Immunology Letters 137 (2011) 38–45 Contents lists available at ScienceDirect Immunology Letters journal homepage: www.elsevier.com/locate/immlet Cytofluorimetric evaluation of N-glycolylated GM3 ganglioside expression on murine leukocytes A. Miranda a,1 , J. de León a,∗,1 , L. Roque-Navarro b , L.E. Fernández c a Tumor Immunology Direction, Centre of Molecular Immunology, 216 esq 15, Atabey, Playa, PO Box 16040, Havana, Cuba b Engineering Antibody Department, Centre of Molecular Immunology, 216 esq 15, Atabey, Playa, PO Box 16040, Havana, Cuba c Innovation Direction, Centre of Molecular Immunology, 216 esq 15, Atabey, Playa, PO Box 16040, Havana, Cuba article info Article history: Received 25 June 2010 Received in revised form 14 January 2011 Accepted 1 February 2011 Available online 13 February 2011 Keywords: NGcGM3 ganglioside Leukocytes Immune system cells activation abstract Gangliosides are considered relevant components of lipid rafts at the plasma membrane. Antigen encounter, immunological synapse assembly and signal transduction modify lipid raft composition and distribution on immune system cells. On the contrary of other gangliosides, differential expression of the N-glycolylated variant of GM3 (NGcGM3) on murine leukocytes has received limited attention. In partic- ular, whether cell activation modulates the expression of NGcGM3 on lymphoid and myeloid cells is still unexplored. Availability of the NGcGM3 specific 14F7 MAb allows us to characterize by cytofluorimetric assays the presence of this molecule on resting and activated immune system cells. On T cells, preferential expression of NGcGM3 was detected on CD4 + single positive thymocytes, peripheral CD4 + lymphocytes and natural occurring regulatory T cells. In comparison with peritoneal B1 cells, reduced expression of NGcGM3 was observed in peritoneal B2 and splenic B cell subpopulations. Of note, activation of CD4 + and NK 1.1 + cells abrogated NGcGM3 expression while LPS-maturated DC increased the ganglioside level at the plasma membrane. Modifications on the presence of NGcGM3 mediated by cell activation did not influence the expression of the N-acetylated variant of GM3 (NAcGM3). In addition to extend previous descriptions of NGcGM3 expression on immunity cell subpopulations, this work highlights the opposite effect of cellular activation over NGcGM3 levels on lymphoid and myeloid cellular series. Obtained results complement the evaluation of a tumor-specific, non-human sialic acid containing ganglioside that has been considered an attractive target for cancer immunotherapy. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Characterization of the particular distribution of molecules on immune cells has constituted a fundamental strategy to study immune system functionality. Differentially expressed molecules significantly influence the capacity of immune cells to react prop- erly to antigenic stimulus, either acting as signal transducers or as natural ligands of pathogens, contributing to regulatory mech- anisms of immune system. Gangliosides are among the molecules expressed on immune cells whose role in the processes of cell–cell adhesion during inflammation and immune response has been described [1,2]. These glycosphingolipids are essential components Abbreviations: mDC, mature dendritic cells; iDC, immature dendritic cells; NK, natural killer; MAb, monoclonal antibody; MFI, mean fluorescence intensity; NAcGM3, N-acetylated GM3 ganglioside; NGcGM3, N-glycolylated GM3 ganglioside; NANA, N-acetyl neuraminic acid; NGNA, N-glycolyl neuraminic acid. ∗ Corresponding author. Tel.: +53 7 271 6810; fax: +53 7 272 0644. E-mail address: yoel@cim.sld.cu (J. de León). 1 These authors contributed equally. of lipid rafts, cholesterol-rich structures that act as anchoring sites for a wide variety of proteins involved in signals transduction and pathogen response [3–6]. Gangliosides are heterogeneously distributed on different types of tissue, depending on the level of cell differentiation [7–9]. Considerable evidence has been accumulated regarding its differ- ential expression on immune cells. Portner et.al. demonstrated that murine B cells stimulated with endotoxin have greater expression of gangliosides than resting B lymphocytes. Addition- ally, resting B cells preferentially carry gangliosides containing NGNA (N-glycolyl neuraminic acid) rather than NANA (N-acetyl neuraminic acid) on activated B cells, indicating modifications in sialic acid metabolism during stimulation [10]. Differential expression of NAcGM1 on thymocytes subpopulations and mature CD4 + and CD8 + murine lymphocytes has also been reported [11]. Unlike the very limited presence of gangliosides carring the N- glycolyl neuraminic acid in human tissues, excepting some tumors [12–14], these molecules are widely distributed among different species of mammals. The expression of N-glycolylated gangliosides has been described in murine leukocytes. Nakamura et al. demon- 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.02.001