NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts

Gamze Isitman; Alexandra Tremblay-McLean; Irene Lisovsky; Julie Bruneau; Bertrand Lebouché; Jean-Pierre Routy; Nicole F. Bernard

doi:10.1371/journal.pone.0164517

Abstract

Natural Killer (NK) cell education, which requires the engagement of inhibitory NK cell receptors (iNKRs) by their ligands, is important for generating self-tolerant functional NK cells. While the potency of NK cell education is directly related to their functional potential upon stimulation with HLA null cells, the influence of NK cell education on the potency of the antibody dependent cellular cytotoxicity (ADCC) function of NK cells is unclear. ADCC occurs when the Fc portion of an immunoglobulin G antibody bridges the CD16 Fc receptor on NK cells and antigen on target cells, resulting in NK cell activation, cytotoxic granule release, and target cell lysis. We previously reported that education via the KIR3DL1/HLA-Bw4 iNKR/HLA ligand combination supported higher KIR3DL1⁺ than KIR3DL1^- NK cell activation levels but had no impact on ADCC potency measured as the frequency of granzyme B positive (%GrB⁺) targets generated in an ADCC GranToxiLux assay. A lower frequency of KIR3DL1⁺ compared to KIR3DL1^- NK cells were CD16⁺, which may in part explain the discrepancy between NK cell activation and target cell effects. Here, we investigated the frequency of CD16⁺ cells among NK cells expressing other iNKRs. We found that CD16⁺ cells were significantly more frequent among NK cells negative for the inhibitory KIR (iKIR) KIR2DL1, KIR2DL3, and KIR3DL1 than those positive for any one of these iKIR to the exclusion of the others, making iKIR⁺ NK cells poorer ADCC effectors than iKIR^- NK cells. The education status of these iKIR⁺ populations had no effect on the frequency of CD16⁺ cells.

Citation: Isitman G, Tremblay-McLean A, Lisovsky I, Bruneau J, Lebouché B, Routy J-P, et al. (2016) NK Cells Expressing the Inhibitory Killer Immunoglobulin-Like Receptors (iKIR) KIR2DL1, KIR2DL3 and KIR3DL1 Are Less Likely to Be CD16+ than Their iKIR Negative Counterparts. PLoS ONE 11(10): e0164517. https://doi.org/10.1371/journal.pone.0164517

Editor: Mark A. Wainberg, McGill University AIDS Centre, CANADA

Received: June 27, 2016; Accepted: September 25, 2016; Published: October 12, 2016

Copyright: © 2016 Isitman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This study received support from the Canadian Institutes for Health Research (http://www.cihr-irsc.gc.ca) HOP-123800 and MOP-142494 and the Fonds de Recherche du Québec-Santé (http://www.frqs.gouv.qc.ca) AIDS and Infectious Diseases Network (http://www.reseausidami.quebec/fr) to NFB. GI was supported by a post-doctoral scholarship from Canadian HIV Trials Network (http://www.hivnet.ubc.ca/) and CIHR (http://www.cihr-irsc.gc.ca). IL was supported by a Ph.D. scholarship from FRQ-S (http://www.frqs.gouv.qc.ca) and CIHR (http://www.cihr-irsc.gc.ca). BL is supported by a Junior I scholarship from FRQ-S (http://www.frqs.gouv.qc.ca). NFB is a member of the RI-MUHC, an institution funded in part by the FRQ-S (http://www.frqs.gouv.qc.ca).

Competing interests: The authors have declared that no competing interests exist.

Introduction

Natural Killer (NK) cells acquire functional competence as they develop through a process known as education, which requires the interaction of inhibitory NK receptors (iNKRs) with their cognate human leukocyte antigen (HLA) ligands on neighboring cells [1–3]. Inhibitory NKRs include inhibitory Killer Immunoglobulin-like Receptors (iKIR), such as KIR2DL1 (2DL1), KIR2DL3 (2DL3), and KIR3DL1 (3DL1), as well as the C-type lectin receptor NKG2A. The 3DL1 receptor interacts with a subset of HLA-A and–B antigens that belong to the Bw4 subset [4,5]. Bw4 antigens differ from the remaining Bw6 HLA-B variants, which do not interact with 3DL1, at amino acids 77–83 of the HLA heavy chain [6]. Thus, NK cells from Bw6 homozygotes with no HLA-A Bw4 alleles can serve as controls for the effect of education though 3DL1 on NK cell function. The 2DL3 receptor interacts with HLA-C group 1 (C1) variants having an asparagine at position 80 of the heavy chain [7,8]. Other HLA-C variants with a lysine at this position belong to the C2 group and are ligands for 2DL1 [8]. The 2DL3 receptor can also bind certain allelic variants of C2, though with lower affinity than 2DL1 [9]. Therefore, 2DL3⁺ NK cells from individuals expressing the C1 ligand are educated, but are either uneducated or less potently educated in individuals expressing only C2 ligands. NKG2A interacts with non-classical major histocompatibility complex class I (MHC-I) HLA-E molecules that present leader peptides from many MHC-I proteins and certain viral derived epitopes [10–13]. NKG2A and HLA-E molecules are highly conserved and their effect on NK cell education is similar from one person to another [14].

NK cell education is a dynamic process whereby functionality can be tuned by the number of iNKRs engaged, the strength of interactions between iNKRs and their ligands and the potential additional engagement of activating NK cell receptors (aNKRs) [9,15–19]. The potency of NK cell education is related to the frequency of NK cells that are activated by stimuli such as HLA null cells and antibody (Ab) dependent NK cell stimulation by recombinant gp120 coated CEM.NKr-CCR5 (CEM) cells in the presence of anti-human immunodeficiency virus (HIV) Envelope (Env) specific Abs [16,18,20–23]. Previous work by our group noted inter-individual variation in NK cell mediated ADCC activity in a flow cytometry based ADCC GranToxiLux (ADCC-GTL) assay, which measured the %GrB⁺ target cells generated in this assay [24,25]. The target cells used in this assay were gp120 coated CEM cells and the source of Ab was HIVIG, immunoglobulin G (IgG) from pooled plasma from HIV infected subjects. We showed that this variability was not due to differences in the ability of 3DL1/HLA-B pairs to educate NK cells [24]. This finding was despite the observation that 3DL1⁺ NK cells that were educated through 3DL1/Bw4 interactions were stimulated by the same gp120 coated CEM target cells in an Ab dependent fashion to higher levels than 3DL1^- NK cells [22,24,26]. Furthermore, we showed that among 3DL1⁺ NK cells, there was a significantly lower frequency of CD16⁺ cells than among 3DL1^- NK cells. As CD16 is crucial to ADCC activity, this would compromise the ability of educated 3DL1⁺ NK cells to act as effector cells in terms of delivering GrB to target cells [24,26].

In this report we expanded on these results by investigating the proportion of CD16⁺ cells among NK cells expressing NKG2A, 2DL1, 2DL3, 3DL1, or none of these iNKR by adopting a gating strategy that focused on NK cells expressing one of these iNKR to the exclusion of the others stained for. We found NK cells exclusively expressing a single iKIR co-expressed CD16 less frequently than did NK cells expressing none of these iNKRs. On the other hand, NK cells exclusively expressing NKG2A only, co-expressed CD16 more frequently than did NK cells negative for these iNKRs. Furthermore, the frequency of CD16 on these single iKIR positive NK cell populations did not differ based on whether or not the NK cells developed in an environment that would have supported their education.

Materials and Methods

Ethics statement and study population

This study was conducted in accordance with the principles expressed in the Declaration of Helsinki. It was approved by the Institutional Review Boards of the Comité d’Éthique du Centre de Recherche du Centre Hospitalier de l’Université de Montréal and the Research Ethics Committee of the McGill University Health Centre—Montreal General Hospital. All individuals provided written informed consent for the collection of samples and subsequent analysis.

We studied 26 HIV seronegative donors. The MHC-I HLA-A, -B, and–C type of each study participants is shown in Table 1. Also shown in this table is the generic 3DL1/S1 genotype, whether a 2DL1 locus was present, whether an allele belonging the 2DL3 group encoded at the 2DL2/L3 locus was present, whether subjects carried a Bw4 allele at either the HLA-A or–B locus, and whether they carried HLA-C alleles encoding antigens belonging to the C1 or C2 group or both.

Download:

Table 1. Study population HLA and KIR genotypes.

https://doi.org/10.1371/journal.pone.0164517.t001

Genotyping

MHC-I alleles were typed using commercial reagents (Atria Genetics Inc., South San Francisco, CA). Genotyping and allotyping of 3DL1/S1 was performed as previously described [27,28]. Presence of 2DL1 and 2DL2/2DL3 loci and alleles belonging to the 2DL3 group was determined using a KIR genotyping kit (One Lambda Inc., Canoga Park, CA). Presence of 2DL1, 2DL2, and 2DL3 alleles was verified by PCR using specific primers and conditions described by Kulkarni et al.[29]

Cells

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Lymphocyte Separation Medium, Wisent, St. Jean Baptiste, QC, Canada) from whole blood obtained by venipuncture into tubes containing EDTA anticoagulant or by leukapheresis, as previously described [18,30]. The cells were then cryopreserved in 10% DMSO, with 90% fetal bovine serum (FBS). After thawing, cells were rested overnight in RPMI media containing 2 mM L-glutamine; 100 IU/ml penicillin; 100 μg/ml streptomycin; 10% FBS (R10) (all from Wisent) at 37°C in a 5% humidified CO₂ incubator at a concentration of 1x10⁶ cells/ml.

CD16 surface staining

Rested, unstimulated PBMCs were cell surface stained to identify the frequency of CD16⁺ expression among CD56^{total/dim/bright} and iNKR^+/- NK cell populations. Following staining with UV Live/Dead Fixable Stain (Invitrogen, Burlington, ON, Canada), surface staining was performed in the dark at room temperature on 1x10⁶ PBMCs per individual with Abs of the following specificities: CD3-BV785 (OKT3), CD56-BV711 (NCAM16.2), 3DL1-BV421 (DX9), CD57-FITC (HCD57), (all from BioLegend, San Diego, CA), NKG2A-PeCy7 (Z199, Beckman Coulter, Mississauga, ON, Canada), CD16-APC-Cy7 (3GB, BD Biosciences, Mississauga, ON, Canada), 2DL1-AlexaFluor700 (143211) and 2DL3-PE (180701; both from R&D Systems, Minneapolis, MN). Stained cells were then washed and fixed with 1% paraformaldehyde solution (PFA, Santa Cruz, Biotechnology, Inc, Dallas, TX). Between 300,000 and 400,000 events per sample were acquired using an LSRFortessa flow cytometer (BD). Data analysis was performed using FlowJo software v10 (Treestar, Ashland OR). Unstained, single color, and fluorescence minus one controls were used for each subject for multi-color compensation and gating purposes.

Statistical analysis

Statistical analysis and graphical presentation were performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA). Wilcoxon matched pairs and Mann-Whitney tests were used to assess the significance of differences on 2 matched or unmatched groups, respectively. Friedman tests with Dunn’s post-test comparisons were used to assess the significance of more than 2 matched groups. Results were reported as medians and inter-quartile ranges (IQR). P-values less than 0.05 were considered significant.

Results

CD16, NKG2A, and CD57 expression in CD56⁺ NK cell populations

NK cells were defined as CD3^-CD56⁺ cells from the live singlet lymphocyte gate. Two NK cell subsets were identified, i.e. the less mature CD3^-CD56^bright cells, which represent approximately 10% of circulating NK cells, and the more differentiated CD3^-CD56^dim population, which comprises the remainder of circulating NK cells [31–34] (S1A Fig). The frequencies of CD16⁺, CD16⁺NKG2A⁺, and CD16⁺CD57⁺ NK cells and their CD16^- counterparts within these three NK cell populations were assessed using the gating strategy shown in Fig 1A.

Download:

Fig 1. Frequency of CD16⁺, NKG2A⁺ and CD57⁺ NK cells and their subsets expressing or not CD16.

(A) Representative gating strategy for determining the frequency of CD16^+/- cells on CD56^total, CD56^dim, and CD56^bright NK cell populations and on the NKG2A^-/+ and CD57^-/+ subsets of these populations. (B) Frequencies of CD16^+/- cells in the CD56^total, CD56^dim, and CD56^bright NK cell populations. Frequencies of expression of NKG2A (C) and CD57 (E) on CD56^total, CD56^dim, and CD56^bright NK cells. A Friedman test was used to assess the significance of matched between group differences. Frequencies of CD16⁺ and CD16^- cells within the NKG2A⁺ (D) and CD57⁺ (F) CD56^total, CD56^dim, and CD56^bright NK cell populations. Each data point represents results for 1 of 26 separate individuals. Bar height and error bars represent the median and interquartile range for the data set. Wilcoxon tests were used to determine significance of within subject differences for the indicated NK subsets linked by a line connecting the data sets. Significant values are shown; “*” = p< 0.05; “**” = p< 0.01; “***” = p< 0.001; “****” = p< 0.0001.

https://doi.org/10.1371/journal.pone.0164517.g001

Consistent with observations made by others, the CD56⁺ and CD56^dim NK populations had higher frequencies of CD16⁺ than CD16^- cells (Fig 1B, left and middle panels and S1 Table), while the opposite was seen for CD56^bright NK cells (right panel) (p = 0.016, p = 0.005, and p = 0.043, respectively, Wilcoxon tests) [33]. NKG2A is an iNKR whose expression declines as NK cells mature [35,36]. The proportion of NKG2A⁺ NK cells was lower in total and CD56^dim than in the CD56^bright population (Fig 1C and S2 Table; p = 0.014 and p<0.0001, respectively, Dunn’s post-tests). Within the total CD56⁺ and CD56^dim NKG2A⁺ populations, there was a higher proportion of CD16⁺ than CD16^- cells, while there was a lower frequency of CD16⁺ than CD16^- cells within the NKG2A⁺CD56^bright population (Fig 1D, and S3 Table p = 0.043, p = 0.002, and p = 0.043, respectively, Wilcoxon).

CD57 is a marker of senescence and terminal differentiation [36–38]. In accordance with this, the frequency of CD57⁺ total CD56⁺ and CD56^dim NK cells was higher than that of CD57⁺CD56^bright NK cells (Fig 1E and S4 Table, p = 0.014 and p<0.0001, respectively, Dunn’s post-tests). A higher proportion of CD57⁺ total CD56⁺, CD56^dim, and CD56^bright NK cells were CD16⁺ than CD16^-, though the difference for CD57⁺CD56^bright NK cells did not achieve statistical significance, likely due to the low frequency of CD57⁺NK cells that were CD56^bright (Fig 1F and S5 Table, p = 0.01, p = 0.0002, and p = 0.4, respectively, Wilcoxon).

CD16 and KIR expression on total CD56⁺ and CD56^dim NK cells

We next assessed the frequency of total CD56⁺ NK cells that were positive or negative for CD16 and that expressed any combination of the iKIR 2DL1, 2DL3, and 3DL1 or none of these IKIR (Fig 2A and S5 Table). The proportion of CD16^- NK cells that expressed at least one of these iKIR was lower than those that expressed none. The frequency of CD16⁺ NK cells that co-expressed or not at least one of these iKIR did not differ significantly.

Download:

Fig 2. CD16 expression on total CD56⁺ NK cells populations expressing all combinations of iKIRs and on CD56^dim single iNKR expressing NK cell populations.

(A) The frequency of KIR⁺ (expressing any Boolean combination of KIR2DL1 (2DL1), KIR2DL3 (2DL3), or KIR3DL1 (3DL1]) subsets in CD16^- and CD16⁺ total CD56⁺ NK cell populations. (B) The gating strategy for assessing the frequencies of single NKG2A, 2DL1, 2DL3, and 3DL1 and CD16 positive CD56^dim NK cells. (C) Frequencies of CD16 positive CD56^dim NK cells that are single positive for NKG2A (n = 26), 2DL1 (n = 25),2DL3 (n = 22), and 3DL1 (n = 22) or negative for all iNKR tested (iNKR^-). Wilcoxon tests were used to determine significance of within subject differences for the indicated NK subsets. Each data point represents results a separate individual. Bar height and error bars represent the median and interquartile range for the data set. Significant values are shown; “**” = p< 0.01; “***” = p< 0.001; “****” = p< 0.0001.

https://doi.org/10.1371/journal.pone.0164517.g002

To further investigate the relationship between iNKR and CD16 expression, we adopted an exclusive gating strategy in order to examine the frequency of CD16⁺ cells among NK cells expressing NKG2A, 2DL1, 2DL3, or 3DL1 to the exclusion of the other iNKR and compared this frequency to that of CD16⁺ NK cells negative for all of these iNKR. For this analysis we used Boolean gating to measure the frequency of NKG2A⁺, 2DL1⁺, 2DL3⁺, 3DL1⁺, and CD16⁺ cells within the CD56^dim NK cell gate (Fig 2B and S6 Table). A higher frequency of CD56^dim NKs expressing NKG2A to the exclusion of the other iNKRs tested (NKG2A⁺2DL1^-2DL3^-3DL1^-) co-expressed CD16, compared to cells negative for all four iNKRs (Fig 2C and S7 Table, 21.1 [15.5, 30.7] and 12.4 [6.4, 5.75], respectively, p = 0.0004 Wilcoxon). We found that a significantly lower proportion of cells expressing each of the three iKIR to the exclusion of the other iNKR (NKG2A^-2DL1⁺2DL3^-3DL1^-, NKG2A^-2DL1^-2DL3⁺3DL1^-, or NKG2A^-2DL1^-2DL3^-3DL1⁺) were CD16⁺ compared to NK cells negative for all four iNKRs tested (Fig 2C and S7 Table, [3.1 (1.6, 5.88)], p<0.0001, [4.41 (2.8, 7.46)], p = 0.008, and [3.84 (1.56, 6.6)], p<0.0001 for comparisons with 2DL1, 2DL3 and 3DL1, respectively Wilcoxon). Together, these results show that NK cells expressing 2DL1, 2DL3, or 3DL1 alone are less likely to be CD16⁺ cells than iNKR^- NK cells. Additionally, CD56^dim NK cells that do not express the KIR studied, but that express NKG2A alone are more likely to co-express CD16 than iNKR^- cells.

The effect of KIR education on CD16 Expression

CD56^dimNKG2A^- NK cells expressing 2DL1, 2DL3, or 3DL1 were stratified according to whether they came from a study subject co-carrying or not a ligand for that KIR, which would support NK cell education through each receptor. We then compared the frequency of CD16⁺ CD56^dim NK cells from subjects whose KIR/HLA genotype would support versus not support education through each receptor (Fig 3 and S8 Table). Educated 2DL1⁺ cells from HLA-C2 carriers were compared to uneducated 2DL1⁺ NK cells from HLA-C1 homozygotes. Likewise, 2DL3⁺ cells from HLA-C1 carriers were compared to those from HLA-C2 homozygotes and 3DL1⁺ cells from Bw4 carriers were compared to 3DL1⁺ cells from Bw6 homozygotes. There were no significant differences in the frequency of CD16⁺ cells in the educated and uneducated groups.

Download:

Fig 3. The frequency of educated and uneducated CD56^dim NK cell populations expressing CD16 and one of the inhibitory KIR KIR2DL1, KIR2DL3, or KIR3DL1.

The frequency of CD16⁺ cells among educated KIR2DL1⁺ (2DL1) NKG2A^-CD56^dim NK cells from HLA-C2 homozygotes (n = 8) versus uneducated HLA-C1 homozygotes (n = 11), educated KIR2DL3⁺ (2DL3) NKG2A^-CD56^dim NK cells from HLA-C1 homozygotes (n = 10) versus uneducated HLA-C2 homozygotes (n = 7), and educated KIR3DL1⁺ (3DL1) NKG2A^-CD56^dim NK cells from Bw4 carriers (n = 8) versus uneducated Bw6 homozygotes (n = 8). The lines and error bars through the datasets represent medians and interquartile ranges. Mann-Whitney tests assessing the significance of differences in the frequency of CD16⁺, single KIR positive cells in educated versus uneducated NK cell subsets found no significant differences.

https://doi.org/10.1371/journal.pone.0164517.g003

Discussion

In this report we show that a lower frequency of CD56^dim NK cells expressing no NKG2A, 2DL1, 2DL3, or 3DL1 were CD16⁺ compared those expressing NKG2A alone, whereas a higher frequency of CD56^dim NKs expressing none of these iNKRs were CD16⁺ compared to those expressing the iKIR 2DL1, 2DL3, or 3DL1 alone. Whether 2DL1, 2DL3, or 3DL1⁺ NK cells developed in a host carrying alleles encoding HLA variants able to educate NK cells expressing these iKIR or not, did not affect the frequency of CD16⁺ cells in these NK cell populations.

CD16 is a low affinity activating Fc receptor (FcRIIIa) on NK cells, monocytes and neutrophils [39]. Its engagement on NK cells leads to the induction of several functions such as cytokine/chemokine secretion and expression of CD107a. Ab dependent NK cell activation also results in the delivery of GrB to target cells and their eventual cytolysis by ADCC. NK cell-mediated ADCC involves the recognition of a cognate antigen on target cells by the fragment variable (Fv) portion of an IgG Ab molecule and the ligation of the fragment crystallizable (Fc) region to CD16 on NK cells.

The higher frequencies of CD16⁺, CD16⁺NKG2A⁺, and CD16⁺CD57⁺ cells than their CD16^- counterparts observed in total CD56⁺ NK cells reflect the higher percentage of CD16⁺ cells among CD56^dim than CD56^bright NK cells in the periphery [32,33]. The higher proportion of CD16^- than CD16⁺ cells within the CD56^bright and NKG2A⁺CD56^bright populations is consistent with CD56^bright NKs being at an earlier stage of differentiation than CD56^dim NK cells [31,36,40]. As NK cells differentiate from CD56^bright to CD56^dim, NKG2A expression declines and KIR expression increases [31,34,35]. CD57⁺ cells have also been reported to proliferate more poorly, express more CD16, and mediate more potent CD16 dependent cytotoxicity [36]. In support of this, we found that CD57⁺CD56^dim NKs were significantly more likely to co-express CD16.

That single iKIR⁺ NK cells are less likely to be CD16⁺ has not been reported previously. This may be partly due to the gating strategies and Ab panels that have been used to detect CD56, CD16, and iKIR on NK cells. Since NKG2A⁺ cells co-express CD16⁺ at a higher frequency than iNKR^- NK cells, if they are included in comparisons of NK cells positive versus negative for particular iKIR they may mask the differences in CD16 frequency in comparisons of single iKIR^+/- NK cell pairs. This may in part explain the results presented in Fig 2A where NKG2A⁺ cells were not excluded and NK cells expressing any combination of the iKIR screened for were gated on. Inclusion of CD56^bright NK cells in such analyses may also mask differences in CD16 expression on iKIR^+/- NK cell pairs. Although they only make up ~10% of the total CD56⁺ NK cell pool they are almost all NKG2A⁺ and rarely KIR⁺ and thus may skew results for the expression of CD16 on iKIR^+/- pairs within the CD56^dim compartment [35].

iKIR expression is required for NK cell education [1,3]. The finding that there are fewer CD16⁺ cells among iKIR⁺ compared to iKIR^- NK cells whether they are from subjects who express HLA ligands that would support the education of these NK cells would make iKIR⁺ NK cells poorer ADCC effector cells than their iKIR^- counterparts, as CD16 is crucial for ADCC activity. This could have contributed to the absence of an impact of educating 3DL1/Bw4 pairs on ADCC potency measured by assessing the frequency of the delivery of GrB to gp120 coated CEM, as previously reported [24]. Using the same data set as the one described in Isitman et al. [24] we stratified the results for %GrB⁺ CEM according to whether the effector cells used in the ADCC-GTL assay came from subjects positive or not for the educating 2DL1/HLA-C2 pair or the 2DL3/HLA-C1 pair. In neither case did we observe differences in the %GrB⁺ CEM cells when effector cells from subjects carrying educating versus non educating combinations were used (not shown).

It is important to note that not all possible iKIR were stained for, which is a limitation of this study. The reason we did not stain for KIR2DL2 is that there is no commercially available Ab that specifically detects this receptor without also detecting 2DL3 with or without KIR2DS2. Abs to other iKIR such as KIR2DL5, KIR3DL2, or KIR3DL3 were not included in the panel. The inhibitory function of these potential iKIR is not well characterized nor is their ligand specificity. In summary, it is possible that subsets of the cells staining as 2DL1⁺, 2DL3⁺, 3DL1⁺, NKG2A⁺, or iNKR^- may co-express these additional iKIR or activating KIR. At present, there is no information on the expression of CD16 on NK cell subsets expressing one or more of these unstained for iKIR.

Interest in ADCC increased when analyses of the results of the RV144 Thai trial revealed that IgG Abs to the V1/V2 low loop of the HIV Env, together with low levels of IgA and elevated levels of ADCC activity, may have contributed to the modest protection against HIV infection observed in this trial [41,42]. This, and the explosion of HIV-Env specific broadly neutralizing Abs, which also mediate ADCC has promoted interest in exploiting Fc-mediated effector functions for protection against HIV infection in a vaccine setting and for HIV therapy [43–47]. Our findings that NK cells that have the potential to be educated through iKIR are less likely to co-express CD16 suggests that non iKIR educated NK cells are the more likely mediators of ADCC. The implication of this observation is that vaccine or therapeutic strategies targeting NK cell mediated ADCC through the induction of ADCC-competent Abs will not be limited by inter-individual genetic differences that determine the potential potency of NK cell education.

Supporting Information

S1 Fig. The gating strategy used for detecting NK cells.

The live lymphocytic singlet population was used to gate on NK cells, which were defined as CD3^-CD56⁺ (CD56^total), CD3^-CD56^dim, and CD3^-CD56^bright. SSC-A = side scatter area; FSC-H = forward scatter height; FSC-A = forward scatter area.

https://doi.org/10.1371/journal.pone.0164517.s001

(TIF)

S1 Table. Data used to create Fig 1B.

Frequency of CD16^+/- cells among total CD56⁺, CD56^dim and CD56^bright NK cells.

https://doi.org/10.1371/journal.pone.0164517.s002

(DOCX)

S2 Table. Data used to create Fig 1C.

Frequency of NKG2A+ cells among total CD56⁺, CD56^dim and CD56^bright NK cells.

https://doi.org/10.1371/journal.pone.0164517.s003

(DOCX)

S3 Table. Data used to create Fig 1D.

Frequency of CD16^+/- NKG2A⁺ cells among total CD56⁺, CD56^dim and CD56^bright NK cells.

https://doi.org/10.1371/journal.pone.0164517.s004

(DOCX)

S4 Table. Data used to create Fig 1E.

Frequency of CD57⁺ cells among total CD56⁺, CD56^dim and CD56^bright NK cells.

https://doi.org/10.1371/journal.pone.0164517.s005

(DOCX)

S5 Table. Data used to create Fig 1F.

Frequency of CD16^+/- CD57⁺ cells among total CD56⁺, CD56^dim and CD56^bright NK cells.

https://doi.org/10.1371/journal.pone.0164517.s006

(DOCX)

S6 Table. Data used to create Fig 2A.

Frequency of CD56⁺ NK cells among Killer Immunoglobulin-like Receptor (KIR)^+/-CD16^+/- cells.

https://doi.org/10.1371/journal.pone.0164517.s007

(DOCX)

S7 Table. Data used to create Fig 2C.

Frequency of CD16⁺ cells among CD56^dim NK cells expressing NKG2A, KIR2DL1 (2DL1), KIR2DL3 (2DL3) or KIR3DL1 (3DL1) to the exclusion of the other inhibitory NK receptors (iNKR) versus none of these iNKR.

https://doi.org/10.1371/journal.pone.0164517.s008

(DOCX)

S8 Table. Data used to create Fig 3.

Frequency of CD56^dim CD16⁺ cells among educated and uneducated KIR2DL1 (2DL1)⁺, KIR2DL3 (2DL3)⁺ and KIR3DL1 (3DL1)⁺ NK cells.

https://doi.org/10.1371/journal.pone.0164517.s009

(DOCX)

Acknowledgments

The authors wish to acknowledge Ms. Rachel Bouchard for contacting study participants and coordinating their recruitment to this study. We are grateful to Ms. Pascale Arlotto and Ms. Josée Girouard for expert nursing skills in obtaining leukophoresis samples from participants and to Ms. Tsoarello Mabanga and Ms. Xiaoyan Ni for expert technical support. We also acknowledge the contribution of the study participants.

This study received support from the Canadian Institutes for Health Research (CIHR) HOP-123800 and MOP-142494 and the Fonds de Recherche du Québec-Santé (FRQ-S) AIDS and Infectious Diseases Network. GI was supported by a post-doctoral scholarship from Canadian HIV Trials Network and CIHR. IL was supported by a Ph.D. scholarship from FRQ-S and CIHR. BL is supported by a Junior I scholarship from FRQ-S. NFB is a member of the RI-MUHC, an institution funded in part by the FRQ-S.

Author Contributions

Conceptualization: NFB GI AT-M IL.
Data curation: GI AT-M IL NFB.
Formal analysis: GI AT-M IL.
Funding acquisition: NFB.
Investigation: GI AT-M IL.
Methodology: GI AT-M IL.
Project administration: NFB.
Resources: JB BL J-PR.
Supervision: GI IL NFB.
Validation: GI AT-M IL NFB.
Visualization: GI AT-M IL NFB.
Writing – original draft: GI AT-M IL NFB.
Writing – review & editing: GI AT-M IL NFB.

References

1. Anfossi N, Andre P, Guia S, Falk CS, Roetynck S, Stewart CA, Breso V, Frassati C, Reviron D, Middleton D, Romagne F, Ugolini S, Vivier E (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25: 331–342. pmid:16901727
- View Article
- PubMed/NCBI
- Google Scholar
2. Kim S, Sunwoo JB, Yang L, Choi T, Song YJ, French AR, Vlahiotis A, Piccirillo JF, Cella M, Colonna M, Mohanakumar T, Hsu KC, Dupont B, Yokoyama WM (2008) HLA alleles determine differences in human natural killer cell responsiveness and potency. Proc Natl Acad Sci U S A 105: 3053–3058. 0712229105 [pii]; pmid:18287063
- View Article
- PubMed/NCBI
- Google Scholar
3. Kim S, Poursine-Laurent J, Truscott SM, Lybarger L, Song YJ, Yang L, French AR, Sunwoo JB, Lemieux S, Hansen TH, Yokoyama WM (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436: 709–713. nature03847 [pii]; pmid:16079848
- View Article
- PubMed/NCBI
- Google Scholar
4. Cella M, Longo A, Ferrara GB, Strominger JL, Colonna M (1994) NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J Exp Med 180: 1235–1242. pmid:7931060
- View Article
- PubMed/NCBI
- Google Scholar
5. Gumperz JE, Litwin V, Phillips JH, Lanier LL, Parham P (1995) The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J Exp Med 181: 1133–1144. pmid:7532677
- View Article
- PubMed/NCBI
- Google Scholar
6. Wan AM, Ennis P, Parham P, Holmes N (1986) The primary structure of HLA-A32 suggests a region involved in formation of the Bw4/Bw6 epitopes. J Immunol 137: 3671–3674. pmid:2431040
- View Article
- PubMed/NCBI
- Google Scholar
7. Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger JL (1993) HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc Natl Acad Sci U S A 90: 12000–12004. pmid:8265660
- View Article
- PubMed/NCBI
- Google Scholar
8. Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N (1998) Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J Immunol 161: 571–577. pmid:9670929
- View Article
- PubMed/NCBI
- Google Scholar
9. Moesta AK, Norman PJ, Yawata M, Yawata N, Gleimer M, Parham P (2008) Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3. J Immunol 180: 3969–3979. 180/6/3969 [pii]. pmid:18322206
- View Article
- PubMed/NCBI
- Google Scholar
10. Braud V, Jones EY, McMichael A (1997) The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol 27: 1164–1169. pmid:9174606
- View Article
- PubMed/NCBI
- Google Scholar
11. Davis ZB, Cogswell A, Scott H, Mertsching A, Boucau J, Wambua D, Le GS, Planelles V, Campbell KS, Barker E (2016) A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells. PLoS Pathog 12: e1005421. PPATHOGENS-D-15-01881 [pii]. pmid:26828202
- View Article
- PubMed/NCBI
- Google Scholar
12. Ulbrecht M, Martinozzi S, Grzeschik M, Hengel H, Ellwart JW, Pla M, Weiss EH (2000) Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis. J Immunol 164: 5019–5022. ji_v164n10p5019 [pii]. pmid:10799855
- View Article
- PubMed/NCBI
- Google Scholar
13. Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, Cerundolo V, Borysiewicz LK, McMichael AJ, Wilkinson GW (2000) Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287: 1031. 8250 [pii]. pmid:10669413
- View Article
- PubMed/NCBI
- Google Scholar
14. Yawata M, Yawata N, Draghi M, Partheniou F, Little AM, Parham P (2008) MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response. Blood 112: 2369–2380. blood-2008-03-143727 [pii]; pmid:18583565
- View Article
- PubMed/NCBI
- Google Scholar
15. Yu J, Heller G, Chewning J, Kim S, Yokoyama WM, Hsu KC (2007) Hierarchy of the human natural killer cell response is determined by class and quantity of inhibitory receptors for self-HLA-B and HLA-C ligands. J Immunol 179: 5977–5989. pmid:17947671
- View Article
- PubMed/NCBI
- Google Scholar
16. Johansson S, Johansson M, Rosmaraki E, Vahlne G, Mehr R, Salmon-Divon M, Lemonnier F, Karre K, Hoglund P (2005) Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules. J Exp Med 201: 1145–1155. jem.20050167 [pii]; pmid:15809355
- View Article
- PubMed/NCBI
- Google Scholar
17. Fauriat C, Ivarsson MA, Ljunggren HG, Malmberg KJ, Michaelsson J (2010) Education of human natural killer cells by activating killer cell immunoglobulin-like receptors. Blood 115: 1166–1174. blood-2009-09-245746 [pii]; pmid:19903900
- View Article
- PubMed/NCBI
- Google Scholar
18. Boulet S, Song R, Kamya P, Bruneau J, Shoukry NH, Tsoukas CM, Bernard NF (2010) HIV protective KIR3DL1 and HLA-B genotypes influence NK cell function following stimulation with HLA-devoid cells. J Immunol 184: 2057–2064. jimmunol.0902621 [pii]; pmid:20061407
- View Article
- PubMed/NCBI
- Google Scholar
19. Brodin P, Karre K, Hoglund P (2009) NK cell education: not an on-off switch but a tunable rheostat. Trends Immunol 30: 143–149. S1471-4906(09)00039-8 [pii]; pmid:19282243
- View Article
- PubMed/NCBI
- Google Scholar
20. Kamya P, Boulet S, Tsoukas CM, Routy JP, Thomas R, Cote P, Boulassel MR, Baril JG, Kovacs C, Migueles SA, Connors M, Suscovich TJ, Brander C, Tremblay CL, Bernard N, Lessard B, Legault D, Maziade PJ, Longpre D, Vezina S, Trottier B, Rouleau D, Turner H, Falutz J, Potter M, Klein MB (2011) Receptor-ligand requirements for increased NK cell poly-functional potential in *h/*y+B57 HIV-1 infected Slow progressors. J Virol. JVI.02652-10 [pii]; pmid:21471235
- View Article
- PubMed/NCBI
- Google Scholar
21. Parsons MS, Zipperlen K, Gallant M, Grant M (2010) Killer cell immunoglobulin-like receptor 3DL1 licenses CD16-mediated effector functions of natural killer cells. J Leukoc Biol. jlb.1009687 [pii]; pmid:20664023
- View Article
- PubMed/NCBI
- Google Scholar
22. Gooneratne SL, Richard J, Lee WS, Finzi A, Kent SJ, Parsons MS (2015) Slaying the Trojan horse: natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T cells. J Virol 89: 97–109. JVI.02461-14 [pii]; pmid:25320293
- View Article
- PubMed/NCBI
- Google Scholar
23. Brodin P, Lakshmikanth T, Johansson S, Karre K, Hoglund P (2009) The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Blood 113: 2434–2441. blood-2008-05-156836 [pii]; pmid:18974374
- View Article
- PubMed/NCBI
- Google Scholar
24. Isitman G, Lisovsky I, Tremblay-McLean A, Parsons MS, Shoukry NH, Wainberg MA, Bruneau J, Bernard NF (2015) Natural killer cell education does not affect the magnitude of granzyme B delivery to target cells by antibody-dependent cellular cytotoxicity. AIDS 29: 1433–1443. 00002030-201507310-00003 [pii]. pmid:26244383
- View Article
- PubMed/NCBI
- Google Scholar
25. Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, Komoriya A, Ochsenbauer C, Kappes JC, Roederer M, Huang Y, Weinhold KJ, Tomaras GD, Haynes BF, Montefiori DC, Ferrari G (2011) High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A 79: 603–612. pmid:21735545
- View Article
- PubMed/NCBI
- Google Scholar
26. Parsons MS, Loh L, Gooneratne S, Center RJ, Kent SJ (2014) Role of education and differentiation in determining the potential of natural killer cells to respond to antibody-dependent stimulation. AIDS 28: 2781–2786. 00002030-201411280-00017 [pii]. pmid:25493604
- View Article
- PubMed/NCBI
- Google Scholar
27. Boulet S, Kleyman M, Kim JY, Kamya P, Sharafi S, Simic N, Bruneau J, Routy JP, Tsoukas CM, Bernard NF (2008) A combined genotype of KIR3DL1 high expressing alleles and HLA-B*57 is associated with a reduced risk of HIV infection. AIDS 22: 1487–1491. pmid:18614872
- View Article
- PubMed/NCBI
- Google Scholar
28. Boulet S, Sharafi S, Simic N, Bruneau J, Routy JP, Tsoukas CM, Bernard NF (2008) Increased proportion of KIR3DS1 homozygotes in HIV-exposed uninfected individuals. AIDS 22: 595–599. pmid:18317000
- View Article
- PubMed/NCBI
- Google Scholar
29. Kulkarni S, Martin MP, Carrington M (2010) KIR genotyping by multiplex PCR-SSP. Methods Mol Biol 612: 365–375. pmid:20033654
- View Article
- PubMed/NCBI
- Google Scholar
30. Boulassel MR, Spurll G, Rouleau D, Tremblay C, Edwardes M, Sekaly RP, Lalonde R, Routy JP (2003) Changes in immunological and virological parameters in HIV-1 infected subjects following leukapheresis. J Clin Apher 18: 55–60. pmid:12874816
- View Article
- PubMed/NCBI
- Google Scholar
31. Yu J, Mao HC, Wei M, Hughes T, Zhang J, Park IK, Liu S, McClory S, Marcucci G, Trotta R, Caligiuri MA (2010) CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood 115: 274–281. blood-2009-04-215491 [pii]; pmid:19897577
- View Article
- PubMed/NCBI
- Google Scholar
32. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH (1986) The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 136: 4480–4486. pmid:3086432
- View Article
- PubMed/NCBI
- Google Scholar
33. Cooper MA, Fehniger TA, Caligiuri MA (2001) The biology of human natural killer-cell subsets. Trends Immunol 22: 633–640. pmid:11698225
- View Article
- PubMed/NCBI
- Google Scholar
34. Sivori S, Cantoni C, Parolini S, Marcenaro E, Conte R, Moretta L, Moretta A (2003) IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors. Eur J Immunol 33: 3439–3447. pmid:14635054
- View Article
- PubMed/NCBI
- Google Scholar
35. Beziat V, Descours B, Parizot C, Debre P, Vieillard V (2010) NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs. PLoS One 5: e11966. pmid:20700504
- View Article
- PubMed/NCBI
- Google Scholar
36. Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, Bjorklund AT, Flodstrom-Tullberg M, Michaelsson J, Rottenberg ME, Guzman CA, Ljunggren HG, Malmberg KJ (2010) Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 116: 3853–3864. blood-2010-04-281675 [pii]; pmid:20696944
- View Article
- PubMed/NCBI
- Google Scholar
37. Lopez-Verges S, Milush JM, Pandey S, York VA, Arakawa-Hoyt J, Pircher H, Norris PJ, Nixon DF, Lanier LL (2010) CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset. Blood 116: 3865–3874. blood-2010-04-282301 [pii]; pmid:20733159
- View Article
- PubMed/NCBI
- Google Scholar
38. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, Casazza JP, Kuruppu J, Migueles SA, Connors M, Roederer M, Douek DC, Koup RA (2003) Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood 101: 2711–2720. pmid:12433688
- View Article
- PubMed/NCBI
- Google Scholar
39. Smalls-Mantey A, Connors M, Sattentau QJ (2013) Comparative efficiency of HIV-1-infected T cell killing by NK cells, monocytes and neutrophils. PLoS One 8: e74858. PONE-D-13-25673 [pii]. pmid:24040353
- View Article
- PubMed/NCBI
- Google Scholar
40. Romagnani C, Juelke K, Falco M, Morandi B, D'Agostino A, Costa R, Ratto G, Forte G, Carrega P, Lui G, Conte R, Strowig T, Moretta A, Munz C, Thiel A, Moretta L, Ferlazzo G (2007) CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 178: 4947–4955. 178/8/4947 [pii]. pmid:17404276
- View Article
- PubMed/NCBI
- Google Scholar
41. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, Frahm N, De Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P, Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH (2012) Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med 366: 1275–1286. pmid:22475592
- View Article
- PubMed/NCBI
- Google Scholar
42. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de SM, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH (2009) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361: 2209–2220. NEJMoa0908492 [pii]; pmid:19843557
- View Article
- PubMed/NCBI
- Google Scholar
43. Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, Buckler-White A, Seaman M, Piatak M Jr., Lifson JD, Dimitrov DS, Nussenzweig MC, Martin MA (2013) Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature 503: 277–280. nature12746 [pii]; pmid:24172896
- View Article
- PubMed/NCBI
- Google Scholar
44. Schoofs T, Klein F, Braunschweig M, Kreider EF, Feldmann A, Nogueira L, Oliveira T, Lorenzi JC, Parrish EH, Learn GH, West AP Jr., Bjorkman PJ, Schlesinger SJ, Seaman MS, Czartoski J, McElrath MJ, Pfeifer N, Hahn BH, Caskey M, Nussenzweig MC(2016) HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science 352: 997–1001. science.aaf0972 [pii]; pmid:27199429
- View Article
- PubMed/NCBI
- Google Scholar
45. Lu CL, Murakowski DK, Bournazos S, Schoofs T, Sarkar D, Halper-Stromberg A, Horwitz JA, Nogueira L, Golijanin J, Gazumyan A, Ravetch JV, Caskey M, Chakraborty AK, Nussenzweig MC (2016) Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo. Science 352: 1001–1004. science.aaf1279 [pii]; pmid:27199430
- View Article
- PubMed/NCBI
- Google Scholar
46. Barouch DH, Whitney JB, Moldt B, Klein F, Oliveira TY, Liu J, Stephenson KE, Chang HW, Shekhar K, Gupta S, Nkolola JP, Seaman MS, Smith KM, Borducchi EN, Cabral C, Smith JY, Blackmore S, Sanisetty S, Perry JR, Beck M, Lewis MG, Rinaldi W, Chakraborty AK, Poignard P, Nussenzweig MC, Burton DR (2013) Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature 503: 224–228. nature12744 [pii]; pmid:24172905
- View Article
- PubMed/NCBI
- Google Scholar
47. Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV (2014) Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell 158: 1243–1253. S0092-8674(14)01055-1 [pii]; pmid:25215485
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Anfossi N, Andre P, Guia S, Falk CS, Roetynck S, Stewart CA, Breso V, Frassati C, Reviron D, Middleton D, Romagne F, Ugolini S, Vivier E (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25: 331–342. pmid:16901727
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Kim S, Sunwoo JB, Yang L, Choi T, Song YJ, French AR, Vlahiotis A, Piccirillo JF, Cella M, Colonna M, Mohanakumar T, Hsu KC, Dupont B, Yokoyama WM (2008) HLA alleles determine differences in human natural killer cell responsiveness and potency. Proc Natl Acad Sci U S A 105: 3053–3058. 0712229105 [pii]; pmid:18287063
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Kim S, Poursine-Laurent J, Truscott SM, Lybarger L, Song YJ, Yang L, French AR, Sunwoo JB, Lemieux S, Hansen TH, Yokoyama WM (2005) Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature 436: 709–713. nature03847 [pii]; pmid:16079848
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Cella M, Longo A, Ferrara GB, Strominger JL, Colonna M (1994) NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J Exp Med 180: 1235–1242. pmid:7931060
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Gumperz JE, Litwin V, Phillips JH, Lanier LL, Parham P (1995) The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J Exp Med 181: 1133–1144. pmid:7532677
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Wan AM, Ennis P, Parham P, Holmes N (1986) The primary structure of HLA-A32 suggests a region involved in formation of the Bw4/Bw6 epitopes. J Immunol 137: 3671–3674. pmid:2431040
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger JL (1993) HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc Natl Acad Sci U S A 90: 12000–12004. pmid:8265660
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N (1998) Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J Immunol 161: 571–577. pmid:9670929
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Moesta AK, Norman PJ, Yawata M, Yawata N, Gleimer M, Parham P (2008) Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3. J Immunol 180: 3969–3979. 180/6/3969 [pii]. pmid:18322206
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Braud V, Jones EY, McMichael A (1997) The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol 27: 1164–1169. pmid:9174606
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Davis ZB, Cogswell A, Scott H, Mertsching A, Boucau J, Wambua D, Le GS, Planelles V, Campbell KS, Barker E (2016) A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells. PLoS Pathog 12: e1005421. PPATHOGENS-D-15-01881 [pii]. pmid:26828202
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Ulbrecht M, Martinozzi S, Grzeschik M, Hengel H, Ellwart JW, Pla M, Weiss EH (2000) Cutting edge: the human cytomegalovirus UL40 gene product contains a ligand for HLA-E and prevents NK cell-mediated lysis. J Immunol 164: 5019–5022. ji_v164n10p5019 [pii]. pmid:10799855
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, Gadola S, Cerundolo V, Borysiewicz LK, McMichael AJ, Wilkinson GW (2000) Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40. Science 287: 1031. 8250 [pii]. pmid:10669413
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Yawata M, Yawata N, Draghi M, Partheniou F, Little AM, Parham P (2008) MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires toward a balance of missing self-response. Blood 112: 2369–2380. blood-2008-03-143727 [pii]; pmid:18583565
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Yu J, Heller G, Chewning J, Kim S, Yokoyama WM, Hsu KC (2007) Hierarchy of the human natural killer cell response is determined by class and quantity of inhibitory receptors for self-HLA-B and HLA-C ligands. J Immunol 179: 5977–5989. pmid:17947671
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Johansson S, Johansson M, Rosmaraki E, Vahlne G, Mehr R, Salmon-Divon M, Lemonnier F, Karre K, Hoglund P (2005) Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules. J Exp Med 201: 1145–1155. jem.20050167 [pii]; pmid:15809355
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Fauriat C, Ivarsson MA, Ljunggren HG, Malmberg KJ, Michaelsson J (2010) Education of human natural killer cells by activating killer cell immunoglobulin-like receptors. Blood 115: 1166–1174. blood-2009-09-245746 [pii]; pmid:19903900
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Boulet S, Song R, Kamya P, Bruneau J, Shoukry NH, Tsoukas CM, Bernard NF (2010) HIV protective KIR3DL1 and HLA-B genotypes influence NK cell function following stimulation with HLA-devoid cells. J Immunol 184: 2057–2064. jimmunol.0902621 [pii]; pmid:20061407
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Brodin P, Karre K, Hoglund P (2009) NK cell education: not an on-off switch but a tunable rheostat. Trends Immunol 30: 143–149. S1471-4906(09)00039-8 [pii]; pmid:19282243
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Kamya P, Boulet S, Tsoukas CM, Routy JP, Thomas R, Cote P, Boulassel MR, Baril JG, Kovacs C, Migueles SA, Connors M, Suscovich TJ, Brander C, Tremblay CL, Bernard N, Lessard B, Legault D, Maziade PJ, Longpre D, Vezina S, Trottier B, Rouleau D, Turner H, Falutz J, Potter M, Klein MB (2011) Receptor-ligand requirements for increased NK cell poly-functional potential in *h/*y+B57 HIV-1 infected Slow progressors. J Virol. JVI.02652-10 [pii]; pmid:21471235
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Parsons MS, Zipperlen K, Gallant M, Grant M (2010) Killer cell immunoglobulin-like receptor 3DL1 licenses CD16-mediated effector functions of natural killer cells. J Leukoc Biol. jlb.1009687 [pii]; pmid:20664023
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Gooneratne SL, Richard J, Lee WS, Finzi A, Kent SJ, Parsons MS (2015) Slaying the Trojan horse: natural killer cells exhibit robust anti-HIV-1 antibody-dependent activation and cytolysis against allogeneic T cells. J Virol 89: 97–109. JVI.02461-14 [pii]; pmid:25320293
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Brodin P, Lakshmikanth T, Johansson S, Karre K, Hoglund P (2009) The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Blood 113: 2434–2441. blood-2008-05-156836 [pii]; pmid:18974374
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Isitman G, Lisovsky I, Tremblay-McLean A, Parsons MS, Shoukry NH, Wainberg MA, Bruneau J, Bernard NF (2015) Natural killer cell education does not affect the magnitude of granzyme B delivery to target cells by antibody-dependent cellular cytotoxicity. AIDS 29: 1433–1443. 00002030-201507310-00003 [pii]. pmid:26244383
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Pollara J, Hart L, Brewer F, Pickeral J, Packard BZ, Hoxie JA, Komoriya A, Ochsenbauer C, Kappes JC, Roederer M, Huang Y, Weinhold KJ, Tomaras GD, Haynes BF, Montefiori DC, Ferrari G (2011) High-throughput quantitative analysis of HIV-1 and SIV-specific ADCC-mediating antibody responses. Cytometry A 79: 603–612. pmid:21735545
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Parsons MS, Loh L, Gooneratne S, Center RJ, Kent SJ (2014) Role of education and differentiation in determining the potential of natural killer cells to respond to antibody-dependent stimulation. AIDS 28: 2781–2786. 00002030-201411280-00017 [pii]. pmid:25493604
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Boulet S, Kleyman M, Kim JY, Kamya P, Sharafi S, Simic N, Bruneau J, Routy JP, Tsoukas CM, Bernard NF (2008) A combined genotype of KIR3DL1 high expressing alleles and HLA-B*57 is associated with a reduced risk of HIV infection. AIDS 22: 1487–1491. pmid:18614872
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Boulet S, Sharafi S, Simic N, Bruneau J, Routy JP, Tsoukas CM, Bernard NF (2008) Increased proportion of KIR3DS1 homozygotes in HIV-exposed uninfected individuals. AIDS 22: 595–599. pmid:18317000
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Kulkarni S, Martin MP, Carrington M (2010) KIR genotyping by multiplex PCR-SSP. Methods Mol Biol 612: 365–375. pmid:20033654
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Boulassel MR, Spurll G, Rouleau D, Tremblay C, Edwardes M, Sekaly RP, Lalonde R, Routy JP (2003) Changes in immunological and virological parameters in HIV-1 infected subjects following leukapheresis. J Clin Apher 18: 55–60. pmid:12874816
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Yu J, Mao HC, Wei M, Hughes T, Zhang J, Park IK, Liu S, McClory S, Marcucci G, Trotta R, Caligiuri MA (2010) CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood 115: 274–281. blood-2009-04-215491 [pii]; pmid:19897577
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH (1986) The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 136: 4480–4486. pmid:3086432
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Cooper MA, Fehniger TA, Caligiuri MA (2001) The biology of human natural killer-cell subsets. Trends Immunol 22: 633–640. pmid:11698225
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Sivori S, Cantoni C, Parolini S, Marcenaro E, Conte R, Moretta L, Moretta A (2003) IL-21 induces both rapid maturation of human CD34+ cell precursors towards NK cells and acquisition of surface killer Ig-like receptors. Eur J Immunol 33: 3439–3447. pmid:14635054
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Beziat V, Descours B, Parizot C, Debre P, Vieillard V (2010) NK cell terminal differentiation: correlated stepwise decrease of NKG2A and acquisition of KIRs. PLoS One 5: e11966. pmid:20700504
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, Bjorklund AT, Flodstrom-Tullberg M, Michaelsson J, Rottenberg ME, Guzman CA, Ljunggren HG, Malmberg KJ (2010) Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 116: 3853–3864. blood-2010-04-281675 [pii]; pmid:20696944
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Lopez-Verges S, Milush JM, Pandey S, York VA, Arakawa-Hoyt J, Pircher H, Norris PJ, Nixon DF, Lanier LL (2010) CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset. Blood 116: 3865–3874. blood-2010-04-282301 [pii]; pmid:20733159
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, Casazza JP, Kuruppu J, Migueles SA, Connors M, Roederer M, Douek DC, Koup RA (2003) Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood 101: 2711–2720. pmid:12433688
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Smalls-Mantey A, Connors M, Sattentau QJ (2013) Comparative efficiency of HIV-1-infected T cell killing by NK cells, monocytes and neutrophils. PLoS One 8: e74858. PONE-D-13-25673 [pii]. pmid:24040353
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref40] 40. Romagnani C, Juelke K, Falco M, Morandi B, D'Agostino A, Costa R, Ratto G, Forte G, Carrega P, Lui G, Conte R, Strowig T, Moretta A, Munz C, Thiel A, Moretta L, Ferlazzo G (2007) CD56brightCD16- killer Ig-like receptor- NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation. J Immunol 178: 4947–4955. 178/8/4947 [pii]. pmid:17404276
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref41] 41. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, Frahm N, De Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P, Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH (2012) Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med 366: 1275–1286. pmid:22475592
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref42] 42. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de SM, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH (2009) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361: 2209–2220. NEJMoa0908492 [pii]; pmid:19843557
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref43] 43. Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, Buckler-White A, Seaman M, Piatak M Jr., Lifson JD, Dimitrov DS, Nussenzweig MC, Martin MA (2013) Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature 503: 277–280. nature12746 [pii]; pmid:24172896
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref44] 44. Schoofs T, Klein F, Braunschweig M, Kreider EF, Feldmann A, Nogueira L, Oliveira T, Lorenzi JC, Parrish EH, Learn GH, West AP Jr., Bjorkman PJ, Schlesinger SJ, Seaman MS, Czartoski J, McElrath MJ, Pfeifer N, Hahn BH, Caskey M, Nussenzweig MC(2016) HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science 352: 997–1001. science.aaf0972 [pii]; pmid:27199429
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref45] 45. Lu CL, Murakowski DK, Bournazos S, Schoofs T, Sarkar D, Halper-Stromberg A, Horwitz JA, Nogueira L, Golijanin J, Gazumyan A, Ravetch JV, Caskey M, Chakraborty AK, Nussenzweig MC (2016) Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo. Science 352: 1001–1004. science.aaf1279 [pii]; pmid:27199430
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref46] 46. Barouch DH, Whitney JB, Moldt B, Klein F, Oliveira TY, Liu J, Stephenson KE, Chang HW, Shekhar K, Gupta S, Nkolola JP, Seaman MS, Smith KM, Borducchi EN, Cabral C, Smith JY, Blackmore S, Sanisetty S, Perry JR, Beck M, Lewis MG, Rinaldi W, Chakraborty AK, Poignard P, Nussenzweig MC, Burton DR (2013) Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature 503: 224–228. nature12744 [pii]; pmid:24172905
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref47] 47. Bournazos S, Klein F, Pietzsch J, Seaman MS, Nussenzweig MC, Ravetch JV (2014) Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell 158: 1243–1253. S0092-8674(14)01055-1 [pii]; pmid:25215485
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Ethics statement and study population

Genotyping

Cells

CD16 surface staining

Statistical analysis

Results

CD16, NKG2A, and CD57 expression in CD56+ NK cell populations

CD16 and KIR expression on total CD56+ and CD56dim NK cells

The effect of KIR education on CD16 Expression

Discussion

Supporting Information

S1 Fig. The gating strategy used for detecting NK cells.

S1 Table. Data used to create Fig 1B.

S2 Table. Data used to create Fig 1C.

S3 Table. Data used to create Fig 1D.

S4 Table. Data used to create Fig 1E.

S5 Table. Data used to create Fig 1F.

S6 Table. Data used to create Fig 2A.

S7 Table. Data used to create Fig 2C.

S8 Table. Data used to create Fig 3.

Acknowledgments

Author Contributions

References

CD16, NKG2A, and CD57 expression in CD56⁺ NK cell populations

CD16 and KIR expression on total CD56⁺ and CD56^dim NK cells