Mecanismos moleculares de ativação das células T γδ humanas na sua interação com células tumorais

Abstract

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.