HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-Like Receptor KIR3DS1

HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-like Receptor KIR3DS1

This information is current as Zahra Kiani, Franck P. Dupuy, Julie Bruneau, Bertrand of September 25, 2021. Lebouché, Cindy X. Zhang, Elise Jackson, Irene Lisovsky, Sandrina da Fonseca, Daniel E. Geraghty and Nicole F. Bernard J Immunol published online 9 May 2018

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 9, 2018, doi:10.4049/jimmunol.1701370 The Journal of Immunology

HLA-F on HLA-Null 721.221 Cells Activates Primary NK Cells Expressing the Activating Killer Ig-like Receptor KIR3DS1

Zahra Kiani,*,† Franck P. Dupuy,* Julie Bruneau,‡,x Bertrand Lebouche´,*,{,‖ Cindy X. Zhang,*,# Elise Jackson,*,# Irene Lisovsky,*,† Sandrina da Fonseca,* Daniel E. Geraghty,** and Nicole F. Bernard*,†,{,††

NK cells elicit important responses against transformed and virally infected cells. Carriage of the gene encoding the activating killer Ig-like receptor KIR3DS1 is associated with slower time to AIDS and protection from HIV infection. Recently, open conformers of the nonclassical MHC class Ib Ag HLA-F were identiﬁed as KIR3DS1 ligands. In this study, we investigated whether the interaction of KIR3DS1 on primary NK cells with HLA-F on the HLA-null cell line 721.221 (221) stimulated KIR3DS1+ NK cells. We used a Downloaded from panel of Abs to detect KIR3DS1+CD56dim NK cells that coexpressed the inhibitory NK cell receptors KIR2DL1/L2/L3, 3DL2, NKG2A, and ILT2; the activating NK cell receptors KIR2DS1/S2/S3/S5; and CCL4, IFN-g, and CD107a functions. We showed that both untreated and acid-pulsed 221 cells induced a similar frequency of KIR3DS1+ cells to secrete CCL4/IFN-g and express CD107a with a similar intensity. A higher percentage of KIR3DS1+ than KIR3DS12 NK cells responded to 221 cells when either inclusive or exclusive (i.e., coexpressing none of the other inhibitory NK cell receptors and activating NK cell receptors detected by the Ab panel) gating strategies were employed to identify these NK cell populations. Blocking the interaction of HLA-F on 221 http://www.jimmunol.org/ cells with KIR3DS1-Fc chimeric protein or anti–HLA-F Abs on exclusively gated KIR3DS1+ cells reduced the frequency of functional cells compared with that of unblocked conditions for stimulated KIR3DS1+ NK cells. Thus, ligation of KIR3DS1 activates primary NK cells for several antiviral functions. The Journal of Immunology, 2018, 201: 000–000.

atural killer cells are effectors of the innate immune Ig-like domains (3D) and whether they have long cytoplasmic tails system that are particularly well adapted for antitumor (L) or short cytoplasmic tails (S). The KIR-L are generally in- N and antiviral activity (1). They can control viral repli- hibitory, whereas KIR-S are activating receptors (14). NK cell

cation at early stages of infection by secreting cytokines and activation status is determined by the integration of activating and by guest on September 25, 2021 chemokines and by killing virally infected cells (1–4). NK cells inhibitory signals received from cell surface receptors binding can also link the innate and adaptive arms of the immune re- their ligands on neighboring cells (15). sponse, leading to NK cell activation of multiple functions, in- The highly polymorphic KIR3DL1/S1 locus is unique among cluding cytotoxicity (5, 6) KIR region genes in that it encodes both activating (KIR3DS1) The killer Ig-like receptors (KIRs) are a large family of receptors and inhibitory (KIR3DL1) receptors. The 84 unique proteins found on subsets of lymphocytes, including NK cells (7). They are encoded by this locus identiﬁed to date can be classiﬁed into encoded by genes within the KIR genetic region that maps to four subgroups based on their cell surface density and sequence chromosome 19q 13.4, within the leukocyte receptor complex (8). similarity: KIR3DL1 inhibitory receptors that are not expressed The KIR region is polygenic, and the KIR genes within this region on the cell surface (KIR3DL1-null), those expressed at a are polymorphic (9–13). KIR nomenclature is based on whether low density (KIR3DL1-low), those expressed at a high density they have 2 extracellular Ig-like domains (2D) or 3 extracellular (KIR3DL1-high), and KIR3DS1 receptors (16–19). The ligand for

*Research Institute of the McGill University Health Centre, Montreal, Quebec H4A Institute of the McGill University Health Centre, an institution funded in part by the 3J1, Canada; †Division of Experimental Medicine, McGill University, Montreal, FRQ-S. Quebec H4A 3J1, Canada; ‡Centre de Recherche du Centre Hospitalier de l’Univer- x Z.K. designed the study, performed the experiments and data analysis, and prepared site´ de Montre´al, Montreal, Quebec H2X 0A9, Canada; Department of Family { the manuscript; F.P.D. performed experiments and edited the manuscript; J.B. and Medicine, University of Montreal, Montreal, Quebec H3T 1J4, Canada; Chronic B.L. provided clinical samples and edited the manuscript; E.J., C.X.Z., S.d.F. and I.L. Viral Illness Service, McGill University Health Centre, Montreal, Quebec H4A ‖ performed the experiments, analyzed data, and edited the manuscript; D.E.G. pro- 3J1, Canada; Department of Family Medicine, McGill University, Montreal, Que- vided the anti–HLA-F mAbs and edited the manuscript; N.F.B. designed the study bec H4A 3J1, Canada; #Department of Microbiology and Immunology, McGill and prepared and revised the manuscript. University, Montreal, Quebec H3A 2B4, Canada; **Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and ††Division of Address correspondence and reprint requests to Dr. Nicole F. Bernard, Research Clinical Immunology, McGill University Health Centre, Montreal, Quebec H3G Institute of the McGill University Health Centre, Glen site, Bloc E, 1001 Decarie 1A4, Canada Boulevard, Room EM3.3238, Montreal, QC H4A 3J1, Canada. E-mail address: [email protected] ORCIDs: 0000-0002-9257-4890 (C.X.Z.); 0000-0001-5311-9409 (N.F.B.). The online version of this article contains supplemental material. Received for publication September 28, 2017. Accepted for publication April 24, 2018. Abbreviations used in this article: 221, 721.221; aNKR, activating NK cell receptor; hIgG1, human IgG1; hmz, homozygous; iKIR, inhibitory KIR; iNKR, inhibitory NK This work was supported by the Canadian Institutes for Health Research (CIHR) cell receptor; KIR, killer Ig-like receptor; MFI, mean ﬂuorescence intensity; MHC-I, Grant MOP-142494 and the Fonds de Recherche du Que´bec-Sante´ (FRQ-S). I.L. was MHC class I; OC, open conformer; PFA, paraformaldehyde. supported by a Ph.D. Scholarship from FRQ-S and CIHR. N.F.B. is a member of the Infectious Diseases, Immunology, and Global Health program of the Research Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701370 2 INTERACTIONS OF HLA-F WITH KIR3DS1 ACTIVATE NK CELLS

KIR3DL1-low and KIR3DL1-high receptors are a subset of Study population and KIR3DL1/S1 genotyping HLA-B and -A isotypes belonging to the HLA-Bw4 group, The study included 10 KIR3DS1 hmz, 2 KIR3DL1 hmz, and 2 KIR3DL1/S1 which is deﬁned by amino acids present at positions 77–83 of the heterozygous donors. Genomic DNA was extracted from PBMCs using HLA H chain (20–22). A dimorphism present at position 80 of QIAamp DNA Blood Mini Kits (Qiagen, Mississauga, ON, Canada) as per HLA-Bw4 H chains dichotomizes these isotypes into those with manufacturer’s directions. KIR3DL1/S1 generic genotyping was performed an isoleucine (*80I) or threonine (*80T) at this position. This by PCR, as described previously (33). dimorphism differentially affects receptor/ligand afﬁnity, inhib- Cells itory strength, and NK cell education potency (23–26). The PBMCs were isolated from leukapheresis samples or from blood draws into remaining HLA-B alleles belonging to the Bw6 group do not vacutainer tubes containing EDTA anticoagulant by density gradient interact with KIR3DL1. KIR3DS1 and KIR3DL1-null receptors centrifugation (Lymphocyte Separation Medium, Wisent Bioproducts, St- have not been reported to interact with HLA*Bw4 Ags (27, 28). Bruno, QC, Canada) and cryopreserved in 10% dimethyl sulfoxide However, the presence of two epitopes, one derived from HIV (Sigma-Aldrich, St. Louis, MO) and 90% FBS (Wisent). The HLA-null cell line 221 was a kind gift from Dr. G. Alter (Ragon Pol and the other from HIV Nef, have been shown to enable Institute, Harvard University, Cambridge, MA). Two hundred and twenty- interactions between KIR3DS1 and one of the HLA Bw4*80I one cells were cultured in RPMI 1640 medium supplemented with 10% Ags, HLA*B57 (29). FBS, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 mg/ml streptomycin Epidemiological studies implicate a role for KIR3DS1 in HIV (R10; all from Wisent). outcomes. HIV-seropositive individuals who are carriers of at least Preparing acid-pulsed 221 cells one copy of KIR3DS1 and HLA-Bw4*80I have a slower time to

AIDS than those expressing the receptor or ligand alone or neither Two million 221 cells were resuspended and incubated in 1 ml R10 (pH 2.2) Downloaded from for 90 s. Acid pulsing was stopped by adding 13 ml of R10 (pH 7.4) to the (30). NK cells from carriers of the KIR3DS1 plus Bw4*80I ge- cells (44). Cells were then washed and resuspended in R10 before use as notype also inhibit viral replication more potently than those from acid-pulsed 221 cells. carriers of the receptor or ligand alone (31, 32). HIV-infected Detection of HLA-F on untreated and acid-pulsed 221 cells CD4+ T cells from carriers of this KIR/HLA genotype stimulate autologous KIR3DS1+ NK cells, but not those expressing inhibi- To detect HLA-F on untreated and acid-pulsed 221 cells, we used the mAb 3D11(45–48). One million 221 cells were resuspended in 100 ml of PBS tory KIR3DL1, to degranulate (31). Furthermore, there is an as- http://www.jimmunol.org/ (Wisent) and 5% FBS (FACS buffer). 3D11 mAb or mouse IgG1 (IgG1) sociation between carriage of the KIR3DS1 homozygous (hmz) isotype control (MOPC-21; BioLegend, San Diego, CA) was added to the genotype and protection from HIV infection because a higher cell suspension for 40 min at 4˚C. Primary Ab binding was detected by frequency of HIV-exposed seronegative subjects than recently incubating washed cells with 1 mg/ml F(ab9)2 goat anti-mouse IgG-eFluor HIV-infected individuals are KIR3DS1 homozygotes, and HIV- 660 (eBioscience, San Diego, CA) secondary Ab for 25 min at 4˚C. A exposed carriers of this genotype seroconvert more slowly than recombinant KIR3DS1-Fc chimeric protein (R&D Systems, Minneapolis, MN) was also used to detect HLA-F on untreated and acid-pulsed 221 do carriers of other KIR3DL1/S1 genotypes (33, 34). KIR3DS1 cells. One million 221 cells were resuspended in 100 ml of FACS buffer has also been associated with outcomes of viral infections other and incubated with KIR3DS1-Fc (25 mg/ml) or with human IgG1 (hIgG1) than HIV (30, 35–37), cancer (38–40), transplantation (41, 42), (ET901; BioLegend) as a negative isotype control for 40 min at 4˚C. and autoimmune diseases (43). Binding of the KIR3DS1-Fc chimera protein and control were detected by by guest on September 25, 2021 adding goat anti-human IgG Fc-PE secondary Ab (eBioscience) for 25 min Garcia-Beltran et al. (28) reported that open conformers at 4˚C. To verify that acid pulsing of 221 cells produced HLA cell surface (OCs) of HLA-F are high-affinity ligands for KIR3DS1. They OCs, the mAbs W6/32, 3D12, and 2M2 (BioLegend) were used to stain found that the interaction of KIR3DS1 with HLA-F was func- untreated and acid-pulsed 221 cells. Although 221 cells are HLA-A, -B, tional and led to activation of cell lines expressing KIR3DS1. In and -C null, they do express HLA-E, which is detected by the pan-HLA– this study, we extended these findings by investigating the ef- specific mAb W6/32, the HLA-E–specific mAb 3D12, and the b2M- specific mAb 2M2. These mAbs do not recognize OCs of HLA-E on fects of HLA-F ligation expressed on the 721.221 (221) cell line acid-pulsed cells. One million 221 cells were resuspended in 100 mlof + with KIR3DS1 on the activation of primary KIR3DS1 NK FACS buffer with W6/32, 3D12, 2M2, or a mouse IgG2a, k isotype control cells. We employed a comprehensive Ab panel able to detect for W6/32 (MOPC-173; BioLegend) or a mouse IgG1, k isotype control several activating NK cell receptors (aNKR) and inhibitory NK for 3D12 and 2M2 (MOPC-21; BioLegend) for 30 min at 4˚C. Primary Ab + binding was detected using F(ab9)2-goat anti-mouse IgG-PE secondary Ab cell receptors (iNKR) to gate on KIR3DS1 NK cells that either (eBioscience). Cells were then washed twice and fixed in 2% parafor- coexpressed (inclusive gating) or not (exclusive gating) other maldehyde (PFA; Santa Cruz Biotechnology, Santa Cruz, CA) until ac- aNKR and iNKR. The novel exclusive gating strategy enabled quisition within 2 h. us to study the function of stimulated KIR3DS1+ NK cells NK cell activation and staining for phenotype and function without the contribution of signaling through other iNKR/ aNKR. Abs able to detect CCL4/IFN-g secretion and CD107a Cryopreserved PBMCs were thawed and rested in R10 for 3 h in a hu- expression were included in the Ab panel to measure the fre- midified 5% CO2 incubator. Rested PBMCs (effector) were cocultured with either untreated or acid-pulsed 221 cells (target) at an E:T ratio of 5:1 in quency of functional NK cell in phenotypically defined NK cell 200 ml of R10 in U-bottom 96-well plates for 6 h at 37˚C in a humidified populations. By comparing these two gating strategies and 5% CO2 incubator. PBMCs cultured alone in R10 served as an unstimu- blocking the interaction of KIR3DS1 and HLA-F, we showed lated negative control, and PBMCs were stimulated with 5.8 mg/ml PMA that KIR3DS1+ ligation by HLA-F on the 221 cell line activated (Sigma-Aldrich); 1.2 mg/ml ionomycin (Sigma-Aldrich) in R10 served as a positive control to ensure that the NK cells being stimulated were viable primary NK cells expressing this receptor. and functional. Brefeldin A (24 mg/ml; Sigma-Aldrich) and monensin (3 mg/ml, GolgiStop; BD Biosciences, Mississauga, ON, Canada) were added Materials and Methods 30 min after the initiation of the coculture. All cells responded to 5.8 mg/ml Ethics statement PMA, 1.2 mg/ml ionomycin stimulation. After stimulation, cells were stained using a fluorochrome-conjugated This study was conducted in accordance with the principles expressed in Ab panel that was validated with single-stained control beads (Comp- the Declaration of Helsinki. It was approved by the Institutional Review Bead; BD Biosciences). Cells were stained for viability using the UV Live/ Boards of the Comite´ d’E´ thique de la Recherche du Centre Hospitalier de Dead staining kit (Invitrogen, Carlsbad, CA), as per manufacturer’s in- l’Universite´ de Montreál and the Research Ethics Committee of the structions. Cells were surface-stained with anti–CD3-BV785 (OKT3), McGill University Health Centre. All subjects provided written informed anti–CD56-BV711 (HDC56), anti–KIR2DL1/S1/S3/S5-FITC (HP-MA4), consent for the collection of samples and subsequent analysis. and anti–CD107a-BV421 (H4A3) (all from BioLegend); anti–KIR3DL1/ The Journal of Immunology 3

S1-PE (REA168) and anti–NKG2A-biotin (REA110) (both from Miltenyi Results Biotec, Auburn, CA); anti–KIR2DL2/L3/S2-FITC (CH-L; BD Biosciences), Expression level of HLA-F on untreated and acid-pulsed anti-KIR3DL2–allophycocyanin (539304), and anti-ILT-2–allophycocyanin (292305) (both from R&D Systems) for 30 min at 4˚C. After washing, binding 221 cells of anti-NKG2A was detected by incubating cells with Qdot 655 Streptavidin Acid pulsing favors OCs of cell surface classical and nonclassical Conjugate (Life Technologies, Burlington, ON, Canada) for 20 min at 4˚C. Cells were then washed and fixed with 2% PFA, permeabilized with Per- MHC class I (MHC-I) Ags, including HLA-F. HLA-F OCs are the meabilization Medium B (Invitrogen), and stained intracellularly with anti– preferred conformation for interactions with KIR3DS1 (28, 49). CCL4-AF700 (D21-1351) and anti-IFN-g–BV510 (B27) (both from BD Fig. 1A and 1B show that 3D11 and KIR3DS1-Fc bound to Biosciences). Samples were then washed twice and fixed with 2% PFA until untreated (top panel) and acid-pulsed (second panel) 221 cells acquisition. with a similar mean fluorescence intensity (MFI). Isotype con- To investigate whether HLA-F on 221 cells can interact with ILT-2 on NK cells to mediate inhibitory signals affecting NK cell function, the anti- trols for these HLA-F–specific reagents bound untreated (third ILT-2–allophycocyanin Ab in the cell surface Ab panel described in the panel) and acid-pulsed (bottom panel) 221 cells with lower MFIs previous paragraph was changed to one conjugated with PE/Cy7 (clone than did 3D11 and KIR3DS1-Fc and with MFIs that did not +/2 GH1/75; BioLegend) so that ILT-2 NK cells could be exclusively substantially differ from each other. To ensure that the acid- gated on and assessed for the frequency of functional cells. To verify that 221 stimulation activated NK cells, we measured the frequency of pulsing regimen produced MHC-I OCs, we stained untreated CD69+ NK cells in gated KIR3DS1+ NK cells that were unstimulated or and acid-pulsed 221 cells with the mAbs W6/32, 3D12, and stimulated exclusively by adding anti–CD69-BV605 (clone FN50; Bio- 2M2. W6/32 recognizes correctly folded classical MHC-1a and Legend) to the Ab mixture used for cell surface staining described in the nonclassical MHC-1b HLA-E and -F Ags, but not their OC previous paragraph. counterparts (28). 3D12 is specific for correctly folded HLA-E, For some experiments, PBMCs were stimulated with EBV-transformed Downloaded from B lymphoblastoid cell lines instead of 221 cells using the same conditions which is expressed at a low intensity of 221 cells. mAb 2M2 is + and Ab panels as described above. The EBV lines were HLA-F as de- specific for b2M, which is present on correctly folded HLA-E but termined by staining with 3D11 as described above for staining 221 cells. not OCs on 221 cells. W6/32 bound untreated 221 cells (top For some experiments, the interaction between KIR3DS1 on NK cells panel) with a higher MFI than it did acid-pulsed 221 cells and HLA-F on 221 cells was blocked by incubating 221 cells in R10 with 25 mg/ml KIR3DS1-Fc chimera protein (R&D Systems) or 25 mg/ml anti- (second panel), which was in the range of the MFI with which HLA-F–specific mAbs 3D11, 4A11, or 6A4 for 50 min at 37˚C in a hu- the isotype control for this mAb bound untreated (third panel) http://www.jimmunol.org/ midified 5% CO2 incubator. For these experiments, PBMCs were also and acid-pulsed (bottom panel) 221 cells (Fig. 1C). 3D12 bound incubated with anti-human CD16 (FcgRIII) F(ab9)2 (3G8; Ancell Corpo- untreated 221 cells (top panel) with a marginally higher MFI ration, Bayport, MN) at 10 mg/ml final concentration for 45 min prior to than it did acid-pulsed 221 cells (second panel). The MFI with coincubation with 221 cells to minimize the effect of signaling through this Fc receptor on NK cell activation. Fc receptor–blocked PBMCs were which the isotype control for this mAb bound untreated (third cocultured with 221 cells for 6 h at 37˚C in a humidified 5% CO2 incu- panel) and acid-pulsed (bottom panel) 221 cells was similar bator. The control conditions for these experiments were unstimulated (Fig. 1D). 2M2 bound untreated 221 cells (top panel) with a PBMCs, PBMCs stimulated with 221 cells that were not preincubated with higher MFI than it did acid-pulsed 221 cells (second panel). The KIR3DS1-Fc chimera protein or anti-HLA-F–specific mAbs, and 221 cells preincubated with hIgG1 isotype control (ET901; BioLegend) at a con- MFI with which the isotype control for this mAb bound un- centration of 25 mg/ml as a control for KIR3DS1-Fc or with 25 mg/ml treated (third panel) and acid-pulsed (bottom panel) 221 cells by guest on September 25, 2021 MOPC-21 as a control for anti-HLA-F–specific mAbs 3D11, 4A11, and was similar (Fig. 1E). 6A4. Following coculture, cells were stained for cell surface and intra- + cellular markers as described above. The percentage of reduction in 221 Untreated and acid-pulsed 221 cells stimulate KIR3DS1 NK KIR3DS1+ or KIR3DL1+ NK cell stimulation in the presence of blocking cells to exhibit similar functional profiles reagents was calculated using the following equation: ((frequency of We next compared the flow cytometry plots generated for total functional cells in the 221-stimulated NK cell population in the presence of a blocking reagent 2 frequency of functional cells in the unstimulated NK CCL4 secretion, total IFN-g secretion, and total CD107a expres- cell population) / (frequency of functional cells in the 221-stimulated NK sion for KIR3DS1+CD56dim NK cells stimulated with untreated or cell population 2 frequency of functional cells in the unstimulated NK cell acid-pulsed 221 cells and for KIR3DS1+CD56dim NK cells left 3 population) 100). unstimulated. The strategy used to gate on functional KIR3DS1+ Flow cytometry analysis CD56dim NK cells is shown in Fig. 2A and 2C. The histograms for these three functional profiles overlapped in KIR3DS1+CD56dim Between 7 3 105 and 1.5 3 106 total events were acquired for each sample using an LSR Fortessa X-20 flow cytometer (BD Biosciences). Results NK cells from a representative individual stimulated with un- obtained from flow cytometry data were analyzed by FlowJo software treated 221 cells (top panel) and acid-pulsed 221 cells (middle (version 10.2; Tree Star, Ashland, OR). We measured the frequency of panel) and had a higher MFI than that seen in unstimulated cells +/2 2 dim KIR3DS1 CD3 CD56 NK cells exhibiting the seven possible com- (bottom panel) (Fig. 3A–C). The frequency of KIR3DS1+CD56dim binations of CCL4, IFN-g, and CD107a function (i.e., trifunctional, three NK cells from 10 KIR3DS1 hmz subjects responding to untreated combinations of bifunctional, and three combinations of monofunctional response patterns). We also assessed the sum of the frequencies of all and acid-pulsed 221 cells was similar for the functional profiles functions tested (total) and the sums of the frequencies of functional characterized by the sum of all functions, total CCL4 secretion, subsets secreting CCL4 (total CCL4), secreting IFN-g (total IFN-g), and total IFN-g secretion, and total CD107a expression (Fig. 3D–G, expressing CD107a (total CD107a). These functional subsets were 2 respectively). assessed for KIR3DS1+ and KIR3DS1 NK cell populations using gating strategies that either included or excluded NK cell populations coex- Collectively, these results show that acid pulsing 221 cells did pressing the NKRs KIR2DL1/L2/L3, KIR2DS1/S2/S3/S5, KIR3DL2, ILT- not improve HLA-F recognition by either an HLA-F–specific mAb 2, and NKG2A. The data presented are background subtracted using or the KIR3DS1-Fc chimera protein and did not enhance the results for matched unstimulated control conditions. ability of 221 cells to stimulate KIR3DS1+CD56dim NK cells. Statistical analysis Given these results, we used untreated 221 cells in subsequent experiments. GraphPad Prism 6 (GraphPad Software, La Jolla, CA) was used to perform + 2 data analysis and graphical presentation. Wilcoxon matched-pairs signed A higher frequency of KIR3DS1 than KIR3DS1 NK cells rank and Friedman tests with Dunn posttests were used to assess the responded to stimulation with 221 cells significance of comparisons of two and more than two groups, respectively, within individual matched data sets. The p values ,0.05 were considered PBMCs were stimulated with 221 cells and then stained with the significant. Ab panel described in the Materials and Methods that included 4 INTERACTIONS OF HLA-F WITH KIR3DS1 ACTIVATE NK CELLS

FIGURE 1. Untreated and acid-pulsed 221 cells express cell surface OCs of HLA-F. The histograms in (A) and (B) show the fluorescence pattern generated by staining untreated (top and third histograms) or acid-pulsed 221 cells (second and bottom histograms) with mAb 3D11 (A) or KIR3DS1-Fc chimera protein (B) (top and second) or with an isotype control (third and bottom). (C)–(E) show the fluorescence pattern generated by staining untreated (top and third histograms) or acid-pulsed (second and bottom histograms) 221 cells with mAbs W6/32 (C), 3D12 (D), or 2M2 (B2M) (E) (top and second) or with isotype control (third and bottom). Binding of 3D11, KIR3DS1-Fc, W6/32, 3D12, 2M2, and isotype controls was detected using fluorochrome- conjugated secondary Abs.

ﬂuorochrome-conjugated Abs able to recognize KIR2DL1/L2/L3/ self-HLA and NKG2A would be educated through the interaction S1/S2/S3/S5, KIR3DL2, ILT-2, and NKG2A on KIR3DS1+/2 of these receptors with their HLA ligands. This would make them CD56dim NK cells. As seen in Fig. 2A, live lymphocyte singlet responsive to stimulation by 221 cells. The level of stimulation 2 dim CD3 CD56 NK cells that inclusively expressed, or not, resulting from missing-self recognition of HLA-null cells by ed- Downloaded from KIR3DS1 were gated on. Boolean gating was used to assess the ucated NK cell subsets can differ from one person to another and frequency of KIR3DS1+ and KIR3DS12 cells that expressed the may confound differences in the stimulation levels of inclusively various combinations of the three functions tested. The frequency gated KIR3DS1+ and KIR3DS12 NK cells. This prompted us to of cells exhibiting the sum of all functions tested, total CCL4 and minimize this possibility by using an exclusive gating strategy to total IFN-g secretion, and total CD107a expression was higher gate out NK cell populations bearing KIR2DL1/L2/L3/S1/S2/S3/ + 2 , +/2 dim among KIR3DS1 than KIR3DS1 NK cells (p 0.0001 for all, S5, KIR3DL2, ILT-2, and NKG2A from KIR3DS1 CD56 http://www.jimmunol.org/ Wilcoxon matched-pairs signed rank test) (Fig. 4A–D). NK cells. The gating strategy used for exclusive KIR3DS1+ and For 10 subjects, we assessed the frequency of trifunctional, three KIR3DS12 NK cell gating is shown in Fig. 2B. combinations of bifunctional, and three combinations of mono- Supplemental Fig. 2 shows that 221 stimulation increased the functional KIR3DS1+/2 NK cells. A higher frequency of inclu- frequency of CD69+ on exclusively gated KIR3DS1+ and sively gated KIR3DS1+ than KIR3DS12 NK cells secreted CCL4 KIR3DL1+ NK cells (p = 0.04 and p = 0.057, respectively, only and both CCL4 and IFN-g (p = 0.027 and p = 0.002, re- Wilcoxon test). Fig. 5 shows for the 10 KIR3DS1 hmz subjects spectively, Wilcoxon test) (Supplemental Fig. 1A, 1B), whereas that even after excluding NK cells expressing the NKR recognized a lower frequency of KIR3DS1+ than KIR3DS12 NK cells by the Ab panel other than KIR3DS1, a higher frequency of + 2 expressed CD107a only (p = 0.039) (Supplemental Fig. 1C). KIR3DS1 than KIR3DS1 NK cells responded to 221 stimula- by guest on September 25, 2021 Inclusively gated KIR3DS1+ and KIR3DS12 NK cells can tion. This was the case when all functions tested were considered express various stochastic combinations of other inhibitory KIR (p = 0.005, Wilcoxon test) as well as when all cells positive for (iKIR), activating KIR, and NKG2A depending on the immu- CCL4, IFN-g, and CD107a were considered (p , 0.04 for all, nogenetic proﬁle of the study subject. NK cells bearing iKIR to Wilcoxon test) (Fig. 5B–D). A higher frequency of exclusively

FIGURE 2. Gating strategy for detecting inclusively and exclusively gated functional KIR3DS1+CD56dim NK cells. Live singlet lymphocytes were gated on, from which CD32CD56dim cells were selected. (A) For inclusive gating, KIR3DS1+ (3DS1+) and KIR3DS12 (3DS12) NK cells were selected for examination of their unstimulated and 221-stimulated functional proﬁles. (B) For exclusive gating, CD32CD56dim NK cells negative for KIR2DL1/L2/L3/ S1/S2/S3/S5, ILT-2, and KIR3DL2 were selected. From these cells, those negative for NKG2A were gated on to identify KIR3DS1+ and KIR3DS12 cells. (C) Inclusively and exclusively gated KIR3DS1+ and KIR3DS12 NK cells were then examined for all possible combinations of CCL4, IFN-g, and CD107a expression by Boolean gating. Functional gates were set on gated total CD32CD56dim cells (NK cells) in unstimulated PBMCs. Unstimulated conditions were used to background subtract 221-stimulated conditions. The Journal of Immunology 5

FIGURE 3. Untreated and acid-pulsed 221 cells stimulate KIR3DS1+ NK cells that exhibit similar functional profiles. The histograms in (A)–(C) show the fluorescence profile of CCL4 secretion (A), IFN-g secretion (B), and CD107a expression (C) following stimulation of KIR3DS1+CD56dim NK cells with Downloaded from untreated (top) or acid-pulsed (middle) 221 cells. The fluorescence profile of unstimulated KIR3DS1+CD56dim NK cells is shown in the bottom panels. These results are a representative example of experiments performed on 10 KIR3DS1 homozygotes. In (D)–(G), the y-axes show the frequency of KIR3DS1+CD56dim NK cells (3DS1+) positive for the sum of the three functions tested (percentage of functional 3DS1+ NK cells) (D), total CCL4 secretion (percentage of total CCL4+ 3DS1+ NK cells) (E), total IFN-g secretion (percentage total IFN-g+ 3DS1+ NK cells) (F), and total CD107a expression (percentage total CD107a 3DS1+ NK cells (G). The frequency of exclusively gated KIR3DS1+CD56dim NK cells stimulated by untreated and acid-pulsed 221 cells is shown in the right- and left-hand bars, respectively, of each panel. Bar height and error bars represent the median and interquartile range of the http://www.jimmunol.org/ background-corrected values generated from NK cells from 10 KIR3DS1 homozygotes. gated KIR3DS1+ than KIR3DS12 NK cells also secreted CCL4 than that for the blocking with hIgG1 control conditions for total only and both CCL4 and IFN-g (p = 0.027 and p = 0.009, re- CD107a expression (p , 0.05, Dunn posttests) (Fig. 6D). By spectively, Wilcoxon test) (Supplemental Fig. 1D, 1E), whereas contrast, differences in the frequency of functional cells stim- the frequency of KIR3DS1+ and KIR3DS12 NK cells expressing ulated with KIR3DS1-Fc–blocked 221 cells was as low as that CD107a only did not differ significantly (p . 0.05) (Supplemental of unstimulated cells for each of these functional subsets Fig. 1F). (Fig. 6A–D). The effect of HLA-F–ILT-2 interactions on the function of ILT-2+ Three HLA-F–specific mAbs were also used to block the in- by guest on September 25, 2021 NK cells. Dulberger et al. (50) recently showed that HLA-F–b2M teraction of HLA-F with KIR3DS1. Blocking with 3D11 was complexes loaded with peptide can interact with the iNKR ILT-2. tested on five KIR3DS1 hmz subjects, whereas blocking with We reasoned that if HLA-F–b2M complexes on 221 cells were 4A11 and 6A4 was tested on three subjects each. Each of these interacting with ILT-2 on NK cells, then a lower percentage of mAbs reduced the frequency of functional cells compared with exclusively gated ILT-2+ than ILT-22 NK cells would be func- that seen for 221-stimulated cells in the presence or absence of tional. Supplemental Fig. 3 shows that there were no significant hIgG1.The differences between stimulated cells with or without differences in the frequency of CCL4- and IFN-g–secreting cells blocking with 3D11 all trended toward but did not achieve in the exclusively gated ILT-2+ versus ILT-22 populations (p . 0. significance (p = values between 0.0625 and 0.12). Precoating 221 05, Wilcoxon test, Supplemental Fig. 3B, 3C). The frequency of cells with these anti–HLA-F mAbs did not reduce the frequency of ILT-2+ with total functionality and secreting CD107a was signif- functional cells to the levels seen for unstimulated PBMCs icantly lower than that of ILT-22 cells (p = 0.004 and p = 0.04, (Fig. 6E–H). Supplemental Fig. 3A, 3D). Blocking experiments with KIR3DS1-Fc were also performed + + on exclusively gated KIR3DS1 NK cells from two KIR3DL1/S1 3DS1 NK cell responses to 221 are due to the interaction of heterozygotes (Supplemental Fig. 4) Blocking the KIR3DS1– KIR3DS1 with HLA-F HLA-F interaction reduced the frequency of functional cells to To determine whether the responsiveness of KIR3DS1+ NK cells 37.8, 0, 16.4, and 74% of that of stimulated PBMCs for the sum of was due to the interaction of this receptor with HLA-F on 221 all and total CCL4, IFN-g, and CD107a functions (Supplemental cells, we blocked this interaction by preincubating 221 cells with Fig. 4A–D). Garcia-Beltran et al. (28) reported that HLA-F is a KIR3DS1-Fc chimera protein before using them to stimulate ligand for KIR3DL1. Burian et al. (49) did not confirm this PBMCs. As shown in Fig. 6, this treatment diminished the fre- finding. KIR3DL1-Fc was used to block interactions between quency of exclusively gated KIR3DS1+ NK cells responding to KIR3DL1 and HLA-F using PBMC responder cells from two 221 cells compared with that observed for responses to 221 cells KIR3DL1 hmz subjects and two KIR3DL1/S1 heterozygotes. The that were not pretreated with KIR3DS1-Fc or were pretreated with frequency of functional KIR3DL1+ NK cells averaged 74.3, 56.3, control hIgG1. The frequency of functional KIR3DS1+ NK cells 46.3, and 99.1% of that of 221-stimulated PBMCs for the sum of under KIR3DS1-Fc blocking conditions was significantly lower all and for total CCL4, IFN-g, and CD107a functions, respectively than that for either the no blocking or blocking with hIgG1 control (Supplemental Fig. 4E–H). None of these differences achieved conditions for total responsiveness, total CCL4 secretion, and total statistical significance, likely due to the small number of subjects IFN-g secretion (p , 0.05 for all, Friedman tests with Dunn tested. posttests) (Fig. 6A–C). The frequency of functional KIR3DS1+ Together, these results show that blocking the interaction of NK cells under KIR3DS1-Fc blocking conditions was also lower KIR3DS1 on NK cells bearing this receptor with HLA-F on 221 6 INTERACTIONS OF HLA-F WITH KIR3DS1 ACTIVATE NK CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. A higher frequency of inclusively gated KIR3DS1+ than KIR3DS12 NK cells responded to stimulation with the HLA-null cell line 221. Shown on the y-axis is the frequency of inclusively gated KIR3DS1+ and KIR3DS12 NK cells exhibiting (A) the sum of all functions tested (percentage of total functional NK cells), (B) the sum of cells secreting CCL4 (percentage of total CCL4+ NK cells), (C) the sum of cells secreting IFN-g (percentage of total IFN-g+ NK cells), and (D) the sum of cells expressing CD107a (percentage of total CD107a+ NK cells). Each data point represents the results of experiments performed using cells from one individual. (A) and (B) include results for 10 KIR3DS1 homozygotes, of whom eight were tested twice for a total of 18 observations. (C) and (D) include results for 10 KIR3DS1 homozygotes, of whom eight were tested on three occasions for a total of 26 ob- by guest on September 25, 2021 servations. The significance of between-group results was assessed using Wilcoxon matched-pairs tests. Significance between-group differences are indicated, ****p # 0.0001. cells reduced the functionality of these cells to a level that was not unstimulated PBMCs (Fig. 7E–H). Differences in the frequency of significantly different from that of unstimulated KIR3DS1+ NK functional cells in the KIR3DS1-Fc blocking conditions compared cells and that was significantly lower than that seen in conditions with unblocked or control hIgG–blocked conditions were statis- where this interaction was not blocked. Furthermore, the exclusive tically significant for all functions except total CD107a expression gating strategy used to detect KIR3DS1+ NK cells minimized the when Wilcoxon tests were used, but not when Friedman tests were possibility that the activation of these cells was due to missing-self used, to assess the significance of between-group differences. recognition of HLA-null cells lacking ligands for iNKR. Thus, the interaction of KIR3DS1 with HLA-F activated primary KIR3DS1+ Discussion NK cells. KIR3DS1 is an activating NKR whose presence is associated with + beneficial HIVoutcomes, such as slow time to AIDS and protection EBV lines express HLA-F and stimulate KIR3DS1 NK cells from HIV infection (30, 33, 52). The ligand for KIR3DS1 was EBV-transformed B cells also express HLA-F (51). We confirmed recently shown to be the nonclassical MHC-Ib molecule HLA-F this expression by staining with 3D11 and used an HLA-F+ EBV– (28, 49). In this study, we confirmed that HLA-F is present on the transformed cell line to stimulate PBMCs from five KIR3DS1 hmz surface of the HLA-null cell line 221. Coculture of 221 cells with subjects using the same conditions as described above. A higher PBMCs activates a higher frequency of CD32CD56dim KIR3DS1+ frequency of exclusively gated KIR3DS1+ than KIR3DS12 CD32 than KIR3DS12 NK cells to express CD69, secrete CCL4 and CD56dim NK cells exhibited the sum of all functions tested, total IFN-g, and express CD107a. We showed that the activation of CCL4 and IFN-g secretion, and total CD107a expression fol- KIR3DS1 by 221 cells occurred in the absence of interrupted lowing stimulation (Fig. 7A–D). However, the difference in the signaling through iKIR because of missing-self recognition or by frequency of functional KIR3DS1+ versus KIR3DS1– only trended activation through several coexpressed aNKR by using a gating toward statistical significance (p = 0.062 for total function, total strategy that excluded KIR3DS1+/2 NK cells coexpressing a panel CCL4- and total IFN-g–secreting cells, and p = 0.12 for total of iNKR and aNKR. We showed that the ligation of KIR3DS1 CD107a-expressing cells, Wilcoxon tests). Blocking with by HLA-F on 221 cells was responsible for the higher fre- KIR3DS1-Fc chimera protein reduced the frequency of EBV- quency of stimulated, exclusively gated CD56dim KIR3DS1+ than transformed B cell–stimulated KIR3DS1+ cells exhibiting all KIR3DS12 NK cells by demonstrating that blocking the interac- functions, total CCL4 and IFN-g secretion, and total CD107a tion of KIR3DS1 with HLA-F reduced the frequency of expression to levels not significantly different from those seen in 221-responsive KIR3DS1+ NK cells. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. A higher frequency of exclusively gated KIR3DS1+ than KIR3DS12 NK cells responded to stimulation with the HLA-null cell line 221. Shown on the y-axis is the frequency of exclusively gated KIR3DS1+ and KIR3DS12 NK cells exhibiting (A) the sum of all functions tested (percentage of total functional NK cells), (B) the sum of cells secreting CCL4 (percentage of total CCL4+ NK cells), (C) the sum of cells secreting IFN-g (percentage of total IFN-g+ NK cells), and (D) the sum of cells expressing CD107a (percentage of total CD107a+ NK cells). Each data point represents the results of

experiments performed using cells from 1 of 10 KIR3DS1 homozygotes. The signiﬁcance of between-group results was assessed using Wilcoxon matched- by guest on September 25, 2021 pairs tests. Signiﬁcance between-group differences are indicated, *p # 0.05, **p # 0.01.

The nature of the ligand for KIR3DS1 has been elusive until with b2M and peptide, is retained intracellularly in resting cells, recently. Epidemiological studies found that carriers of KIR3DS1 and can be expressed on the cell surface of activated cells as an and HLA-Bw4*80I combinations progress to AIDS more slowly OC without peptide or b2M, although its cell surface expression than carriers of the receptor or ligand alone (30). However, in the may increase as MHC-Ia is upregulated (44, 55–58). HLA-F is absence of HLA-Bw4-80I alleles, KIR3DS1 homozygosity is as- present on the surface of B lymphoblastoid cell lines such as 221, sociated with more rapid progression to AIDS (30). Studies in- on EBV-transformed B cells, and on HIV-infected cells (28). vestigating NK cell–mediated inhibition of HIV replication found Goodridge et al. (44) reported that HLA-F can be expressed in- that effector cells from carriers of this KIR/HLA genotype com- dependently of the endoplasmic reticulum peptide binding path- bination suppressed HIV replication in autologous infected CD4+ ways because of its altered cytoplasmic domain. Based on T cells more effectively than carriers of the receptor or ligand differential Ab staining for HLA-F, they concluded that at alone (31, 32). Despite this, attempts to demonstrate a direct in- least three different forms of HLA-F may be expressed over the teraction between KIR3DS1 and HLA-Bw4*80I Ags have failed course of lymphocyte activation. They confirmed that HLA-F is (27, 28). An exception to this was the observation that KIR3DS1 expressed on activated lymphocytes independently of TAP could bind the HLA-Bw4*80I Ag HLA*B57 if certain HIV- and tapasin. Thus, HLA-F can be spontaneously expressed as an derived peptides were present to facilitate this interaction (29). OC (58). Garcia-Beltran et al. used fusion proteins of the extracellular do- Mild acid treatment of cells dissociates b2M and peptide from main of several KIR gene products with the Fc portion of IgG1 to HLA H chains producing OCs (51). We compared the ability of screen a panel of 100 recombinant MHC-I proteins. KIR3DS1-Fc the HLA-F–specific mAb 3D11 and a KIR3DS1-Fc chimera bound no HLA-A, -B, or -C Ags. Rather, the preferred ligand for protein to recognize HLA-F on untreated and acid-pulsed 221 KIR3DS1 was HLA-F OC (28). Several other KIR3D receptors cells and found that both reagents bound HLA-F on untreated and shared with KIR3DS1 the ability to bind HLA-F OCs (28, 53). acid-pulsed 221 cells with a similar MFI. We also showed that Surface plasmon resonance, pull-down experiments, heterodimeriza- untreated and acid-pulsed 221 cells stimulated a similar frequency tion experiments, and HLA-F tetramers were used to show that of KIR3DS1+ NK cells to produce CCL4 and IFN-g and express KIR3DS1 binds to OCs of HLA-F (28, 49). CD107a with similar intensities. We verified that the procedure HLA-F differs from classical MHC-Ia proteins in several ways. used for acid pulsing produced OCs by showing that the mAbs MHC-Ia proteins are highly polymorphic, form complexes with W6/32 and 3D12 recognized HLA-E on untreated but not on acid- b2M and short peptides, and are expressed at the cell surface of pulsed 221 cells. W6/32 and 3D12 only bind correctly folded most human cells (54). HLA-F is monomorphic, binds weakly HLA-E. Furthermore, the b2M-specific 2M2 mAb served as a 8 INTERACTIONS OF HLA-F WITH KIR3DS1 ACTIVATE NK CELLS Downloaded from

FIGURE 6. Blocking KIR3DS1 on NK cells suppresses the activation of KIR3DS1+ NK cells stimulated by 221 cells. Shown on the y-axis is the frequency of exclusively gated KIR3DS1+CD32CD56dim NK cells exhibiting (A and E) the sum of all functions tested (percentage of total functional NK cells), (B and F) the sum of cells secreting CCL4 (percentage of total CCL4+ NK cells), (C and G) the sum of cells secreting IFN-g (percentage of total http://www.jimmunol.org/ IFN-g+ NK cells), and (D and H) the sum of cells expressing CD107a (percentage of total CD107a+ NK cells) following no stimulation (PBMC), stimulation with 221 cells (221), stimulation with 221 cells precoated with human IgG1 (hIgG1) Ab (A–D), or stimulation with mouse IgG1 (MIgG1) (E–H) and stimulation with 221 cells precoated with KIR3DS1-Fc chimera protein (3DS1-Fc) (A–D) or the anti–HLA-F mAbs 3D11, 4A11, or 6A4 (E–H). For (A)–(D), bar heights and error bars represent the median and interquartile range of results generated using cells from eight KIR3DS1 homozygotes. The significance of between-group results was assessed using Friedman tests with Dunn posttests. The boxes over the 3DS1-Fc blocking conditions show the percentage of reduction in the frequency of functional cells from that seen for cells stimulated with 221 cells with no blocking. Statistically significant between-group comparisons are shown by a line linking groups. For (E)–(H), bar heights represent the medians and interquartile range of five observations for PBMC, PBMC plus 221, MIgG1, and the blocking condition using mAb 3D11; three observations were assessed for blocking with the mAbs 4A11 and 6A4. The boxes over the mAbs blocking conditions show the percentage of reduction in the frequency of functional cells from that seen for cells stimulated with

221 cells in the same experiment. Signiﬁcance between-group differences are indicated, *p # 0.05, **p # 0.01. by guest on September 25, 2021

control for acid-pulsed stripping of b2M from HLA-E on 221 cells panel of iNKR and aNKR. Abs to KIR2DL5 were not included in because it only recognized unpulsed cells. These findings contrast this panel because we found that coexpression of KIR2DL5 with with what has been shown by Garcia-Beltran et al. (28). They KIR3DS1 did not modulate the responsiveness of NK cells showed that Jurkat cells expressing KIR3DS1 were triggered more expressing KIR3DS1 without KIR2DL5 to 221 stimulation (68). potently by acid-pulsed than untreated 221 cells. Despite this, the Abs to KIR2DS4 were not included in the panel because none of same group showed that cell-sized coated beads loaded with HLA- the 10 KIR3DS1 hmz study subjects carried KIR2DS4, as the gene F activated KIR3DS1-expressing Jurkat cells to a similar extent, encoding this receptor is in negative linkage disequilibrium with whether acid pulsed or not. Discrepant results may be due to KIR3DS1 frequently found on telomeric KIR region group B disparate sensitivities of the cell types responding to stimulatory haplotypes (69, 70). The observation that exclusively gated cells expressing HLA-F (primary NK cells versus KIR3DS1- KIR3DS1+ NK cells responded better to 221 stimulation than transduced Jurkat cells) or to the nature of the stimulatory sig- KIR3DS12 NK cells narrowed the possibilities for the identity of nal (untreated and acid-pulsed 221 cells versus HLA-F–coated the NKR responsible for 221-dependent KIR3DS1+ activation. beads). At least in our hands, the HLA-F present on the surface of The loss of exclusively gated KIR3DS1+ NK cell activation when 221 cells is in a conformation able to interact with KIR3DS1 the interaction between KIR3DS1 and HLA-F was blocked con- present on primary NK cells isolated from KIR3DS1 hmz subjects, firmed that the interaction between this receptor/ligand pair and acid pulsing improves neither HLA-F’s stimulatory capability is functional and responsible for the activation of primary nor its recognition by HLA-F–specific reagents. KIR3DS1+ NK cells. In contrast, mAbs to HLA-F were less ef- The interaction of iNKR with their HLA ligands is necessary for fective at blocking the activation of KIR3DS1+ NK cells. The NK cell education, which in turn confers NK cells with the reason for this is unclear, as the concentrations of mAbs used were functional potential to become activated if they encounter trans- saturating in binding assays. It is possible that these mAbs have a formed, stressed, or virus-infected self-cells with downmodulated higher off rate than the KIR3DS1-Fc chimera protein, exposing ligands for these iNKR (59, 60). Some activating KIRs may also HLA-F to KIR3DS1. Alternately, this result may suggest that participate in NK cell education, but these interactions should tune KIR3DS1 recognizes other ligands on 221 cells whose interaction down NK cells’ responsiveness to altered self-cells (61, 62). HLA- with KIR3DS1 is not blocked by these Abs. Combining more than null cells such as 221 are well known to stimulate educated NK one mAb for blocking did not further reduce KIR3DS1+ NK cell cells because they lack the ligands for several iNKR, which activation. We cannot formally exclude the possibility that re- transmit inhibitory signals (63–67). Reducing the effect of ceptors other than KIR3DS1 are involved in the activation of missing-self recognition on NK cell activation was the impetus for KIR3DS1+ NK cells. However, the reduction in the frequency of excluding KIR3DS1+ and KIR3DS12 NK cells coexpressing a activated KIR3DS1+ NK cells to levels at or below those seen for The Journal of Immunology 9 Downloaded from

FIGURE 7. EBV-transformed B cells express HLA-F, which interacts with KIR3DS1 to stimulate exclusively gated KIR3DS1+ NK cells. (A–D) Shown on the y-axis is the frequency of exclusively gated KIR3DS1+ and KIR3DS12 CD32CD56dim NK cells exhibiting (A) the sum of all functions tested (percentage of total functional NK cells), (B) the sum of cells secreting CCL4 (percentage of total CCL4+ NK cells), (C) the sum of cells secreting IFN-g (percentage of total IFN-g+ NK cells), and (D) the sum of cells expressing CD107a (percentage of total CD107a+ NK cells). Each data point represents the results of experiments performed using cells from one of five KIR3DS1 homozygotes. The significance of between-group results was assessed using Wilcoxon matched-pairs tests. (E–H) Shown on the y-axis is the frequency of exclusively gated KIR3DS1+CD32CD56dim NK cells exhibiting (E) the sum http://www.jimmunol.org/ of all functions tested (percentage of total functional NK cells), (F) the sum of cells secreting CCL4 (percentage of total CCL4+ NK cells), (G) the sum of cells secreting IFN-g (percentage of total IFN-g+ NK cells), and (H) the sum of cells expressing CD107a (percentage of total CD107a+ NK cells) following no stimulation (PBMC), stimulation with an EBV-transformed B cell line (EBV), stimulation with EBV-transformed B cells precoated with hIgG1 Ab, and stimulation with EBV-transformed B cells precoated with KIR3DS1-Fc chimera protein (3DS1-Fc). For (E)–(H), bar heights and error bars represent the median and interquartile range of results generated using cells from five KIR3DS1 homozygotes. The significance of between-group results was assessed using Friedman tests with Dunn posttests. The boxes over the 3DS1-Fc blocking conditions show the percentage of reduction in the frequency of functional cells from that seen for cells stimulated with EBV-transformed B cells with no blocking. Statistically significant between-group comparisons are shown by a line linking groups. Significance between-group differences are indicated, *p # 0.05, **p # 0.01. by guest on September 25, 2021 unstimulated PBMCs when the interaction between KIR3DS1 and express HLA-F and that KIR3DS1+ NK cells suppress HIV rep- HLA-F is blocked by KIR3DS1-Fc chimera protein suggests that lication in autologous CD4+ T cells (28, 31, 32). The frequency of the contribution of other NK receptors to activation is unlikely or KIR3DS1+ cells in KIR3DS1 hmz subjects was reported to be minimal. nearly twice as high as that in KIR3DL1/S1 heterozygotes, with a We found that 221 cells that were not acid pulsed did induce a median of 61% (range 41–81%) in a population of five KIR3DS1 significantly lower frequency of exclusively gated ILT-2+ than ILT- hmz subjects (74). For the 10 KIR3DS1 hmz subjects we studied, 22 NK cells exhibiting the sum of all functions and expressing the mean 6 SD of KIR3DS1+CD56dim NK cells was 46.0 6 CD107a. However, differences in the frequency of functional cells 20.2% (range 7.5–75.9%). It is interesting to speculate that the were small (i.e., 34 6 12.4% versus 36.4 6 12.3% for the sum high frequency of KIR3DS1+ cells in KIR3DS1 hmz donors may of all functions for ILT-2+ versus ILT-22 cells, respectively, and be a factor in the association of this genotype with protection from 13.0 6 5.4% versus 14.6 6 6.1% for total CD107a-expressing infection. Mature NK cells express prestored IFN-g transcripts, ILT-2+ versus ILT-22 cells, respectively). Between-group differ- granzyme, and perforin and are ready to lyse targets within min- ences were not significant for the frequency of total CCL4 and utes of activation, which would be a desirable characteristic of total IFN-g secretion. Thus, there is some evidence for inhibition cells mediating early responses to HIV that may prevent the es- of the function of ILT-2+ cells by HLA-F on 221. The low level of tablishment of infection (72). Secretion of the chemokines CCL3, inhibition and the absence of differences in 3D11 and KIR3DS1- CCL4, and CCL5 by activated NK cells also mediate antiviral Fc staining in acid-pulsed versus unpulsed cells would support the effects by blocking HIV entry (71). KIR3DS1 is also maintained conclusion that much of the HLA-F on 221 cells are OCs and not on the surface of NK cells in HIV-infected individuals (74). The in complexes with b2M and peptide, which is the form that can high frequency of NK cells bearing activating KIR3DS1 receptors interact with ILT-2 (50). in individuals carrying the gene encoding this receptor is consis- Activated KIR3DS1+ NK cells secrete CCL4 and IFN-g and tent with the epidemiological data suggesting a critical role for express CD107a. It is notable that CCL4 is a chemokine able to this receptor in controlling HIV-1 pathogenesis and contributing to bind the CCR5 coreceptor for HIV entry, blocking HIV infection the prevention of HIV infection (28, 30–34, 52). of new target cells (71). IFN-g is a critical cytokine for innate and adaptive immune responses against viral infections (72), and Acknowledgments CD107a is a marker of NK cell degranulation, a surrogate marker We acknowledge Xiaoyan Ni and Tsoarello Mabanga for expert technical for target cell lysis (73). Production of these molecules by stim- assistance and Pascale Artotto and Joseé Girouard for nursing support + ulated KIR3DS1 NK cells may be a mechanism underlying the obtaining leukapheresis samples. We thank the study participants without association of KIR3DS1 homozygosity with a reduced risk of HIV whose generous donation of biological specimens this work would not infection (33, 34, 52). Others have shown that HIV-infected cells have been possible. 10 INTERACTIONS OF HLA-F WITH KIR3DS1 ACTIVATE NK CELLS

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(B) (C) (A) 80 80 80 * * ** 60 60 60 NK Cells +  40 40 40 onlyNK Cells only NK Cells + IFN- + +

20 20

%CCL4 20 %CD107 %CCL4

0 0 0 3DS1+ 3DS1- 3DS1+ 3DS1- 3DS1+ 3DS1-

(D) (E) (F) 80 80 80

60 * 60 ** 60 NK cells +  40 40 40 Only NK cells onlyNK Cells IFN- + + +

%CCL4 20 20 20 %CD107 %CCL4

0 0 0 3DS1+ 3DS1- 3DS1+ 3DS1- 3DS1+ 3DS1-

Figure S1. Differences in the frequency of 221 stimulated KIR3DS1+ than KIR3DS1- NK cells secreting CCL4 only, secreting both CCL4 and IFN-γ and expressing CD107a only. Shown on the y-axis is the frequency of inclusively gated (A-C) and exclusively gated (D-F) KIR3DS1+ (3DS1+) and KIR3DS1- (3DS1-) cells positive for CCL4 secretion only (% CCL4+ only NK cells) (A, D), positive for CCL4 and IFN-γ secretion (% CCL4+IFN-γ+ NK cells) (B, E) and positive for CD107a expression only (% CD107a+ only NK cells) (C, F). Each data point represents results from 1 of a total of 10 KIR3DS1 hmz. The significance of between-group results was assessed using Wilcoxon matched pairs tests. Significant between-group differences are shown by * - p ≤ 0.05 and ** - p ≤ 0.01. Figure S2

(A) 80 (B) 80

60 60 p=0.03 NK cells NK cells NK + 40 + 40 p=0.12 3DL1 +

%CD69+3DS1 20 %CD69

0 0 1 d 2 1 te 2 d 2 la te 2 u m ula ti m ti ns U ns U

Figure S2. 221 stimulation induces an increased the frequency of CD69+ NK cells. Shown on the y-axis is the frequency of exclusively gated CD69+KIR3DS1+ CD56dim NK cells (left panel) and CD69+KIR3DL1+CD56dim NK cells (right panel) on unstimulated and 221 stimulated cells. Results from 5 and 4 observations were used to generate results for KIR3DS1+ and KIR3DL1+ NK cells, respectively. A Wilcoxon matched pars test was used to assess the significance of differences between unstimulated and stimulated conditions. P-values for these comparisons are shown over the lines linking the 2 test conditions. Figure S3

(A) (B)

80 p=0.004 80 p=0.12

60 60 NK cells NK + 40 40

%Total Fnx NK cells NK Fnx %Total 20 20 %TotalCCL4

0 0 + - 2 + - T 2 T2 L 2 T IL I T L (D) IL I (C) 80 80

p=0.4 p=0.04 60 60 NK cells NK NK cells NK + +  40 40

%TotalIFN- 20 20 %Total CD107a %Total

0 0 - + 2 2 - T T + L IL 2 T2 I T L IL I Figure S3. Comparisons of the frequency of functional ILT-2+ versus ILT-2- cells following stimulation with 221 cells. Shown on the y-axis is the frequency of exclusively gated ILT-2+ and ILT-2- NK cells exhibiting (A) the sum of all functions tested (% Total Fxn NK cells), (B) the sum of cells secreting CCL4 (% Total CCL4+ NK cells), (C) the sum of cells secreting IFN-γ (% Total IFN-γ+ NK cells) and (D) the sum of cells expressing CD107a (% Total CD107a+ NK cells). Each data point represents results from 1 of a total of 9 KIR3DS1 hmz. The significance of between-group results was assessed using Wilcoxon matched pairs tests. P-values for comparisons between exclusively gated ILT-2+ and ILT-2- NK cells are shown over the lines linking the 2 populations in each panel. Figure S4

(A) (B) (C) (D)

100 lls

lls 100 100

100 lls 80 80 80 37.8% 80 0.0% 16.4% 74.0% 60 60 60 60

40 40 40 40

20 20 20 20 %Fnx 3DS1+ NK cells NK 3DS1+ %Fnx 0

0 ce NK IFN+3DS1+ %Total 0 0 %Total CCL4+3DS1+ NK ce NK CCL4+3DS1+ %Total

C 1 1 c ce NK CD107+3DS1+ %Total 2 G F C 1 1 c C 1 1 c 1 1 c 2 g - G F 2 F G F I 1 M 22 g - M 2 G - MC 22 PBM h S B I 1 B Ig 1 B Ig 1- D P h S P h P h S 3 D DS D 3 3 3 (E) (H) (F) (G) 100 lls 100

lls 100 100 lls 80 74.3% 80 80 80 56.3% 46.5% 99.2% 60 60 60 60

40 40 40 40

20 20 20 20 %Fnx 3DL1+ NK cells 0 0 0 %Total IFN+3DL1+ NK ce NK IFN+3DL1+ %Total %Total CD107+3DL1+ NK ce NK CD107+3DL1+ %Total

%Total CCL4+3DL1+ NK ce NK CCL4+3DL1+ %Total 0 C 1 1 c 1 c 2 G F C 1 c C F M 2 g - F 1 1 c M 221 G - B I 1 M 221 G - 2 F B Ig 1 P h L B Ig 1 MC 2 G - P h L D P h L B Ig 1 D 3 D P h L 3 3 D 3

Figure S4. The effect of blocking KIR3DS1 or KIR3DL1 on NK cells from KIR3DL1/S1 heterozygotes and KIR3DL1 on NK cells from KIR3DL1 homozygotes stimulated by 221 cells. Shown on the y-axis is the frequency of exclusively gated KIR3DS1+CD3-CD56dim NK cells (A-D) or exclusively gates KIR3DL1+CD3-CD56dim NK cells (E-H) exhibiting (A, E) the sum of all functions tested (% Total Fxn NK cells), (B, F) the sum of cells secreting CCL4 (% Total CCL4+ NK cells), (C, G) the sum of cells secreting IFN-γ (% Total IFN-γ+ NK cells) and (D, H) the sum of cells expressing CD107a (% Total CD107a+ NK cells) following no stimulation (PBMC), stimulation with 221 cells (221), stimulation with 221 cells pre-coated with human IgG1 antibody (hIgG1) and stimulated with 221 cells pre-coated with KIR3DS1-Fc chimera protein (3DS1-Fc) (A- D) or the KIR3DL1-Fc (3DL1-Fc) (E-H). Bar heights and error bars represent the median and interquartile range of KIR3DS1+ cells from 2 KIR3DL1/S1 heterozygotes carrying no HLA-Bw4 alleles (A-D) and KIR3DL1+ NK cells from 2 KIR3DL1/S1 heterozygotes and 2 KIR3DL1 homozygotes carrying no HLA-Bw4 alleles. The boxes over the 3DS1-Fc and 3DL1-Fc blocking conditions show the percent reduction in the frequency of functional cells from that seen for cells stimulated with 221 cells with no blocking.