Costimulatory Molecules CD80 and CD86 NK Cell Triggering by The

NK Cell Triggering by the Human Costimulatory Molecules CD80 and CD86 Julia L. Wilson, Jehad Charo, Alfonso Martín-Fontecha, Paolo Dellabona, Giulia Casorati, Benedict J. Chambers, This information is current as Rolf Kiessling, Maria-Teresa Bejarano and Hans-Gustaf of September 26, 2021. Ljunggren J Immunol 1999; 163:4207-4212; ; http://www.jimmunol.org/content/163/8/4207 Downloaded from

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. NK Cell Triggering by the Human Costimulatory Molecules CD80 and CD861

Julia L. Wilson,* Jehad Charo,† Alfonso Martı´n-Fontecha,* Paolo Dellabona,‡ Giulia Casorati,‡ Benedict J. Chambers,* Rolf Kiessling,† Maria-Teresa Bejarano,* and Hans-Gustaf Ljunggren2*

NK cell-mediated effector functions are regulated by a delicate balance between positive and negative signals. Receptors trans- mitting negative signals upon engagement with target cell MHC class I molecules have been characterized in detail in recent years. In contrast, less information is available about receptor-ligand interactions involved in the transmission of positive or “triggering” signals to NK cells. Recently, it has been described that murine NK cells are triggered by the costimulatory molecules CD80, CD86, and CD40. Using NK cell lines derived from PBMC as effectors, we demonstrate that the human CD80 and CD86 gene products can function as triggering molecules for NK cell-mediated cytotoxicity. Expression of human CD80 or CD86 molecules in murine Downloaded from B16.F1 melanoma cells rendered these significantly more susceptible to lysis by human NK cell lines. Blocking of the transfected gene products with specific mAb reduced lysis levels to that of nontransfected control cell lines. Triggering of human NK cells by CD80 and CD86 appeared to be independent of CD28 and CTLA-4, at least as determined by the reagents used in the present study, because the expression of these molecules could not be detected on the NK cell lines by either flow cytometry or in redirected lysis assays. Thus, human NK cells may use receptors other than CD28 and CTLA-4 in their interactions with CD80 and CD86 http://www.jimmunol.org/ molecules. Alternatively, interactions may involve variants of CD28 (and possibly CTLA-4) that are not recognized by certain anti-CD28 mAb. The Journal of Immunology, 1999, 163: 4207–4212.

he outcome of NK cell-mediated effector functions, in- through which NK cells mediate Ab-dependent cellular cytotox- cluding “spontaneous” cytotoxicity and cytokine release, icity (ADCC) against targets coated with IgG (9, 10). Several other T elicited upon contact with other cells depends on a deli- cell-surface molecules have been shown to activate NK cells upon cate balance between inhibitory and stimulatory signals (1–3). NK specific stimulation, though the biological significance of these cells express MHC class I-specific receptors that suppress NK cell- responses have been less clear. Perhaps the best studied molecule mediated cytotoxicity upon interaction with their ligands. Several in this respect is the rodent NKR-P1 molecule (11). Other candi- by guest on September 26, 2021 inhibitory class I binding receptors have been identified. Human date activation molecules include the mouse 2B4 molecule and NK cells express Ig superfamily members of the killer inhibitory LAG-3 (12, 13). The NK-TR1 molecule may play a significant role 3 receptors (KIR) family (4) and the lectin-like receptor CD94, in signal transduction during natural killing but not in ADCC (14). which forms disulfide-linked heterodimers with members of the Several other NK cell-expressed molecules have been reported to NKG2 family (5). The latter CD94/NKG2 receptors are most activate NK cells. Of these, the best characterized are CD2, mouse likely also expressed by murine NK cells (6, 7), which also express CD69 and Ly6, and rat gp42 molecules (15–17). the relatively well-characterized lectin-like Ly49 inhibitory recep- Most of the identified KIR have immunoreceptor tyrosine in- tors (8). hibitory motifs (ITIM) in their cytoplasmic tail that prevent NK Relatively less is known about activating or “triggering” receptors on NK cells and their corresponding ligands. Probably the best cell activation (18). However, additional KIR lacking ITIM have defined activation receptor is the Fc␥RIII (CD16) molecule, been identified that trigger NK cell-mediated lysis upon interaction with appropriate HLA alleles (19, 20). In a similar manner, CD94/ NKG2A heterodimers contain an ITIM and inhibit NK cell lysis upon interaction with its ligand HLA-E (21, 22). In contrast, *Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden; †Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden; and ‡Labora- CD94/NKG2C heterodimers lack a corresponding ITIM and may tory Di Immunochimica, DIBIT, H.S. Raffaele, Milan, Italy function as activating receptors upon association with KARAP/ Received for publication May 21, 1999. Accepted for publication August 4, 1999. DAP12 (23). The biological role of these MHC class I binding The costs of publication of this article were defrayed in part by the payment of page activating receptors, and corresponding Ly49 receptors in mice charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. that lack ITIM (24), is not yet defined. 1 This work was supported by grants from the Swedish Cancer Society, the Swedish Two recently identified candidates for non-MHC binding recep- Medical Research Council, the Petrus and Augusta Hedlunds Stiftelse, the Tobias tors are the human NKp44 and NKp46 molecules described by the Stiftelsen, the Lars Hiertas Stiftelse, and the Karolinska Institutet. J.L.W. has been Moretta groups (25–27). NKp44 is expressed by NK cells upon in supported by a long-term Marie Curie Biotechnology Fellowship. A.M.-F. has been supported by a Cancerfonden Postdoctoral Fellowship. vitro cultivation in IL-2. NKp46 is expressed by both resting and 2 Address correspondence and reprint requests to Dr. Hans-Gustaf Ljunggren, Micro- activated NK cells. The available data strongly support the notion biology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm, Swe- that NKp44 and NKp46 trigger NK cell-mediated cytotoxicity den. E-mail address: [email protected] (25–27). NKp44 and NKp46 are coupled to the intracytoplasmic 3 Abbreviations used in this paper: KIR, killer cell inhibitory receptor; ITIM, immunoreceptor tyrosine inhibitory motif; LAK, lymphokine activated killer cell; ADCC, signal transduction machinery via association with KARAP/ Ab-dependent cellular cytotoxicity; rh, recombinant human; h, human; m, murine. DAP12 or CD3␰, respectively (27).

Previous reports from our own and other laboratories have dem- Table I. Cell surface CD80 and CD86 expression on cell lines used in onstrated that murine NK cells are triggered to lyse tumor target the present study cells expressing the costimulatory molecules CD80, CD86, and CD40 (28–31) and that human peripheral blood-derived NK cells mAb Staining can be triggered by CD40 (32). In addition, it has been demon- Cell Line Background Anti-CD80 Anti-CD86 strated that the human NK leukemia cell line YT2C2 is capable of killing B7-1-transfected tumor cell lines in a CD28-dependent B16.F1 3a 56 fashion (33). In the present study, we have addressed the ability of B16.F1-hCD80 5 230 7 B16.F1-hCD86 4 13 257 human peripheral blood-derived NK cell lines to be triggered by B16.F1-hCD80/hCD86 9 216 266 human CD80 and CD86 costimulatory molecules expressed on a tumor cell lines. We demonstrate that the expression of human Mean fluorescence intensity. CD80, CD86, or CD80/CD86 molecules in murine B16.F1 melanoma cells increases their susceptibility to lysis by human NK cell lines. Blocking of the transfected gene products with specific mAb 1000 U/ml recombinant human IL-2 (rhIL-2, Peprotech, London, U.K.) or ␣ reduced susceptibility to lysis to levels comparable to that of non- 2000 U/ml rhIFN- (Peprotech) and cultured at 37°C for 48 h. NK cell lines were generated by resuspending nonadherent cells in IMDM medium transfected control lines. Triggering of human NK cells by CD80 (Life Technologies) supplemented with 5% heat-inactivated FCS, 5% heat- and CD86 appeared to be independent of CD28 and CTLA-4, at inactivated ABϩ human serum, and 10% leukocyt- conditioned medium least as determined by the reagents used within this study, because (36). Then, 2.5 ϫ 105 cells/well were cultured in 24-well plates at 37°C, in ϫ 5 expression of these molecules could not be detected on the cell the presence of 0.5 10 irradiated (5000 rad) RPMI 8866 cells (37). Downloaded from Following 7 days of culture, cells were collected and enriched for NK cells surface of the NK cells used as assessed by either flow cytometry by negative depletion. Cells were incubated for 45 min at 4°C with satu- or in redirected lysis assays. However, these data do not exclude rating amounts of anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 the possibility that NK cells may express variants of CD28 (or (OKT8) hybridoma supernatants. Cells were washed twice with PBS be- possibly CTLA-4) that are not recognized by the mAb used within fore resuspending in rabbit complement (Pel-Freez, Rodgers, AR) and in- ϫ 5 this study (34). cubated at 37°C for 1 h. After washing, 1 10 recovered cells were further expanded in 24-well plates in complete medium supplemented with 200 U/ml rhIL-2 in the presence of 1 ϫ 105 RPMI 8866 cells to generate http://www.jimmunol.org/ Materials and Methods NK cell lines. Typical lines were Ͼ99% positive for the human NK cell Cell lines marker CD56 and negative for CD3 and other T cell markers. In some experiments, the NK cell lines were cultured for 48 h in media supple- K562 (human erythroleukemia), P815 (murine mastocytoma), RPMI 8866 mented with 100 ng/ml rhIL-12 (Peprotech) before use as effector cells in cells (human EBV-transformed B cell line, kindly provided by G. Trinch- cytotoxicity assays. ieri, Philadelphia), and the B16.F1 murine melanoma (C57BL/6) were cultured in complete medium consisting of RPMI 1640 media (Life Technol- NK cell-mediated cytotoxicity ogies, Ta¨by, Sweden) supplemented with 10% heat-inactivated FCS, 2 mM 51 L-glutamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin. B16.F1 Cytotoxicity of cells was measured in a standard 4-h Cr release assay 51 cells transfected with vectors containing the human CD80 and CD86 mol- using Na2 CrO4-labeled cells as targets. Experiments were conducted in ecules have been described (35). B16.F1 cells transfected with human triplicate at various E:T ratios. The percentage specific 51Cr release (spe- by guest on September 26, 2021 CD80 (B16.F1-hCD80) were maintained in complete media supplemented cific lysis) was calculated according to the formula: % specific lysis ϭ with 500 ␮g/ml G418 (Saveen, Malmo¨, Sweden); and B16.F1 cells trans- [(experimental release Ϫ spontaneous release)/(maximum release Ϫ spon- fected with human CD86 (B16.F1-hCD86) were maintained in media sup- taneous release)] ϫ 100. plemented with 20 ␮g/ml mycophenolic acid (Sigma, Stockholm, Swe- In Ab blocking studies, 1 ϫ 106 target cells were preincubated for 30 den), 20 ␮g/ml xanthine (Sigma), and hypoxanthine, aminopterin, and min at room temperature with 25 ␮g/ml of either anti-CD80 or anti-CD86 thymidine (1ϫ HAT; Life Technologies). The B16.F1 cell lines transfected or isotype control mAb. Cells were washed in complete media before use with both CD80 and CD86 (B16.F1-hCD80/CD86) were maintained in in the cytotoxicity assay. media containing 500 ␮g/ml G418, 20 ␮g/ml mycophenolic acid, 20 ␮g/ml xanthine, and 1ϫ HAT. Redirected ADCC Monoclonal Abs and flow cytometry Purified NK cells were incubated, in round-bottom 96-well plates, together with 51Cr-labeled P815 cells to achieve a final E:T ratio of 40:1 in a total FITC- and PE-labeled mAbs against CD3 (clone HIT3a), CD16 (clone volume of 150 ␮l. Monoclonal Ab specific for CD28, CTLA-4, CD16, and 3G8), CD28 (L293), CTLA-4 (BNI3), CD56 (B159), CD80 (L307.4), isotype-matched controls were added at a final concentration of 200 ng/ml. CD86 (2331), HLA-DR (L243), and isotype-matched labeled controls were Cultures were incubated for 4 h and harvested as in a standard chromium obtained from Becton Dickinson (Stockholm, Sweden). An additional anti- release assay. CD28 mAb (YTH913.12; Serotec, Novakemi, Stockholm, Sweden) was also used. Abs were used to routinely screen transfected cell lines for Results expression of the transgene product by flow cytometry. Purity of NK lines was also assessed by flow cytometry. For blocking studies, purified anti- CD80-, CD86-, and CD80/CD86-transfected B16.F1 cell lines human CD80 (DAL-1) and anti-human CD86 (BU63) mAb were used Transfection of murine melanoma B16.F1 cells with cDNA for (Serotec). For staining, cells were washed and resuspended in PBS with 1% human CD80 (hCD80), human CD86 (hCD86), or both hCD80 heat-inactivated FCS and 0.01% NaN3. Abs were diluted in this buffer and used at a final concentration of between 2 and 20 ␮g/ml. Incubations with and hCD86 resulted in stable cell surface expression of these mol- Abs were conducted for 30 min on ice. Following the final washing, la- ecules (35). Table I shows the expression of hCD80 and hCD86 on beled cells were fixed with 1% formaldehyde solution (Sigma), and 10,000 control and transfected B16.F1 cells. Expression of MHC class I cells were analyzed by flow cytometry on a FACScan flow cytometer using molecules was not affected by the hCD80 or hCD86 gene products CellQuest software (Becton Dickinson). (data not shown). The levels of CD80 and CD86 expressed by the NK polyclonal populations and NK cell lines transfected cell lines were found to correspond roughly to those Lymphokine-activated killer (LAK) cells and NK cell lines were generated expressed by monocyte-derived dendritic cells (data not shown). from human blood. Briefly, human PBMC were isolated from heparinized venous blood or cytopheresis buffy coats from healthy adult volunteer do- Lysis of CD80-, CD86-, and CD80/CD86-transfected B16.F1 nors by Ficoll-Hypaque density centrifugation (Lymphoprep, Nycomed, cells by human LAK cells Oslo, Norway). PBMC were depleted of adherent cells by plastic adher- ence for 45 min at 37°C. The recovered cells were resuspended at a con- Human LAK cells, stimulated with either rhIFN-␣ or rhIL-2, do centration of 1 ϫ 106 cells/ml in complete medium supplemented with not efficiently lyse B16.F1 melanoma cells. In contrast, similar The Journal of Immunology 4209

Blocking of CD80 and CD86 with specific mAbs impaired NK cell line triggering of lysis To verify that the increased sensitivity of the CD80- and CD86- transfected cell lines to NK cell line-mediated lysis was a direct consequence of cell-surface costimulatory molecule expression, experiments were performed in which the hCD80, hCD86, or hCD80/hCD86 molecules were blocked with specific mAb. The increased lysis of the transfected target cells could be inhibited completely by addition of specific mAb against hCD80 or hCD86, demonstrating that the observed effect was not an indirect consequence of the expression of the transgene (Fig. 2). FIGURE 1. LAK cell-mediated lysis of the B16.F1-hCD80, B16.F1- NK cell-mediated killing of CD80/CD86-expressing targets hCD86, and B16.F1-hCD80/hCD86 cell lines. LAK cells were stimulated occurred in the absence of CD28 and CTLA-4 with either rhIFN-␣ (left) or rhIL-2 (right). Target cells were B16.F1 (ࡗ), B16.F1-hCD80 (F), B16.F1-hCD86 (Œ), and B16.F1-hCD80/hCD86 (f). The NK cell lines used in the present study were analyzed pheno- One representative of five experiments is shown. typically using flow cytometry and were found to be CD3ϪCD14ϪCD19ϪCD56ϩ. The IL-2-propagated CD56ϩ NK cell lines did not express any detectable levels of CD28 or CTLA-4 (data not shown). As T cells up-regulate these molecules upon Downloaded from effector cells readily killed B16.F1 cells transfected with the activation, it could be argued that this is also the case with NK hCD80, hCD86, or hCD80/hCD86 costimulatory molecules. Sig- cells. Thus, the NK cell lines were analyzed for expression of nificantly higher levels of lysis were observed for the transfected CD28 and CTLA-4 after stimulation with high doses of rhIL-2 cells when compared with corresponding untransfected cell lines (1,000 U/ml) (Fig. 3, A and B) or with rhIL-2 plus rhIL-12 (1000 (Fig. 1) or vector-transfected control cell lines (data not shown). U/ml and 100 ng/ml, respectively) (Fig. 3, C and D). Using two separate anti-CD28 mAb (L293 and YTH913.12), we could not http://www.jimmunol.org/ Lysis of CD80-, CD86-, and CD80/CD86-transfected B16.F1 detect any levels of expression of CD28 and CTLA-4 following cells by human NK cell lines activation (Fig. 3, A and C; data not shown). Control experiments Because LAK cell cultures stimulated by high levels of IL-2 are a using PHA and Con A T cell blasts demonstrated that these Abs potential source of cytotoxic T cells, we wished to address the lytic were able to recognize appropriate cell-surface molecules (data not activity of pure NK cells. Thus, long-term CD16ϩCD56ϩCD3Ϫ shown). NK cell lines were generated. These lines readily killed targets NK cells can mediate reverse ADCC against NK-resistant P815 transfected with the hCD80, hCD86, and hCD80/hCD86 costimu- cells following CD16 cross-linking. Addition of anti-CD28 and anti-CTLA-4 mAb into reverse ADCC assays did not lead to any latory molecules while the wild-type parent cell line or vector con- by guest on September 26, 2021 trol cells were killed at significantly lower levels (Table II, and induction of lysis of the P815 cells. This absence of lysis provides data not shown). Notably, however, the hCD80/hCD86 double further support for the notion that NK cell recognition of hCD80 or transfectant cells were not killed better than any of the single trans- hCD86 is not mediated by either CD28 or CTLA4 (Fig. 4). fectants (Table II). It was reported that murine NK cells stimulated with IL-12 exhibited augmented responses to murine CD80 Discussion (mCD80)- and murine CD86 (mCD86)-transfected tumor cell lines There is an emerging consensus that NK cell recognition and sub- (30). Therefore, additional experiments were performed in which sequent effector mechanisms are regulated by both activating and the IL-2-propagated NK cell lines were stimulated with rhIL-12. inhibitory signals. In this study, using purified populations of hu- These effector cells did not display any detectable increased ability man NK cells, we demonstrate that the human CD80 and CD86 to lyse hCD80- or hCD86-transfected target cells compared with gene products function as triggering signals for NK cell-mediated cell lines stimulated with IL-2 alone (data not shown). cytotoxicity.

Table II. NK cell line-mediated lysis of B16.F1 target cells

Target Cell Lines

Expt. E:T Ratio B16.F1 B16.F1-hCD80 B16.F1-hCD86 B16.F1-hCD80/hCD86

1 100Ϻ125a 59 62 57 33Ϻ114353827 11Ϻ1 2 15 15 10 3Ϻ10 5 4 3 2 100Ϻ139726062 33Ϻ122654943 11Ϻ119443039 3Ϻ1 7 30 14 16 3 100Ϻ133597468 33Ϻ114356154 11Ϻ1 2 17 37 35 3Ϻ10 5 1114

a Percent speciﬁc lysis. 4210 NK CELL TRIGGERING BY HUMAN CD80 AND CD86 Downloaded from FIGURE 2. Lysis of B16.F1-hCD80, B16.F1-hCD86, and B16.F1-hCD80/hCD86 cell lines is inhibited by Abs speciﬁc for hCD80 or hCD86. Each target cell line (indicated on the top of the panel) was assessed for killing by an NK cell line in the absence or presence of blocking Abs. Target cells without blocking Abs (ࡗ), target cells with isotype-matched control Abs (f), target cells with anti-hCD80 mAbs (Œ), target cells with anti-hCD86 mAbs (F), and target cells with both anti-hCD80 and anti-hCD86 (ϫ) are shown. The combination of anti-hCD80 and anti-hCD86 mAbs was performed only with the B16.F1-hCD80/hCD86 cell line. One representative of three experiments is shown. http://www.jimmunol.org/

The present results are in line with observations in the murine of lysis by MHC class I, or lack of other necessary triggering system, demonstrating that CD80, CD86, and CD40 can trigger molecules. mouse NK cells to mediate lysis (28–31). Furthermore, it has been Previous data from our groups have suggested that murine co- demonstrated that human NK cells can be triggered by interactions stimulatory molecules trigger NK cells in a CD28- and CTLA-4- between CD40 on the target cell and CD40L expressed by NK independent way (29, 31). However, murine (39, 40) and human cells (32). However, it should be noted that introduction of the studies (33, 41, 42) suggest that when CD28 is expressed by NK human CD80 gene into a previously negative human squamous cells, such as the human YT cell line, then this molecule may

cell carcinoma of the head and neck was found not to be associated transmit triggering signals through ligation with CD80. Thus, for by guest on September 26, 2021 with increased susceptibility to lysis by human NK cells (38). This CD28-expressing NK cell effectors, killing of target cells express- led the authors to conclude that CD80 molecules are not involved ing costimulatory molecules may be in part dependent upon trig- in triggering of adult NK cells. While this conclusion may be valid gering via this receptor. However, this does not exclude a potential for this particular cell line, it may not exclude a triggering effect of CD28/CTLA-4-independent component operating in parallel. The CD80, or other costimulatory molecules, when introduced into present results demonstrate that triggering of NK cells by CD80- other tumors. NK cell insensitivity despite expression of costimu- and CD86-expressing target cells may occur independently of latory molecules can be explained in many ways, e.g., lack of CD28. The failure to detect cell-surface expression of both CD28 appropriate expression of relevant adhesion molecules, inhibition and CTLA-4 by either redirected lysis experiments or direct ﬂow cytometric analysis argues against the involvement of these molecules, at least in a form detectable by the present reagents used.

FIGURE 4. NK cell lines mediate redirected lysis against P815 cells in FIGURE 3. Human peripheral blood-derived NK cell lines do not stain the presence of anti-CD16 mAbs but not in the presence of anti-CD28 or positive for either CD28 or CTLA-4. Two-color dot-plots show CD56PE- anti-CTLA-4 mAbs. A, NK cell-mediated lysis of P815 alone (ࡗ)orinthe CD28FITC (A and C) and CD56PE-CTLA-4FITC (B and D) ﬂuorescence presence of anti-CD16 mAb. B, NK cell-mediated lysis of P815 in the on NK cells stimulated with IL-2 (A and B) or with IL-2 plus IL-12 (C presence of isotype control mAbs (ࡗ), anti-CD28 mAbs (f), or anti- and D). CTLA4 mAbs (Œ). The Journal of Immunology 4211

The absence of detectable levels of CD28 and CTLA-4 on human 4. Moretta, A., and L. Moretta. 1997. HLA class I specific inhibitory receptors. NK cell lines derived from peripheral blood is also in line with Curr. Opin. Immunol. 9:694. 5. Lo´pez-Botet, M., M. Carretero, T. Belloń,J.J.Pe´rez-Villar, M. Llano, and observations by others (38). However, we cannot exclude that very F. Navarro. 1998. The CD94/NKG2 C-type lectin receptor complex. Curr. Top. low expression levels of CD28 and/or CTLA-4 (undetectable by Microbiol. Immunol. 230:41. 6. Vance, R. E., J. R. Kraft, J. D. Altman, P. E. Jensen, and D. H. Raulet. 1998. the present means of analysis) or the existence of variants of CD28 Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major and/or CTLA-4 on NK cells could account for some of the ob- histocompatibility complex (MHC) class I molecule Qa-1(b). J. Exp. Med. 188: served effects. Indeed, a recent publication by Galea-Lauri et al. 1841. 7. Salcedo, M., P. Bousso, H. G. Ljunggren, P. Kourilsky, and J. P. Abastado. 1998. has suggested that detection of the CD28 molecule on NK cells The Qa-1b molecule binds to a large subpopulation of murine NK cells. Eur. depends entirely upon the mAb used (34). The authors suggested J. Immunol. 28:4356. that NK cells may express a variant isotype of CD28, as two of 8. Takei, F., J. Brennan, and D. L. Mager. 1997. The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol. Rev. 155:67. four anti-CD28 mAbs positively stained NK cells whereas all four 9. Takai, T., M. Li, D. Sylvestre, R. Clynes, and J. V. Ravetch. 1994. FcR␥ chain stained T cells. Incidentally, the two mAbs that failed to stain NK deletion results in pleiotrophic effector cell defects. Cell 76:519. cells were also the two mAb used throughout this study. This dif- 10. Hazenbos, W. L., J. E. Gessner, F. M. Hofhuis, H. Kuipers, D. Meyer, I. A. Heijnen, R. E. Schmidt, M. Sandor, P. J. Capel, M. Daeron, ference of mAb binding could be due to posttranslational modifi- J. G. van de Winkel, and J. S. Verbeek. 1996. Impaired IgG-dependent anaphy- cations, splice variants of the CD28 molecule, or other alterations. laxis and Arthus reaction in Fc␥RIII (CD16) deficient mice. Immunity 5:181. 11. Chambers, W. H., N. L. Vujanovic, A. B. DeLeo, M. W. Olszowy, Galea-Lauri et al. observed that there was considerable variation in R. B. Herberman, and J. C. Hiserodt. 1989. Monoclonal antibody to a triggering the levels of CD28 expression between NK lines and also individ- structure expressed on rat natural killer cells and adherent lymphokine-activated uals despite mRNA levels remaining steady. NK cells may also killer cells. J. Exp. Med. 169:1373. 12. Mathew, P. A., B. A. Garni-Wagner, K. Land, A. Takashima, E. Stoneman, express novel CD80/CD86 binding receptors. In this respect, it M. Bennett, and V. Kumar. 1993. Cloning and characterization of the 2B4 gene Downloaded from should be mentioned that a novel receptor, functionally related to encoding a molecule associated with non-MHC-restricted killing mediated by CD28, has been described recently (43). It is possible that such activated natural killer cells and T cells. J. Immunol. 151:5328. 13. Miyazaki, T., A. Dierich, C. Benoist, and D. Mathis. 1996. Independent modes of CD28-related receptors could account for the observed effects. Al- natural killing distinguished in mice lacking Lag3. Science 272:405. ternatively, it cannot be excluded that the CD80 and CD86 mol- 14. Ortaldo, J. R., A. T. Mason, L. H. Mason, R. T. Winkler-Pickett, P. Gosselin, and ecules are recognized by several low-affinity “pattern recognition S. K. Anderson. 1997. Selective inhibition of human and mouse natural killer tumor recognition using retroviral antisense in primary natural killer cells: in- receptors” that may not easily be identified using conventional volvement with MHC class I killer cell inhibitory receptors. J. Immunol. 158: http://www.jimmunol.org/ flow cytometric techniques. 1262. 15. Seaman, W. E., E. C. Niemi, M. R. Stark, R. D. Goldfien, A. S. Pollock, and It is known that NK cells can be triggered by several different J. B. Imboden. 1991. Molecular cloning of gp42, a cell-surface molecule that is receptors (2), thus the triggering observed with targets expressing selectively induced on rat natural killer cells by interleukin 2: glycolipid mem- CD80 and CD86 could operate in parallel with other activating brane anchoring and capacity for transmembrane signaling. J. Exp. Med. 173: 251. molecules. Susceptible targets may interact with NK cells through 16. Karlhofer, F. M., and W. M. Yokoyama. 1991. Stimulation of murine natural multiple different receptors simultaneously. It has been proposed killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL- that NK cell activation results from signals through one or more 2-activated NK cells possess additional specific stimulation pathways. J. Immu- nol. 146:3662. receptors that are not NK cell specific such as adhesion molecules, 17. Siliciano, R. F., J. C. Pratt, R. E. Schmidt, J. Ritz, and E. L. Reinherz. 1985. including e.g., CD2, LFA-1, and CD28 (18). However, NK cell Activation of cytolytic T lymphocyte and natural killer cell function through the by guest on September 26, 2021 specificity has been claimed for the recently identified NKp44 and T11 sheep erythrocyte binding protein. Nature 317:428. 18. Lanier, L. L., B. Corliss, and J. H. Phillips. 1997. Arousal and inhibition of NKp46 receptors (26, 27). Furthermore, NKR-P1 expression ap- human NK cells. Immunol. Rev.155:145. pears to be NK cell specific, with the exception of the expression 19. Biassoni, R., C. Cantoni, M. Falco, S. Verdiani, C. Bottino, M. Vitale, R. Conte, A. Poggi, A. Moretta, and L. Moretta. 1996. The human leukocyte antigen of NK1.1 on murine “NKT” cells (44). Thus, NK cell triggering (HLA)-C-specific “activatory” or “inhibitory” natural killer cell receptors display may be mediated by NK cell specific as well as more widely used highly homologous extracellular domains but differ in their transmembrane and receptor-ligand interactions. intracytoplasmic portions. J. Exp. Med. 183:645. 20. Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, C. Bottino, In conclusion, the present, as well as recently published (28– and L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) 33), observations on NK cell interaction with costimulatory mol- receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182: ecules suggest that these molecules should be put on the list for 875. 21. Braud, V. M., D. S. Allan, C. A. O’Callaghan, K. So¨derstro¨m, A. D’Andrea, candidate NK cell-activating ligands on target cells. As to the co- G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, L. L. Lanier, and stimulatory molecules, the biological relevance of the ability of A. J. McMichael. 1998. HLA-E binds to natural killer cell receptors CD94/ NK cells to recognize these molecules remains to be explored. NKG2A, B and C. Nature 391:795. 22. Lee, N., M. Llano, M. Carretero, A. Ishitani, F. Navarro, M. Lopez-Botet, and Perhaps NK cells communicate in a specific manner with profes- D. E. Geraghty. 1998. HLA-E is a major ligand for the natural killer inhibitory sional APC via these molecules (45). Indeed, a recent study in a receptor CD94/NKG2A. Proc. Natl. Acad. Sci. USA 95:5199. 23. Lanier, L. L., B. Corliss, J. Wu, and J. H. Phillips. 1998. Association of DAP12 murine model suggested that cell-to-cell contact between DC and with activating CD94/NKG2C NK cell receptors. Immunity 8:693. resting NK cells resulted in a substantial increase in both NK cell 24. Nakamura, M. C., P. A. Linnemeyer, E. C. Niemi, L. H. Mason, J. R. Ortaldo, cytotoxicity and IFN-␥ production (46). Moreover, we have ob- J. C. Ryan, and W. E. Seaman. 1999. Mouse Ly-49D recognizes H-2Dd and activates natural killer cell cytotoxicity. J. Exp. Med. 189:493. served that monocyte-derived dendritic cells and Langerhans-like 25. Sivori, S., M. Vitale, L. Morelli, L. Sanseverino, R. Augugliaro, C. Bottino, cells are efficiently lysed by autologous NK cells (J.L.W. et al., L. Moretta, and A. Moretta. 1997. p46, a novel natural killer cell-specific surface unpublished observations). molecule that mediates cell activation. J. Exp. Med. 186:1129. 26. Cantoni, C., C. Bottino, M. Vitale, A. Pessino, R. Augugliaro, A. Malaspina, S. Parolini, L. Moretta, A. 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