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Veterinary immunology and immunopathology2012; 147(1-2); 60-68; doi: 10.1016/j.vetimm.2012.04.003

Generation and characterization of monoclonal antibodies to equine NKp46.

Abstract: The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.
Copyright © 2012 Elsevier B.V. All rights reserved.
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Publication Date: 2012-04-07 PubMed ID: 22551980PubMed Central: PMC3354985DOI: 10.1016/j.vetimm.2012.04.003Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • N.I.H.
  • Extramural
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • Non-P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research involves the generation and testing of monoclonal antibodies that identify equine NKp46, a marker of NK cells in the horse’s immune system. The research will aid in a better understanding of the equine NK cell population which is largely unexplored.

Methodology

  • The research involved expressing the extracellular region of equine NKp46 with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46). This was used to generate mouse monoclonal antibodies (mAbs).
  • The generated mAbs were first screened using an Enzyme-Linked Immunosorbent Assay (ELISA) to test their ability to recognize NKp46 and to ensure they didn’t recognize IL-4 or the similar immunoreceptor CD16.
  • Nine mAbs which passed this screening were selected and tested for their ability to identify full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein.

Findings

  • The selected mAbs were found to recognize a population of lymphocytes, that were morphologically similar to NKp46+ cells in humans and cattle, via flow cytometric analysis.
  • Using representative mAb 4F2 in a study on nine horses, the researchers found that 0.8-2.1% of PBL were labelled, correlating with mean fluorescence intensity consistent with gene expression data.
  • MAb 4F2+ PBL were enriched by magnetic cell sorting and were observed to express higher levels of NKp46 mRNA than 4F2- cells, as determined by quantitative RT-PCR.
  • PBL from five horses that had CD3 removed contained more 4F2+ cells than unsorted PBL.
  • Through another ELISA test, researchers discovered that the nine mAbs recognized three different epitopes, indicating that they can be used to identify various forms of the NKp46 antigen.

Conclusion

  • The generated and characterized monoclonal antibodies to equine NKp46 would provide better tools to understand the largely uncharacterized equine NK cell population.

Cite This Article

APA
Noronha LE, Harman RM, Wagner B, Antczak DF. (2012). Generation and characterization of monoclonal antibodies to equine NKp46. Vet Immunol Immunopathol, 147(1-2), 60-68. https://doi.org/10.1016/j.vetimm.2012.04.003

Publication

Veterinary immunology and immunopathology
ISSN: 1873-2534
NlmUniqueID: 8002006
Country: Netherlands
Language: English
Volume: 147
Issue: 1-2
Pages: 60-68

Researcher Affiliations

Noronha, Leela E
  • Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States.
Harman, Rebecca M
    Wagner, Bettina
      Antczak, Douglas F

        MeSH Terms

        • Animals
        • Antibodies, Monoclonal / biosynthesis
        • Antibodies, Monoclonal / immunology
        • CHO Cells
        • Cricetinae
        • Cricetulus
        • Epitopes
        • Flow Cytometry
        • Horses / immunology
        • Interleukin-4 / genetics
        • Mice
        • Natural Cytotoxicity Triggering Receptor 1 / analysis
        • Natural Cytotoxicity Triggering Receptor 1 / genetics
        • Natural Cytotoxicity Triggering Receptor 1 / immunology
        • Recombinant Fusion Proteins / biosynthesis

        Grant Funding

        • F32 HD055794 / NICHD NIH HHS
        • R01 HD049545 / NICHD NIH HHS
        • F32 HD 055794 / NICHD NIH HHS

        References

        This article includes 30 references
        1. Almeida-Oliveira A, Smith-Carvalho M, Porto LC, Cardoso-Oliveira J, Ribeiro Ados S, Falcão RR, Abdelhay E, Bouzas LF, Thuler LC, Ornellas MH, Diamond HR. Age-related changes in natural killer cell receptors from childhood through old age.. Hum Immunol 2011 Apr;72(4):319-29.
          pubmed: 21262312doi: 10.1016/j.humimm.2011.01.009google scholar: lookup
        2. Appleton JA, Gagliardo LF, Antczak DF, Poleman JC. Production of an equine monoclonal antibody specific for the H7 hemagglutinin of equine influenza virus.. Vet Immunol Immunopathol 1989 Dec;23(3-4):257-66.
          pubmed: 2629195doi: 10.1016/0165-2427(89)90139-6google scholar: lookup
        3. Biassoni R, Bottino C, Cantoni C, Moretta A. Human natural killer receptors and their ligands.. Curr Protoc Immunol 2002 Feb;Chapter 14:14.10.1-14.10.23.
          pubmed: 18432872doi: 10.1002/0471142735.im1410s46google scholar: lookup
        4. Biassoni R, Cantoni C, Pende D, Sivori S, Parolini S, Vitale M, Bottino C, Moretta A. Human natural killer cell receptors and co-receptors.. Immunol Rev 2001 Jun;181:203-14.
          pubmed: 11513142doi: 10.1034/j.1600-065x.2001.1810117.xgoogle scholar: lookup
        5. Biassoni R, Pessino A, Bottino C, Pende D, Moretta L, Moretta A. The murine homologue of the human NKp46, a triggering receptor involved in the induction of natural cytotoxicity.. Eur J Immunol 1999 Mar;29(3):1014-20.
          pubmed: 10092106doi: 10.1002/(SICI)1521-4141(199903)29:03<1014::AID-IMMU1014>3.0.CO;2-Ogoogle scholar: lookup
        6. Bloushtain N, Qimron U, Bar-Ilan A, Hershkovitz O, Gazit R, Fima E, Korc M, Vlodavsky I, Bovin NV, Porgador A. Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46.. J Immunol 2004 Aug 15;173(4):2392-401.
          pubmed: 15294952doi: 10.4049/jimmunol.173.4.2392google scholar: lookup
        7. Connelley T, Storset AK, Pemberton A, MacHugh N, Brown J, Lund H, Morrison IW. NKp46 defines ovine cells that have characteristics corresponding to NK cells.. Vet Res 2011 Feb 23;42(1):37.
          pmc: PMC3055825pubmed: 21345198doi: 10.1186/1297-9716-42-37google scholar: lookup
        8. De Maria A, Biassoni R, Fogli M, Rizzi M, Cantoni C, Costa P, Conte R, Mavilio D, Ensoli B, Cafaro A, Moretta A, Moretta L. Identification, molecular cloning and functional characterization of NKp46 and NKp30 natural cytotoxicity receptors in Macaca fascicularis NK cells.. Eur J Immunol 2001 Dec;31(12):3546-56.
          pubmed: 11745374doi: 10.1002/1521-4141(200112)31:12<3546::aid-imm㕆>3.0.co;2-wgoogle scholar: lookup
        9. de Mestre A, Noronha L, Wagner B, Antczak DF. Split immunological tolerance to trophoblast.. Int J Dev Biol 2010;54(2-3):445-55.
          pmc: PMC2879498pubmed: 19876828doi: 10.1387/ijdb.082795adgoogle scholar: lookup
        10. Gazit R, Gruda R, Elboim M, Arnon TI, Katz G, Achdout H, Hanna J, Qimron U, Landau G, Greenbaum E, Zakay-Rones Z, Porgador A, Mandelboim O. Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1.. Nat Immunol 2006 May;7(5):517-23.
          pubmed: 16565719doi: 10.1038/ni1322google scholar: lookup
        11. Halfteck GG, Elboim M, Gur C, Achdout H, Ghadially H, Mandelboim O. Enhanced in vivo growth of lymphoma tumors in the absence of the NK-activating receptor NKp46/NCR1.. J Immunol 2009 Feb 15;182(4):2221-30.
          pubmed: 19201876doi: 10.4049/jimmunol.0801878google scholar: lookup
        12. Johnson WC, Bastos RG, Davis WC, Goff WL. Bovine WC1(-) gammadeltaT cells incubated with IL-15 express the natural cytotoxicity receptor CD335 (NKp46) and produce IFN-gamma in response to exogenous IL-12 and IL-18.. Dev Comp Immunol 2008;32(8):1002-10.
          pubmed: 18329100doi: 10.1016/j.dci.2008.01.011google scholar: lookup
        13. Joncker NT, Fernandez NC, Treiner E, Vivier E, Raulet DH. NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model.. J Immunol 2009 Apr 15;182(8):4572-80.
          pmc: PMC2938179pubmed: 19342631doi: 10.4049/jimmunol.0803900google scholar: lookup
        14. Jozaki K, Shinkai H, Morozumi T, Tanaka-Matsuda M, Eguchi-Ogawa T, Wada Y, Uenishi H. Cloning, expression, and polymorphisms of natural killer cell receptor NCR1 in pigs.. Anim Biotechnol 2010 Jul;21(3):156-63.
          pubmed: 20665287doi: 10.1080/10495391003617663google scholar: lookup
        15. Kulberg S, Boysen P, Storset AK. Reference values for relative numbers of natural killer cells in cattle blood.. Dev Comp Immunol 2004 Jul 1;28(9):941-8.
          pubmed: 15183034doi: 10.1016/j.dci.2004.02.004google scholar: lookup
        16. Lanier LL. NK cell recognition.. Annu Rev Immunol 2005;23:225-74.
          pubmed: 15771571doi: 10.1146/annurev.immunol.23.021704.115526google scholar: lookup
        17. Lunn DP, McClure JT, Schobert CS, Holmes MA. Abnormal patterns of equine leucocyte differentiation antigen expression in severe combined immunodeficiency foals suggests the phenotype of normal equine natural killer cells.. Immunology 1995 Mar;84(3):495-9.
          pmc: PMC1415121pubmed: 7751035
        18. Mandelboim O, Lieberman N, Lev M, Paul L, Arnon TI, Bushkin Y, Davis DM, Strominger JL, Yewdell JW, Porgador A. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells.. Nature 2001 Feb 22;409(6823):1055-60.
          pubmed: 11234016doi: 10.1038/35059110google scholar: lookup
        19. Noronha LE, Harman RM, Wagner B, Antczak DF. Generation and characterization of monoclonal antibodies to equine CD16.. Vet Immunol Immunopathol 2012 Apr 15;146(2):135-42.
          pmc: PMC3684017pubmed: 22424928doi: 10.1016/j.vetimm.2012.02.006google scholar: lookup
        20. Noronha LE, Huggler KE, de Mestre AM, Miller DC, Antczak DF. Molecular evidence for natural killer-like cells in equine endometrial cups.. Placenta 2012 May;33(5):379-86.
          pmc: PMC3319276pubmed: 22357194doi: 10.1016/j.placenta.2012.01.018google scholar: lookup
        21. Ponassi M, Cantoni C, Biassoni R, Conte R, Spallarossa A, Pesce A, Moretta A, Moretta L, Bolognesi M, Bordo D. Structure of the human NK cell triggering receptor NKp46 ectodomain.. Biochem Biophys Res Commun 2003 Sep 19;309(2):317-23.
          pubmed: 12951052doi: 10.1016/j.bbrc.2003.08.007google scholar: lookup
        22. Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, Moretta L, Moretta A. p46, a novel natural killer cell-specific surface molecule that mediates cell activation.. J Exp Med 1997 Oct 6;186(7):1129-36.
          pmc: PMC2211712pubmed: 9314561doi: 10.1084/jem.186.7.1129google scholar: lookup
        23. Storset AK, Slettedal IO, Williams JL, Law A, Dissen E. Natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs.. Eur J Immunol 2003 Apr;33(4):980-90.
          pubmed: 12672064doi: 10.1002/eji.200323710google scholar: lookup
        24. Viveiros MM, Antczak DF. Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.. Dev Comp Immunol 1999 Sep;23(6):521-32.
          pubmed: 10512462doi: 10.1016/s0145-305x(99)00030-0google scholar: lookup
        25. Wagner B, Hillegas JM, Babasyan S. Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses.. Vet Immunol Immunopathol 2012 Apr 15;146(2):125-34.
          pubmed: 22405681doi: 10.1016/j.vetimm.2012.02.007google scholar: lookup
        26. Wagner B, Hillegas JM, Antczak DF. A monoclonal antibody to equine interleukin-4.. Vet Immunol Immunopathol 2006 Apr 15;110(3-4):363-7.
          pubmed: 16480777doi: 10.1016/j.vetimm.2006.01.001google scholar: lookup
        27. Wagner B, Radbruch A, Rohwer J, Leibold W. Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE.. Vet Immunol Immunopathol 2003 Mar 20;92(1-2):45-60.
          pubmed: 12628763doi: 10.1016/s0165-2427(03)00007-2google scholar: lookup
        28. Wagner B, Robeson J, McCracken M, Wattrang E, Antczak DF. Horse cytokine/IgG fusion proteins--mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines.. Vet Immunol Immunopathol 2005 May 1;105(1-2):1-14.
          pubmed: 15797470doi: 10.1016/j.vetimm.2004.11.010google scholar: lookup
        29. Walzer T, Jaeger S, Chaix J, Vivier E. Natural killer cells: from CD3(-)NKp46(+) to post-genomics meta-analyses.. Curr Opin Immunol 2007 Jun;19(3):365-72.
          pubmed: 17442558doi: 10.1016/j.coi.2007.04.004google scholar: lookup
        30. Yu J, Mitsui T, Wei M, Mao H, Butchar JP, Shah MV, Zhang J, Mishra A, Alvarez-Breckenridge C, Liu X, Liu S, Yokohama A, Trotta R, Marcucci G Jr, Benson DM, Loughran TP Jr, Tridandapani S, Caligiuri MA. NKp46 identifies an NKT cell subset susceptible to leukemic transformation in mouse and human.. J Clin Invest 2011 Apr;121(4):1456-70.
          pmc: PMC3069763pubmed: 21364281doi: 10.1172/JCI43242google scholar: lookup

        Citations

        This article has been cited 9 times.
        1. Jaworska J, de Mestre AM, Wiśniewska J, Wagner B, Nowicki A, Kowalczyk-Zięba I, Wocławek-Potocka I. Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares-A Preliminary Study. Animals (Basel) 2022 Nov 30;12(23).
          doi: 10.3390/ani12233373pubmed: 36496894google scholar: lookup
        2. Antczak DF, Allen WRT. Placentation in Equids. Adv Anat Embryol Cell Biol 2021;234:91-128.
          doi: 10.1007/978-3-030-77360-1_6pubmed: 34694479google scholar: lookup
        3. Patel RS, Tomlinson JE, Divers TJ, Van de Walle GR, Rosenberg BR. Single-cell resolution landscape of equine peripheral blood mononuclear cells reveals diverse cell types including T-bet(+) B cells. BMC Biol 2021 Jan 22;19(1):13.
          doi: 10.1186/s12915-020-00947-5pubmed: 33482825google scholar: lookup
        4. Tallmadge RL, Wang M, Sun Q, Felippe MJB. Transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses. PLoS One 2018;13(9):e0202646.
          doi: 10.1371/journal.pone.0202646pubmed: 30183726google scholar: lookup
        5. Grøndahl-Rosado C, Bønsdorff TB, Brun-Hansen HC, Storset AK. NCR1+ cells in dogs show phenotypic characteristics of natural killer cells. Vet Res Commun 2015 Mar;39(1):19-30.
          doi: 10.1007/s11259-014-9624-zpubmed: 25434421google scholar: lookup
        6. Connelley TK, Longhi C, Burrells A, Degnan K, Hope J, Allan AJ, Hammond JA, Storset AK, Morrison WI. NKp46+ CD3+ cells: a novel nonconventional T cell subset in cattle exhibiting both NK cell and T cell features. J Immunol 2014 Apr 15;192(8):3868-80.
          doi: 10.4049/jimmunol.1302464pubmed: 24639352google scholar: lookup
        7. Mair KH, Müllebner A, Essler SE, Duvigneau JC, Storset AK, Saalmüller A, Gerner W. Porcine CD8αdim/-NKp46high NK cells are in a highly activated state. Vet Res 2013 Mar 1;44(1):13.
          doi: 10.1186/1297-9716-44-13pubmed: 23452562google scholar: lookup
        8. Holmes CM, Wagner B. Characterization of Nasal Mucosal T Cells in Horses and Their Response to Equine Herpesvirus Type 1. Viruses 2024 Sep 25;16(10).
          doi: 10.3390/v16101514pubmed: 39459849google scholar: lookup
        9. Bude SA, Lu Z, Zhao Z, Zhang Q. Pseudorabies Virus Glycoproteins E and B Application in Vaccine and Diagnosis Kit Development. Vaccines (Basel) 2024 Sep 20;12(9).
          doi: 10.3390/vaccines12091078pubmed: 39340108google scholar: lookup
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