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MicroRNA determinants of the balance between effector and regulatory T cells in vivo

Periodic Reporting for period 4 - DevoTed_miR (MicroRNA determinants of the balance between effector and regulatory T cells in vivo)

Reporting period: 2020-01-01 to 2021-02-28

THE PROBLEM
This project aims to dissect the microRNA (miRNA) networks that control the differentiation of effector and regulatory T cell subsets in vivo, in various experimental models of infection and autoimmunity. We are focusing on three critical mediators of T cell functions: interferon- (IFN-) and interleukin-17A (IL-17), highly pro-inflammatory cytokines; and Foxp3, the transcription factor that confers suppressive properties to regulatory T cells. We envisage the identification of specific miRNAs that can modulate the balance between effector and regulatory T cell subsets, and thus impact on protective immune responses and/ or immune-mediated pathology.

THE IMPORTANCE
This project will provide major conceptual and experimental advances towards manipulating miRNAs either to boost immunity or to treat autoimmunity. This is particularly relevant given the increase in incidence of cancer and autoinflammatory diseases (such as multiple sclerosis or Crohn’s disease) in our society. MiRNAs are an exciting prospect, especially by being easily manipulated, to address this unmet medical need via manipulation of pro-/ anti-inflammatory cytokines.

OBJECTIVES
This project has 5 main objectives:
1. Characterize the miRNA repertoires of in vivo-generated effector and regulatory T cell subsets, isolated from infection or autoimmune models established in a reporter mouse for Ifng, Il17 and Foxp3.
2. Define the individual miRNAs that impact selectively on effector or regulatory T cell differentiation, based on loss- and gain-of-function experiments.
3. Determine the impact of miRNA expression modulation on effector or regulatory T cell subsets in vivo, using infection and autoimmune models, thus attesting the physiological relevance of the miRNA-mediated mechanisms.
4. Dissect the external cues and intracellular mechanisms that regulate candidate miRNA expression in specific effector or regulatory T cell subsets.
5. Identify the mRNA networks controlled by candidate miRNAs using a combination of bioinformatics and biochemical assays, and couple the effects of miRNA and mRNA manipulation on effector or regulatory T cell subsets in vivo.

CONCLUSIONS
This project provided major advances to our understanding of miRNA-mediated regulation of T cell differentiation by identifying novel miRNA regulators of effector T cell functions in vivo:
1. In the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), miR-122 is a negative regulator of the pathogenic phenotype of Th17 cells, acting as a brake of pathogenicity (as seen in peripheral lymphoid organs) that is lost in the central nervous system. These data demonstrate the therapeutic potential of miRNAs that modulate CD4+ T cell differentiation in autoimmunity.
2. In CD8+ T cells, we found that miR-181a limits IFN- production by suppressing the expression of the transcription factor Id2, which in turn promotes the Ifng expression program. Importantly, upon murid herpes virus 4 (MuHV-4) challenge, miR-181a-deficient mice showed a more vigorous IFN-+ CD8+ T cell response, and were able to control viral infection significantly more efficiently than control mice. Collectively, these data established a novel role for miR-181a in regulating IFN-–mediated effector CD8+ T cell responses, with important implications for anti-viral immunity.
3. In gamma-delta (gd) T cells, we identified various miRNAs involved in the differentiation of the their main effector subsets producing IFN-γ and/ or IL-17. This included the first miRNA shown to play a non-redundant role in gd T cell differentiation: miR-146a. We found miR-146a to limit the functional plasticity of IL-17-producing gd T cells, namely their acquisition of IFN-γ expression. This impacted on immunity to intracellular bacteria, but may also be relevant to other diseases where IFN-γ plays pivotal roles, such as cancer, viral infection or severe malaria.
(i) CD4+ T cells
We observed that silencing of miR-122-5p, a miRNA we found enriched in Th17 cells, precipitated the onset of EAE, a mouse model of multiple sclerosis. Additionally, we observed that treatment with miR-122-5p antagomiR increased the frequency and absolute numbers of IL-17+ CD4+ T cells in central nervous system (CNS) infiltrates (analysed at day 13 post immunization) and in the draining lymph nodes (analysed at day 28 post immunization). Together with some additional data on the dynamics of miR-122 expression, our data (in preparation for publication) suggest that miR-122 is a negative regulator of the pathogenic phenotype of Th17 cells, acting as a brake of pathogenicity (as seen in peripheral lymphoid organs) that is lost in the CNS where disease unfolds.

(ii) CD8+ T cells
We found that miRNAs constitute a post-transcriptional brake to IFN- expression by CD8+ T cells, as the genetic interference with the Dicer processing machinery resulted in the increased production of IFN-by both thymic and peripheral CD8+ T cells. We further found that miR-181a limits IFN- production by suppressing the expression of the transcription factor Id2, which in turn promotes the Ifng expression program. Importantly, upon murid herpes virus 4 (MuHV-4) challenge, miR-181a-deficient mice showed a more vigorous IFN-+ CD8+ T cell response, and were able to control viral infection significantly more efficiently than control (miR-181a+/+) mice. Collectively, these data established a novel role for miR-181a in regulating IFN-–mediated effector CD8+ T cell responses in vitro and in vivo. These results were published in Journal of Molecular Medicine (Amado et al. 2020).

(iii) Gamma-delta T cells
We found miR-146a to be specifically enriched in CD27- CCR6+ gd T cells producing IL-17. To further explore the functional role of miR-146a in gd T cell differentiation, we pursued a loss-of-function approach using miR-146a-deficient mice in mixed (1:1) bone marrow (BM) and mixed (1:1) neonatal thymus chimeras. gd27- T cells derived from miR-146a-deficient progenitors had a higher frequency of IFN-g+ IL-17+ (double producers) cells than their WT counterparts. Furthermore, we detected a significantly increased frequency and cell number of double producers in L. monocytogenes infected miR-146a-deficient compared to wild type controls. Altogether, these results indicate that miR-146a acts as a cell-intrinsic break to IFN-g production by gd27- T cells, thereby limiting their functional plasticity. These results were published in Science Immunology (Schmolka et al. 2018).
MiR-146a is, to our knowledge, the first miRNA to play a non-redundant role in the differentiation of pro-inflammatory gd T cell subsets. Its impact on IFN-g production may have important implications in diseases where this cytokine promotes disease progression, such as ulcerative colitis/ Crohn’s disease; or in others where it plays key protective roles, such as cancer. We expect similar impact from our unpublished data on miR-122 in CD4+ T cells in autoimmune diseases, particularly multiple sclerosis.
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