Identificação de iGb3 e iGb4 em células de melanoma murino B16F10- Nex2 e efeito antitumoral de células dendríticas primadas com iGb3 mediado por células iNKT

• Autor(a) principal: Dias, Bianca Rachid [UNIFESP]
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://repositorio.unifesp.br/handle/11600/9699
• topic: Dendritic cells; INKT cells; Glycosphingolipids iGb3 and iGb4; GM3; B16F10-Nex2 melanoma; Melanoma B16F10-Nex2; Células iNKT; Glicoesfingolipídeos iGb3, iGb4 e GM3; Células dendríticas; Imuno vigilância

CD1d-restricted iNKT cells are protective against the murine melanoma B16F10- Nex2 growing subcutaneously in syngeneic animals. This is inferred from the fast tumor growth in animals genetically deficient in CD1d (CD1d-KO), which showed a survival time significantly shorter than that of WT animals equally challenged with tumor cells. CD1d belongs to a family of glycoproteins resembling MHC class I, involved in the presentation, chiefly in dendritic cells, of lipidic antigens to iNKT cells. In the present work we focus on the identification of an endogenous lipid component expressed in melanoma cells able to induce an immunosurveillance response based on iNKT. We also investigated the possibility of animal protection against tumor development by using dendritic cells primed with the endogenous lipid. The extraction of membrane lipid components was carried out from in vivo grown tumors, thus avoiding artifacts from the in vitro grown cultures. Three different extraction protocols were tested (A, B, C), and 14 different fractions were obtained and tested for the activation of hybridoma DN32.D3, a cell line of immortalized murine NKT cells. Fraction F3 of protocol A (F3A) activated hybridoma DN32.D3 to produce IL-2. For an efficient presentation of lipids from this fraction we successfully used bone marrow dendritic cells (BMDC) on in vitro and in vivo assays. F3A and NKT-cell activating glycolipids, agalactosylceramide (a-GalCer) and isoglobohexosylceramide (iGb3), were tested. In the attempt to isolate the stimulatory component present in the melanoma F3A fraction, HPTLC was used and revealed with several specific reagents for sialic acid residues, neutral sugars, phosphate and total lipids. The fraction was also separated in silica columns, immunostained with Bandeiraea simplicifolia BS-1 lectin and analyzed by mass spectrometry. Ganglioside GM3 and neutral glycosphingolipids iGb3, Gb3, iGb4 and Gb4 were identified by ESI-LIT-MS (electrospray ionization- linear ion trap- mass spectrometry). By incubation of iGb3 with BMDC and these with DN32.D3 cells, the latter were activated to produce IL-2. GM3 consistently inhibited the activation of iNKT cells. Cytotoxicity assays were then carried out, in which we found that NKT cells stimulated by BMDC, primed with a-GalCer or iGb3, encircled the B16F10-Nex2 tumor cells as visualized by fluorescent microscopy. NKT cells promoted lysis of up to 40% of tumor cells. In vivo tests showed that mice injected endovenously with murine melanoma cells and treated with dendritic cells primed with a-GalCer or iGb3, had lungs with 4-fold fewer nodules than an equally challenged but untreated group. The present results show that the murine melanoma B16F10-Nex2 expresses iGb3 and its precursor iGb4, being able to activate NKT cells and conferring them a cytotoxic activity in vitro against melanoma. Such lipid (iGb3) was also protective in vivo reducing the melanoma pulmonary metastases when presented by dendritic cells used in cellular therapy protocol. This is the fist work showing effectively that an endogenous CD1d-restricted glycolipid able to activate iNKT cells display a protective antitumor effect, using cellular therapy with dendritic cells.