Exploring the impact of gamma-delta T cell subsets in motor performance and Parkinson´s disease progression

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MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

Publication . Gordino, Gisela; Costa Pereira, Sara; Corredeira, Patrícia; Alves, Patrícia; Costa, Luis; Gomes, Anita Q.; Silva-Santos, Bruno; Ribot, Julie

γδ T cells are a conserved population of lymphocytes that contributes to anti-tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL-2 or IL-15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA-181a as a key modulator of human γδ T cell differentiation. We observe that miR-181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR-181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR-181a overexpression restricts their levels of NKG2D and TNF-α. Upon in silico analysis, we identify two miR-181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR-181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next-generation immunotherapies.

2021Artigo científico

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Regulation of human γδ T cell type 1 functional differentiation by microRNAs

Publication . Gordino, Gisela Silva; Ribot, Julie Cécile Caroline; Santos, Bruno Miguel de Carvalho e Silva; Gozzelino, Raffaella

γδ T cells have been proposed as a first line of immune defense, acting in response to a variety of stress-inducible or pathogen-associated metabolites. Their functions include a potent cytolytic and inflammatory activity against a wide range of malignant cells. However, γδ T cell-based clinical trials in cancer patients have only achieved objective responses between 10-33%. Therefore, a better understanding of the mechanisms involved in regulating γδ T cell activation and functional differentiation may be critical for their improving their outcomes in clinical settings. Data from the host laboratory has previously shown γδ T cell anti-tumor properties to be selectively acquired upon stimulation with interleukin (IL)-2 – but not with IL-7 – which induced the de novo expression of the transcription factors (TFs) T-bet and eomesodermin, required for the cytotoxic type 1 program. Critically, while the transcriptional key-players behind γδ T cell acquisition of cytotoxic / type 1 effector functions have been extensively explored, the post-transcriptional mechanisms that modulate γδ T cell anti-tumoral functions are still poorly understood. This PhD study aimed to explore an additional layer in the regulation of γδ T cell type 1 differentiation, by characterizing the post-transcriptional mechanisms mediated by microRNAs (miRs) involved in this process. We identified miR-181a(-5p and -2-3p) and miR-135b-5p to be either downregulated or upregulated, respectively, in differentiated (IL-2 cultured) γδ thymocytes, when compared with their immature (IL-7 cultured) counterparts. Interestingly, we observed a significant inverse correlation between miR-181a(-5p and -2-3p) expression and the protein levels of three important hallmarks of γδ T cell acquisition of type 1 effector program, namely tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and natural killer group 2 member D (NKG2D), suggesting a miR-181a-mediated suppression of γδ T cell differentiation. On the other hand, miR-135b-5p was positively correlated with these differentiation hallmarks, thus suggesting that this miR could promote γδ T cell acquisition of type 1 effector functions. Of note, while the miR candidates levels correlated with both NKG2D and type 1 cytokine production in in vitro-differentiated γδ thymocytes, these associations were no longer sustained for the type 1 cytokine levels in mature γδ peripheral blood lymphocytes (PBLs) ex vivo, indicating that miR-181a(-5p and -2-3p) and miR-135b-5p regulatory effect likely depends on γδ T cell maturation status. In line with this, while miR-181a overexpression significantly decreased TNF-α and NKG2D protein levels in artificially (in vitro-)differentiated γδ T cells, it only impaired the expression of NKG2D in naturally differentiated γδ PBLs, whereas type 1 cytokine production remained unaffected. Also noteworthy was the fact that miR-135b-5p overexpression in γδ PBLs elicited a mild decrease in TNF-α and NKG2D messenger (m)RNA levels, while it did not affect TNF-α, IFN-γ, or NKG2D protein levels. In silico data coupled with a Gene Set Enrichment Analysis (GSEA) identified two potential miR-181a-5p and -2-3p targets, Map3k2 and Notch2, which are linked with T cell development and differentiation processes; and two miR-135b-5p targets, Il10 and Cd39, associated with the establishment of an immunosuppressive phenotype in T cells. Importantly, Map3k2 and Notch2 levels were downregulated in γδ T cells overexpressing miR-181a; while miR-135b-5p overexpression in γδ PBLs impaired Cd39 (but not Il10) levels. Consistently, our luciferase reporter assays validated the direct binding between miR-181a and its two targets, as well as between miR-135b-5p and its Cd39 target. Finally, by assessing these miRs species profile in mature γδ PBLs isolated from metastatic cancer patients versus healthy donors, we found miR-181a(-5p and -2-3p) to be highly expressed in prostate cancer patients, and this pattern associated with lower NKG2D levels. On the other hand, we consistently observed a significant decrease in miR-135b-5p expression for all the cancer patients cohorts, which also associated with a decrease in NKG2D levels in these patients. Furthermore, we found CD39 and IL-10 expression to be increased in γδ PBLs from metastatic cancer patients, and both these molecules expression negatively correlated with NKG2D levels in these patients. Thus, we believe our study constitutes an important addition to the molecular mechanisms that govern the acquisition of type 1 effector functions of human γδ T cells, especially at the post-transcriptional level, which is less explored. Altogether our work highlights the critical role of miR-mediated mechanisms in modulating human γδ T cell type 1 functional differentiation / anti-tumor responses and open new avenues for the design of new (or improved) γδ T cell-based clinical protocols, towards their effective application in cancer immunotherapy.

2022-06Tese de doutoramento

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/128866/2017