We analyzed herein whether members of the tetraspanin superfamily are involved in human immature dendritic cell (DC) functions such as foreign antigen internalization, phagocytosis, and cell migration. We show that CD63, CD9, CD81, CD82, and CD151 are present in immature DCs. Whereas CD9 and CD81 are mostly expressed at the cell surface, CD63 and CD82 are also located in intracellular organelles. Complexes of monoclonal antibody (Mab) FC-5.01-CD63 or Fab-5.01-CD63 were rapidly translocated "outside-in" and followed the endocytic pathway through early endosomes and lysosomes, reaching major histocompatibility complex (MHC) class IIenriched compartments (MIICs) in less than one hour. Internalization of CD63 was also observed during Saccharomyces cerevisiae phagocytosis. Moreover, an association of CD63 with the -glycan receptor dectin-1 was observed. Mabs against CD9, CD63, CD81, and CD82 enhanced by 50% the migration induced by the chemokines macrophage inflammatory protein-5 (MIP- 5 IntroductionDendritic cells (DCs) comprise a family of professional antigenpresenting cells (APCs) that are sentinels of the immune system. 1 In this regard, DCs have been shown to efficiently stimulate both naive B and T cells and to elicit primary immune responses. 2,3 Their remarkable effectiveness is due to their ability to capture, process and present antigens along with costimulatory signals, and to migrate to secondary lymphoid tissues. 4 Different stages of maturation are responsible for the different functions of DCs. Immature DCs, widely present in peripheral tissues, efficiently uptake antigens but express moderate levels of major histocompatibility complex (MHC) class II and costimulatory molecules. In contrast, mature DCs poorly acquire antigens but express higher levels of MHC class II and costimulatory molecules, are able to migrate into lymph nodes, and become potent activators of resting T cells. [2][3][4][5] Since antigen internalization and processing, as well as cell migration, are essential properties of DCs, the study of the possible involvement of tetraspanins in these processes is a challenging question, as it could bring new insight into the physiologic role of these molecules in DCs and other cells. In recent years, considerable interest has arisen in the expanding tetraspanins family, which are integral membrane proteins with 2 extracellular domains (EC1 and EC2) that are variably glycosylated. 6 The most conspicuous members of this family are CD9, 7 CD63/lamp-3, 8 CD81/TAPA-1, 9 CD82/KAI1, 10 and CD151. 11 Tetraspanins form several specific complexes implicated in a variety of cellular processes such as migration, adhesion, proliferation, and signal transduction, initially leading to the idea that these proteins could play a "molecular facilitator" role. 6 There is growing evidence that points to a possible role of tetraspanins in antigen processing and presentation. In fact, it has been recently found that CD63 is modified after translation during maturation of DCs, and this event is accompanied by morph...
HLA-E is a non-classical MHC molecule whose expression by tumour cells has been recently reported in several human cancer types. We studied HLA-E expression in colorectal cancer patients, its clinical significance and prognostic value, as well as characterized its expression in colorectal cancer cell lines. We analysed HLA-E expression at the transcript level by qRT-PCR in micro-dissected samples and at the protein level by semiquantitative immunohistochemistry on paraffin-embedded tissue sections from 42 biopsies of colorectal cancer patients. We observed that HLA-E transcript and protein are spontaneously overexpressed in a significant proportion of colorectal tumour biopsies, as compared to normal mucosae. We also found a negative correlation between HLA-E expression and the CD57 + cells infiltrate. Moreover, we analysed HLA-E expression in several colorectal cancer cell lines and demonstrated that IFN-γ upregulates the expression of membrane HLA-E in vitro. Interestingly, we demonstrated that colorectal cancer cell lines overexpressing HLA-E at the cell surface inhibited NK-mediated cell lysis. Although IFN-γ regulatory role needs further investigation, we provide evidence suggesting that this cytokine, within the tumour microenvironment, could promote HLA-E translocation to the surface of tumour epithelial cells. Furthermore, we showed that upregulation of HLA-E could be a marker of shorter disease-free survival in Dukes' C patients and we suggest that this molecule renders tumours less susceptible to immune attack.
Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-γ secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.
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