Browsing by Author "Silva-Santos, Bruno"
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- Control of T cell effector functions by miRNAsPublication . Inácio, Daniel P.; Amado, Tiago; Silva-Santos, Bruno; Gomes, Anita QuintalThe differentiation of effector T cells is a tightly regulated process that relies on the selective expression of lineage-defining master regulators that orchestrate unique transcriptional programs, including the production of distinct sets of effector cytokines. miRNAs are post-transcriptional regulators that are now viewed as critical players in these gene expression networks and help defining cell identity and function. This review summarises the role of individual miRNAs in the regulation of the differentiation of effector T cell subsets, including CD4+ T helper cells, cytotoxic CD8+ T cells and innate-like NKT cells. Moreover, we refer to miRNAs that have been identified to affect simultaneously two or more effector T cell populations, impacting on the balance between effector T cells in vivo, thus constituting potential biomarkers or targets for therapies aiming at boosting immunity or controlling autoimmunity.
- Cross-regulation between cytokine and microRNA pathways in T cellsPublication . Amado, Tiago; Schmolka, Nina; Metwally, Hozaifa; Silva-Santos, Bruno; Gomes, Anita QuintalmicroRNA (miRNA) mediated regulation of protein expression has emerged as an important mechanism in T-cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines, which are both key input signals and major products of T-cell function. Here, we summarize the current data on the molecular cross-talk between cytokines and miRNAs: how cytokines regulate miRNA expression, and how specific miRNAs control cytokine production in T cells. We also describe the inflammatory consequences of deregulating the miRNA/cytokine axis in mice and humans. We believe this topical area will have key implications for immune modulation and treatment of autoimmune pathology.
- Dissecting the IFN-g versus IL-17-specific mRNAomes of effector gd T lymphocytesPublication . Inácio, Daniel; Pamplona, Ana; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Gomes, Anita Q.; Silva-Santos, BrunoThe ability of murine γδ T cells to rapidly produce the pro-inflammatory cytokines interleukin-17 (IL-17) or interferon-γ (IFN-γ) underlies their crucial roles in several pathophysiological contexts, from infection to cancer or autoimmunity. This functional capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a significant fraction of γδ T cells migrate to peripheral sites already committed to producing either IL-17 (gd17) or IFN-γ (gdIFN). While several studies have studied these gd T cell subtypes using surface markers that enrich for effector function, we still lack a characterization of the mRNA transcriptomes that specifically associate with IL-17 or IFN-g production by gd T cells. To overcome this limitation, in this study, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure IL-17+, IFN-γ+, and the remaining IL-17-IFN-g-(DN) γδ T cell populations from the peripheral lymphoid organs to perform RNA sequencing and identify the subset-specific mRNAomes. Overall, we detected the expression of 12822 genes in gd T cells, with a significant number of genes being enriched in gd17 when compared with gdIFN and gdDN cells. Among these, 936 genes were differentially expressed between the three populations, with gd17 and gdIFN cells displaying the most distinct mRNAomes, which highlights their functional specialization, and gdIFN being more similar to DN than gd17 cells. A more detailed analysis of the top 30 differentially expressed genes among the most expressed genes by gd17 and gdIFN cells revealed that the majority of the signature genes increase their expression levels in the periphery upon their egress from the thymus, suggesting that these effector subsets only terminate their differentiation process at peripheral sites. Notably, gd17-associated signature genes are specifically expressed in this subset, unlike gdIFN signature genes, which are also often expressed by gdDN T cells, thus suggesting a developmental relationship between these two subpopulations. Collectively, our data allowed us to identify distinct mRNA signatures directly associated with cytokine expression in γδ T cells, several of which we are now further studying in disease models to identify potential new roles in pathophysiology.
- Dissecting the IFN-g versus IL-17-specific transcriptomes of effector gd T lymphocytes: a new role for signalling adaptor ThemisPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Lesourne, Renaud; Gomes, Anita Q.; Silva-Santos, BrunoThe crucial role of murine γδ T cells in several (patho)physiological contexts stems from a complex process of ‘developmental preprogramming’ in the thymus, after which a significant fraction of γδ T cells populate peripheral sites already endowed with the capacity to secrete either IL-17 or IFN-γ1. However, despite the relevance of these γδ T cell effector subsets, we still lack knowledge on the transcriptomes that specifically associate with IL-17 or IFN-γ production. To address this, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure peripheral IL-17-producing (γδ17) or IFN-γ-producing (γδIFN) γδ T cells to perform RNA-sequencing.
- Dissecting the role of microRNAs in effector versus regulatory CD4+ T cell differentiation during (auto)immune responses in vivoPublication . Cunha, Carolina; Romero, Paula Vargas; Inácio, Daniel; Pais, Ana Teresa; Pelicano, Catarina; Sobral, Daniel; Costa, Marina; Mensurado, Sofia; Sousa, Natacha Gonçalves; Papotto, Pedro; Enguita, Francisco; Gomes, Anita Q.; Silva-Santos, BrunoIntroduction: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They have been implicated in the regulation of the differentiation and function of CD4+ T cell subsets, key players in host defense against pathogens, but also responsible for immune-mediated diseases depending on the correct vs incorrect balance, respectively, between pro-inflammatory effector CD4+T cells, including the IFN-γ-producers T helper 1 (Th)1 and the IL-17-producers Th17 cells, and anti-inflammatory regulatory T cells (Treg). While individual miRNAs were found to regulate the differentiation of specific CD4+ T cell populations, an approach based on in vivo responses is still missing and is key to understanding how miRNA networks control this balance in pathophysiology. Methodology: We have established a triple reporter mouse for Ifng, Il17, and Foxp3, and subjected it to experimental autoimmune encephalomyelitis (EAE). We performed miRNA-seq analysis on Th1, Th17, and Treg cells isolated from the spleen and lymph nodes (LNs) at the peak plateau stage to identify miRNA candidates specifically expressed in one of the cell populations. We have in vivo modulated their expression levels using antagomiRs observed the course of EAE progression and characterised their upstream regulation in vitro in either Th1 or Th17 differentiation conditions. Results: The miRNA-seq data has allowed the identification of 110 miRNAs differentially expressed between effector (Th1 and Th17) and regulatory (Treg) subsets. From those, 9 were specifically upregulated in one population versus the others. In vivo miRNA modulation showed that silencing miR-122 precipitated the onset of EAE, whereas overexpressing miR-1247 decreased the severity of the disease. Cytokine-regulated miR-1247 and miR-122 expression levels are inversely associated with pathogenic signatures of Th1 and Th17 cells between lymphoid and central nervous systems. Discussion: Our results suggest that miR-122 and miR-1247 act as peripheral brakes to CD4+ T cell pathogenicity that are overruled in the inflamed target organ. These findings may have important implications for autoimmune diseases.
- Dissection of the IFN-γ versus IL-17-specific transcriptomes of γδ T cells: a new role for signaling adaptor ThemisPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Lesourne, Renaud; Gomes, Anita Q.; Silva-Santos, BrunoThe crucial role of murine γδ T cells in several (patho)physiological contexts stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a significant fraction of γδ T cells populate peripheral sites already endowed with the capacity to secrete either IL-17 or IFN-γ. However, despite the relevance of these γδ T cell effector subsets, we still lack knowledge on the transcriptomes that specifically associate with IL-17 or IFN-γ production. To address this, we established a double reporter IL-17-GFP:IFN-γ-YFP mouse strain, which allowed us to isolate pure peripheral IL-17-producing (γδ17) or IFN-γ-producing (γδIFN) γδ T cells to perform RNA-sequencing. This led to the identification of the distinct transcriptomes of γδ17 and γδIFN cells, which surprisingly diverged in 6337 differentially (over 1.5-fold) expressed genes. Pathway and gene ontology analyses indicated that γδ17 cells differ from γδIFN cells in their selective ability to sense and integrate external cues, whereas γδIFN stands out in replication, transcription, and translation processes. A detailed analysis of the top differentially expressed genes between γδ17 and γδIFN cells revealed that most of the signature genes of each subset increased their expression levels in the periphery (compared to the thymus), suggesting that γδ17 and γδIFN cells only terminate their differentiation process at peripheral sites. Among the top differentially expressed genes, we found Themis, a T cell-specific gene involved in the regulation of TCR signal strength, to be enriched in γδIFN cells. Importantly, we found that Themis deficiency leads to a dysregulated effector γδ T cell peripheral compartment at steady state, which upon infection with Plasmodium berguei ANKA sporozoites confers Themis-deficient mice full protection from experimental cerebral malaria, a γδIFN-dependent pathology. Accordingly, we observed a less activated and less proliferative γδIFN population in the peripheral lymph nodes of infected Themis-deficient mice compared to Themis-sufficient controls. This work demonstrates the relevance of the characterization of the γδIFN and γδ17 transcriptomes to uncover new players in the regulation of γδ T cell effector functions, which may open new avenues for their manipulation in disease settings.
- Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory gd T cell subsetsPublication . Schmolka, Nina; Serre, Karine; Grosso, Ana R.; Rei, Margarida; Pennington, Daniel J.; Gomes, Anita Q.; Silva-Santos, BrunoTwo distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.
- Epigenetic mechanisms in the regulation of lymphocyte differentiationPublication . Schmolka, Nina; Silva-Santos, Bruno; Gomes, Anita Q.Epigenetics refers to heritable phenotype changes that do not involve alterations in the DNA sequence. While DNA methylation and histone modifications represent the classical epigenetic mechanisms, RNA-based interference can also be integrated into the epigenetic machinery. In particular, microRNAs play important roles in regulating gene expression at the posttranscriptional level. Thus, from the nucleus to the cytoplasm, various “epigenetic” mechanisms can control cell fate decisions and differentiation. This has been extensively documented in lymphocyte biology, from a commitment to the B- and T-cell lineages to the differentiation of multiple effector lymphocyte subsets. Here we review the accumulated knowledge that fundaments our understanding of how classical epigenetics and RNA-based regulatory mechanisms impact B- and T-cell differentiation.
- Identification of a panel of ten cell surface protein antigens associated with immunotargeting of leukemias and lymphomas by peripheral blood γδ T cellsPublication . Gomes, Anita Q.; Correia, Daniel V.; Grosso, Ana R.; Lanca, Telma; Ferreira, Cristina; Lacerda, João F.; Barata, João T.; Silva, Maria Gomes da; Silva-Santos, BrunoBackground: Vγ9Vδ2 T lymphocytes are regarded as promising mediators of cancer immunotherapy due to their capacity to eliminate multiple experimental tumors, particularly within those of hematopoietic origin. However, Vγ9Vδ2 T-cell based lymphoma clinical trials have suffered from the lack of biomarkers that can be used as prognostic of therapeutic success. Design and Methods: We have conducted a comprehensive study of gene expression in acute lymphoblastic leukemia and non-Hodgkin’s lymphomas, aimed at identifying markers of susceptibility versus resistance to Vγ9Vδ2 T cell-mediated cytotoxicity. We employed cDNA microarrays and quantitative real-time PCR to screen 20 leukemia and lymphoma cell lines, and 23 primary hematopoietic tumor samples. These data were analyzed using state-of-the-art bioinformatics, and gene expression patterns were correlated with susceptibility to Vγ9Vδ2 T cell-mediated cytolysis in vitro. Results: 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. Furthermore, some of these candidates displayed a striking variance of expression among primary follicular lymphomas and T-cell acute lymphoblastic leukemias. Conclusions: Our results suggest that hematopoietic tumors display a highly variable repertoire of surface proteins that can impact on Vγ9Vδ2 cell-mediated immune targeting. The prognostic value of the proposed markers can now be evaluated in the upcoming Vγ9Vδ2 T cell-based lymphoma/leukemia clinical trials.
- A key role for microRNAs in regulating IL-17 versus IFN-g production by gamma-delta T cellsPublication . Amado, Tiago; Schmolka, N.; Sobral, Daniel; Enguita, Francisco; Inácio, Daniel; Silva-Santos, Bruno; Gomes, Anita Q.γδ T cells are an important source of the pro-inflammatory cytokines IL-17 and IFN-γ under (patho)physiologic conditions. In the mouse, CD27+ γδ T cells are committed to IFN-γ expression, whereas their CD27- counterparts make IL-17 but are capable of co-expressing both cytokines under inflammatory conditions. We aim to characterize a novel layer of microRNA-mediated regulation of effector γδ T cell differentiation. First, by comparing the microRNA pools of the two CD27-based γδ T cell subsets, we found that miR-146a was selectively enriched in CD27- γδ T cells and restricted their IFN-γ production by targeting Nod1 mRNA. Next, to overcome the caveat of using surface markers, which do not allow isolation of pure populations of IL-17 or IFN-γ producing γδ T cells, we used a double reporter IL-17-GFP: IFNg-YFP mouse strain. Pure IL-17+ or IFN-γ+ γδ T cell populations were isolated from peripheral lymphoid organs and subjected to next-generation sequencing analysis of both microRNA and mRNA repertoires. This allowed us to identify, for the first time, miRNA and mRNA signatures directly associated with cytokine expression, rather than TCR Vγ usage of maturation markers. Furthermore, differentially expressed miRNAs and mRNAs were bioinformatically integrated into networks that allowed the identification of 6 miRNAs predicted to target key determinants of the IL-17 program; and 3 miRNA candidates for the IFN-γ program of γδ T cells. Ongoing molecular assays provide an unprecedented functional characterization of the impact of microRNAs on the identity and differentiation of effector γδ T cell subsets.
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