Giacoma De Tullio

MicroRNAs (miRNAs) are small non-coding RNAs that control the expression of many target messenger RNAs (mRNAs) involved in normal cell functions (differentiation, proliferation and apoptosis). Consequently their aberrant expression and/or functions are related to pathogenesis of many human diseases including cancers. Haematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate commitment, differentiation, proliferation, and apoptosis of hematopoietic stem cells (HSC). Alterations on this network could affect the normal haematopoiesis, leading to the development of haematological malignancies such as lymphomas. The incidence of lymphomas is rising and a significant proportion of patients are refractory to standard therapies. Accurate diagnosis, prognosis and therapy still require additional markers to be used for diagnostic and prognostic purpose and evaluation of clinical outcome. The dysregulated expression or function of miRNAs in various types of lymphomas has been associated with lymphoma pathogenesis. Indeed, many recent findings suggest that almost all lymphomas seem to have a distinct and specific miRNA profile and some miRNAs are related to therapy resistance or have a distinct kinetics during therapy. MiRNAs are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum where they are highly stable and quantifiable within the diagnostic laboratory at each consultation. Accordingly they could be specific biomarkers for lymphoma diagnosis, as well as useful for evaluating prognosis or disease response to the therapy, especially for evaluation of early relapse detection and for greatly assisting clinical decisions making. Here we summarize the current knowledge on the role of miRNAs in normal and aberrant lymphopoiesis in order to highlight their clinical value as specific diagnosis and prognosis markers of lymphoid malignancies or for prediction of therapy response. Finally, we discuss their controversial therapeutic role and future applications in therapy by modulating miRNA.

BACKGROUND: Many aspects of lymphoma pathophysiology indicate mutual interactions between the host immune system and lymphoma cells. These interactions may either promote or control lymphomagenesis. An unconventional subset of CD4−CD8− double-negative T cells (DNTs) has been recently described to contribute specifically to anti-tumor immunity. Indeed, DNTs are involved in immune regulation and tolerance as well as in host defence and inflammation, acting as both regulatory T cells and/or cytotoxic T cells. DNTs are T lymphocytes expressing either αβ or γδ T-cell receptor (TCR) and lacking of CD4,CD8 and CD56.In healthy human donors and murine models, they constitute about the 1-5% of lymphocytes in the peripheral blood and in lymphoid organs. No data are available on the role of DNT cells in human anti-lymphoma immunity. Translating information from murine models, expanded DNT cells would not impair host immunity against lymphoma and perhaps stimulate it. On the other hand, DNT cells also demonstrated to have a direct in vitro anti-tumor activity against lymphoma. Few data are available on the prognostic significance of DNTs in lymphomas, on their interaction with other immune cells and on their functional attitude. AIMS: The aim of this study is to assess the frequency and the functional attitude of circulating DNTs in Lymphoma patients and healthy donors as controls, in order to assess the role of DNTs on clinical outcome. METHODS: For phenotypic and functional characterization of DNTs peripheral blood samples of 30 Lymphoma patients and 16 healthy donors were prospectively collected. The staining of circulating DNT subset was performed with the following conjugated monoclonal antibodies (MoAbs) for surface and intracellular markers: CD3,CD4,CD8,CD56,CD45,TCRαβ,CD45Ra,CD45Ro,CCR7, CD27,CD28, CD30,CD69,GITR,CD95,CD178,CD152,IFN-γ,TNF-α,granzymeB,perforin.Isotype-matched MoAbs will be used as staining controls. For functional studies, DNTs were purified from PBMCs of patients through a negative selection by using a specific MACS microbeads and then cultured for 2 week in complete medium supplemented with anti-CD3 (OKT3), rhIL-2 and rhIL-4. Data was acquired using a 8-colour flow cytometer and analyzed using Kaluza software. Data were compared among the groups using the Mann-Whitney non parametric test or Kruskal–Wallis one-way analysis of variance. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. RESULTS: We observed a significant decrease (p = 0.006) of αβ-DNTs in the peripheral blood of patients with untreated lymphoma (20.5 ± 4.8 SE, ) (Mean + SE) as compared with healthy controls (31.3 ± 3.4),and their number correlated with disease relapse/progression (fig.1-2).In Hodgkin's Lymphoma patients the αβ-DNT frequencies were significantly increased as compared with other histotypes (fig. 3). Interestingly, after ex vivo expansion, DNTs acquired an immunemodulatory cytokine profile, characterized by the secretion of IFN-γ and granzyme B which are known as central component of anti-tumor immune responses (fig.4) CONCLUSIONS: To date, no data has been reported on DNT phenotypic and functional characterization in Lymphoma patients. Our study has demonstrated for the first time that αβ-DNTs could play an important role in both the development and the progression of lymphomas. In addition, based on our preliminary results,it is likely that ex-vivo expanded DNTs exert an anti-tumor activity,thus suggesting their possible use as new strategy for adoptive immune-therapy.