whether HLA-I-presented peptides can induce expansion or contraction of KIR + NK cell populations binding HLA-I/peptide complexes. Using HLA-C * 03:04/peptide tetramer-staining of primary human KIR2DL2/3 + NK cells derived from healthy participants in a YFV vaccine trial and from HCV-and HIV-1infected individuals, we tested the hypothesis that KIR2DL2/3 + NK cells binding to HLA-C * 03:04 molecules presenting a specific viral peptide would differ in frequency between the different study groups. The study cohorts included individuals carrying or lacking HLA-C * 03, and the selected peptides were previously shown to be naturally processed and presented by HLA class I, as indicated by described specific CD8+ T cell responses against the tested epitopes (7,20,21). The results from this study did not support the initial hypothesis, but rather show that KIR2DL2/3 + NK cells follow a consistent peptide-dependent hierarchy in their binding to HLA-C * 03:04, which is independent of the underlying viral infection or vaccination, and also of whether a study subject encoded for HLA-C * 03 or not, but was determined by the binding affinity of the respective HLA-C * 03:04/peptide complex to KIR2DL2/3. These findings are in line with a previous study comparing binding of HLA-B57 tetramers refolded with a Dengue virus (DENV)-derived peptide in DENV-infected vs. uninfected individuals, demonstrating comparable frequencies of HLA-B57/DENV peptide tetramer + CD56 dim NK cells (22). Furthermore, Colantonio et al. observed staining of KIR2D + NK cells with tetramers refolded by the SIV-derived peptide GAG 71−79 GY9 in a previously non-SIV-exposed rhesus macaque (23). These data show that HLA-C group 1/peptide tetramers can be used to identify KIR2DL2/3+ NK cell populations, but also suggest that an antigen-dependent expansion or contraction of the respective NK cell populations does not occur.
Expansion of individual NK cell subsets was reported for different viral infections. This included expansion of NK cells expressing Ly49H in MCMV-infected mice (16), NKG2C in HCMV-and HCV-infected individuals (13,24), and KIR3DS1 in acute HIV-1 infections (15). Furthermore, contraction of NK cells was shown to occur in infections with HIV-1, HCV, and varicella zoster virus (25)(26)(27). However, the molecular mechanisms driving these expansions or contractions remain elusive, with some studies suggesting a role of licensing through interactions between HLA-I and inhibitory KIRs (15,28). To examine whether KIR2DL2/3 are able to mediate NK cell expansion or contraction in vivo, we longitudinally assessed the frequency of KIR2DL2/3 + NK cells able to bind a YFV-derived peptide presented by HLA-C * 03:04 in individuals receiving the YFV-17D vaccine. In contrast to a study reporting an increased expression of KIR2DL3 mRNA following vaccination with YFV-17D at day 7 (29), we observed stable frequencies of both total KIR2DL2/3 + NK cells as well as tetramer + KIR2DL2/3 + NK cells for all four tested time points. The described gene induction of KIR2DL3 after YFV vaccination might therefore have been restricted to other KIR-expressing cell populations, such as T cells, as only bulk KIR2DL3 mRNA levels on whole blood were quantified in the study by Gaucher et al. As previous studies also suggested an influence of HCMV infection on NK cell expansion (14,16,30), we assessed the HCMV status of our study subjects, and did not observe any differences in frequencies of tetramer + KIR2DL2/3 + NK cells between HCMV + and HCMV neg individuals. As expansion of NK cells following viral infections has been largely described for activating NK cell receptors, including KIR3DS1 and NKG2C in humans and Ly49H in mice (14,15,30,31), the capacity of NK cells to expand might be attributed to DAP-12, the adaptor molecule mediating signal transduction of activating receptors (30). Signaling of inhibitory receptors in contrast is mediated through ITIMs located in the intracellular tail of the receptor (32). Notably these expansions are not purely dependent on KIR-HLA interactions, as in TAP-deficient patients an expansion of NKG2C+ NK cells was observed albeit the very low levels of HLA class I (33). Our data suggest that engagement of the inhibitory receptor KIR2DL2/3 by HLA-C * 03:04/peptide complexes is not inducing KIR2DL2/3 + NK cell expansion. This is further supported by the observation that frequencies of HLA-C * 03:04/peptide complexbinding KIR2DL2/3 + NK cells did not differ between HLA-C * 03 + and HLA-C * 03 neg individuals. However, these data need to be interpreted in the context of the modest size of the study cohorts used, and confirmatory studies in larger cohorts are required to better control for additional factors that may influence KIR + NK cell frequencies, such as specific KIR2DL2/3 and HLA-C subtypes, as well as CMV serostatus. Furthermore, not all KIR2DL2/3 + NK cells bound to HLA-C * 03:04/peptide tetramers, suggesting that additional factors, such as KIR2DS2 genotype, surface KIR-expression levels or the ability of KIRs to cluster on the cell surface might impact HLA-C binding.
The results from our study show that binding of KIR + NK cells to HLA-C * 03:04/peptide complex is impacted by the biochemical properties of the HLA-I-bound peptide. We determined the binding affinities of the different tested peptides to HLA-C * 03:04 and the avidity of HLA-C * 03:04/peptide tetramers to KIR2DL2/3, and observed that the higher binding of YFV/HLA-C * 03:04-NS2A 4−13 -compared to HIV/HLA-C * 03:04-Gag 296−304 -and HCV/HLA-C * 03:04-Core 136−144 -tetramers to KIR2DL2/3 + NK cells was associated with the higher binding avidity of YFV/HLA-C * 03:04-NS2A 4−13 complexes to KIR2DL2/3. This observation is in line with a previous study mapping binding-avidity and peptide-selectivity of Mamu-KIR3DL05 to the same domain of the receptor, thereby emphasizing the influence of the HLA-I bound peptide on KIR avidity (23). Furthermore, our results show that the binding of YFV/HLA-C * 03:04-NS2A 4−13 -, HIV/HLA-C * 03:04-Gag 296−304and HCV/HLA-C * 03:04-Core 136−144 -tetramers to KIR2DL2/3 + NK cells was independent of the binding affinity of the corresponding peptide to HLA-C * 03:04. Binding of inhibitory KIRs to HLA class I molecules during NK cell development has been shown to play a critical role in determining the functionality of KIR + NK cells, a process referred to as NK cell licensing or education (34,35). To assess the relevance of KIR2DL3 + /HLA-C * 03:04 interactions for the functionality of KIR2DL3 + NK cells, we compared the ability of tetramer + and tetramer neg KIR2DL3 + NK cells to degranulate in one individual, using the described KIR2DL3-specific antibody (clone #180701) (17). The results showed that the ability of KIR2DL3 + NK cells to bind to YFV/HLA-C * 03:04-NS2A 4−13tetramers correlated with a higher functional capacity of these cells, which is consistent with NK cell licensing/education. We furthermore observed that the capacity of tetramer + KIR2DL3 + NK cells was influenced by the engagement of KIR2DL3 to its ligand. Around 35% of tetramer + KIR2DL3 + NK cells degranulated in response to classical HLA-I deficient 221 cells. When these tetramer + KIR2DL3 + NK cells were exposed to 221 cells presenting either the YFV/NS2A 4−13 -or the HCV/Core 136−144 -peptide via HLA-C * 03:04, degranulation was reduced, while 221 cells presenting the HIV/Gag 296−303 -peptide inhibited NK cell degranulation less. These data were in line with the low binding of HIV/HLA-C * 03:04-Gag 296−304 -tetramers to KIR2DL3 + NK cells. We furthermore observed a tetramer + and tetramer neg population within KIR2DL3 + NK cells, which might suggest differential HLA-C * 03:04-binding to different KIR2DL3 subtypes, as most NK cells will only express one of the two alleles (36). However, one limitation of studying KIR + NK cells is the cross-reactivity of several of the available antibodies, in particular between closely related activating and inhibitory KIRs. While the antibody used to identify KIR2DL3 + NK cells (clone #180701) was described not to cross-react with KIR2DL2 and KIR2DS2 (17), we cannot completely rule out crossreactivity with these receptors. In conclusion, the findings in this study demonstrate consistent hierarchies in the frequencies of KIR2DL2/3 + NK cells binding HLA-C * 03:04/peptide complexes that were not changed by an underlying viral infection or vaccination.

AUTHOR CONTRIBUTIONS
MZ and SL performed experiments and analyzed the data. FG, JS, CH, and AR recruited patients and processed patient materia. WG-B helped with assay design. SL and MA designed the study. MZ, SL, and MA wrote the manuscript. All authors provided continuous critical review of the data and commented on the manuscript.