by HLA-B7 Variants and Target Cells NK Cells + Regulated ...

NK Cell Natural Cytotoxicity and IFN-g Production Are Not Always Coordinately Regulated: Engagement of DX9 KIR1 NK Cells by HLA-B7 Variants and Target Cells1 Zoya B. Kurago,*† Charles T. Lutz,2*§ Kelly D. Smith,3*‡ and Marco Colonna¶ DX9 mAb-binding killer cell-inhibitory receptors (KIR) recognize HLA-B molecules that express the Bw4 public serologic epitope. We assessed DX91 NK cell fine specificity recognition of HLA-B7 variants and HLA-B27 alleles by 51Cr release natural cytotoxicity assays and by flow cytometry and enzyme-linked immunospot (ELISPOT) IFN-g synthesis and release assays. 721.221 target cell expression of Bw41 HLA-B27 alleles specifically inhibited DX91 NK cell natural cytotoxicity and IFN-g synthesis and release. A triple substitution of HLA-B7 at residues 80, 82, and 83 known to induce expression of the Bw4 serologic epitope also specifically inhibited DX91 NK cell natural cytotoxicity and IFN-g responses. Single HLA-B7 amino acid substitution variants were recognized in the same decreasing rank order by DX91 NK cells and Bw4-reactive mAbs: G83R > R82L > N80T 5 HLA-B7. Natural cytotoxicity inhibition was reversed by the presence of blocking DX9 mAb. Natural cytotoxicity and IFN-g production were coordinately regulated by a panel of HLA-B7 variants expressed on 721.221 cells, suggesting that these two effector functions are inhibited by the same KIR-mediated signaling mechanisms. In contrast, some NK cell clones killed 721.221 and K562 target cells equally well but released much more IFN-g in response to K562 target cells. Differential regulation of natural cytotoxicity and IFN-g release shows that NK cell effector functions respond to distinct signals. The Journal of Immunology, 1998, 160: 1573–1580.

N

K cells are important effector cells in immune responses to virus, intracellular bacteria, allogeneic cells, and tumor cells (1). The major NK cell effector mechanisms are cytotoxicity and cytokine release. Ab-dependent cellular cytotoxicity is triggered by CD16 engagement of IgG bound to target cells (1). Natural cytotoxicity is not known to be triggered by a specific receptor-ligand interaction and may result from a summation of many weak stimulatory interactions (2). Cytotoxicity eliminates infected or transformed cells. NK cells also secrete cytokines, including IFN-g, TNF-a, and granulocyte-macrophage-CSF (1). Cytokines recruit other inflammatory cells to the site of infection or tumor growth. IFN-g may be particularly important, inasmuch as it controls viral replication, activates macrophages, enhances MHC class I and class II Ag presentation, and directs Agspecific immune responses and T cell cytokine secretion toward the cellular Th1 type (3). Mechanisms of NK cell signaling are being actively investigated (4), but it is not known whether target

Departments of *Pathology, †Oral Pathology, Radiology, and Medicine, and ‡Microbiology and the §Immunology and Molecular Biology Graduate Programs, University of Iowa, Iowa City, IA 52242, and ¶The Basel Institute for Immunology, Basel, Switzerland Received for publication August 7, 1997. Accepted for publication October 23, 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Roy J. Carver Charitable Trust, National Institutes of Health DE11139, and the Basel Institute for Immunology, which was founded and is supported by Hoffmann-La Roche Ltd., CH-4002, Basel, Switzerland. ZBK and KDS were supported by National Institutes of Health Training Grants KI6 DE00175 and GM073397.

cell induction of natural cytotoxicity and cytokine secretion are always coordinately regulated. Both Ab-dependent cellular cytotoxicity and natural cytotoxicity are inhibited by NK cell killer cell-inhibitory receptor (KIR)4 engagement (2, 4, 5). Transduction of KIR expression by recombinant vaccinia virus imparts the ability of the appropriate HLA class I molecules to inhibit natural cytotoxicity (6). KIR and HLA class I molecules directly bind (6 – 8), forming 1:1 complexes in vitro (8). The known KIRs belong to an increasingly large family of cell surface molecules that are expressed on many NK cells and on T cell subsets. KIR are diverse in extracellular regions, expressing two (the p58 group) or three (the p70 group) Ig-like domains. Both groups display tandem YxxL immunoreceptor tyrosine-based inhibitory motifs in the cytoplasmic tails that interact with SHP-1 and SHP-2, SH2-containing phosphotyrosine phosphatases (9 –12). KIR interaction with phosphatases appears to be critical for transduction of negative signals. NK cell activation represents a balance between inhibitory and activatory signals, given that KIR-mediated negative signals can be overwhelmed by multiple positive signals from CD2, CD16, CD69, and DNAX accessory molecule-1 molecules (2). The HLA-binding sites of various KIR are being mapped. NK1 and NK2 p58 KIR family members recognize complementary sets of HLA-C alleles that differ at residues 77 and 80 (13). Biassoni et al. (14) provided data that both residue 77 and 80 were important, whereas Mandelboim et al. (15) mapped NK1 and NK2 KIR differentiation of HLA-C alleles solely to residue 80. The DX9 mAb binds to NKB1 and related p70 KIR (2, 16). Natural cytotoxicity by DX91 NK cells is inhibited by target cell display of HLA-B27 and related Bw41 HLA-B alleles (17). Bw4- and Bw6-reactive Abs (18, 19) and DX91 NK cell recognition (17, 20) clearly map

2 Address correspondence and reprint requests to Dr. Charles T. Lutz, Department of Pathology, University of Iowa, Iowa City, IA 52242-1182. E-mail address: [email protected] 3 Current address: Department of Pathology, University of Washington Medical Center, Seattle, WA 98195.

Copyright © 1998 by The American Association of Immunologists

4 Abbreviations used in this paper: KIR, killer-inhibitory receptors; SCS, iron-supplemented calf serum; ELISPOT, enzyme-linked immunospot.

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to residues 77 to 83 in naturally occurring HLA-B alleles. Thus, the NK1, NK2, and DX91 KIR recognize similar a1 a-helix sites. Attempts have been made to identify specific HLA-B residues that are critical for KIR recognition, but general conclusions have been frustrated by the fact that both DX91 and DX92 NK cells are inhibited by HLA-B27 molecules (16, 21). On the basis of allele comparison, Cella et al. (22) deduced that residue 80 isoleucine was critical for Bw41 HLA molecule inhibition of NK cell natural cytotoxicity. Luque et al. (23) found that several HLA-B27 subtypes, but not B*2702, inhibited natural cytotoxicity in a subset of HLA-B27-recognizing NK cells. Sequence comparison and sitedirected mutagenesis of the protective B*2705 subtype indicated that residue 80 Thr, but not Ile, was critical for KIR recognition (23). Recently, Gumperz et al. (20) found that residues 82 and 83 contributed to Bw41 HLA-B allele recognition by DX91 KIR. NK cell-mediated cytotoxic functions are expressed rapidly on contact with susceptible target cells (1). In contrast, NK cell cytokine secretion requires a distinct and relatively long term activation program which includes gene transcription (24 –26). Thus, it is important to investigate how cytokine production is affected by KIR engagement. Although KIR engagement is well established to prevent NK cell-mediated cytotoxicity, little is known about how KIR signaling affects other NK cell effector functions. Bellone et al. (27) showed that PHA blast target cells that failed to elicit NK cell natural cytotoxicity also failed to stimulate NK cell cytokine release. KIR inhibition of these effector functions was not demonstrated. More recently, D’Andrea et al. (28) showed that KIR engagement on effector T cells inhibited both superantigeninduced cytotoxicity and cytokine production. Experiments in mice infected with murine CMV suggested that cytokines differentially control NK cell-mediated cytotoxic functions and IFN-g secretion (29, 30). “Split responses” by individual NK cells has not been documented to our knowledge. Therefore, it was important to measure both natural cytotoxicity and cytokine release by NK cell clones. Using unmutated and variant HLA molecules, we tested the hypothesis that inhibition of DX91 NK cell natural cytotoxicity and IFN-g production show the same fine specificity recognition pattern. In addition, we investigated whether NK cell natural cytotoxicity and cytokine effector functions can be separated using distinct target cells.

Materials and Methods Preparation of NK cell lines and clones NK cell lines and clones were generated under one of three sets of conditions. 1) PBMC from a Bw41 Bw62 (HLA-A2, 3; B38, 51; DR4, 11) healthy donor were sorted to 90 to 95% purity for CD32 cells using antiCD3 mAb 7D6-FITC (IgG1, a generous gift of Dr. T. Waldschmidt, University of Iowa) on a FACS 440 (Becton Dickinson, San Jose, CA). Cells were expanded for 1 week in RPMI 1640/10% FBS (HyClone Laboratories, Inc., Logan, UT) with L-glutamine, HEPES, and gentamicin in the presence of irradiated autologous PBMC (1 3 105), 721.221 lymphoblasts (5 3 104) with or without K562 cells (5 3 104), and 100 U/ml IL-2 (Hoffmann-La Roche via the AIDS Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health). Rapidly proliferating NK cell lines were cloned by limiting dilution at 1 to 3 cells per well in flat-bottom 96-well plates in the presence of the stimulation mix described above. The clones and the K8 cell line were stimulated weekly using the same protocol, and they were tested for cytotoxicity 8 to 10 days after stimulation. All cells were analyzed by FACS on several occasions for surface expression of CD56, CD3, and CD8, using NKH1-RD1 (phycoerythrin-labeled anti-CD56 mAb, Coulter Corp., Miami, FL), FITC-7D6 (anti-CD3), and FITC-3B5 (anti-CD8, a generous gift of Dr. T. Waldshmidt). NK cell clones with a CD32, CD561, and CD81 or CD82 phenotype were tested for cytolytic activity. Line K8 was 60% NK cells (CD32 CD561) and 40% T cells (CD32 CD561), with .99% of the DX91 cells expressing the NK cell phenotype. 2) PBMC were sorted for staining by mAb DX9 (IgG1, a generous gift of Dr. L. L. Lanier, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA), and cloned by

limiting dilution at 1 to 3 cells per well in 96-well flat-bottom plates in RPMI 1640/15% FBS, L-glutamine, nonessential amino acids, 2-ME, sodium pyruvate, gentamicin, and 500 U/ml IL-2, in the presence of irradiated 721.221 lymphoblasts and autologous PBMC (2 3 104 each). IL-2 was added every 3 to 4 days at 300 to 500 U/ml, while complete stimulation was provided every 5 to 7 days, as needed. In addition to phenotyping, clones were stained with mAb DX9, mAb GL183 (anti-p58/p50 KIR NK2 IgG1, Coulter/Immunotech, Westbrook, ME), and HP3D9 (antiCD94 IgG1, ascites, a generous gift of Dr. M. Lo´pez-Botet, Hospital de la Princessa, Madrid, Spain). CD32CD561DX91GL1831 or GL1832 clones were tested for functional activity 5 to 10 days after stimulation. All clones tested were DX91 at similar staining intensities. 3) Clones were also generated and maintained as described (13).

Target cells and cytotoxicity assays 721.221 lymphoblasts were transfected, maintained, and routinely analyzed for surface HLA class I expression as described elsewhere (31, 32). K562 is a HLA class I-negative NK cell-sensitive erythroleukemia cell line. Standard 5-h 51Cr release assays were performed at a variety of E:T ratios. For blocking experiments, mAbs DX9 (5 mg/ml) and HP3D9 (1: 12,000 dilution of ascites) were added to effector cells for 30 min, on ice, before the addition of target cells.

Enzyme-linked immunospot (ELISPOT) assays An established protocol was modified slightly (33). Briefly, Immulon 2 (Dynatech Laboratories, Inc., Chantilly, VA) flat-bottom plates were precoated with anti-IFN-g mAb M700A (Endogen, Woburn, MA) at 8 to 10 mg/ml, the plates were blocked with 10% iron-supplemented calf serum (SCS, HyClone) in PBS and stored at 220°C until the day of the experiment. Before the assays, plates were equilibrated at 37°C with assay medium (10% SCS in RPMI 1640 with HEPES and gentamicin). All cells were washed three to four times in assay medium and counted. Effectors mixed with 12 to 60 3 104 targets were plated in duplicate or in triplicate in 100 to 200 ml of assay medium per well at 37°C and 7.5% CO2 for 18 to 25 h. Control wells received either targets or effectors only, or effectors with IL-2. After incubation, plates were washed and developed for bound IFN-g using sequential incubations with IFN-g-specific biotinylated mAb M701B (Endogen), Extravidin-alkaline phosphatase and 5-bromo-4chloro-3-indolyl phosphate-nitroblue tetrazolium substrate (Sigma Chemical Co., St. Louis, MO). After overnight incubation with the substrate, plates were washed and photographed using high contrast film. The IFN-g spots were counted directly off the plate or off the photographs at a final magnification of 310.

Detection of intracellular IFN-g Effectors were used 7 to 10 days after stimulation. Effectors were washed up to 24 h before the assays to remove stimulants. Target cells were washed three to four times immediately before the assay. Effectors were mixed with assay medium only, with IL-2 (200 U/ml) and IL-12 (10 ng/ml) (Sigma Chemical Co.), or with target cells at an E:T ratio of 1:2 in 10% FBS-RPMI 1640 with HEPES and gentamicin in sterile round-bottom tubes, centrifuged at 1000 rpm for 1 min, and incubated at 37°C and 7.5% CO2 for 11 to 12 h. Brefeldin A (5 mg/ml) (Sigma Chemical Co.) was added during the final 5 to 6 h to inhibit cytokine secretion. Cells were prepared for flow cytometry using a slightly modified protocol developed by PharMingen, San Diego, CA. Cells were washed, incubated for 20 min with DX9-FITC (PharMingen), with CD56-specific NKH1-CY5 (Coulter and Amersham Life Sciences, Inc., Arlington Heights, IL), or with mouse IgG1-FITC (Fisher Scientific, Pittsburgh, PA). After a washing, cells were fixed overnight with 2% paraformaldehyde, 2.5% SCS, 0.1% sodium azide in PBS. On the following day, cells were treated with permeabilization buffer (0.1% saponin (Sigma Chemical Co.), 1% heat-inactivated FCS, 0.1% sodium azide, PBS) for 30 min, washed, and incubated for 40 min with phycoerythrin-labeled IFN-g-specific mAb B27 (PharMingen) or mouse IgG1 (Fisher Scientific) in permeabilization buffer. Cells were washed with permeabilization buffer, resuspended in 5% SCS, 0.1% sodium azide, PBS, and analyzed by FACS 440.

Results

DX91 NK cells and Bw4-reactive Abs recognize HLA alleles and variants with the same hierarchy To map the fine specificity of DX91 KIR recognition, we tested NK cell clones with HLA-A,B,C2 721.221 target cells that expressed equivalent levels of transfected Bw41 and Bw61 HLA alleles and HLA-B7 variants (Fig. 1). Cell surface levels of these

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FIGURE 1. Transfected 721.221 cells express comparable levels of surface HLA class I molecules, as measured with mAb W6/32. In several experiments, untransfected and pHeBo-transfected 721.221 cells bound similar levels of W6/32 mAb. Transfectants were used only if they expressed similar HLA class I levels within 48 h of each cytotoxicity and IFN-g assay.

and other HLA-B7 variants were similar to those of the unmutated HLA-B7, when transfectants were tested on several occasions (Refs. 31, 32, 34, and 35 and data not shown). The same transfectants had been used to map Bw4-reactive and Bw6-reactive mAb epitopes in a previous study (31). Despite variable recognition of HLA-B7 and 721.221 target cells, the cytotoxic activity by all DX91 NK cell clones was inhibited by target cell expression of Bw41 B*2705 (Figs. 2 and 3A, top) and B*2702 (Fig. 3A, top) molecules. DX92 NK cells were not inhibited by Bw41 HLA-B27 alleles and HLA-B7 variants (Fig. 3, bottom panels of A and B). Although Bw41 alleles were usually strongly inhibitory, some DX91 clones were inhibited weakly (see below). Similar variability has been observed by other investigators (17) and may be due to variable NK cell expression of activatory receptors that overcome KIR-mediated inhibitory signals (2). The B7/80,82,83 variant, which matches B*2705 amino acid at residues 80, 82, and 83, also inhibited DX91 NK cell cytotoxicity, often to the same degree as B*2705. Single residue variant B7/G83R inhibited killing nearly as well as the B7/80,82,83 variant (Figs. 2 and 3B, top). B7/R82L was recognized by some clones (Fig. 2, clone 12U1; data not shown), but usually to a lesser degree than B7/G83R (Fig. 2). B7/N80T was recognized no better than unmutated HLA-B7. NK clone 12U1 was less cytotoxic against 721.221 target cells but discriminated between unmutated HLA-B7 and the B7/80,82,83 variant at higher E:T ratios. This clone did not discriminated between single residue HLA-B7 variants. Clone 12U1 expressed a mAb GL183-reactive KIR (data not shown) and was strongly inhibited by target cell HLA-Cw3 expression, consistent with reports from many other laboratories (14, 15, 36). Control HLA-B7 variants with mutations at residues 79 and 114 did not affect natural cytotoxicity by DX91 clone 5F12 (Fig. 3B). DX91 NK cells recognized the HLA-B7 variants with the same hierarchy as a panel

FIGURE 2. DX91 NK cell clones recognize B*2705, B7/80,82,83, and B7/G83R. Cytotoxicity was measured against 721.221 transfectants and K562 target cells. Right and left panels are from the same experiment for each NK cell clone. Results are representative of one to six experiments for each clone and of 13 additional DX91 and/or Bw4-specific NK cell clones. E:T ratios for clones 12D1, 12B3, and 12R1 are identical and are shown below the 12R1 panel.

of Bw4-reactive mAbs (Table I), suggesting that the KIR recognition structure and the serologic epitope are closely related. Bw41 alleles and variants are recognized by DX91 KIR To investigate the role of DX91 KIR in NK cell recognition of the HLA-B7 variants, receptor engagement was blocked by DX9 mAb. When sufficient numbers of NK cells were available, we also used HP3D9 anti-CD94 mAb, which matches DX9 in IgG1 isotype and staining density of the NK cell clones tested (data not shown). The presence of DX9 mAb in the 51Cr release assay largely restored the ability of DX91 NK cells to kill transfectants that expressed B*2705 or the B7/80,82,83 variant (Fig. 4). Although clone 12 M1 was only weakly inhibited by B*2705, DX9 mAb restored natural cytotoxicity against the B*2705 transfectant (Fig. 4). This indicates that DX91 KIR engagement was responsible for the weak B*2705-inhibitory effect. DX9 mAb-mediated restoration of killing of the B7/G83R transfectant was complete for clone 12B3 and partial for the other NK cell clones (Fig. 4). CD94-specific mAb HP3D9 did not alter the killing of the B*2705, B7/80,82,83, or B7/G83R transfectants and had little effect on the other transfectants compared with the pHeBo vector transfectant (Fig. 4). The effects of CD94 vary between NK cell clones and depend on coexpressed NKG2 isoforms (37–39). The minimal effect by mAb HP3D9 shows that mAb DX9 blocking was specific. Protection by the B7/R82L variant was low and variable, and

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COORDINATE REGULATION OF NK CELL EFFECTOR FUNCTIONS Table I. DX91 KIR- and Bw4-reactive mAb share fine specificity recognition patternsa 721.221 Transfectant

Bw4-Reactive mAb Binding

DX91 KIR Recognition

B*2705 HLA-B7 B7/80,82,83 B7/N80T B7/R82L B7/G83R

6/6 0/6 6/6 0/6 4/6 5/6

1111 2 111 2 1/2 11

a Summary of Bw4-reactive mAb binding (31) and of DX91 KIR recognition, assessed either by natural cytotoxicity or by IFN-g production.

FIGURE 3. DX91, but not DX92, NK cell clones recognize B7/80,82,83 and B7/G83R variants. Cytotoxicity was measured using three DX91 NK cell clones (MK1.29, 3D2, and 5F12) and two DX92 NK cell clones (MK1.34 and HP1) at the E:T ratios indicated. A, 721.221 target cell transfectants expressed B*2702, B*2705, HLA-B7 (B7), and B7/80,82,83; B, 721.221 target cell transfectants expressed HLA-B7 (B7) and the HLA-B7 variants indicated. Results shown in A and B are from the same assay and are representative of at least three independent experiments.

reversal of protection by DX9 mAb also varied (Fig. 4). On occasion, B7/N80T transfectants were killed less efficiently than HLA-B7 transfectants, but DX9 mAb did not influence the level of killing. DX9 mAb did not increase killing of HLA-B7, -A3, or -Cw3 transfectants. Therefore, inhibition of DX91 NK cell natural cytotoxicity paralleled the effect of the DX9 mAb. Both decreased in rank order: B*2705 $ B7/80,82,83 . B7/G83R . B7/R82L . B7/N80T 5 HLA-B7. DX91 KIR inhibit NK cell IFN-g production and natural cytotoxicity with the same fine specificity To investigate whether target cell HLA class I inhibits NK cell IFN-g production we measured IFN-g content in individual cells by flow cytometry (see Materials and Methods). All the NK cell

clones examined had significant amounts of intracellular IFN-g when incubated with media alone, and different target cells caused only small changes (data not shown). Therefore, we used K8, a polyclonal cell line that contained 60% (CD32CD561) NK cells and 40% (CD31 CD562) T cells at the time of analysis. Similar to DX91 NK clones, the K8 cells killed Bw41 cells less well than Bw42 cells (data not shown). Virtually all (99%) of the DX91 cells expressed CD56 (data not shown). Cell surface staining allowed us to simultaneously measure intracellular IFN-g content and DX91 KIR or CD56 expression. When incubated with media alone, the K8 polyclonal cells did not produce IFN-g (Fig. 5A). After incubation with transfected 721.221 cells, a proportion of both DX91 and DX92 K8 cells produced IFN-g (Fig. 5A). Gating on the DX91 population as illustrated in Fig. 5A, we quantitated effector cell response to various HLA alleles and HLA-B7 variants (Fig. 5B). About 40 to 50% of DX91 cells produced IFN-g in response to IL-2 and IL-12 cytokines in the absence of target cells, and in response to incubation with K562 cells, or Cw3- or pHeBotransfected 721.221 cells (Fig. 5B). Target cell HLA-B7 or HLA-A3 expression inhibited IFN-g synthesis modestly, perhaps due to engagement of CD94 or other inhibitory receptors on DX91 NK cells. IFN-g synthesis by the DX91 population was markedly inhibited by B*2705 and B7/80,82,83, and to a lesser extent by G83R and B7/R82L (Fig. 5B). In this experiment, B7/G83R and B7/R82L were about equally effective (Fig. 5B); in other assays with the K8 line, B7/G83R was more inhibitory (data not shown). The B7/N80T variant did not inhibit DX91 cell IFN-g synthesis more than unmutated HLA-B7 (Fig. 5B). Essentially identical results were obtained in other experiments when we gated on CD561DX91 effector cells (data not shown). These data indicate that DX91 KIR engagement inhibits NK cell IFN-g synthesis. The fine specificity recognition of the HLA-B7 variants was similar for natural cytotoxicity and a panel of Bw4-reactive mAb (Table I). We also investigated whether target cell HLA expression also inhibits IFN-g release. We used the ELISPOT assay because it requires few NK cells, is sensitive, and measures the relative number of responding cells. The assay was robust, with K562 cells typically inducing 150 to 350 IFN-g ELISPOTs per 500 NK cells (Fig. 6 and data not shown). In contrast, K562 supernatants induced no NK cell IFN-g release (data not shown). Fewer IFN-g ELISPOTs were induced by 721.221 target cells (Fig. 6), but the differences between transfectants could be quantitated. Clone 12R1 produced .40 IFN-g ELISPOTs per 500 effector cells in response to pHeBo vector-transfected 721.221 cells (Fig. 6). HLAB7, B7/N80T, and B7/R82L transfectants induced more ELISPOTs, indicating that these transfected HLA molecules did not inhibit clone 12R1 IFN-g release. In contrast, the B7/80,82,83 and B7/G83R variants reproducibly inhibited clone 12R1 IFN-g release (Fig. 6). This pattern closely parallels the pattern of clone

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FIGURE 4. Cytotoxicity of DX91 NK cell clones against 721.221 transfectants was tested in the presence of DX9 mAb (5 mg/ml) or media alone. When sufficient NK cells were available, CD94-specific HP3D9 mAb (1:12,000 dilution of ascites) also was used. Results are representative of at least two independent experiments per clone and of three other DX91 clones.

FIGURE 5. HLA-B7 variants inhibit DX91 NK cell IFN-g production. The K8 cell line was incubated as described in Materials and Methods in media alone, IL-2 1 IL-12, with K562 target cells (lower right), or with 721.221 cells transfectants, as indicated. A, Intracellular IFN-g and surface DX9 staining. Forward and side light scatter gates were applied to exclude dead cells and .90% of the target cells. The gated DX92 population contains some NK cells, all of the T cells, and a variable number of target cells. B, Single-color histogram plots of intracellular IFN-g staining of DX91 cells. Indicated are the percentages of DX91 cells that express intracellular IFN-g, gated as shown in A.

FIGURE 6. HLA-B7 variants inhibit DX91 NK cell IFN-g release. NK cell clones (500 per well) were incubated with K562 cells or transfected 721.221 cells, and ELISPOTs were counted, as described in Materials and Methods. A and B are from the same experiment for each clone, plotted on different scales. Error bars represent SDs from duplicate wells.

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FIGURE 7. NK cells differentially regulate natural cytotoxicity and IFN-g release in response to K562 and 721.221 target cells. NK cell clone 12D1 was tested simultaneously in a 51Cr release assay (A) and an IFN-g ELISPOT assay (B), as described in Materials and Methods.

12R1 natural cytotoxicity (Figs. 2 and 4). Both HLA-A3 and HLACw3 inhibited clone 12R1 IFN-g release (Fig. 6), even though HLA-Cw3 caused little (Fig. 2) or no (Fig. 4) inhibition of clone 12R1 natural cytotoxicity. This may indicate that the ELISPOT assay is more sensitive than the 51Cr release assay to HLA class I-mediated inhibition. The HLA-Cw3-mediated inhibition was not due to clone 12R1 expression of mAb GL183-reactive NK2 KIR (data not shown). Although B7/80,82,83 caused little or no inhibition of clone 12 M1 natural cytotoxicity (Fig. 4), B7/80,82,83 reproducibly inhibited clone 12 M1 IFN-g release (Fig. 6). This may be consistent with greater sensitivity of the ELISPOT assay. Clone 12U1 killed all 721.221 transfectants relatively less well than K562 target cells, but B7/80,82,83 inhibition of natural cytotoxicity could be appreciated in all assays in which higher E:T ratios were used (Fig. 2). Relative to HLA-B7 and other HLA-B7 variants, the B7/80,82,83 variant inhibited clone 12U1 IFN-g release. Clone 12U1 expresses mAb GL183-reactive NK2 KIR (data not shown), and both natural cytotoxicity and IFN-g release were inhibited by target cell HLA-Cw3 expression (Figs. 2 and 6). We conclude that KIR inhibit both NK cell IFN-g synthesis and release. The DX91 KIR fine specificity pattern of HLA-B7 variant recognition is similar for both aspects of IFN-g production. NK cell cytotoxicity and IFN-g release are differentially regulated To compare NK cell effector functions in the absence of KIR signaling, we used 721.221 and K562 target cells that express little or no HLA class I. Natural cytotoxicity and IFN-g release were often regulated congruously. For example, K562 cells were superior to pHeBo-transfected 721.221 cells in inducing both natural cytotoxicity (Fig. 2) and IFN-g release (Fig. 6) by clone 12U1. However, other NK cells responded differentially to the target cells, and these differences became more accentuated with prolonged in vitro passage. Clone 12D1 (Fig. 7A) and other long term NK cell clones (data not shown) killed the two targets equally well. However, these NK cells released much more IFN-g in response to K562 cells than to pHeBo-transfected 721.221 cells (Fig. 7B and data not shown). For example, clone 12D1 generated at least 50-fold more IFN-g ELISPOTs in response to K562 cells than to 721.221 cells (Fig. 7B). These results clearly show that natural cytotoxicity and IFN-g release are differentially regulated in NK cells.

Discussion DX91 KIR recognize HLA-B27 and other Bw41 HLA-B alleles (16). Single HLA-B7 amino acid substitutions showed a hierarchy of binding by Bw4-reactive mAbs: B*2705 $ B7/80,82,83 . B7/ G83R . B7/R82L . B7/N80T 5 HLA-B7 5 no binding (31).

Remarkably, DX91 NK cells showed the same recognition hierarchy as the mAb panel. We observed complete or partial reversal of protection by DX9 mAb; this is consistent with the conclusion that DX91 KIR engaged the HLA-B7 variants and transmitted an inhibitory signal. These conclusions are similar to recently published findings with mutants of the Bw61 B*1502 allele: the R82L variant was marginally protective and the G83R variant was more protective than unmutated B*1502, but not as protective as the closely related Bw41 B*1513 allele (20). The strong protection offered by the B7/80,82,83 variant indicates that residue 77 asparagine, which is invariant in Bw41 HLA-B alleles, is not required for recognition by DX91 KIR. The B7/N80T variant did not protect from DX91 NK cell natural cytotoxicity, ruling out an effect of residue 80 threonine, at least in the context of HLA-B7. These and other results emphasize the importance of the a1 a-helix for KIR recognition. HLA-C-specific p58 KIR recognition has been mapped to HLA-C residues 77 and 80 (14), although the importance of residue 77 has been disputed (15). In addition to allele-specific amino acids, shared HLA-C residues 73, 76, and 90 located on the outer face of the a1 a-helix also are important (36). DX91 KIR are distinct from other HLA-B27-sensitive NK receptors that recognize B*2705, but not B*2702 (23). These latter KIR are sensitive to B*2705 mutations at a1 a-helix residue 80. Although the fine specificities for many KIR are distinct, most or all KIR recognize the same general region of the a1 a-helix. In apparent contrast to this rule, KIR NKAT4 recognizes HLA-A3, but not HLA-A2 or HLA-Aw68, despite identity in residues 77– 83. It is possible that NKAT4 binds other HLA regions. Alternatively, other HLA regions may affect a1 a-helix conformation. Likewise, DX91 NK cells are not inhibited by target cell Bw41 HLA-A molecules, despite sharing identical residues 77– 83 with many Bw41 HLA-B molecules (17). This indicates that other residues, directly or via conformation effects, contribute to DX91 KIR-HLA binding. Several considerations indicate that DX91 KIR directly bind residue 83 arginine on Bw41 HLA-B molecules. The B7/G83R variant binds 10 of 11 HLA-B7-reactive mAbs, including two Bw6-reactive mAbs that are affected by the B7/80,82,83 mutation (31). This indicates that the G83R mutation does not induce large conformation changes. Potential influence of the B7/G83R mutation on the nearby N-linked carbohydrate at amino acid residue 86 is not likely responsible for DX91 KIR recognition, as natural cytotoxicity by DX91 NK cells was not affected by the absence of HLA carbohydrate (17). In B*2705, residue 83 arginine is exposed to solvent and points away from the peptide binding groove (40), making it likely that a KIR could contact this residue. This is consistent with the model proposed by Mandelboim et al. (36) that p58 KIR contact the outer surface of the HLA-C a1 a-helix. Consistent with a lack of effect on peptide binding, the B7/G83R mutation did not alter recognition by 12 of 12 peptide-specific alloreactive CTL clones (34, 35). HLA-B7 and B*2705 bind largely nonoverlapping sets of peptides (41). This indicates that DX91 KIR recognition of Bw41 HLA-B molecules is largely independent of HLA-bound peptide. The role of peptide in NK cell MHC class I recognition has been controversial. Mouse NK cell recognition of MHC class I molecules is largely independent of the identity of bound peptide (42). In contrast, human NK cell recognition of B*2705 and HLA-A11 depends on specific bound peptide (43, 44). Although peptide specificity was not stringent, NK cells did not recognize B*2705-bound peptides with highly charged amino acids at residues P7 and P8 (45). These and other data suggest that inappropriate peptide side chains interfere with KIRMHC interactions by charge hindrance or conformational effects.

The Journal of Immunology The relative degree of inhibition by the HLA-B7 variants differed between DX91 NK cell clones. Not all DX91 KIR are identical in amino acid sequence (2). Therefore, it is possible that different KIR have distinct fine specificity, despite equivalent binding of DX9 mAb. However, a more likely explanation stems from the observation that NK cell clones express myriad inhibitory and activatory receptors, some of which are clonally distributed. Both inhibitory and activatory members of the KIR gene family have been described (46). Expression of DX91 KIR does not correlate with expression of other KIR or other molecules by NK cell clones (47). CD94 activates or inhibits NK cell effector functions (37), depending on association with NKG2 subtypes (38, 39). CD94 does not recognize Bw41 molecules but does recognize HLA-B7 and other HLA alleles (38, 48). Therefore, some HLA-B7 variants may bind both DX91 KIR and CD94 or other molecules, with the effect on NK cell function dependent on the CD94-associated NKG2 isoform and on the number and strength of the NK cell receptor-target cell ligand interactions. NK cells perform two major effector functions, cytotoxicity and cytokine release. KIR engagement inhibits NK cell Ab-dependent cytotoxicity and natural cytotoxicity (2, 4, 5). KIR inhibition of natural cytotoxicity likely is effected rapidly and is released in a matter of seconds, because NK1 NK cells efficiently killed susceptible 51Cr-labeled target cells in the presence of resistant HLA-C1 unlabeled target cells (49). Likewise, preincubation of unlabeled Bw41 B*5801-transfected 721.221 cells with a DX91 NK cell clone for a few minutes to a few hours did not decrease natural cytotoxicity of 51Cr-labeled 721.221 targets (L. Lanier, DNAX, personal communication). Because cytotoxicity inhibition is so transient, KIR signaling probably inhibits cytotoxic granule release. KIR effects on perforin and granzyme biosynthesis have not been reported. NK cells and T cells increase IFN-g transcription and steady state mRNA levels in response to IL-2 and other stimuli (24 –26). However, it has been reported that IL-2 alone does not stimulate NK cell IFN-g release, indicating that IFN-g biosynthesis also is controlled at or beyond the level of protein synthesis (25). Because of the multiple levels of control in NK cells, it was of interest to study how KIR engagement affected IFN-g production. Previous studies have suggested that NK cell cytotoxicity and cytokine production are sometimes differentially regulated. Neutralization of IL-12 during murine CMV infection abrogated NK cell IFN-g secretion but did not alter NK cell-mediated cytotoxicity (29, 30). In contrast, neutralization of IFN-a decreased NK cell-mediated cytotoxicity but did not alter NK cell IFN-g secretion (29). These results show that cytokines differentially influence NK cell effector functions, at least in the setting of viral infections in vivo. However, because these studies were performed with whole animals and polyclonal populations, the differential effects of IL-12 and IFN-a neutralization may have been due to actions on distinct NK cell subpopulations rather than “split responses” by single NK cells. Therefore, it was important to measure both natural cytotoxicity and IFN-g production by single NK cell clones. Using flow cytometry and ELISPOT assays, we found that HLA variants inhibited DX91 NK cell IFN-g production in the same rank order as natural cytotoxicity. These data indicate that KIR signals coordinately inhibit NK effector cell functions that are conducted quickly (cytotoxicity) and over a longer time (IFN-g synthesis). Our results also show that natural cytotoxicity and IFN-g release can be regulated separately in NK cells. Some NK cell clones efficiently and equally killed K562 and 721.221 target cells, but secreted IFN-g better in response to K562 cells than to 721.221 cells. This “split response” became more marked after long term

1579 culture of the NK cell clones. It is not clear why 721.221 cells induced less NK cell IFN-g production over time, but it may be related to the long term culture of the NK cells with 721.221 stimulator cells. K562 cells express very low levels of HLA class I molecules (Refs. 50 –52 and data not shown). Likewise, 721.221 cells bind very little anti-HLA class I mAb and do not express HLA-A,B,C class Ia molecules (53, 54) or HLA-G class Ib molecules (55) which are known to engage KIR (56). Therefore, the inability of 721.221 cells to induce NK cell IFN-g release probably was not due to HLA class I-induced KIR- or CD94-inhibitory signals. K562 cells efficiently stimulate NK cell functions in many assay systems (1, 57), and a recombinant soluble form of the stimulatory NKG2-C molecule binds K562 cells, but not all B lymphoblasts (58). We propose that K562 cells and 721.221 B lymphoblasts differ in expression of membrane ligands or in induced secreted molecules that stimulate NK cell IFN-g release. Regardless of the stimulatory molecules involved, the NK cell “split response” shows that the target cell induced signals for cytotoxicity and for IFN-g release are distinct. Overall, our results indicate that stimulatory and inhibitory receptor control of NK cell functions is complex and operates at multiple levels.

Acknowledgments We thank Hoffmann-La Roche and the AIDS Reagent Program for rIL-2; Tom Waldschmidt, Lewis Lanier, and Miguel Lo´pez-Botet for gifts of Abs; and Brian Mace and Paul Reimann for technical assistance.

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