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Mecanismos moleculares de ativação das células T γδ humanas na sua interação com células tumorais
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Resumo(s)
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
Palavras-chave
Linfócitos T Leucemia Linfoma Imunoterapia Teses de doutoramento - 2012