NK Cell-Depleting Anti-Asialo GM1 Ab Exhibits a Lethal Off-Target Effect on Basophils In Vivo This information is current as Hideto Nishikado, Kaori Mukai, Yohei Kawano, Yoshiyuki of September 24, 2021. Minegishi and Hajime Karasuyama J Immunol published online 13 April 2011 http://www.jimmunol.org/content/early/2011/04/12/jimmun ol.1100370 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/04/12/jimmunol.110037 Material 0.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published April 13, 2011, doi:10.4049/jimmunol.1100370 The Journal of Immunology NK Cell-Depleting Anti-Asialo GM1 Ab Exhibits a Lethal Off-Target Effect on Basophils In Vivo Hideto Nishikado,*,1 Kaori Mukai,*,1,2 Yohei Kawano,* Yoshiyuki Minegishi,*,† and Hajime Karasuyama*,† NK cells are innate immune lymphocytes and play a key role in both innate and adaptive immunity. Their pivotal functions in vivo have been illustrated in mice by means of their ablation with NK cell-depleting Abs, particularly anti-asialo GM1 (ASGM1). In this study, we show that the whole population of basophils constitutively expresses ASGM1 as well as CD49b (DX5) as does the NK cell population and was ablated in vivo by anti-ASGM1 as efficiently as by a basophil-depleting anti-Fc«RIa Ab. Anti-ASGM1– mediated basophil depletion was operative as for NK cell depletion in various mouse strains, irrespective of NK1 allotype and MHC H2 haplotype, including C57BL/6, BALB/c, C3H, and A/J mice. These results identified basophils as a previously un- recognized target of anti-ASGM1–mediated cell depletion and raised concern about possible contribution of basophils, rather Downloaded from than or in addition to NK cells, to some of phenotypes observed in anti-ASGM1–treated mice. Indeed, regardless of the presence or absence of NK cells in mice, anti-ASGM1 treatment abolished the development of IgE-mediated chronic cutaneous allergic inflammation as efficiently as did the treatment with basophil-depleting Ab. Given the fact that basophils have recently been shown to play crucial roles in a variety of immune responses, our finding of the off-target effect on basophils issues a grave warning about the use of anti-ASGM1 and underscores the need for careful interpretation of phenotypes observed in anti- ASGM1–treated mice. The Journal of Immunology, 2011, 186: 000–000. http://www.jimmunol.org/ atural killer cells provide innate defense against viruses spleen from various mouse strains (4). The expression of ASGM1 and tumor cells by killing target cells and producing is not strictly confined to NK cells among hematopoietic cells and N immunoregulatory cytokines (1–3). Two types of NK is detected on a subpopulation of NKT, CD8+ T, and gd T cells (9, cell-depleting Abs, a polyclonal Ab specific to asialo GM1 10) and some activated form of CD4+ T cells, macrophages, and (ASGM1) (4–6) and an mAb specific to NK1.1 (7), have com- eosinophils under certain experimental conditions (11–13). Nev- monly been used to elucidate in vivo functions of NK cells in ertheless, anti-ASGM1–mediated NK cell depletion still remains mice. Anti-ASGM1–mediated NK cell depletion is effective in a a powerful tool to analyze in vivo functions of NK cells. by guest on September 24, 2021 variety of mouse strains, whereas anti-NK1.1–mediated NK cell Basophils are basophilic granulocytes and represent ,1% of depletion works only in certain strains such as C57BL/6 and SJL peripheral blood leukocytes (14). Interestingly, basophils in mice and not in many other strains lacking the NK1.1 allotype, in- express typical NK markers CD49b (DX5) and NKR2B4 (2B4) cluding BALB/c, C3H, and A/J mice (8). Anti-ASGM1 activity on their surface (15–17) and produce granzyme B-like serine pro- was first identified .30 y ago in antiserum produced by immu- tease, mouse mast cell protease-8 (18, 19), even though cytotoxic nizing rabbits with mouse brain tissues and shown to be respon- activity of basophils remains to be investigated. Basophils also sible for antiserum-mediated elimination of NK activity in the display several characteristics shared by tissue-resident mast cells, including surface expression of the FcεRI, and the release of *Department of Immune Regulation, Tokyo Medical and Dental University Graduate allergy-related chemical mediators such as histamine in response School, Tokyo 113-8519, Japan; and †Japan Science and Technology Agency, Core to various stimuli (14). Because of these mast cell-like phenotypes Research for Evolutional Science and Technology, Tokyo Medical and Dental Uni- versity Graduate School, Tokyo 113-8519, Japan and their rarity, basophils have often erroneously been considered 1H.N. and K.M. contributed equally to this work. as minor relatives of mast cells or as blood-circulating precursors 2Current address: Department of Pathology, Stanford University School of Medicine, of tissue-resident mast cells (20). However, recent studies using Stanford, CA. basophil-depleting Abs (21–23) and genetically engineered mice Received for publication February 3, 2011. Accepted for publication March 15, 2011. deficient only in basophils (24, 25) have illuminated nonredundant This work was supported by research grants from the Japan Science and Technology roles for basophils in acquired immunity regulation, protective Agency, Core Research for Evolutional Science and Technology, the Japanese Min- immunity to pathogens, and immunological disorders such as al- istry of Education, Culture, Sports, Science and Technology, Takeda Science Foun- dation, the Mitsubishi Foundation, the Naito Foundation, and the Uehara Memorial lergy and autoimmunity (26–36). Foundation. In the current study, we found that the whole population of Address correspondence and reprint requests to Dr. Hajime Karasuyama, Department basophils in mice homogeneously expresses high levels of ASGM1 of Immune Regulation, Tokyo Medical and Dental University Graduate School, 1-5- and that in vivo administration of anti-ASGM1 readily ablates most 45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail address: karasuyama.mbch@ tmd.ac.jp of basophils, in addition to NK cells, in a variety of mouse strains, The online version of this article contains supplemental material. regardless of NK1 allotype and MHC H2 haplotype. These un- Abbreviations used in this article: APCy, allophycocyanin; ASGM1, asialo GM1; expected findings raise concern about the possible contribution BMPC, bone marrow protective cell; IgE-CAI, IgE-mediated chronic allergic inflam- of basophils, rather than or in addition to NK cells, to some of mation; TNP, 2,4,6-trinitrophenol. phenotypes observed in anti-ASGM1–treated mice. Indeed, treat- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ment of mice with anti-ASGM1 abolished the development of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100370 2 ANTI-ASIALO GM1 ABLATES BASOPHILS IN ADDITION TO NK CELLS IgE-mediated chronic cutaneous allergic inflammation as efficiently Statistical analysis as did the treatment with basophil-depleting Ab. The statistical significance of differences between groups was calculated with a two-tailed Student t test or ANOVA followed by Ryan’s test. A p Materials and Methods value ,0.05 was considered statistically significant. Mice Results C57BL/6, BALB/c, C3H mice (6–10 wk old) were purchased from CLEA Japan (Tokyo, Japan). A/J mice were from Japan SLC (Hamamatsu, Ja- NK cell-depleting anti-ASGM1 but not anti-NK1.1 Ab ablates pan), and rag22/2gc2/2 C57BL/6 mice were from Taconic Farms (Ger- basophils in vivo mantown, NY). All animal studies were approved by the Institutional Preparation of a highly purified basophil population for functional Animal Care and Use Committee of Tokyo Medical and Dental University. analysis is extremely difficult, particularly in mice, due to the Abs and other reagents rarity of basophils and the lack of murine basophil-specific Ab that ε leaves basophils unstimulated. Although enrichment of basophils FITC-conjugated anti-CD3 (145-2C11) and anti-rabbit IgG, FITC-conju- + ε + gated streptavidin, PE-conjugated anti-TCRgd (GL3), PE-conjugated (CD49b Fc RIa ) without their activation can be achieved in streptavidin, allophycocyanin (APCy)-conjugated anti-CD8a (53-6.7) and mice by magnetic sorting using anti-CD49b–bound beads, baso- CD49b (HMa2), and APCy-conjugated streptavidin were purchased from phils represent only 5–20% of the CD49b+ cells in the spleen, ε BD Pharmingen (San Diego, CA). PE-conjugated anti-Fc RIa (MAR-1) bone marrow, and peripheral blood, whereas the rest of cells are and biotinylated anti-CD3ε (145-2C11) were purchased from eBioscience (San Diego, CA). Unconjugated anti-FcεRIa (MAR-1), APCy-Cy7–con- mainly NK and NKT cells. We thought that Ab-mediated de- ε pletion of NK/NKT cells in vivo before the cell preparation could jugated streptavidin, PE-Cy7–conjugated anti-CD3 (145-2C11), and PE- Downloaded from Cy7–conjugated streptavidin were purchased from Biolegend (San Diego, increase the efficiency of basophil enrichment. When adminis- CA). Anti-CD16/32 (2.4G2) and anti-NK1.1 (PK136) were prepared from tered i.p., both anti-ASGM1 and anti-NK1.1 were indeed effec- hybridoma culture supernatants in our laboratories.