Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/5276
Título: Mecanismos moleculares de ativação das células T γδ humanas na sua interação com células tumorais
Molecular mechanisms of human γδ T cell activation and tumor cell recognition
Autor: Correia, Daniel Vargas, 1981-
Orientador: Santos, Bruno Silva, 1973-
Palavras-chave: Linfócitos T
Teses de doutoramento - 2012
Data de Defesa: 2011
Resumo: The immune system of jawed vertebrates includes various lymphocyte populations capable of recognizing and eliminating tumor cells, which constitutes the basis of cancer immunotherapy. γδ T lymphocytes are innate-like cells that account for 1-15% of human peripheral blood lymphocytes (PBL), and represent the majority of T cells in epithelial tissues of healthy individuals. Moreover, it is well established that both Vδ1+ and Vδ2+ γδ T cell subsets are endowed with strong, MHC-unrestricted cytotoxicity against tumor cells of diverse tissue origin. The unique responsiveness of Vγ9Vδ2 T cells, the dominant subset of γδ PBLs, to non-peptidic prenyl-pyrophosphate antigens (phosphoantigens), constitutes the basis of current γδ T cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of Vγ9Vδ2 T cells have remained unclear. We have here characterized the cellular and molecular events triggered in Vγ9Vδ2 T cells by the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). We compared the molecular signatures produced by HMB-PP stimulation, with those of canonical T cell receptor (TCR) complex signaling, induced by anti-CD3ε monoclonal antibody (OKT3) treatment. HMB-PP activated the MEK/Erk and PI-3K/Akt signaling pathways as rapidly and efficiently as OKT3, and induced an almost identical transcriptional profile in Vγ9+ T cells. Moreover, MEK/Erk and PI-3K/Akt activities were indispensable for the cellular effects of HMB-PP, including γδ T cell activation, proliferation and anti-tumor cytotoxicity, which were also abolished upon antibody blockade of the Vγ9+ TCR during the stimulation period. Thus, our data provided a detailed characterization of HMB-PP as a putative Vγ9Vδ2 TCR agonist. The elucidation of the molecular mechanisms responsible for Vγ9Vδ2 T cell activation, permitted the subsequent dissection of the mechanisms involved in tumor cell recognition. Several hematological tumor cell lines, identified as susceptible or resistant to fully-activated (HMB-PP-treated) Vγ9Vδ2 T cells, were screened for the identification of potential determinants of tumor cell targeting, and biomarkers that could be useful for Vγ9Vδ2 T cell-based cancer clinical trials. We performed a comprehensive transcriptomics study using cDNA microarrays and quantitative real-time PCR, in acute lymphoblastic leukemia and non-Hodgkin’s lymphoma cell lines and primary samples. We identified a panel of 10 genes encoding cell surface proteins that were statistically differentially expressed between “γδ-susceptible” and “γδ-resistant” hematopoietic tumors. Within this panel, 3 genes (ULBP1, TFR2 and IFITM1) were associated with increased susceptibility to Vγ9Vδ2 T-cell cytotoxicity, whereas the other 7 (CLEC2D, NRP2, SELL, PKD2, KCNK12, ITGA6 and SLAMF1) were enriched in resistant tumors, suggesting that hematological tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 T cell-mediated immunotargeting. Among the candidate biomarkers, we established ULBP-1 as a non-redundant determinant of Vγ9Vδ2 T cell recognition of hematological tumors. Furthermore, we observed a frequent down-modulation of ULBP1 expression in primary samples from leukemia and lymphoma patients, which associated with resistance to Vγ9Vδ2 T cell cytotoxicity in vitro. Aiming to overcome potential immune escape mechanisms (such as loss of ULBP-1 expression), we screened a series of T cell-activating compounds in order to elicit γδ T cell-mediated killing of resistant tumors. Phytohemagglutinin (PHA), a plant-derived mitogen, combined with IL-2, induced the differentiation of a novel, highly cytolytic subset of Vδ2(-) Vδ1+ PBLs expressing natural cytotoxicity receptors (NCRs). Thus, NKp30, NKp44 and NKp46 could be selectively upregulated in Vδ1+ cells by AKTdependent signals provided synergistically by γc cytokines (IL-2 or IL-15) and TCR stimulation. Specific gain-of-function and loss-of-function experiments demonstrated that NKp30 makes the most important contribution to leukemia cell recognition. Thus, NKp30+ Vδ1+ T-cells constitute a novel, inducible and specialized killer lymphocyte population whose potential for cancer immunotherapy should be evaluated in future clinical trials.
Descrição: Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2012
URI: http://hdl.handle.net/10451/5276
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