Human NK Cells (CD244)-Expressing Targets Activates CD48 Stimulation

CD48 Stimulation by 2B4 (CD244)-Expressing Targets Activates Human NK Cells

This information is current as Birgitta Messmer, Philipp Eissmann, Sebastian Stark and of September 27, 2021. Carsten Watzl J Immunol 2006; 176:4646-4650; ; doi: 10.4049/jimmunol.176.8.4646 http://www.jimmunol.org/content/176/8/4646 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD48 Stimulation by 2B4 (CD244)-Expressing Targets Activates Human NK Cells1

Birgitta Messmer, Philipp Eissmann, Sebastian Stark, and Carsten Watzl2

Human NK cells can be activated by a variety of different cell surface receptors. Members of the SLAM-related receptors (SRR) are important modulators of NK cell activity. One interesting feature of the SRR is their homophilic interaction, combining receptor and ligand in the same molecule. Therefore, SRR cannot only function as activating NK cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that does not show homophilic interaction. Instead, 2B4 is activated by binding to CD48, a GPI-anchored surface molecule that is widely expressed in the hemopoietic system. In this study, we show that 2B4 also can function as an activating NK cell ligand. 2B4-expressing target cells can efﬁciently stimulate NK cell cytotoxicity and IFN-␥ production. Using soluble receptor fusion proteins and SRR-transfected cells, we show that 2B4 does not bind to any other

SRR expressed on NK cells, but only interacts with CD48. Lysis of 2B4-expressing target cells can be blocked by anti-CD48 Abs Downloaded from and triggering of CD48 in a redirected lysis assay can stimulate NK cell cytotoxicity. This demonstrates that 2B4 can stimulate NK cell cytotoxicity and cytokine production by interacting with NK cell expressed CD48 and adds CD48 to the growing number of activating NK cell receptors. The Journal of Immunology, 2006, 176: 4646–4650.

he activity of NK cells is regulated by a ﬁne balance be- certain circumstances 2B4 can even mediate the inhibition of NK

tween signals from activating and inhibitory cell surface cell function in mouse and human NK cells (19–21). Interestingly, http://www.jimmunol.org/ T receptors (1). Inhibitory receptors for MHC class I mol- 2B4 and CD48 are also expressed on multipotent hemopoietic pro- ecules guarantee the self-tolerance of NK cells (2). NK cell acti- genitor cells and, together with other members of the SRR, are the vation results in cell-mediated cytotoxicity and cytokine secretion only receptors whose expression can distinguish hemopoietic stem and is important for the immune response against viral infections cells and progenitor cells (22). These ﬁndings suggest that the and transformed cells (3). A variety of different surface receptors interaction between 2B4 and CD48 is involved in the regulation of can induce NK cell activation. The natural cytotoxicity receptors many cellular processes in the hemopoietic system. NKp30, NKp44, and NKp46 and the NKG2D receptor have been In this study, we show that 2B4 cannot only work as an acti- described as the major NK cell activating receptors (4). Members vating NK cell receptor, but that it can also function as an acti- of the signaling lymphocyte activation molecule SLAM-related vating NK cell ligand. 2B4 expression on target cells can stimulate by guest on September 27, 2021 receptors (SRR)3 are important in the modulation and costimula- NK cell cytotoxicity and IFN-␥ production through the engage- tion of NK cell responses and can also regulate the function of ment of CD48 on NK cells. This demonstrates that the 2B4-CD48 other lymphocytes (5). The SRR expressed by all NK cells are 2B4 interaction can generate bidirectional signals through 2B4 (CD244), NTB-A (NK-T-B cell Ag), and CS1 (CRACC). NTB-A and CD48. and CS1 are their own ligands and can lead to NK cell activation by homophilic interaction (6–8). 2B4 binds to CD48 (9), a GPI- Materials and Methods anchored Ig superfamily member, which is widely expressed in the Cells, Abs, and fusion proteins hemopoietic system and is also present on all NK cells. The cells used in this study were: NK92-C1 (cultured in Alpha medium, 2B4 is an important modulator for the activity of NK cells and 12.5% FCS, 12.5% horse serum, 50 ␮M 2-ME, 2 mM glutamine and pen- other lymphocytes (10–12). It can effectively costimulate the sig- icillin/streptomycin); 293 (cultured in DMEM, 10% FCS, penicillin/strep- nals of other activating NK cell receptors (13) and can enhance the tomycin); BaF3 (cultured in RPMI 1640, 10% FCS, 50 mM 2-ME, penicillin/streptomycin); P815 (cultured in IMDM, 10% FCS, penicillin/ cytotoxic activity of Ag-speciﬁc T cells (14). 2B4 is expressed by streptomycin); and primary human NK cells, isolated and cultured as human eosinophils and can activate their effector functions (15). described (23). T cell clones were generated by limiting dilution of NK The interaction between 2B4 and CD48 is important for the pro- cell-depleted PBL. Clones were stimulated by autologous PBL as feeder liferation of mouse NK and T cells and is necessary for the gen- cells in the presence of PHA and 100 U/ml IL-2 and restimulated by al- eration of effector functions in mouse NK cells (16–18). Under logeneic PBL after 1 wk. Wells positive for growth were then expanded and tested by surface staining. BaF3 and 293 cells were stably transfected with the cDNAs for CD48, 2B4, NTB-A, CS1, or GFP using a retroviral Institute for Immunology, University of Heidelberg, Heidelberg, Germany system. The Abs used were anti-CD48 (clone 4H9; Santa Cruz Biotech- nology), anti-CD48 (clone J4-57; Beckman Coulter), anti-2B4 (C1.7) (pro- Received for publication July 12, 2005. Accepted for publication February 3, 2006. vided by G. Trinchieri, The Wistar Institute, Philadelphia, PA), goat anti- The costs of publication of this article were defrayed in part by the payment of page mouse IgG PE-conjugated (Jackson ImmunoResearch Laboratories), and charges. This article must therefore be hereby marked advertisement in accordance MOPC21 (Sigma-Aldrich) as an isotype control. The monoclonal anti- with 18 U.S.C. Section 1734 solely to indicate this fact. NTB-A (NT-7) and anti-CRACC (CS1.4), and anti-isoleucine zipper 1 This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB405 (ILZ)-11 have been previously described (6, 24). The isoleucine fusion A9. proteins ILZ-CD4, ILZ-2B4, ILZ-CD48, ILZ-NTB-A, and ILZ-CS1 have 2 Address correspondence and reprint requests to Dr. Carsten Watzl, Institute for been generated as described previously (24). Immunology, Im Neuenheimer Feld 305, University of Heidelberg, 69120 Heidel- FACS analysis berg, Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: SRR, SLAM-related receptor; ILZ, isoleucine For surface staining, the cells were incubated with the indicated mAbs (10 zipper. ␮g/ml) or ILZ-fusion proteins (1 ␮g/ml) in 50 ␮l of FACS buffer (PBS, 2%

FCS) for 20 min on ice. All washing steps were performed with cold FACS buffer. Ab-labeled cells were then stained with PE-conjugated goat anti- mouse Ab (1/200). ILZ fusion protein stained cells were incubated with an anti-ILZ-11 Ab (5 ␮g/ml) followed by PE-conjugated goat anti-mouse Ab. Killing assay Target cells were grown to mid-log phase, and 5 ϫ 105 cells were labeled in 100 ␮l of CTL medium (IMDM with 10% FCS and penicillin/streptomycin) with 100 ␮Ci 51Crfor1hat37°C. Cells were washed twice in CTL medium and resuspended at 5 ϫ 104 cells/ml in CTL medium. A total of 5000 target cells/well was used in the assay. Effector cells were resuspended in CTL medium, distributed on a V-bottom 96-well plate, and mixed with labeled target cells at different E:T ratios. Maximum release was determined by incubating target cells in 1% Triton X-100. For spontaneous release, targets were incubated without effectors in CTL medium alone. For a redirected lysis assay Abs were used at a final assay concentration of 0.5 ␮g/ml. For blocking experiments, Abs were used at a final assay concentration of 10 ␮g/ml. All samples were done in triplicates, and IL-2 was used at 100 IU/ml final concentration in the whole assay. After a 1-min centrifugation at 1000 rpm, plates were incubated for 4 h at 37°C. Supernatant was harvested and 51Cr release was measured in a gamma counter. Percentage of specific release was calculated as ((experimental release Ϫ spontaneous release)/(maximum release Ϫ spontaneous Downloaded from release)) ϫ 100. IFN-␥ release assay The 96-well plates were coated with anti-ILZ-11 Ab (10 ␮g/ml in PBS) for 16 h at 4°C. Plates were washed with sterile PBS and incubated with ILZ-fusion proteins (5 ␮g/ml in PBS) for 1 h at room temperature. Plates were washed again and incubated with 1 ϫ 105 NK cells per well. Target http://www.jimmunol.org/ cells were used at 1 ϫ 105 cells/well. All samples were done in triplicates. FIGURE 1. 2B4-expressing target cells stimulate NK cell-mediated cy- Plates were incubated for 20 h at 37°C and supernatants were harvested. totoxicity. A, Analysis of stable Baf3 and 293 transfectants. Stably trans- Quantification of IFN-␥ was performed using a quantitative sandwich fected Baf3 or 293 cells were stained with an isotype control Ab (open ELISA (Quantikine kit; R&D Systems) according to the manufacturer’s histogram) or an Ab against the transfected surface receptor (black histo- instructions. gram). For GFP transfectants (black histogram) the fluorescence was com- Results pared with nontransfected cells (open histogram). B and C, Killing assay using NK92 cells as effectors. The indicated Baf3 (B) or 293 (C) transfec- To investigate the functional consequences of stimulating different tants were used as targets in a 4-h 51Cr release assay with NK92 as effector SRR with their respective ligands on target cells we generated cells. The assays were repeated at least three times and representative re- by guest on September 27, 2021 stable mouse Baf3 transfectants expressing human NTB-A, CD48, sults are shown. and CS1 (Fig. 1A). As a control we also generated Baf3 cells expressing 2B4 or GFP (Fig. 1A). As expected, the expression of CD48, NTB-A, or CS1 resulted in increased killing of the Baf3 expressed on the target cells stimulates cytotoxicity by engaging cells by the human NK cell line NK92 (Fig. 1B), most likely CD48 on the NK cells. CD48 is expressed by all NK cells as well through the stimulation of 2B4, NTB-A, or CS1, respectively. In- as the NK92 cell line, and ILZ-2B4 can specifically bind to NK92 terestingly, also the 2B4-expressing Baf3 cells were much better cells (Fig. 2C) and also to primary NK cells (data not shown). targets when compared with the control transfected Baf3-GFP To prove that the interaction between 2B4 and CD48 is respon- cells (Fig. 1B). To confirm that 2B4 expression on target cells can sible for the enhanced killing of 2B4-expressing target cells, we enhance their lysis, we generated human 293 cells stably express- tried to block the killing of 293-2B4 cells with different Abs. Two ing 2B4 or GFP as a control (Fig. 1A). Also in this system, 2B4 different anti-CD48 Abs were both effective in completely block- expression significantly enhanced the lysis of the 293 cells (Fig. ing the killing of 293-2B4 cells (Fig. 3), demonstrating that CD48 1C). This result suggests that 2B4 can also function as an activat- is essential for the killing of 293-2B4 targets. Also an anti-2B4 Ab ing ligand for human NK cells. significantly reduced the lysis, possibly by masking 2B4 on the The only known ligand for human 2B4 is CD48 (9). To confirm target cells (Fig. 3). As the NK92 cells used in this assay are this interaction and to exclude the possibility that 2B4 stimulates FcR-negative, binding of the anti-2B4 Ab to the 293-2B4 cells NK cell activation by binding to another member of the SRR, we cannot induce Ab-dependent cellular cytotoxicity. 2B4 is present stained the 2B4-transfected Baf3 and 293 cells with different sol- on the target and the NK cells. Therefore, the amount of Ab used uble ILZ receptor fusion proteins (24). Only ILZ-CD48 bound may not be enough to completely mask 2B4 on the target cells, specifically to the Baf3-2B4 and 293-2B4 cells, whereas no bind- explaining the lack of a more complete block. ing of ILZ-2B4, NTB-A, or CS1 could be observed (Fig. 2A). This These data suggest that CD48 can function as an activating NK demonstrates that the transfected 2B4 only binds to CD48 and does cell receptor. To directly test this idea, we performed a redirected not interact with 2B4, NTB-A, or CS1, which are the only SRR lysis assay using FcR-positive P815 cells as targets. Two different expressed by human NK cells. To test whether the binding of anti-CD48 Abs were both able to stimulate NK cell cytotoxicity ILZ-CD48 is specific for 2B4-expressing cells we tested the bind- when compared with an isotype control Ab (Fig. 4A). This dem- ing to the different Baf3 and 293 transfectants. ILZ-CD48 only onstrates that engagement of CD48 can lead to NK cell cytotox- bound the Baf3-2B4 and 293-2B4 cells (Fig. 2B), confirming the icity. To test whether the engagement of CD48 by soluble 2B4 or specificity of this interaction. More importantly, soluble ILZ-2B4 2B4-expressing target cells can also stimulate cytokine release by only bound to Baf3-CD48 and 293-CD48 cells, but not to any NK cells, we incubated NK92 cells in wells coated with ILZ-2B4 other transfectant (Fig. 2B), demonstrating that 2B4 specifically or CD4 as a control, or with 2B4- or GFP-expressing Baf3 and 293 and exclusively interacts with CD48. This suggests that the 2B4 cells. Engagement of NK cells by coated ILZ-2B4 clearly induced 4648 2B4 AS ACTIVATING NK CELL LIGAND

FIGURE 3. Killing of 293–2B4 cells can be blocked by anti-CD48 Abs. 293-GFP or 293–2B4 cells were used at the indicated E:T ratio in a killing assay with the FcR-negative NK cell line NK92 in the presence of a control Ab (IgG1) or two different Abs against CD48 or an anti-2B4 Ab. One representative of three different experiments is shown.

Discussion Downloaded from NK cells can be activated by a variety of different surface receptors. In this study we have shown that 2B4 expression on target cells can lead to the activation of NK cells, resulting in cytotoxicity FIGURE 2. The 2B4 cells only interacts with CD48. A, Baf3–2B4 or and IFN-␥ production. The 2B4-expressing target cells bound only 293–2B4 cells were stained with the indicated ILZ fusion proteins, fol- soluble CD48 receptor and soluble 2B4 only interacted with lowed by an anti-ILZ Ab (ILZ-11) and a PE-labeled goat anti-mouse Ab. CD48-expressing cells. This demonstrates the specificity of the http://www.jimmunol.org/ B, The indicated cells were stained with ILZ-2B4 or ILZ-CD48 as de- 2B4-CD48 interaction and excludes that 2B4-expressing target scribed in A. C, NK92 cells were stained with an anti-CD48 Ab (left)or cells stimulate NK cells by interacting with other members of the ILZ-2B4 (right). Open histogram represents control staining. SRR present on NK cells, such as 2B4 itself, NTB-A, or CS1. CD48 can therefore be added to the growing list of stimulating NK cell receptors. Our data confirm a recent report by Mathew et al. (26) demonstrating that 2B4-transfected K562 cells are better the production of IFN-␥ (Fig. 4B). Also, the 2B4-expressing 293 killed by NK92 cells than by untransfected K562 targets. and Baf3 cells stimulated substantially more IFN-␥ production Members of the SRR are mostly homophilic, combining recep- than the GFP-transfected control cells (Fig. 4B). This demonstrates tor and ligand in the same molecule. Interactions between SRR- by guest on September 27, 2021 that engagement of NK cells by 2B4 can stimulate not only cyto- expressing cells can therefore generate bidirectional signals that toxicity, but also the production of cytokines. affect both interacting cells. 2B4 is the only SRR that does not The experiments shown so far were performed using the NK cell show homophilic binding but specifically interacts with CD48. line NK92. This cell line is highly cytotoxic and is thought to best resemble primary NK cells. It is also the first NK cell line used clinically (25). To investigate, whether 2B4 is also an activating ligand for nontransformed NK cells, we tested purified primary NK cells for their lysis of 293 cells expressing 2B4 or GFP as a control. The presence of 2B4 significantly enhanced the lysis of 293 cells (Fig. 5A). This demonstrates that 2B4 can also function as an activating ligand for purified primary NK cells. However, when comparing NK cells, from different donors, we observed this effect only in ϳ45% of the cases (6 of 13 different donors tested). In the other primary NK cells, there was no difference in cytotoxicity when comparing 2B4 with GFP-expressing 293 cells, even though the NK cells from all donors expressed comparable levels of CD48. This finding demonstrates that additional factors or receptors may play a role in the stimulation of NK cell cytotoxicity by 2B4-expressing target cells. To test whether naturally expressed 2B4 can also stimulate NK cells, we made use of the fact that a subpopulation of CD8-positive T cells express 2B4. We therefore generated T cell clones and screened them for 2B4 expression (Fig. 5B). We then compared FIGURE 4. Stimulation of CD48 induces NK cell cytotoxicity and ␥ 2B4-positive and 2B4-negative T cell clones as targets in a killing IFN- production. A, Redirected lysis of P815 cells using NK92 cells in the assay in the presence of a control Ab or an Ab against 2B4. As presence of a control Ab (IgG1) or two different anti-CD48 Abs. A representative of three different experiments is shown. B, NK92 cells were effectors we used NK92 cells because these cells lack FcRs and incubated in wells coated with the indicated ILZ fusion proteins or with the can therefore not mediate Ab-dependent cellular cytotoxicity. The indicated target cells. After 20 h, the supernatants were harvested and anti-2B4 Ab reduced target cell lysis only in the case of 2B4- IFN-␥ was measured by ELISA. All samples were done in triplicates. positive T cell clones, demonstrating that endogenously expressed Mean and SD are shown. The experiment was performed twice with similar 2B4 can also stimulate NK cell cytotoxicity. results. The Journal of Immunology 4649

GPI-anchored surface molecules are enriched in specialized mem- brane domains called lipid rafts (28). Rafts have been shown to be important for the activation of NK cells and other lymphocytes (23, 29–31). The clustering of rafts by the engagement of CD48 could be sufﬁcient to generate a positive signal leading to NK cell activation. A similar activity of CD48 has been described for the stimulation of T cells (32). The fact that not all primary NK cells can be stimulated by 2B4-expressing target cells suggests that additional cofactors may be necessary for CD48-mediated NK cell activation. We have not yet found any signiﬁcant difference between NK cells that can be stimulated by 2B4-expressing target cells and NK cells that cannot. Finding the reason for this difference and studying the signal transduction of CD48 in future studies will give us a better understanding of the molecular mechanism of the 2B4-CD48 interaction.

Disclosures The authors have no financial conflict of interest. Downloaded from References FIGURE 5. The 2B4 cells can also function as an activating ligand for 1. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274. 2. Long, E. O., D. F. Barber, D. N. Burshtyn, M. Faure, M. Peterson, purified primary NK cells. A, The 293 transfectants were used as targets in S. Rajagopalan, V. Renard, M. Sandusky, C. C. Stebbins, N. Wagtmann, and 51 a 4-h Cr release assay with purified human NK cells as effector cells. The C. Watzl. 2001. Inhibition of natural killer cell activation signals by killer cell assay was repeated with NK cells from 13 different donors. Six donors immunoglobulin-like receptors (CD158). Immunol. Rev. 181: 223–233. showed a similar activity as shown. B, T cell clones were stained with an 3. Hamerman, J. A., K. Ogasawara, and L. L. Lanier. 2005. NK cells in innate http://www.jimmunol.org/ immunity. Curr. Opin. 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