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The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction

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Abstract

The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence.

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Abbreviations

18:1 Biotinyl PE:

1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl)

Abs:

antibodies

aIgG1:

anti-IgG1

aKi-67:

anti-Ki-67

aNGcGM3:

anti-NGcGM3

Bc:

B lymphocytes or B cells

CIM:

Centro de Inmunología Molecular

GM3:

monosialodihexosyl-ganglioside

hs:

hours

IgGs:

immunoglobulins G

iNKT:

invariant natural killer T cells

MeOH:

methanol

min:

minutes

NAc:

N-Acetylneuraminic acid

NAcGM3:

N-Acetyl-GM3

NGc:

N-Glycolylneuraminic acid

NGcGM3:

N-Glycolyl-GM3

NKT:

natural killer T cells

ON:

overnight

PFA:

paraformaldehyde

SEM:

standard error of the mean

TCR:

T cell receptor

TMC:

tonsillar mononuclear cells

β-GluCer:

β-Glucosylceramide

α-GalCer:

α-Galactosylceramide

References

  1. Fishman PH, Brady RO (1976) Biosynthesis and function of gangliosides. Science 194(4268):906–915

    Article  CAS  PubMed  Google Scholar 

  2. Varki A (2011) Colloquium paper: uniquely human evolution of sialic acid genetics and biology. Proc Natl Acad Sci USA 107(Suppl 2):8939–8946

    Google Scholar 

  3. Varki A, Kannagi R, Toole BP (2009) Glycosylation changes in cancer. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology, 2nd edn. Cold Spring Harbor, New York (Chapter 44)

    Google Scholar 

  4. Fernandez LE, Gabri MR, Guthmann MD, Gomez RE, Gold S, Fainboim L, Gomez DE, Alonso DF (2010) NGcGM3 ganglioside: a privileged target for cancer vaccines. Clin Dev Immunol 2010:814397. doi:10.1155/2010/814397

    Article  PubMed  PubMed Central  Google Scholar 

  5. Guthmann MD, Castro MA, Cinat G, Venier C, Koliren L, Bitton RJ, Vazquez AM, Fainboim L (2006) Cellular and humoral immune response to N-Glycolyl-GM3 elicited by prolonged immunotherapy with an anti-idiotypic vaccine in high-risk and metastatic breast cancer patients. J Immunother 29(2):215–223

    Article  CAS  PubMed  Google Scholar 

  6. Alfonso M, Diaz A, Hernandez AM, Perez A, Rodriguez E, Bitton R, Perez R, Vazquez AM (2002) An anti-idiotype vaccine elicits a specific response to N-Glycolyl sialic acid residues of glycoconjugates in melanoma patients. J Immunol 168(5):2523–2529

    Article  CAS  PubMed  Google Scholar 

  7. Alfonso S, Diaz RM, de la Torre A et al (2007) 1E10 anti-idiotype vaccine in non-small cell lung cancer: experience in stage IIIb/IV patients. Cancer Biol Ther 12:1847–1852

    Article  Google Scholar 

  8. Scursoni AM, Galluzzo L, Camarero S et al (2011) Detection of N-Glycolyl GM3 ganglioside in neuroectodermal tumors by immunohistochemistry: an attractive vaccine target for aggressive pediatric cancer. Clin Dev Immunol 2011:245181. doi:10.1155/2011/245181

    Article  PubMed  PubMed Central  Google Scholar 

  9. Padler-Karavani V, Yu H, Cao H, Chokhawala H, Karp F, Varki N, Chen X, Varki A (2008) Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: potential implications for disease. Glycobiology 18(10):818–830

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tarlinton D, Good-Jacobson K (2013) Diversity among memory B cells: origin, consequences, and utility. Science 341(6151):1205–1211

    Article  CAS  PubMed  Google Scholar 

  11. Rajewsky K (1996) Clonal selection and learning in the antibody system. Nature 381(6585):751–758

    Article  CAS  PubMed  Google Scholar 

  12. Galli G, Nuti S, Tavarini S, Galli-Stampino L, De Lalla C, Casorati G, Dellabona P, Abrignani S (2003) CD1d-restricted help to B cells by human invariant natural killer T lymphocytes. J Exp Med 197(8):1051–1057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dellabona P, Padovan E, Casorati G, Brockhaus M, Lanzavecchia A (1994) An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J Exp Med 180(3):1171–1176

    Article  CAS  PubMed  Google Scholar 

  14. Lantz O, Bendelac A (1994) An invariant T cell receptor alpha chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8-T cells in mice and humans. J Exp Med 180(3):1097–1106

    Article  CAS  PubMed  Google Scholar 

  15. Venkataswamy MM, Porcelli SA (2009) Lipid and glycolipid antigens of CD1d-restricted natural killer T cells. Semin Immunol 22(2):68–78

    Article  PubMed  PubMed Central  Google Scholar 

  16. Vinuesa CG, Chang PP (2013) Innate B cell helpers reveal novel types of antibody responses. Nat Immunol 14(2):119–126

    Article  CAS  PubMed  Google Scholar 

  17. Cox D, Fox L, Tian R, Bardet W, Skaley M, Mojsilovic D, Gumperz J, Hildebrand W (2009) Determination of cellular lipids bound to human CD1d molecules. PLoS ONE 4(5):e5325. doi:10.1371/journal.pone.0005325

    Article  PubMed  PubMed Central  Google Scholar 

  18. Zemmour J, Little AM, Schendel DJ, Parham P (1992) The HLA-A, B “negative” mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutation in the translation initiation codon. J Immunol 148(6):1941–1948

    CAS  PubMed  Google Scholar 

  19. Exley M, Garcia J, Balk SP, Porcelli S (1997) Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8-T cells. J Exp Med 186(1):109–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Shiratsuchi T, Schneck J, Kawamura A, Tsuji M (2009) Human CD1 dimeric proteins as indispensable tools for research on CD1-binding lipids and CD1-restricted T cells. J Immunol Methods 345:49–59

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Webb TJ, Li X, Giuntoli RL, Lopez PH, Heuser C, Schnaar RL, Tsuji M, Kurts C, Oelke M, Schneck JP (2012) Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer. Cancer Res 72:3744–3752

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lombardi V, Stock P, Singh AK, Kerzerho J, Yang W, Sullivan BA, Li X, Shiratsuchi T, Hnatiuk NE, Howell AR, Yu KO, Porcelli SA, Tsuji M, Kronenberg M, Wilson SB, Akbari O (2010) A CD1d-dependent antagonist inhibits the activation of invariant NKT and prevents development of allergen-induced airway hyperreactivity. J Immunol 184:2107–2115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Singh AK, Harrison SH, Schoeniger JS (2000) Gangliosides as receptors for biological toxins: development of sensitive fluoroimmunoassays using ganglioside-bearing liposomes. Anal Chem 72:6019–6024

    Article  CAS  PubMed  Google Scholar 

  24. Perez ME, Billordo LA, Baz P, Fainboim L, Arana E (2014) Human memory B cells isolated from blood and tonsils are functionally distinctive. Immunol Cell Biol 92(10):882–887

    Article  CAS  PubMed  Google Scholar 

  25. Tahir SM, Cheng O, Shaulov A, Koezuka Y, Bubley GJ, Wilson SB, Balk SP, Exley MA (2001) Loss of IFN-γ production by invariant NK T cells in advanced cancer. J Immunol 167(7):4046–4050

    Article  CAS  PubMed  Google Scholar 

  26. Exley M, Porcelli S, Furman M, Garcia J, Balk S (1998) CD161 (NKR-P1A) costimulation of CD1d-dependent activation of human T cells expressing invariant V alpha 24 J alpha Q T cell receptor alpha chains. J Exp Med 188(5):867–876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Schwarting R, Gerdes J, Niehus J, Jaeschke L, Stein H (1986) Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67. J Immunol Methods 90(1):65–70

    Article  CAS  PubMed  Google Scholar 

  28. Bendelac A, Rivera MN, Park SH, Roark JH (1997) Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 15:535–562

    Article  CAS  PubMed  Google Scholar 

  29. Hong S, Scherer DC, Singh N, Mendiratta SK, Serizawa I, Koezuka Y, Van Kaer L (1999) Lipid antigen presentation in the immune system: lessons learned from CD1d knockout mice. Immunol Rev 169:31–44

    Article  CAS  PubMed  Google Scholar 

  30. Singh N, Hong S, Scherer DC, Serizawa I, Burdin N, Kronenberg M, Koezuka Y, Van Kaer L (1999) Cutting edge: activation of NK T cells by CD1d and alpha-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype. J Immunol 163(5):2373–2377

    CAS  PubMed  Google Scholar 

  31. Padler-Karavani V, Hurtado-Ziola N, Pu M et al (2011) Human xeno-autoantibodies against a non-human sialic acid serve as novel serum biomarkers and immunotherapeutics in cancer. Cancer Res 71(9):3352–3363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Haig NA, Guan Z, Li D, McMichael A, Raetz CR, Xu XN (2011) Identification of self-lipids presented by CD1c and CD1d proteins. J Biol Chem 286(43):37692–37701

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Brennan PJ, Tatituri RV, Brigl M, Kim EY, Tuli A, Sanderson JP, Gadola SD, Hsu FF, Besra GS, Brenner MB (2011) Invariant natural killer T cells recognize lipid self-antigen induced by microbial danger signals. Nat Immunol 12:1202–1211

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kain L, Webb B, AndersonL BL, Deng S, Costanzo A, Holt M, Self K, Zhao M, Teyton A, Kronenberg M, Everett C, Bendelac A, Zajonc DM, Savage PB, Teyton L (2014) The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian alpha-linked glycosylceramides. Immunity 41(4):543–554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Heitmann D, Ziehr H, Muthing J (1998) Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 710:1–8

    Article  CAS  PubMed  Google Scholar 

  36. Muindi K, Cernadas M, Watts GF et al (2010) Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphingolipids presented by CD1d [corrected]. Proc Natl Acad Sci 107(7):3052–3057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Park JE, Wu DY, Prendes M, Lu SX, Ragupathi G, Schrantz N, Chapman PB (2008) Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand. Immunology 123(1):145–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gritzapis AD, Dimitroulopoulos D, Paraskevas E, Baxevanis CN, Papamichail M (2002) Large-scale expansion of CD3(+)CD56(+) lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants. Cancer Immunol Immunother 51(8):440–448

    CAS  PubMed  Google Scholar 

  39. Giroux M, Denis F (2005) CD1d-unrestricted human NKT release chemokines upon Fas engagement. Blood 105(2):703–710

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We are indebted to Cecilia Venier and Marcelo Guthmann for their technical assistance and critical reading of the manuscript, respectively. We also thank Facundo Batista, who kindly supplied reagents when we needed them most due to the governmental restrictions on importing goods. We thank Luis Enrique Fernandez and his group at the CIM, Havana, Cuba, for generously providing liposomes and 14F7 mAbs. This research was funded by grants from the following Argentinean governmental agencies: ANPCyT (PICT PRH 00078, granted to scientists returning to the country), CONICET (PIP 0101, granted to young investigators), and UBA (UBACyT 20020110200026, granted to newly formed groups). María Virginia Gentilini and María Eugenia Pérez were recipients of CONICET postdoctoral and postgraduate scholarships, respectively. We thank Otolaryngology and Hemotherapy Services at Clinical Hospital for providing samples.

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Authors and Affiliations

  1. Institute of Immunology, Genetics and Metabolism (INIGEM), Clinical Hospital, University of Buenos Aires, National Council for Scientific and Technological Research, Av Córdoba 2351, C1120AAF, Buenos Aires, Argentina

    M. Virginia Gentilini, M. Eugenia Pérez, Pablo Mariano Fernández, Leonardo Fainboim & Eloísa Arana

  2. Department of Immunogenetics, School of Exact Sciences, University of Misiones, Posadas, Misiones, Argentina

    M. Eugenia Pérez

  3. Department of Immunology, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina

    Pablo Mariano Fernández, Leonardo Fainboim & Eloísa Arana

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  1. M. Virginia Gentilini
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  2. M. Eugenia Pérez
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Correspondence to Eloísa Arana.

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Gentilini, M.V., Pérez, M.E., Fernández, P.M. et al. The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction. Cancer Immunol Immunother 65, 551–562 (2016). https://doi.org/10.1007/s00262-016-1812-y

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