Negative Regulation of Expression and Function of Fc γRIII by CD3ζ in Murine NK Cells This information is current as Hisashi Arase, Tadahiro Suenaga, Noriko Arase, Yoshimitsu of September 26, 2021. Kimura, Katsuhiko Ito, Ritsuko Shiina, Hiroshi Ohno and Takashi Saito J Immunol 2001; 166:21-25; ; doi: 10.4049/jimmunol.166.1.21 http://www.jimmunol.org/content/166/1/21 Downloaded from References This article cites 31 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/166/1/21.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Negative Regulation of Expression and Function of Fc␥RIII by CD3␨ in Murine NK Cells1 Hisashi Arase,2 Tadahiro Suenaga, Noriko Arase,3 Yoshimitsu Kimura,4 Katsuhiko Ito, Ritsuko Shiina, Hiroshi Ohno,5 and Takashi Saito6 Fc␥RIII is involved in Ab-dependent cell-mediated cytotoxicity (ADCC) and cytokine production by NK cells. Signaling and expression of Fc␥RIII are dependent on FcR␥. Although NK cells express not only FcR␥ but also CD3␨, the role of CD3␨ in NK cell function remains unclear. Here, we found that the expression of Fc␥RIII on NK cells from CD3␨-deficient mice is unexpectedly up-regulated compared with that on cells from normal mice. Furthermore, ADCC and IFN-␥ production upon Fc␥RIII-cross- linking by NK cells from CD3␨-deficient mice were also up-regulated. Up-regulation of the surface expression of Fc␥RIII on CD3␨-deficient NK cells is not mediated by transcriptional augmentation of either Fc␥RIII or FcR␥ gene because there was no significant difference in the expression of mRNA for Fc␥RIII and FcR␥. Transfection of CD3␨ into a cell line expressing Fc␥RIII Downloaded from and FcR␥ induced a decrease in the cell surface expression of Fc␥RIII. These findings reveal a negative regulatory role of CD3␨ in Fc␥RIII-mediated function of murine NK cells. The Journal of Immunology, 2001, 166: 21–25. atural killer cells are activated upon recognition of a mice do not express Fc␥RIII or show ADCC function (9, 10). variety of target cells and exhibit natural cytotoxicity. Furthermore, NK cells from Fc␥RIIIϪ/Ϫ mice also fail to exhibit N Recently, a number of receptors were found to be in- ADCC, confirming that Fc␥RIII is the IgG Fc receptor responsible http://www.jimmunol.org/ volved in the activation of NK cells, although the exact features of for this function in NK cells (11). the receptors responsible for natural cytotoxicity remain unclear The CD3␨-chain is one of the components of the TCR-CD3 (1, 2). NK cells also show Ab-dependent cell-mediated cytotoxic- complex and possesses three tyrosine-based activation motifs ity (ADCC)7 upon cross-linking of IgG Fc␥R with Ab (3–5). (ITAM) in its cytoplasmic domain. ITAMs of CD3␨ are rapidly Among different Fc␥Rs, NK cells mainly express the low affinity tyrosine phosphorylated upon TCR cross-linking and transduce ac- receptor for IgG termed Fc␥RIII (CD16). Recently, human tivation signals in T cells (12). In addition, CD3␨ is required for Fc␥RIII on NK cells has been shown to be involved in direct the cell surface expression of the TCR complex and plays a crucial recognition of specific targets in the absence of Ab (6), although its role in the regulation of the assembly and intracellular transport of physiological function is still unclear. Therefore, Fc␥RIII ex- the TCR-CD3 complex. Indeed, the expression of TCR is severely by guest on September 26, 2021 pressed on NK cells is involved in ADCC and partly in Ab-inde- impaired in CD3␨-deficient cells and mice (13–17). pendent natural cytotoxicity. CD3␨ is also expressed in both human and murine NK cells Fc␥RIII is expressed on the cell surface in association with despite the fact that NK cells do not express TCR and seem to be FcR␥ (Fc⑀RI␥), which was originally identified as a signaling involved in NK cell activation (18, 19). CD3␨ is phosphorylated component of the high affinity IgE receptor (Fc⑀RI) complex (7, upon cross-linking of Fc␥RIII and is thought to be involved in 8). Because Fc␥RIII cannot be expressed on the cell surface in the signal transduction through Fc␥RIII in human NK cells (20, 21). absence of FcR␥, NK cells obtained from FcR␥-deficient (Ϫ/Ϫ) However, it is known that there is a significant difference between human and murine CD3␨ in Fc␥RIII expression. Human CD3␨ as well as FcR␥ can be associated with Fc␥RIII and are involved in Department of Molecular Genetics, Chiba University Graduate School of Medicine, the surface expression of Fc␥RIII, whereas murine Fc␥RIII can Chiba, Japan associate only with FcR␥, not with CD3␨. (22). In addition, no Received for publication July 7, 2000. Accepted for publication September 27, 2000. significant functional defects have been reported in NK cells from Ϫ/Ϫ The costs of publication of this article were defrayed in part by the payment of page CD3␨ mice (17). From these analyses, it has been widely be- charges. This article must therefore be hereby marked advertisement in accordance lieved that CD3␨ does not play any important role in the activation with 18 U.S.C. Section 1734 solely to indicate this fact. of murine NK cells. 1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry ␥ of Education, Science, and Culture. In the present study we found that Fc RIII expression on NK cells from CD3␨Ϫ/Ϫ mice is greatly enhanced. Furthermore, 2 Current address: Department of Microbiology and Immunology, University of Cal- ifornia San Francisco, CA 94143. Fc␥RIII-mediated functions of NK cells from CD3␨-deficient mice 3 Current address: Hooper Foundation, University of California, San Francisco, CA were also up-regulated. These findings demonstrate a novel func- 94143. tion of CD3␨ in murine NK cells in regulation of the expression 4 Current address: Mitsui Pharmaceuticals Inc., Mobara 297-0017, Japan. and function of Fc␥RIII. 5 Current address: Division of Molecular Membrane Biology, Cancer Research In- stitute, Kanazawa University, Kanazawa 920-0934, Japan. Materials and Methods 6 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Mice Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan. E-mail address: [email protected] The establishment and characterization of CD3␨Ϫ/Ϫ mice were described ␨Ϫ/Ϫ 7 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; previously (14). CD3 mice were maintained in our animal facility and Ϫ/Ϫ GFP, green fluorescence protein; IRES, internal ribosomal entry site; ITAM, immu- back-crossed to C57BL/6 mice seven times. FcR␥ mice were generated noreceptor tyrosine-based activation motif; sIg, surface Ig. from C57BL/6-derived embryonic stem cells as previously described (10). Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 22 NEGATIVE REGULATION OF Fc␥RIII BY CD3␨ IN NK CELLS Preparation of NK cells streptavidin-peroxidase (Vecstain Elite ABC kit; Vector, Burlingame, CA), an ECL system (Amersham International, Aylesbury, U.K.). NK cells were purified as previously described (23). Briefly, splenocytes were mixed with anti-CD4 mAb (GK1.5) and anti-CD8 mAb (53.6.7) fol- Transfection lowed by incubation with magnetic beads (Advanced Magnetics, Cam- bridge, MA) coupled with goat anti-mouse IgG Ab and goat anti-rat IgG cDNAs for Fc␥RIII and FcR␥ were subcloned into pMx retrovirus expres- Ab (Cappel, Organon Teknika, West Chester, PA) to remove surface Igϩ sion vector (provided by Dr. T. Kitamura, University of Tokyo, Tokyo, (sIgϩ) B cells and CD4ϩ and CD8ϩ T cells. The residual cells were then Japan). These cDNAs were transiently transfected into BOSC23 packaging incubated with PE-anti-NK1.1 mAb and FITC-anti-CD3 mAb (PharMin- cells using Lipofectamine Plus (Life Technologies, Gaithersburg, MD). gen, San Diego, CA). The stained cells were sorted into NK1.1ϩ CD3Ϫ Culture supernatants were collected at 2 days after transfection, and NIH- cells by FACStarPlus (Becton Dickinson, Mountain View, CA). The purity 3T3 cells were infected by addition of the supernatants. Fc␥RIII-expressing of the sorted cells was always Ͼ99%. cells were purified by FACStarPlus, and a single-cell clone stably express- ing Fc␥RIII was obtained. CD3␨ was subcloned into internal ribosomal Cell culture and stimulation entry site (pIRES)-green fluorescence protein (EGFP) expression vector (Clontech, Palo Alto, CA). Mutant CD3␨ in which isoleucine in the trans- Purified NK cells were cultured in RPMI 1640 supplemented with 10% membrane region was substituted to leucine (I46L-CD3␨) was generated ␮ ϫ Ϫ5 FCS, kanamycin (100 g/ml), and 5 10 M 2-ME. NK cells were by recombinant PCR and subcloned into pIRES-EGFP vector. CD3␨- expanded by culturing freshly purified NK cells for 5–7 days in the pres- IRES-GFP, I46L-CD3␨-IRES-GFP, and IRES-GFP (vector only) were ence of 1000 U/ml human rIL-2 (Ajinomoto, Kawasaki, Japan) and were transiently transfected into the Fc␥RIII transfectants.