Browsing by Author "Pamplona, Ana"
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- 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.
- 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.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Silva, Marta; Pamplona, Ana; Enguita, Francisco; 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 (patho)physiological contexts. This capacity stems from a complex thymic process of ‘developmental pre-programming’, after which a large fraction of γδ T cells migrates to peripheral sites already committed to producing IL-17 or IFN-γ. We have previously found that miR-146a maintains the cell identity of peripheral IL-17-committed gδ T cells by inhibiting IFN-g production. To further address the role of microRNAs in γδ T cell differentiation, we isolated pure IL-17+ and IFN-γ+ γδ T cell populations from the peripheral lymphoid organs of a double reporter IL-17-GFP: IFN-γ-YFP mouse strain to perform small RNA-sequencing. This allowed us to identify distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten microRNAs differentially expressed between IL-17+ and IFN-γ+ subsets to further characterize. We first analyzed the expression pattern of each candidate microRNA in γδ T cell subsets throughout mouse ontogeny and upon gain-of-function studies in in vitro γδ T cell cultures. Our results indicate that while some microRNAs regulate γδ T cell development in the thymus, other candidates modulate their peripheral effector functions. More specifically, using a miR-181a deficient mouse model, we have found that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the IL-17/IFN-γ balance towards the IL-17-pathway in neonatal life, which is further maintained in the periphery during adult life. On the other hand, miR-7a-5p and miR-139-5p, overexpressed in peripheral IFN-g+ γδ T cells, regulate peripheral γδ T cell effector functions, either promoting functional plasticity or acting as an IFN-γ auto-repressor, respectively. Finally, miR-322-5p and miR-450b-3p, overexpressed in IL-17+ γδ T cells, may have therapeutic potential by modulating IFN-γ levels, which are critical in anti-tumoral and antiviral responses.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Sobral, Daniel; Cunha, Carolina; Silva, Marta; Pamplona, Ana; Enguita, Francisco; 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 (patho)physiological contexts. This capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a large fraction of γδ T cells migrate to peripheral sites already committed to producing either IL-17 or IFN-γ. To globally address the role of microRNAs in effector γδ T cell differentiation, we established a double reporter IL-17-GFP: IFN-γ-YFP mouse strain and isolated pure IL-17+ and IFN-γ+ γδ T cell populations from peripheral lymphoid organs to perform small RNA-sequencing. This allowed us to identify distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten candidate microRNAs differentially expressed between IL-17+ and IFN-γ+ γδ T cells to functionally study further. Our results indicate that while some microRNAs, such as miR-128-3p and miR181a-5p, regulate γδ T cell development in the thymus, other candidates, including miR-7a-5p, miR-139-5p, miR-322-5p, and miR-450b-3p, modulate peripheral γδ T cell effector functions. Furthermore, using a miR-181a deficient mouse model, we have demonstrated that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the in vivo IL-17/IFN-γ balance towards the IL-17 pathway in the neonatal thymus, which is further maintained in the periphery during adult life. These data demonstrate the impact of microRNAs on the development, differentiation, and functional identity of effector γδ T cell subsets, which may open new avenues for their manipulation in disease settings.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; Pamplona, Ana; Enguita, Francisco; 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 (patho)physiological contexts. This capacity stems from a complex process of ‘developmental pre-programming in the thymus, after which a large fraction of γδ T cells migrate to peripheral sites already committed to producing either the IL-17 or IFN-γ. We have previously found that one microRNA, miR-146a, maintains peripheral γδ T cell identity by inhibiting IFN-g production by the IL-17-committed CD27− gδ T cell subset. To further and more globally address the role of microRNAs in effector γδ T cell differentiation, we established a double reporter IL17-GFP:IFN-γ-YFP mouse strain and isolated pure IL-17+ and IFN-γ+ γδ T cell populations from the peripheral lymphoid organs to perform small RNA-sequencing. This allowed us to identify clearly distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten candidate microRNAs differentially expressed between IL-17+ and IFN-γ+ γδ T cells to study further. We characterized the detailed expression pattern of each candidate microRNA in γδ T cell subsets throughout mouse ontogeny and upon gain-of-function studies in in vitro cultures of γδ T cells. Our results indicate that while some microRNAs, such as miR-128-3p and miR181a-5p, regulate γδ T cell development in the thymus, other candidates, including miR-7a-5p, miR-139-5p, miR-322-5p, and miR-450b-3p, modulate peripheral γδ T cell effector functions. More specifically, using a miR-181a deficient mouse model, we have found that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the in vivo IL-17/IFN-γ balance towards the IL-17 pathway in neonatal life, which is further maintained in the periphery during adult life. On the other hand, miR-7a-5p and miR-139-5p, overexpressed in peripheral IFN-g+ γδ T cells, regulate peripheral γδ T cell effector functions, either acting as an IFN-γ auto-repressor (miR-139-5p) or promoting functional plasticity (miR-7a-5p). Finally, miR-322-5p and miR-450b-3p, overexpressed in IL-17+ γδ T cells, may have therapeutic potential by modulating the production of IFNγ, whose levels are critical in anti-tumoral and anti-viral responses. These data demonstrate the impact of microRNAs on the differentiation and functional identity of effector γδ T cell subsets, which may open new avenues for their manipulation in disease settings.
- A key role for microRNAs in the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; Enguita, Francisco; Pamplona, Ana; 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 (patho)physiological contexts. This capacity stems from a complex thymic process of ‘developmental pre-programming’, after which a large fraction of γδ T cells migrate to peripheral sites already committed to producing IL-17 or IFN-γ. We have previously found that miR-146a maintains the cell identity of peripheral IL-17-committed gδ T cells by inhibiting IFN-g production. To further address the role of microRNAs in γδ T cell differentiation, we isolated pure IL-17+ and IFN-γ+ γδ T cell populations from the peripheral lymphoid organs of a double reporter IL-17-GFP:IFN-γ-YFP mouse strain to perform small RNA-sequencing. This allowed us to identify distinct microRNA signatures associated with cytokine expression in γδ T cells, from which we selected ten microRNAs differentially expressed between IL-17+ and IFN-γ+ subsets to further characterise. We first analyzed the expression pattern of each candidate microRNA in γδ T cell subsets throughout mouse ontogeny and upon gain-of-function studies in in vitro γδ T cell cultures. Our results indicate that while some microRNAs regulate γδ T cell development in the thymus, other candidates modulate their peripheral effector functions. More specifically, using a miR-181a deficient mouse model, we have found that miR-181a, highly expressed in immature γδ T cell subsets in the thymus, shifts the IL-17/IFN-γ balance towards the IL-17-pathway in neonatal life, which is further maintained in the periphery during adult life. On the other hand, miR-7a-5p and miR-139-5p, overexpressed in peripheral IFN-g+ γδ T cells, regulate peripheral γδ T cell effector functions, either promoting functional plasticity or acting as an IFN-γ auto-repressor, respectively. Finally, miR-322-5p and miR-450b-3p, overexpressed in IL-17+ γδ T cells, may have therapeutic potential by modulating IFN-γ levels, which are critical in anti-tumoral and antiviral responses.
- microRNAs are key regulators of the development and functional differentiation of γδ T cell subsetsPublication . Inácio, Daniel; Amado, Tiago; Silva, Marta; Sobral, Daniel; Cunha, Carolina; Enguita, Francisco; Pamplona, Ana; 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 and non-redundant roles in several (patho)physiological contexts, such as tissue homeostasis, infection, autoimmunity and cancer. This capacity stems from a complex process of ‘developmental pre-programming’ in the thymus, after which a large fraction of γδ T cells migrate to peripheral sites already committed to producing IL-17 or IFN-γ, unlike their ab T cell counterparts1. So far, several miRNAs have been implied in the control of the differentiation and IFN-γ and IL-17 levels by ab Th1 and Th17 cells, respectively2. However, little is known about the action of these post-transcriptional regulators on γδ T cell differentiation. Schmolka et al. showed that miR-146a is selectively enriched in IL-17-biased CD27- γδ T cells and restricts their co-production of IFN-γ by targeting Nod1 mRNA, therefore regulating γδ T cell plasticity3. This isolated work illustrates the need of a more comprehensive study of the miRNA repertoires of γδ T cells and of the regulatory networks they take part in the control of IFN-γ and IL-17 production by these cells.
- Signature cytokine-associated transcriptome analysis of effector γδ T cells identifies subset-specific regulators of peripheral activationPublication . Inácio, Daniel; Amado, Tiago; Pamplona, Ana; Sobral, Daniel; Cunha, Carolina; Santos, Rita F.; Oliveira, Liliana; Rouquié, Nelly; Carmo, Alexandre M.; Lesourne, Renaud; Gomes, Anita; Silva-Santos, Brunoγδ T cells producing either interleukin-17A (γδ17 cells) or interferon-γ (γδIFN cells) are generated in the mouse thymus, but the molecular regulators of their peripheral functions are not fully characterized. Here we established an Il17a-GFP:Ifng-YFP double-reporter mouse strain to analyze at unprecedented depth the transcriptomes of pure γδ17 cell versus γδIFN cell populations from peripheral lymph nodes. Within a very high fraction of differentially expressed genes, we identify a panel of 20 new signature genes in steady-state γδ17 cells versus γδIFN cells, which we further validate in models of experimental autoimmune encephalomyelitis and cerebral malaria, respectively. Among the signature genes, we show that the co-receptor CD6 and the signaling protein Themis promote the activation and proliferation of peripheral γδIFN cells in response to T cell antigen receptor stimulation in vitro and to Plasmodium infection in vivo. This resource can help to understand the distinct activities of effector γδ T cell subsets in pathophysiology.
- γδ-T cells promote IFN-γ–dependent Plasmodium pathogenesis upon liver-stage infectionPublication . Ribot, Julie C.; Neres, Rita; Zuzarte-Luís, Vanessa; Gomes, Anita Quintal; Mancio-Silva, Liliana; Mensurado, Sofia; Pinto-Neves, Daniel; Santos, Miguel M.; Carvalho, Tânia; Landry, Jonathan J. M.; Rolo, Eva A.; Malik, Ankita; Silva, Daniel Varón; Mota, Maria M.; Silva-Santos, Bruno; Pamplona, AnaCerebral malaria (CM) is a major cause of death due to Plasmodium infection. Both parasite and host factors contribute to the onset of CM, but the precise cellular and molecular mechanisms that contribute to its pathogenesis remain poorly characterized. Unlike conventional αβ-T cells, previous studies on murine γδ-T cells failed to identify a nonredundant role for this T cell subset in experimental cerebral malaria (ECM). Here we show that mice lacking γδ-T cells are resistant to ECM when infected with Plasmodium berghei ANKA sporozoites, the liver-infective form of the parasite and the natural route of infection, in contrast with their susceptible phenotype if challenged with P. berghei ANKA-infected red blood cells that bypass the liver stage of infection. Strikingly, the presence of γδ-T cells enhanced the expression of Plasmodium immunogenic factors and exacerbated subsequent systemic and brain-infiltrating inflammatory αβ-T cell responses. These phenomena were dependent on the proinflammatory cytokine IFN-γ, which was required during liver stage for modulation of the parasite transcriptome, as well as for downstream immune-mediated pathology. Our work reveals an unanticipated critical role of γδ-T cells in the development of ECM upon Plasmodium liver-stage infection.