TYPE Original Research PUBLISHED 18 November 2022 DOI 10.3389/fimmu.2022.1027855 Disruption of the HLA-E/NKG2X OPEN ACCESS axis is associated with EDITED BY Hugh Thomson Reyburn, Spanish National Research Council uncontrolled HIV infections (CSIC), Spain REVIEWED BY Luis Romero-Martı́n 1,2, Clara Duran-Castells 1,2, Enrique Martin-Gayo, Fundación de la Universidad Mireia Olivella 3, Mı́riam Rosás-Umbert 1, Marta Ruiz-Riol 1,4, Autónoma de Madrid, Spain Francisco Borrego, Jorge Sanchez 5, Dennis Hartigan-O´Connor 6,7, Biocruces Bizkaia Health Research Beatriz Mothe 1,3,8, Àlex Olvera 1,3,4*† and Christian Brander 1,3,4,9*† Institute, Spain 1 IrsiCaixa AIDS Research Institute, Hospital Germans Trias i Pujol, Institute for Health Science *CORRESPONDENCE Research Germans Trias i Pujol (IGTP), Badalona, Spain, 2 Universitat Autonoma de Barcelona (UAB), Àlex Olvera Barcelona, Spain, 3 University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain, aolvera@irsicaixa.es 4 CIBERINFEC, Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Christian Brander Madrid, Spain, 5 Impacta, Lima, Peru, 6 California National Primate Research Center and Department cbrander@irsicaixa.es of Medical Microbiology and Immunology, University of California, Davis, Davis, CA, United States, † These authors have contributed 7 Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, equally to this work San Francisco, CA, United States, 8 Fundació Lluita contra la Sida, Infectious Disease Department, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 9 ICREA, Barcelona, Spain SPECIALTY SECTION This article was submitted to NK and Innate Lymphoid Cell Biology, a section of the journal Frontiers in Immunology The contribution of the HLA-E/NKG2X axis in NK-mediated control of HIV RECEIVED 25 August 2022 infection remains unclear. We have studied the relationship between HLA-E ACCEPTED 18 October 2022 expression and phenotypical as well as functional characteristics of NK cells, in PUBLISHED 18 November 2022 the context of chronic HIV infection and in an in vitro model of acute infection. CITATION High viremia in HIV+ individuals was related to increased HLA-E expression, Romero-Martı´n L, Duran-Castells C, Olivella M, Rosás-Umbert M, and changes in NK subpopulations, especially a reduction of the CD56bright as Ruiz-Riol M, Sanchez J, well as an increase in adaptive NK subpopulation. Uncontrolled HIV infection Hartigan-O´Connor D, Mothe B, Olvera À and Brander C (2022) was also characterized by a reversion of the NKG2A/NKG2C expression ratio Disruption of the HLA-E/NKG2X and a loss of positive and negative regulation of NK mediated by HLA-E. This axis is associated with uncontrolled was reflected in a lower cytotoxic, degranulation and cytokine production HIV infections. Front. Immunol. 13:1027855. capacity, especially in CD56bright and adaptive NK. In line with these results, doi: 10.3389/fimmu.2022.1027855 HLA-E expression showed a positive correlation with viral growth inhibition in COPYRIGHT an in vitro model of acute infection at day 7, which was lost after 14 days of © 2022 Romero-Martı´n, Duran-Castells, culture. Using HLA-E expressing K562 cells, we determined that only one out of Olivella, Rosás-Umbert, Ruiz-Riol, Sanchez, Hartigan-O´Connor, Mothe, 11 described HIV-derived HLA-E epitopes increased HLA-E surface stability. In Olvera and Brander. This is an open- spite of that, eight of the 11 epitopes were capable of increasing degranulation access article distributed under the terms of the Creative Commons and three drove differences in NK-cell mediated cell lysis or cytokine secretion. Attribution License (CC BY). The use, In conclusion, our results indicate that HLA-E molecules presenting HIV- distribution or reproduction in other derived epitopes may sensitize target cells for NK lysis in early HIV infection. forums is permitted, provided the original author(s) and the copyright However, prolonged exposure to elevated HLA-E expression levels in vivo may owner(s) are credited and that the lead to NK cell dysfunction and reduced viral control In chronic infection. original publication in this journal is cited, in accordance with accepted academic practice. No use, KEYWORDS distribution or reproduction is HIV, Natural Killer cells, HLA-E, NKG2A, NKG2C permitted which does not comply with these terms. Frontiers in Immunology 01 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 Introduction relevant function in regulating NK function through the HLA-E/ NKG2X axis. HLA-E is a non-classical class Ib histocompatibility Natural killer (NK) cells are innate lymphoid cells that molecule with very limited polymorphism. Only two alleles represent approximately 5-10% of circulating peripheral blood (HLA-E*01:01 and HLA-E*01:03) have been demonstrated to mononuclear cells (PBMC) and can be divided in a broad be properly expressed on the cell surface and together spectrum of NK-cell subsets. In brief, NK cells were first cover >99% of the global allelic diversity of HLA-E (15). Given classified into two subpopulations based on the expression of this conservation, targeting HLA-E epitope presentation has CD56 and CD16, named CD56bright (CD56bright CD16-) and been considered for developing universal immunotherapies CD56dim (CD56dim CD16+), and different phenotypical and and vaccines (16). Structural differences between the dominant functional characteristics were attributed to these subsets (1). HLA-E alleles are very limited since a single residue in position More recent studies have reported a novel, memory-like, 107 within the a2 domain, differentiates HLA-E*01:01 adaptive NK subset that expresses the maturation marker (arginine) from HLA-E*01:03 (glycine) (17). The presence of CD57 and responds specifically to certain pathogens (2). In the positive amino acid in this position in HLA-E*01:01 is addition, in persistent infections, sustained NK stimulation can thought to cause the lower surface stability of HLA-E*01:01 lead to a skewed exhausted phenotype with reduced cytokine compared to HLA-E*01:03 (18). In addition, this change may production (3–5). To date, NK cell exhaustion has been mainly also induce a subtle structural shift in the binding-cleft a2 helix, studied in the context of tumor-associated NK cells and to resulting in an altered repertoire of presented epitopes. HLA-E application of strategies to reverse their exhaustion and was first described as part of the innate immune system and was augment anti-tumor activity (6–9). reported to inhibit NK mediated lysis of healthy cells by In general, NK-cell stimulation is the result of a balance presenting peptides derived from the leader sequences of HLA between activating and inhibitory signals to which they are class I molecules to the CD94/NKG2A heterodimer (19). exposed, signals that can vary depending on the nature of the Growing evidence supports now also a role of HLA-E in the binding cell (10). NK cells express diverse receptors, which adaptive immune response through the presentation of usually use classical or non-classical MHC-class I molecules as pathogen-derived epitopes to specific CD8+ T-cells (20, 21). ligands. They can be divided in inhibitory receptors, which Several viruses have developed mechanisms to evade NK trigger NK activation if they cannot detect MHC molecules killing of infected target cells, such as the expression of peptides presenting canonical self-derived peptides on the surface of mimicking human canonical self-epitopes to effectively stabilize target cells (referred to as “the missing self”), and their inhibitory NK receptors (22) or the modulation of surface activating counterparts that activate NK cells upon recognition molecules, such as HLA class I, to counteract NK recognition of MHC class I molecules presenting non-self-peptides (11). The via the missing self-axis (23). For instance, HCMV-derived C-type lectin-like receptor superfamily (NKG2) are NK UL40 peptide, presented on HLA-E, transduces an inhibitory receptors with most of its members (NKG2A, B, C, E and H) signal through CD94/NKG2A recognition (24). HCMV has also binding non-classical MHC class Ib molecules (including HLA- been described to alter NK cells subsets by driving the E). Binding of HLA-E to the heterodimer formed by CD94 and proliferation of a memory-like CD57+NKG2Chigh NK cells NKG2A has an inhibitory effect on NK cell function, since subset, with an imprint in the IFNG locus that leads to a NKG2A contains two immune receptor tyrosine-based strong IFN-g production upon NKG2C engagement (25, 26). inhibitory motifs (ITIM) that transduce an inhibitory signal Finally, it has been shown that the NKG2A/NKG2C ratio is (12). However, if HLA-E is downregulated on target cells (the inverted in NK cells derived from people living with HIV and co- missing self), or the bound epitope repertoire on HLA-E infected with HCMV (27). prevents effective interaction with CD94/NKG2A, the loss of A number of past studies support an important role of HLA- ligand interaction induces NK activation due to a lack of the E and NK cells activity also in the context of HIV infection. First, inhibitory signal (7, 8). On the other hand, when CD94 forms the HIV Nef protein has been suggested to downregulate surface heterodimers with NKG2C, E or H, these receptors have an expression of HLA-E, impairing the recognition of infected cells activating effect on the NK cell. This different function is due to a by either the T cell receptors or NKG2X/CD94 and thereby positively charged residue within the transmembrane regions of avoiding cell lysis (28). Still, mostly through elution studies, five NKG2C, E and H that allows them to associate with the HIV-1 derived epitopes have been reported to be presented by immunoreceptor tyrosine-based activating motif (ITAM) of HLA-E: RMYSPVSIL (RL9), PEIVIYDYM (PM9), TALSEGATP DAP12 (13). Of note, NKG2C expression is increased at later (TP9), RIRTWKSLV (RV9) (29) and AISPRTLNA (AA9) (30), stages of NK cell maturation and binds HLA-E with lower while some others have been reported as potential HLA-E affinity than NKG2A (14). Among the myriad of NK epitope candidates since they show slight effects on HLA-E receptors, we focused our study on these two NK receptors stabilization: EKIKALVEI (EI9), MYSPVSILD (MD9), (NKG2A and NKG2C) due to their marked opposite, but very NEEAAEWDR (NR9), QMAVFIHNF (QF9) and Frontiers in Immunology 02 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 YFSVPLDEG (YG9) (29). However, among these HIV-1-derived 1 acquisition (n=8, acute), iii) low risk HIV seronegative HLA-E binding peptides, only AA9 has been shown to have a individuals (n=24, seronegative), iv) chronically HIV infected functional impact on the NK response by increasing the individuals one year before receiving cART (n=11, chronics), v) activation of NKG2A + NK cells. These data have been chronically infected individuals after receiving cART for at least interpreted to suggest that HIV-1 might alter the presented one year (n=6, treated) and an additional validation cohort of HLA-E peptide repertoire and trigger NK cells degranulation by long-term non-progressors, HIV infected individuals not blocking the inhibitory signal of the NKG2A receptor (30). Thus, receiving cART (n=23, LTNP) (Supplementary Table 2). The understanding the role of HLA-E/NKG2X regulation in the NK study was approved by the Comitè Ètic d’Investigació Clı́nica of mediated elimination of HIV-1 infected cells and how the Hospital Germans Trias i Pujol (CEIC EO-12-042), and all availability and presentation of viral epitopes can interfere in participants provided written informed consent. Fresh these processes, may be critical to our understanding of HIV leukocyte fraction from blood donations (n=18) from HIV- control. These mechanisms may be especially relevant when seronegative individuals was obtained from the Catalan Blood considering proposed NK cell therapies, such as the infusion of and Tissue Bank (Banc de Sang i Teixit, BST). ex vivo expanded specific NK cell populations and chimeric antigen receptor (CAR)-expressing NK cells (31, 32), or the use of latency reversing agents to eliminate HIV infected cells by HIV-derived HLA-E binders Kick-and-Kill strategies (33). peptides selection In the present work, we hypothesized that an increased HLA-E surface expression in untreated, chronically HIV-1 A total of 14 HLA-E binding epitopes (Table 1) were infected, viremic individuals leads to a chronic NK stimulation synthesized (Snypeptide) and tested in our study, including: 2 through the HLA-E/NKG2X axis, while HIV-derived epitopes described canonical HLA-E epitopes, one derived from the could further modulate the HLA-E/NKG2X interaction. Such HLA-Cw1 signal peptide (VL9-Cw1) (41) and another derived modulation may eventually cause a disruption of the balance from CMV (VL9-CMV) (42), and 12 non-canonical peptides between NKG2A and NKG2C expression and induce an derived from EBV (SL9) (43) and HIV (AA9, KG9, MD9, PM9, impaired NK response. We employed peptide binding studies, QE9, RL9, RV9, SN9, TP9, VI9, YG9). HIV-derived peptides NK functional tests and structural analysis of HLA-E*01:01 and were selected based on reported HLA-E surface stabilization HLA-E*01:03 to test this hypothesis and to address how the activity (MD9, PM9, RV9, TP9, YG9) (29), alteration of NK HLA-E/NKG2X axis relates to NK activity and consequent degranulation (AA9) (30) and HIV homologies of described SIV clinical outcome of untreated HIV infection. epitopes shown to be the targeted by SIV-specific Mamu-E restricted T-cell responses in rhesus macaques (KG9, QE9, RL9, SN9, VI9) (44). Material and methods Cell samples Structural analysis of HLA-E and TCR/ NKG2 receptors interaction HIV-infected subjects (n=51) recruited at the Hospital Germans Trias i Pujol (Badalona, Spain) and the IMPACTA A molecular model was constructed for HLA-E*01:01 and clinics in Lima (Peru) were included in this study. These HLA-E*01:03 bound to PM9 and RL9 peptides based on the included individuals that were not receiving antiretroviral 6GL1 X-ray crystal structure. The initial molecular model of treatment and had i) low HIV plasma viral loads (HIV-low, HLA-E-peptides interacting with NKG2A/2C-CD94 and TCR n=31) of <10,000 HIV RNA copies/mL (range: 25 to 9,999; was constructed based on available X-ray crystal structure in the median: 1,877 HIV RNA copies/mL) and CD4 counts that Protein Data Bank (3CDG and 5W1V). The initial models were ranged from 434 to 1,343 cells/mm3 (median: 762 cells/mm3) Energy Minimized using Gromacs 5.0 (45). or ii) high HIV plasma viral loads (HIV-high, n=20) of >50,000 HIV RNA copies/mL (range: 50,295 to 1,200,000; median: 239,511 HIV RNA copies/mL) and CD4 counts that ranged Peptide-pulse HLA-E surface from 11 to 726 cells/mm 3 (median: 284 cells/ mm 3 ) stabilization assay (Supplementary Table 1). Additional unrelated cohorts were included as comparison and validation groups (n=54): i) highly- HLA-E*(01:03) transfected K562 cell lines (HLA-E/VL9- exposed seronegative individuals, sampled six months prior to K562), co-expressing the HLA-B*0702 signal sequence derived infection (n=6, pre-HIV), ii) acute/recently HIV-1 infected VL9 peptide (VMAPRTVLL) (46), were kindly provided by Dr. individuals, sampled within a median of 6 months from HIV- Joosten (University of Leiden, The Netherlands) and originally Frontiers in Immunology 03 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 TABLE 1 HLA-E binding epitopes tested in this study. ID Sequence Organism Protein Reference VL9-B7 VMAPRTVLL Human HLA-B7 Leader sequence 34 VL9-Cw1 VMAPRTLIL Human HLA-Cw1 Leader sequence 35 VL9-CMV VMAPRTLIL CMV UL40 36 E SL9 SQAPLPCVL EBV BZLF1 37 HIV AA9 AISPRTLNA HIV Gag p24 38 HIV KG9 KRIKCFNCG HIV Gag p24 39 HIV MD9 MYSPVSILD HIV Gag p24 40 HIV PM9 PEIVIYQYM HIV Pol Retrotranscriptase 40 HIV QE9 QMLKETINE HIV Gag p24 39 HIV RL9 RMYSPTSIL HIV Gag p24 40 HIV RV9 RIRTWKSLV HIV Vif 40 HIV SL9 SEELRSLYN HIV Gag p24 39 HIV TP9 TALSEGATP HIV Gag p17 40 HIV VI9 VGEIYKRWI HIV Gag p24 39 HIV YG9 YFSVPLDEG HIV Pol Protease 40 produced by Dr. E. Weiss (Ludwig-Maximilians-Universitat, cultured in R10 medium (RPMI supplemented with 10% FBS, Munich, Germany) (42). These cell lines were cultured in 100 U/mL penicillin and 100 ug/mL streptavidin), together IMDM supplemented with 10% fetal bovine serum (FBS) and with the different ratios of NK cells isolated from the HIV 200 µg/mL of G418 (I10 medium) at 26°C 5% CO2 for three seronegative individuals, for 4 hours at 37°C and 5% CO2. hours, prior to being pulsed with 20 µg/mL of canonical and Cultures were then run on a Canto II BD Cytometer and the non-canonical HIV-derived peptides, for 16 hours at 26°C 5% percentage of killing of labelled target cells calculated as 100-% CO2. Cells were then washed twice with I10 medium and rested calcein-AM+ K562 cells. In each condition, an exponential for 2 hours at 37°C 5% CO2. Cells were then stained with 3D12 regression curve was determined by the percentage of killing PE anti-human HLA-E (BioLegend) and fixed (Fix&Perm Cell resulting from the different effector to target ratios in the co- Permeabilization Kit, Thermofisher). At least 100,000 events culture. Results were expressed in lytic units (48) calculated as were collected on a LSRII BD cytometer and analyzed using the number of NK cells contained in 106 PBMC able to lyse FlowJo v10 software. HLA-E surface stabilization was expressed 20% of 100,000 target cells. as (% HLA-E+ cells in peptide-pulsed cells/% HLA-E+ cells in non-pulsed cells) and (HLA-E MFI in peptide-pulsed cells/HLA- E MFI in non-pulsed cells). NK degranulation assay NK degranulation activity was measured by flow cytometry NK cytotoxicity assay at a fix 5:1 NK effector to target cell ratio. Targets were HLA- E*01:03/VL9-K562 cells pulsed with canonical and non- Peripheral blood mononuclear cells (PBMC) were isolated canonical (HIV-derived) peptides. NK cells sorted from from cryopreserved leukocyte fractions using Lymphoprep™ cryopreserved PBMC and peptide-pulsed target cells were co- (Stemcell) density gradient. NK cells were isolated by magnetic cultured for 4 hours in the presence of Golgi Stop, Golgi Plug bead separation (MACS Miltenyi Biotec) from PBMC, and APC anti-human CD107 (Biolegend) and stained for surface following the manufacturer’s instructions. NK cytotoxic markers: SK7 Amcyan anti-CD3 (BD), 5.1H11 BV785 anti- capacity was measured at 10:1, 5:1, 2.5:1, and 1:1 NK effector CD56 and B73.1 BV-605 anti-CD16 (Biolegend). to target cell ratio (HLA-E*0103/VL9-K562 targets). Target Subsequently, cells were fixed, permeabilized (Fix&Perm Cell cells were pulsed with canonical and non-canonical HIV- Permeabilization Kit, Thermofisher) and stained with 6401.1111 derived peptides and labeled using calcein-AM to perform a FITC anti-human TNF-a and B27 PE-Cy7 anti-human IFN-g retention assay as previously described (47). Briefly, 12,500 (BD). Approximately 20,000 events per samples were acquired peptide-pulsed HLA-E/VL9-K562 cells were labelled for 30 on a Canto II BD Cytometer and analyzed using FlowJo minutes at 37°C 5% CO2 with calcein-AM at 10 nM and then v10 software. Frontiers in Immunology 04 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 NK cells subpopulations and magnetic bead separation (MACS NK isolation kit, Miltenyi determination of NKG2A and Biotec). The assay was performed in triplicates by culturing NKG2C expression 50,000 HIV-infected or uninfected autologous CD4+ T-cells alone or in the presence of unstimulated autologous NK cells To discriminate different NK cell subpopulations, isolated (2:1, 1:1, 1:2 and 1:10 NK effector to CD4 + T-cells target cell NK cells from cryopreserved PBMC from HIV seronegative ratio). NK cells were previously rested for 2 hours at 37°C 5% (n=12), HIV-high (n=8) and HIV-low (n=8) individuals were CO2 after magnetic sorting in R10 medium, supplemented with stained with SK7 Amcyan anti-CD3 (BD), 5.1H11 BV-785 anti- 50 U/mL of IL-2 and 1 ng/mL of IL-15. After one week (day 7 of CD56, B73.1 BV-605 anti-CD16, HNK-1 PerCP-Cy5 anti-CD57 co-culture), half of the medium was collected and replaced with (Biolegend), 131411 PE anti-NKG2A and 134591 APC anti- 50 µL of R10 supplemented with 100 U/mL of IL-2 and 2 ng/mL NKG2C (R&D Systems). Samples were run on a LSRII BD of IL-15. At day 14, the culture supernatant was harvested and Cytometer and analyzed using FlowJo v10 software. NK cell NK cells from the long-term culture characterized by flow subpopulations were classified as (i) CD56-CD16- (CD3-CD56- cytometry (as described before). HIV-1 p24 Gag concentration CD16-CD57-), (ii) CD56dim (CD3-CD56dimCD16+CD57-), (iii) in the supernatant was measured at day 7 and day 14 by adaptive NK cells (CD3 - CD56dim CD16 + CD57 + ) and (iv) commercially available HIV-1 p24 ELISA kit (Innogenetics) in CD56 bright (CD3 - CD56 bright CD16 - CD57 - ). NKG2A and accordance with manufacturer’s instructions. Samples were read NKG2C surface expression were evaluated in all NK on a Perkin Elmer Ensight Plate reader. NK-mediated inhibition subpopulations, by the gating strategy summarized in Figure of viral replication was calculated as %inhibition = [(HIV-1 p24 S1A. This data set, together with data from CD3-negative cells Gag concentration in CD4+ T-cells cultured alone)-(HIV-1 p24 from the sorted NK cell fraction used in the NK-VIA Gag concentration in CD4+ T-cells cultured with NK cells)]/ experiments, were downsampled to obtain 3,000 events/fcs file. (HIV-1 p24 Gag concentration in CD4+ T-cells cultured An unsupervised tSNE dimensional reduction was performed on alone)x100. FlowJo 10.5.2 using the default settings of a nearest neighbours of 15 and a minimum Euclidean distance of 0.5. Number of clusters was determined by X-Shift (Number nearest neighbors: Total HIV-1 DNA quantification 132, Euclidean distance metric) and FlowSOM and was applied to determine the level of expression of CD56, CD16, CD57, Total HIV-1 DNA was quantified in PBMC lysates by NKG2A and NKG2C in a total of 11 clusters in the CD3- droplet digital PCR (ddPCR) in duplicate as described negative population. The abundance of each cluster was previously (39). Briefly, two different primer/probe sets, evaluated comparing samples from HIV seronegative, HIV- annealing to the 5′ long terminal repeat and Gag regions, high and HIV-low individuals. respectively were used to circumvent sequence mismatch in the individual´s proviruses, and the RPP30 housekeeping gene was quantified in parallel to normalize sample input. Raw NK-mediated viral inhibition assay ddPCR data were analyzed using the QX100 droplet reader and QuantaSoft v.1.6 software (Bio-Rad). NK cell mediated viral inhibition activity was measured in 12 HIV-negative individuals. Briefly, the CD4+ T cell enriched fraction was obtained from freshly isolated PBMC using NK DNA/RNA extraction and real-time PCR and CD8+ T-cell specific magnetic beads depletion (MACS NK Isolation kit and CD8+ T-cell isolation kit, Miltenyi Biotec) and DNA and RNA were simultaneously extracted using the stimulated with PHA (5 µg/mL) in R10. HLA-E surface AllPrep DNA/RNA Mini kit (QIAGEN) from cell pellets from expression was measured on the CD3+ CD4+ population by the 51 HIV infected individuals and the 12 HIV seronegative flow cytometry (as mentioned before) after three days of individuals. Retrotranscription (SuperScript II first-strand stimulation. Gating strategy is shown in Figure S1B. This synthesis Supermix) was performed on those samples and the fraction was then infected by spinoculation (720g for 2h at 25° resulting cDNA was used for reverse transcription-PCR using C) with HIV-1 BaL and HIV-1 NL4-3 laboratory-adapted strains TaqMan gene expression assay for detection of HLA-E at a multiplicity of infection (MOI) of 0.01. On day 2, the day (Hs03045171_m1) and TBP (Hs99999910_m1) all from before starting the co-culture, one vial of cryopreserved PBMC Applied Biosystems in a 7500 Real-Time PCR Systems from the same individual was thawed and cultured overnight (Applied Biosystems™). Relative expression was calculated as with 20 U/mL of IL-2, prior to positive NK cells sorting by 2-DCt (where Ct is the median threshold cycle from 3 replicates). Frontiers in Immunology 05 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 HLA-E genotyping <0.0001). This difference was confirmed in unrelated validation cohorts, where a remarkable reduction of HLA-E expression was An in-house developed strategy based on Next Generation found in individuals showing control of HIV infection in Sequencing (NGS) was used for HLA-E genotyping of human comparison to untreated HIV infected individuals with high PBMC and HLA-E expressing K562 cell lines at the Laboratori viral loads (MW p-value = 0.0006) (Figure 1A). In addition, d’Histocompatibilitat i Immunogenètica, Banc de Sang i Teixits when compared to HIV seronegative individuals, the levels of (BST). This strategy consists of a long-range PCR to amplify the HLA-E expression were significantly higher in individuals with HLA-E gene from 5’ UTR to 3’ UTR. Library preparation chronic HIV infection, receiving cART (MW p-value = 0.0106) (enzymatic fragmentation, adapter ligation, and barcoding) or untreated (MW p-value <0.0001). Moreover, we observed not was performed using the NGSgo-LibrX and NGSgo-IndX kits only a direct positive correlation between HLA-E expression and (GenDx) according to the manufacturer’s instructions. The final plasma viral load (r = 0.5681, p-value <0.0001), but also between denatured library was sequenced using a MiSeq system with a HLA-E expression and proviral HIV copy numbers (r = 0.3828, 300-cycle MiSeq Reagent Kit (Illumina). HLA-E genotype p-value = 0.0086, Figure 1B). determination was performed using NGSengine software We next clarified whether the single amino acid difference (GenDX, version 2.21.0.20156) and the IPD-IMGT/HLA between HLA-E*01:01 and HLA-E*01:03 resulted in differential database as a reference (version 3.38.0). Raw sequencing data associations with virological parameters. HLA-E allele haplotype are available at the European Nucleotide Archive under the did not impact HLA-E gene expression and did not show any accession number ERA18504000. association with HIV plasma viral load when all samples were compared together (data not shown). However, when we compared the impact of the allele polymorphisms between Statistical analysis HLA-E*01:01 and *01:03 on plasma viral load only in individuals with lower HIV viral load (HIV-low, n=31), a GraphPad Prism version 7 for Mac OS X (La Jolla, CA) was significantly higher proportion of individuals with used for statistical analyses. For comparison between groups, undetectable plasma viral load (<50 HIV RNA copies/mL) non-parametric Mann-Whitney (MW) test was performed for were HLA-E heterozygous (c2, p-value = 0.0186, Figure 1C). unpaired data and Wilcoxon test (W) for paired data. c2 analysis These data suggest that higher levels of HLA-E expression in was used to detect differences in the frequency of individuals PBMC are related to uncontrolled chronic HIV infection, while with plasma viral loads below the detection level. Multiple reduced expression is associated with relative control of HIV comparisons were performed by one-way ANOVA, two-way viremia. In addition, there was a benefit of HLA-E*01:01/01:03 ANOVA for unpaired data and Friedman’s test (F) for paired heterozygosity as individuals with low and undetectable viral data with post-hoc false discovery rate correction (FDR). HLA-E loads were enriched for a heterozygous genotype. mRNA expression levels measured by RT-PCR were corrected for CD4+ T cell counts when possible. Correlation between two measures was analyzed using the non-parametric Spearman Uncontrolled chronic HIV infection is rank test, the corresponding correlation p-value was also related to an imbalance of the CD56bright calculated. Statistical significance criteria were set at p- NKG2A+ and adaptive value <0.05 and q-value <0.15. (CD16+CD56dimCD57+) NKG2C+ NK cell subpopulations In the context of infectious diseases, HLA-E expression has Results been linked to activation of NK cells with antiviral capacity. Since our analyses indicated that elevated HLA-E expression was Association of HLA-E expression level related with high plasma viral loads, we examined whether this and allele genotype with HIV finding could reflect changes in specific NK cell subsets. For this disease progression purpose, we compared the prevalence of NK subsets characterized by the surface expression of NK subset markers In order to detect a potential relationship between HLA-E CD56, CD16, CD57 and NKG2A and NKG2C in seronegative gene expression and the course of HIV infection, we evaluated (SN), HIV-low (HL) and HIV-high (HH) individuals. the HLA-E mRNA levels in total PBMC from HLA-E genotyped, Dimensional reduction was conducted after multiparameter HIV-seropositive individuals with different levels of viremia and flow cytometry based on CD56, CD16, CD57, NKG2A and CD4+ T cell counts. Significantly different HLA-E mRNA levels NKG2C (Figure 2A) and 11 clusters were defined. Figure 2B were observed between HIV-high and HIV-low groups, even represent the relative expression of the surface markers for each when correcting for CD4+ T cell counts (Figure 1A, MW p-value cluster. We then compared the abundance of each cluster among Frontiers in Immunology 06 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C FIGURE 1 Role of HLA-E allele haplotypes and expression level during HIV infection. (A) HLA-E mRNA expression, measured by qPCR and corrected by CD4 count, is reduced in HIV-low individuals (n=31) compared to HIV-high (n=20) individuals (left). HLA-E expression progressively increases during natural course of HIV infection (right). (B) HLA-E expression levels correlated positively with plasma viral loads and HIV viral reservoir size (determined as copy number of integrated proviral DNA). HLA-E allelic differences showed no difference in terms of HLA-E expression, plasma viral load and reservoir size. (C) Frequency of HLA-E genotypes within the HIV-low group (n=31). Mann-Whitney test was performed to compare differences between groups (A), Spearman’s rank correlation coefficient was applied to evaluate correlations (B) and Chi-square was performed to determine the significance of HLA-E haplotype frequencies among HIV natural controllers (C). Results were considered statistically significant when p-value <0.05 while trends are indicated in grey. Frontiers in Immunology 07 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C FIGURE 2 Unsupervised analysis of the NK cell repertoire. (A) tSNE dimensional reduction was performed in the CD3 negative fraction of magnetically- sorted NK-cells, based on the expression of CD56, CD16, CD57, NKG2A and NKG2C in a cohort of HIV seronegative individuals (SN, n=18), HIV- low (HL, n=8) and HIV-high (HH, n=8). (B) A total of 11 clusters was identified by X-shift and FLOWSOM was applied to determine their specific markers. (C) Comparison of clusters differentially represented in the different groups. Comparisons between groups were evaluated by Mann- Whitney test for non-parametric data. Results were considered statistically significant when p-value <0.05 while trends are shown in grey. groups. Cluster 1 (CD56-CD16-CD57-) was significantly more increased in HL compared to SN (MW, p = 0.0008). Cluster abundant in HH compared to SN (MW, p <0.0001) and HL 10, corresponding to CD16+ adaptive NK cells (CD56+CD16+ (MW, p = 0.0070). By contrast, cluster 2 (CD56bright NKG2A++) + CD57 +++ NKG2C + ) were less abundant in SN and HL was significantly reduced in HH compared to SN (MW, p = compared to HH (MW, SN vs HH p-value = 0.0031; HL vs 0.0006) and HL (MW, p = 0.0011). Cluster 5 (CD56-CD16- HH, p-value = 0.0030), linking the expansion of these cells to CD57+NKG2A+NKG2C-) was increased in HL compared to HH chronic uncontrolled HIV infection (Figure 2C). This imbalance (MW, p = 0.0047) and cluster 8 (CD56-CD16+CD57+) was in the abundance of CD56bright and adaptive NK cell subsets was Frontiers in Immunology 08 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 also associated with differences in the expression of HLA-E impacted by chronic HIV infection since HIV infected receptors NKG2A and NKG2C, suggesting that HLA-E individuals, and individuals with high viral loads (HH expression drives this distinct NK differentiation and thus individuals) in particular, showed a loss of CD56bright and influences their capacity to support control of HIV infection. an increase of the terminally-differentiated adaptive NK cells To further explore this hypothesis, the proportions of CD56- (characterized here as CD56dimCD16+CD57+). The HLA-E/ CD16-, CD56bright (CD56brigthCD16-), CD56dim NKG2X axis also showed severe alterations depending on CD4 (CD56dimCD16+) and adaptive NK (CD56dimCD16+CD57+) count and plasma viral load, suggesting that the impaired populations, together with their expression of the HLA-E antiviral activity of these NK-subsets could be driven by receptors NKG2A and NKG2C in SN, HL and HH were high levels of HLA-E expression during uncontrolled determined. The median % of these subpopulations in HL, HIV infection. HH and SN is represented as a radar chart in Figure 3A. HH showed a significant loss of CD56bright NK cells compared to HL (MW p-value = 0.0011) and SN (MW p-value = 0.0004). In NK cells exposed to high levels of HLA-E addition, in total NK cells, the NKG2A/NKG2C ratio was show an impaired function significantly increased in SN compared to HH (MW p-value = 0.0008), highlighting the reduced expression of NKG2A in Given the significant changes observed in NK-cell subset chronic uncontrolled HIV infection. The subpopulation distribution, their NKG2X receptor expression and their analysis indicated this difference to stem mainly from correlations with HLA-E expression, we next determined the CD56bright NK cells (MW p-value = 0.0027) and adaptive NK ability of NK cells isolated from different patient groups to cells (MW p-value = 0.0051). The shift towards NKG2C respond to signals through HLA-E/NKG2X. To measure NK expression was less pronounced in HL compared to SN. Still, a response to “missing-self”, we determined cytotoxicity significant reduction was observed in NKG2A/NKG2C ratios in (measured as % of killed K562), degranulation (% of CD107a+ CD56bright compared to SN individuals (MW, p-value = 0.0111) NK cells) and cytokine production (IFN-g+ and TNF-a+ NK cells) (Figure 3B). Overall, these data indicate that CD56bright NK cells after coculture with HLA-null K562 cells. When testing total NK are reduced in individuals with higher viremia, while adaptive cells derived from SN, HL and HH, we observed a significant NK cells are more abundant in those samples. NKG2A/NKG2C reduction in NK cytotoxic capacity in HIV infected individuals ratio was related to uncontrolled infections and observed not compared with SN, true for both HL (MW p-value = 0.0176) and, only in the whole NK cell population, but also in CD56bright and especially, for HH (MW p-value = 0.0008, Figure 4A). A direct adaptive NK cells subsets. comparison between HL and HH demonstrated stronger NK cell We next tested for potential relationships between NK cell cytotoxic activity in HL, although this did not reach statistical subsets size, the relative levels of the NKG2A and NKG2C significance (MW, p-value = 0.077). Impaired NK cell function in expression in each of these subset as well as the NKG2A/ HIV-infected individuals was also reflected by reduced IFN-g NKG2C ratio and the CD4 count, plasma viral load and HLA- production in both HL (MW, p-value = 0.0007) and HH (MW, p- E mRNA levels (Figure 3C). CD4 count correlated positively value = 0.0007) compared to SN (Figure 4A). No differences in with NKG2A (r=0.61, p-value=0.002) surface expression in degranulation levels nor TNF-a+ cells were observed. We tested total NK cell fraction, NKG2A expression and NKG2A/ then if these impairments were associated with the expression of NKG2C ratio in adaptive NK cells (r=0.51, p-value=0.048 HLA-E by correlating the different NK effector functions with the and r=0.57, p-value=0.024, respectively) and the abundance basal level of HLA-E mRNA expression in the PBMC sample used of CD56bright NK cells (r=0.67, p-value=0.006). Plasma viral to isolate the NK cells. Indeed, HLA-E expression levels were load correlated negatively with NKG2A expression in total NK associated with a detrimental effect on NK cell function since we cells (r= -0.527, p-value=0.038) as well as with the observed negative correlations between HLA-E expression and preservation of CD56 bright subpopulation (r= -0.776, p- NK cytotoxicity (r= -0.6341, p-value=0.0015), degranulation value=0.001). Of note, HLA-E mRNA correlated positively (%CD107a+, r= -0.4647, p-value=0.0293) and IFN-g production with NKG2C expression in total NK cell fraction (r=0.367, p- (r= -0.5744, p-value=0.0052, Figure 4B). value=0.033) and CD56bright (r=0.508, p-value=0.002) NK To gain further insights whether HLA-E expression was cells while it correlated negatively with NKG2A expression associated with dysregulated NK function in specific NK subsets, in adaptive NK cells (r=-0.427, p-value=0.012). As a we repeated the effector function analysis focusing on the two consequence, HLA-E expression was correlated with an NK subsets showing the most significant alterations in chronic imbalance in the NKG2A/C ratio in adaptive NK cells (r= HIV infection, namely CD56bright and adaptive NK cells -0.344, p-value=0.046), indicating a link between the increased (Figure 4C). The HIV uninfected group (SN) showed indeed expression of NKG2C, its ligand HLA-E and uncontrolled superior capacity of CD56bright cells to degranulate (MW, SN vs HIV infection. Furthermore, the results indicate that NK cell HL p-value= 0.0047, SN vs HH p-value = 0.0047), secrete IFN-g subsets and the expression of NKG2X receptors are severely (MW, SN vs HL p-value = 0.0310, SN vs HH p-value = 0.0093) Frontiers in Immunology 09 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C FIGURE 3 Manual gating analysis of the NK cell fraction. (A) Magnetically sorted NK cells from HIV seronegative (SN, n=18), HIV-low (HL, n=8) and HIV- high (HH, n=8) were characterized by manual gating. Four different subpopulations were characterized as CD56-CD16-, CD56bright (CD56bright + CD16-), CD56dim (CD56dimCD16+) and adaptive NK cells (CD56dimCD16+CD57+). Differences between groups (SN, HL, HH) were analyzed by non-parametric Mann-Whitney test and represented in the radar chart. (B) NKG2A/NKG2C expression ratio in total NK cell fraction (left), CD56bright (middle) and adaptive NK cells (right) in the different groups (SN-black, HL-purple, HH-blue). (C) Correlogram showing Spearman’s rank test correlation of NKG2A and NKG2C expression in total NK cells, CD56bright and adaptive NK cells with clinical parameters (CD4 count, plasma viral load) and HLA-E mRNA expression. Only significant correlations are shown. Results were considered statistically significant when p- value <0.05. or produce TNF-a (MW, SN vs HL p-value = 0.0047, SN vs HH higher IFN-g (MW, SN vs HL p-value = 0.0003, SN vs HH p-value = 0.0127). No significant differences in CD56bright p-value =0.0007) and TNF-a production (MW, SN vs HL functional responses were observed between HL and HH, p-value = 0.0007, SN vs HH p-value = 0.0023) in uninfected although this subset was significantly reduced in HH. individuals compared to chronically HIV infected individuals. Similarly, the adaptive NK subset showed a significantly Interestingly, degranulation activity was not reduced in this Frontiers in Immunology 10 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C D FIGURE 4 Analysis of NK cell cytotoxic activity, degranulation and cytokine secretion upon HLA-E stimulation. Magnetically sorted NK cells from HIV seronegative (SN, black, n=6), HIV-Low (HL, purple, n=8) and HIV-high (HH, blue, n=8) were cocultured with K562 cell lines (lacking the expression of HLA-E) at an effector:target ratio of 5:1. After four hours, NK functional responses were measured by flow cytometry. (A) Total NK cell functions: cytotoxicity against K562 (top left), degranulation measured as % of CD107a+ (bottom left), production of IFN-g (top right) and TNF-a (bottom right). (B) Correlogram of HLA-E expression in SN, HL and HH with markers of total NK functionality. C) Functional response in the CD56bright (top) and adaptive NK cells (bottom) in terms of degranulation (% of CD107a+) (left), production of IFN-g (middle) and TNF-a (right). (D) Correlogram of HLA-E level of expression with CD56bright and adaptive NK cell functions. Comparisons between groups were evaluated by Mann-Whitney test for non-parametric data while correlations were performed by Spearman’s rank correlation test. Results were considered statistically significant when p-value <0.05 while trends are shown in grey. adaptive NK subset which was also not reduced in size in HIV production of IFN-g and TNF-a (r=-0.5305, p-value=0.0111; infected individuals. In line with the data obtained in the total r = -0.5598, p-value=0.0067, respectively). Overall, these results NK population, HLA-E mRNA expression in the source PBMC indicate that higher levels of HLA-E expression in chronic HIV was negatively corelated (Figure 4D) with the degranulation infection are not only associated with shifts in the NKG2A to capacity of CD56bright (r= -0.4692, p-value=0.0276), IFN-g and NKG2C expression ratios, but also with reductions in cytotoxic, TNF-a production in CD56bright (r=-0.5538, p-value=0.0075; degranulation and cytokine production effector functions in the r = -0.4363, p-value=0.0424, respectively) and adaptive NK cells most affected NK subsets. Frontiers in Immunology 11 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 Short-term NK-mediated HIV Effect of HIV-derived peptides on viral growth inhibition is related HLAE*01:03 surface stabilization to HLA-E expression and consequent ability to alter NK cells activation In order to discern whether the reduced NK cell effector response was the cause or consequence of uncontrolled HIV Since HLA-E expression levels were associated with infection, we modeled acute HIV infection in an in vitro changes in the NKG2A/NKG2C ratio as well as the infection model. Using this model, we assessed antiviral abundance of the different NK subpopulation, we explored activity of NK cells in an in vitro viral replication inhibition the possibility that a change in the HLA-E peptide repertoire assay (NK-VIA) by coculturing sorted NK cells from HIV- impacts the signaling through the HLA-E/NKG2X axis and negative individuals with in vitro HIV infected autologous drives these alterations. We thus analyzed the role of HIV- CD4+ T-cells. For the two HIV strains tested, a strong derived, HLA-E-binding epitopes in the activation of NK cells inhibitory capacity of NK cells was observed at day 7 from HIV seronegative individuals (n=5), using HLA-E*01:03 (Figure 5A) of culture. However, this activity was significantly transfected K562 cells, co-expressing the canonical peptide reduced by day 14 for HIV-1NL4-3 (W p-value = 0.0106) and VL9-B7 and pulsed with canonical and non-canonical HLA-E HIV-1BaL. (W p-value = 0.0164). In parallel, the 14-day culture binding peptides. We measured HLA-E surface stability as well showed marked changes in NK subpopulations, with a loss of as HLA-E restricted NK cytotoxicity, degranulation and CD56 dim and a gain of CD56bright and CD56 neg subsets cytokine production (IFN-g and TNF-a). The effect of each (Figure S2). The expression of NKG2A was increased in all peptide compared to the canonical, endogenously expressed, NK subsets at day 14, while NKG2C was increased in CD56neg VL9-B7 epitope was determined and compared between and CD56dim (Figure S3) but reduced in adaptive NK cells peptides (Figure 6). (Figure 5B) in line with observations in long-term chronic The presence of the described canonical HLA-E binding HIV infection. Furthermore, in adaptive NK cells, coculture epitopes increased the surface expression when compared with HIV-1BaL infected CD4+ T-cells maintained NKG2C with VL9-B7 endogenous expression (Friedman test, VL9- expression at higher levels compared to uninfected cultures, Cw1 p-value = 0.0002, CMV p-value = 0.001). The same was reflected by a lower NKG2A/NKG2C ratio (Two-way ANOVA, seen for the non-canonical EBV-derived SL9 peptide p-value = 0.0463 Figure 5B), suggesting HIV infection might be (Friedman test, E SL9, p-value = 0.0196). Interestingly, only indirectly driving NKG2C expression on NK cells. one of the 11 HIV-derived candidate epitopes (H RL9) When measuring viral HIV Gag p24 in culture supernatants increased the surface expression (Friedman test, p-value = over time, strong correlations were observed at day 7 between 0.0439, Figure 6A). NK-mediated inhibition of viral replication and surface HLA-E Given the different potency of described HLA-E presented expression (Figure 5C), for both, HIV-1NL4-3 (r= 0. 8322; peptides to stabilize surface expression, we assessed whether p-value = 0.0013) and HIV-1BaL (r= 0.7167; p-value = 0.0369). these differences would translate to more or less effective The level of inhibition also correlated with the basal HLA-E sensitization of target cells to NK cell recognition. Indeed, mRNA expression in the PBMC used to isolate autologous NK HLA-E*01:03 expressing K562 target cells pulsed with three and CD4+ T cells (Figure 5D), in the case of HIV-1NL4-3 (r= HIV-derived peptides increased the cytotoxic activity of NK cells 0.6434; p-value = 0.0278). This was in line with the direct significantly (Friedman test, KG9 p-value = 0.0339, MD9 correlation between HLA-E mRNA level in the sample and p-value = 0.0284 and PM9 p-value = 0.0403, Figure 6B). These surface HLA-E expression (Figure S4, r= 0,6643; p-value = three and all but two exogenously added HIV-derived epitopes 0,0219). There was no difference between HLA-E allele triggered significant increases in NK degranulation (Friedman genotype and NK-mediate control in our model of test, AA9 p-value = 0.0310, KG9 p-value = 0.0002, MD9 acute infection. p-value = 0.0011, PM9 p-value = 0.0011, QE9 p-value = Together, these data show that high levels of HLA-E 0.0027, RV9 p-value = 0.0019, SN9 p-value = 0.0033, TP9 p- expression mediate at first, an effective viral inhibition by NK value = 0.0162, Figure 6C). Finally, two of the three HIV-derived cells. However, our data also suggest that long-term exposure to peptides that induced cytotoxicity also were able to increase the HIV in untreated chronic infection as well as in extended (day number of TNF-a+ NK cells (Friedman test, KG9 p-value = 14) co-cultures with infected CD4 T cells, drives NKG2C 0.0162 and PM9 p-value = 0.0133, Figure 6D). expression which, in turn, may cause reduced NK functions Together, eight of the eleven tested HIV peptides induced due to its diminished affinity for HLA-E presented peptides increases in at least one of the tested NK functions when compared to NKG2A (14). presented by HLA-E*01:03. Remarkably, two of the HIV Frontiers in Immunology 12 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C D FIGURE 5 Short term NK-mediated inhibition of viral replication is related to HLA-E expression and diminishes over time. (A) Inhibition of viral replication comparing two different laboratory adapted strains (HIV-1NL4-3 in pink and HIV-1BaL in blue) and two different timepoints (day 7 in dark grey and day 14 in light grey). (B) Long-term NK-CD4+ T-cell coculture induced changes on the NK HLA-E receptors favoring the expression of the inhibitor NKG2A. HIV infection resulted in higher levels of NKG2C expression as well as a lower level of NKG2A/NKG2C ratios in adaptive NK cells. (C) NK cell control of the viral infection correlated with HLA-E surface expression and basal mRNA levels (D). Color code indicates sample genotype. Univariate analysis was performed using Wilcoxon test for paired data (A), while multiple comparisons were performed by one-way ANOVA (B). Correlation was calculated using Spearman ‘s rank correlation coefficient (C, D). Results were considered statistically significant when p-value <0.05. peptides (KG9 and PM9) presented by HLA-E*01:03 Structural model of the antigen stimulated NK cells and increased three of the four cellular presentation of HIV-derived peptides functions measured (cytotoxicity, degranulation and TNF-a through HLA-E to NK cells production). These data indicate that HLA-E*01:03 might be sensitive to changes in the HLA-E repertoire and undergo In order to further understand the interplay between HLA-E changes that can be recognized by NKG2X receptors on alleles (*01:01 or *01:03), NKG2X receptors and HIV-derived NK cells. peptides that triggered NK responses, a structural model was Frontiers in Immunology 13 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 A B C D E FIGURE 6 Effect of HIV-1 derived peptides on HLA-E*01:03 surface stabilization and consequent ability to alter NK functionality. HLA-E *01:03 transfected K562 cell line co-expressing the VL9 epitope was pulsed with canonical and non-canonical HLA-E binding peptides. (A) Peptide-pulsed cells induced HLA-E surface stabilization, measured by flow cytometry and recorded as %HLA-E+ K562 cells. Peptide-pulsed cells modified the NK cytotoxic capacity (B), degranulation (C) and cytokine secretion (D, IFN-g (left) and TNF-a (right)). Multiple comparisons with the control VL9 epitope were performed by Friedman’s test for paired data with post-hoc False discovery rate. Significant differences are marked in the graphs with * and (A-D) correspond to p-values<0.05. Table (E) represents significant p-values when FDR indicated q-values<0.15. constructed. The structural model of HLA-E-CD94-NKG2X NKG2A/C interface and thus, differences in NKG2X affinity for (Figure 7) illustrates that the presentation of HIV-derived the HLA-E/epitope complex may be determined by the ASILP PM9 and RL9 peptides differs in the binding mode compared motif in NKG2C/SIISP in NKG2A that change the CD94 to canonical VL9 peptides. In particular, the first residue (P1) in interface, similarly to what happens when the canonical VL9 both, the PM9 and RL9 epitopes is exposed to NKG2A, peptide is presented (49). Interestingly, HLA-E*01:01 and *01:03 providing evidence for a direct interaction between the differ only in residue Arg/Gly 107, which is outside the epitope presented epitope and NKG2X position Arg215, which binding pocket. It is however located next to another charged localizes close to position 197 in NKG2A and NKG2C, which residue (Arg108) which is responsible for the interaction with is occupied by a Glu and Lys residue, respectively. Additionally, NK cells receptors NKG2A and NKG2C through Asp202 position P5 in the presented epitope points towards the CD94- (Figure 7). Hence, the NKG2A and NKG2C molecules can be Frontiers in Immunology 14 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 FIGURE 7 CD94-NKG2(A/C) interaction with HLA-E-VL9/PM9 peptide. VL9 (in yellow) and PM9 (in orange) peptides protrude from HLA-E (in green) allowing for an additional interaction with Arg215 from NKG2A/2C (in grey). Residue 197 (Glu residue in NKG2A and Lys residue in NKG2C, respectively) is close to Arg 215. The ASILP and SIISP sequence motifs (in magenta) presented by NKG2A and NKG2C, respectively, are in close proximity to position P5 of VL9 and PM9/RL9 peptide. HLA-E (in green) differs in Arg/Gly 108 (in cyan), which interacts with NKG2A/2C Asp 201 through Arg 108 (in blue). expected to be very sensitive to minor changes in HLA-E and HLA-E on the surface of target cells is especially interesting, allotypes affecting this position (Arg108) (Figure 7). given the conserved nature of the HLA-E locus compared to Thus, our structural model identifies interactions between non- classical HLA class I loci (HLA-A, -B, or –C) and the variable canonical HIV-1-derived HLA-E presented peptides and NKG2X expression of different NKG2X throughout the NK receptors in an allele-dependent manner, providing the potential differentiation process. The conserved nature of HLA-E has explanation for differential NK-cell recognition of infected cells also the potential to render NK-mediated recognition and through the HLA-E/NKG2X axis. In this model, Position 5 in the elimination of virally infected cells through HLA-E an antiviral presented epitope may further determine the affinity to the activator mechanism that extends across all the human population, not (NKG2C) or inhibitory (NKG2A) receptor. restricted to individuals expressing a specific classical HLA class I genotype. For these reasons, in the present study we aimed to determine the relevance of NK cell activation through the HLA- Discussion E/NKG2X axis in the natural control of HIV infection. Previous studies reported a link between HLA-E expression NK cells play a crucial role in antiviral immunity, a role and HIV infection and showed that infected CD4+ T-cells had an evidenced by the increased susceptibility to viral infections when increased surface HLA-E expression compared to their NK cells are impaired or depleted (38, 40, 50). Aside from uninfected counterparts (51). In our initial study population, surface molecules such as Fc-receptors that are involved in we observed a progressive increase of HLA-E mRNA expression antibody-mediated effector functions, NK exert a large portion along the HIV disease course and documented a strong positive of their activity by detecting changes in HLA surface expression correlation between increased HLA-E expression and plasma or in the HLA-presented epitope repertoire, caused by viral viral loads and proviral DNA in cART-naïve individuals with infections. The importance of these defense mechanisms is chronic HIV infection. While the precise mechanisms of such supported by the observation that viruses causing chronic increased expression remain to be defined, it is possible that infections, like CMV, EBV and HIV, have developed several different soluble factors, such as IL-27, could be potential drivers. mechanisms to escape such NK responses, including the In fact, it has been demonstrated that IL-27 is i) capable of reduction of HLA expression on infected cells, expressing upregulating surface expression of HLA-E (52), ii) significantly epitopes mimicking human HLA canonical epitopes or increased in individuals with high plasma viremia (35) and iii) influencing NK subsets and their immune-receptor repertoire associated with ineffective NK-cell responses (36). Such an effect (25, 26). Of these NK receptors, the interaction between NKG2X may not be limited to drive HLA-E expression on infected T Frontiers in Immunology 15 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 cells, but may also extend to other cell types susceptible to HIV HCMV, NK subset shifts are equally associated with an infection, such as macrophages and other cells, chronically epigenetic imprint that modulates NK cells function and exposing NK to sustained elevated levels of HLA-E. drives the expansion of mature, memory-like, NKG2C+CD57+ Importantly, in our study we also controlled for HLA-E NK cells (26, 54). In our case, we observed that uncontrolled allelic differences in terms of HIV disease progression and NK HIV infection in the absence of ART was associated with a cells activation. This aspect has been largely neglected in the similar increase in HLA-E mRNA expression and loss of the past, probably due to the fact that the two worldwide distributed highly active CD56 bright NK subset, accompanied by an alleles, HLA-E*01:01 and *01:03, only differ in one amino acid. expansion of terminally differentiated, adaptive NK cells. To However, it is well known that such small differences can cause validate these observations in HIV infection, we extended our functional differences and, for instance in the case of HLA-B*58, analyses to HIV-uninfected individuals and found common have been associated with opposite risk for HIV disease patterns in NK cells subsets between HIV seronegative and progression (37). Indeed, in our cohort, HLA-E heterozygosity HIV-low that differed from those in HIV-high individuals. (HLA-E*01:01/01:03) was significantly more common in This included the maintenance of NKG2A expressing individuals with naturally controlled HIV infection and with CD56bright NK cells and a reduction in the frequency of the undetectable viral loads. Such an advantage of heterozygous terminally differentiated NKG2C+ adaptive NK cells compared HLA genetics has been described in the past for classical HLA to highly viremic individuals. Notably, we also observed a cluster class I alleles and for rare allele variants (34), but not for the less of CD56dimCD57brightNKG2C+ which was increased in HIV polymorphic HLA-E locus. This observation also suggests that patients compared to uninfected samples, suggestive of a the functional differences between the two alleles may be progressive increase of NKG2C+ adaptive NK cells and a loss pronounced enough to provide heterozygous individuals an of CD56bright NK cells in progressive HIV disease. To validate advantage to control HIV more effectively than HLA-E these findings, we measured the expression of NKG2A and homozygous individuals. The lack of K562 cells expressing NKG2C in four different NK cells subpopulations defined as HLA-E*01:01 only, prevented us from further clarifying the CD56-CD16-, CD56bright, CD56dim and adaptive NK cells inherent ability of each allele to be stabilized on cell surface by (CD56dimCD16+CD57+). We confirmed that HIV-high showed binding epitopes. a profound loss of CD56 bright NK cells and identified a A past study by Natterman et al. also demonstrated that HIV significant reversion of the NKG2A/NKG2C balance in total infection led to reduced NK cell activity (51). To understand NK cells from HIV infected individuals’ cells, which was mainly whether and how higher HLA-E levels were related to reduced driven by the CD56bright and adaptive subsets. While some HIV control, we investigated the relationship between the preceding studies reported NKG2C+NKG2A- NK cells as a potential to activate NK cells through the HLA-E/NKG2X axis marker of lower viral set point (55) and an increased killing of and controlled and uncontrolled HIV infection. We observed HIV infected CD4+ T-cells by autologous NK cells when that the HLA-E/NKG2X axis was profoundly impacted by blocking NKG2A (51), both studies were based on acute HIV chronic HIV infection, especially among individuals with high infection ex vivo or in vitro, respectively. Still, both studies viral loads. In particular, NK cells from HIV infected individuals established a link between the course of HIV disease and showed a severely impaired activation capacity through HLA-E HLA-E/NKG2X regulation of NK cell activity. “missing-self” mechanism as they were less activated when Our results are in line with the observed reversion of the exposed to target cells lacking HLA-E. We also found that NKG2A/NKG2C ratio in NK cells from HIV infected HIV infected individuals, and especially HIV-high, showed a individuals with HCMV co-infection (27). HCMV is a highly switch of the HLA-E receptors NKG2A/NKG2C ratio, in line disseminated virus within the human population, with an with previous studies (27). Thus, our data suggest that increased estimated worldwide seroprevalence of 83% (95%UI: 78-88) expression of NKG2C, together with the chronic nature of HIV (56). Although we could not determine the CMV status for all infection, causes constant activation of NK cells, ultimately participants in the present study, its high prevalence suggests leading to an impaired state. HLA-E is thought to play into that the vast majority of these samples were HCMV infected as this mechanisms by its relative lower affinity of NKG2C well and some of the alterations on NK cell phenotype and compared to NKG2A, causing loss of NK regulation (14). function could also be related to latent CMV infection. We This interpretation is also in line with the findings of Merino observed an impaired NK cell response upon stimulation et al (5), who showed that chronic in vitro stimulation of NK through missing self paradigm along with this consistent cells through the HLA-E/NKG2X axis, using plate-bound switch from NKG2C to NKG2A expression. Further studies agonistic antibodies, promoted the expansion of adaptive NK assessing NK cell exhaustion in a defined co-infection cohort will cells and induced the expression NKG2C and CD57. These cells be needed to conclusively determine the functional also present an epigenetic reprogramming that causes an consequences of the reverted receptor ratio. exhausted phenotype and profound NK dysfunction (5), which In our ex-vivo model of NK mediated inhibition of HIV may be difficult to be restored (53). In other infections like replication, we observed a positive correlation between NK- Frontiers in Immunology 16 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 mediated antiviral activity and HLA-E expression within the first Some of these structural particularities may support the benefits seven days of culture. After fourteen days of culture however, of HLA-E heterozygosity in HIV control as heterozygous might NK mediated viral control was significantly reduced and the have a broader and more differentiated ability to present more/ correlation with HLA-E expression was lost. We observed a shift different epitopes and thus trigger differential NK responses. The towards NKG2C expression in HIV-1BaL infected cultures expanded structural insights in the present study may also concomitant with a reduced NK killing activity in longer-term explain the reported 6-fold lower affinity for the HLA-E- cultures. These results are in line with previous in vitro HIV peptide complex to NKG2C and contribute to the observed studies, were the continuous stimulation through the HLA-E/ switch from NKG2A to NKG2C expression, with its NKG2X axis reduced NK functionality (5). Importantly, the in downstream functional consequences on NK-mediated vitro data directly support our ex-vivo data and a scenario where antiviral activity. elevated HLA-E expression in response to HIV infection will Overall, our data are in line with a mechanism where chronic drive a shift in the NKG2A/NKG2C balance on NK cells, which HIV infection with high HLA-E expression levels, which is eventually will hamper their ability to exert effective especially pronounced in individuals with uncontrolled HIV antiviral activity. infections, continuously stimulates NK cells through the As HLA-E surface expression levels as well as signaling via NKG2X receptors, driving a change in NK subpopulations NGK2X receptors can be influenced by the epitope-repertoire ratios from CD56bright NK cells towards a functionally less presented by HLA-E, we tested whether HIV-derived epitopes active profile. These CD56bright NK cells are traditionally presented by HLA-E could affect NK cells activation, potentially considered as regulatory and their role in HIV control could by displacing the canonical VL9 epitope and thus leading to NK- go beyond the direct killing of infected cells (58). These changes mediated cell killing by the “missing-self” mechanism. We first appear to go hand in hand with a profound disruption of the used single HLA-E transfected K562 cells, pulsed with canonical regulation of the “missing-self” NK activation pathway through and non-canonical HIV-1-derived peptides and competing with the HLA-E/NKG2X axis, which can be further influenced by an endogenously expressed VL9-B7 epitope. Although only one changes in the HLA-E presented epitope repertoire due to HIV HIV-derived peptide increased HLA-E surface levels, we infection. Indeed, HL maintained higher levels of cytotoxicity observed profound differences in the activation of NK cells and TNF-a production upon HLA-E stimulation compared to derived from HIV seronegative individuals in response to HH although there were not significant differences in NK cell peptide-pulsed target cells, suggesting that low levels of HLA-E degranulation. This could indicate a mechanism of cell death viral antigen presentation could trigger NK cell activation as induction that might differ from the release of cytolytic granules. have been shown for classical HLA class I molecules (57). These Our results are further supported by previous studies that have analyses could not be repeated in NK cells from HIV infected suggested different effects of HIV viremia on NK cell number individuals due to the high cell demand for such assays. and function during either acute or chronic stages of the Furthermore, our results did not discriminate whether this infection in a HLA-E dependent manner (56). Our data are activation occurs through the abrogation of NKG2A also in line with studies showing NK cells from HIV infected engagement and the consequent missing-self signaling or individuals to be in an unresponsiveness state and express through NKG2C signaling or a combination of both transcriptomic signatures indicative of higher exhaustion mechanisms. Further studies including specific receptor markers (59), loss of surface NK cytotoxicity receptors blocking may help to determine the exact mechanism of NK expression (60) and a consequent reduction in cytolytic cells sensing of HIV infected cells through the HLA-E/ capacity, despite showing elevated levels of activation markers NKG2X axis. (61). This dysfunctionality has been proposed to contribute to Finally, we tested structural models to structurally support the loss of HIV control, and which may not be readily restored the functional impact of HLA-E repertoire, NKG2X receptor upon ART treatment (62). These findings are especially relevant switch and HLA-E allele polymorphism on NK cells mediated for potential therapeutic applications targeting NK cells such as control of HIV infection. Our analyses indicate that the enhancing antibody dependent cell cytotoxicity (ADCC)67, differences between NKG2A and NKG2C influences the enhancement of NK activity using TLR ligands68,69 or the interaction with HLA-E alleles and the bound epitopes in infusion of engineered NK cells expressing CAR receptors different ways: (1) through the ASILP/SIISP motif on NGK2X (35). Indeed, there are some reports indicating that NK cells that interacts with position P5 of the bound epitope and which function can be rescued with the addition of IL-15 (63), at least could directly determine the affinity to the activator (NKG2C) or in vitro, and also that the exhausted transcriptomic signature inhibitory (NKG2A) receptor to the HLA-E/epitope complex; observed in uncontrolled HIV infection could be partly restored (2) through E/K 197 residues that map close to presented after vaccination (60). The functional data in our present study epitopes, such as PM9 and RL9, and (3) through Asp202 that and the novel structural insights into how HLA-E allele interacts with Arg 108, located in close proximity to Arg107 and polymorphism can impact NKG2X receptor recognition may representing the only change between HLA-E*01:01 and *01:03. thus allow to therapeutically improve the antiviral capacity of Frontiers in Immunology 17 frontiersin.org Romero-Martı́n et al. 10.3389/fimmu.2022.1027855 NK cells and to guide future therapeutic use of these cells in HIV grant European AIDS Vaccine Initiative 2020 (EAVI2020) cure strategies. #GA681137 (CB). The work was also partly supported by grant PI17/01465 (AO) from the Instituto de Salud Carlos III, co-financed by the Fondo Europeo de Desarrollo Regional Data availability statement (FEDER) “Una manera de hacer Europa”. CB is a senior ICREA research professor. The data presented in the study are deposited in the European Nucleotide Archive (ENA) repository, accession number ERA18504000. Acknowledgments We would like to thank the team of Dr. J. Miguel Ló pez- Ethics statement Botet (Departament de Ciències Experimentals i de la Salut, UPF/IMIM, Barcelona) for their advice starting The studies involving human participants were reviewed and this project. approved by Comitè Ètic d’Investigació Clı́nica of Hospital Germans Trias i Pujol (CEIC EO-12-042). The patients/ participants provided their written informed consent to Conflict of interest participate in this study. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could Author contributions be construed as a potential conflict of interest. Conceptualization: L-RM, MR-R, CB, AO. Methodology: LR-M, CD-C, MO, MR-U. Investigation: LR-M, CD-C, MR-U. Publisher’s note Visualization: LR-M, CB, AO. Supervision: CB, AO. Resources: BM, CB, DH-O'C. Writing – original draft: LR-M. Writing – All claims expressed in this article are solely those of the review & editing: CD-C, MO, MR-U, MR-R, JS, BM, DH-O'C, authors and do not necessarily represent those of their affiliated AO, CB. All authors contributed to the article and approved the organizations, or those of the publisher, the editors and the submitted version. reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Funding The present study was supported by the USA National Supplementary material Institutes of Health (NIH) - National Institute of Allergy and Infectious Diseases - P01 Research Program Project Grant The Supplementary Material for this article can be found 1P01AI131568-01 (CB) and funding from the European online at: https://www.frontiersin.org/articles/10.3389/ Union’s Horizon 2020 research and innovation program under fimmu.2022.1027855/full#supplementary-material References 1. Freud AG, Mundy-Bosse BL, Yu J, Caligiuri MA. The broad spectrum of 5. Merino A, Zhang B, Dougherty P, Luo X, Wang J, Blazar BR, et al. Chronic human natural killer cell diversity. Immunity (2017) (5):820–833. doi: 10.1016/ stimulation drives human NK cell dysfunction and epigenetic reprograming. J Clin j.immuni.2017.10.008 Invest. (2019) 29(9):3770–3785. doi: 10.1172/JCI125916 2. Brillantes M, Beaulieu AM. Memory and memory-like NK cell responses to 6. 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