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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

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

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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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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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Keywords

  • CD1d
  • B cells
  • Invariant natural killer T cells
  • Tumor antigen
  • Gangliosides