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DevoTed_miR Report Summary

Project ID: 646701
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - DevoTed_miR (MicroRNA determinants of the balance between effector and regulatory T cells in vivo)

Reporting period: 2015-07-01 to 2016-12-31

Summary of the context and overall objectives of the project

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 exper-imental 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 identifica-tion 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 inflammatory or autoim-mune diseases in our society. Diseases such as type I diabetes, multiple scle-rosis or Crohn’s disease constitute huge burdens and urge new therapeutic developments. 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 regula-tory 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 regula-tory T cell subsets in vivo, using infection and autoimmune models, thus attest-ing the physiological relevance of the miRNA-mediated mechanisms.
4. Dissect the external cues and intracellular mechanisms that regulate candi-date miRNA expression in specific effector or regulatory T cell subsets.
5. Identify the mRNA networks controlled by candidate miRNAs using a combi-nation of bioinformatics and biochemical assays, and couple the effects of miRNA and mRNA manipulation on effector or regulatory T cell subsets in vivo.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have been analyzing the miRNA repertoires of CD4+ T cell subsets (Th1, Th17 and Treg), CD8+ and  T cells. The most advanced sub-project (already developed in Aims 1, 2, 3 and 5) is the latter, which we will detail in this report. The other sub-projects are still at the level of Aim 1 for CD4+ T cell subsets; and Aim 2 for CD8+ T cells.

Aim 1: Identification of the miRNA repertoires of specific in vivo-generated effector T cell (Teff) subsets
To address the role of miRNAs in the differentiation of effector, IFN versus IL-17 producing  T cells, we have performed miRNA expression profiling (6th Gen miRNA array, Exiqon). The two functional  T cell subsets were ex-vivo isolated from peripheral lymphoid organs (lymph node and spleen) using two surface markers, CD27 and CCR6, given that CD27+  T cells produced IFN whereas CD27- CCR6+  T cells made IL-17, as we previously described (Ribot et al. Nat Immunol 2009). We identified 35 miRNAs differentially ex-pressed miRNAs between 27+ and 27-CCR6+ cells. Strikingly, we found only two related miRNAs, miR-146a and miR-146b, to be overexpressed in 27-CCR6+ T cells when compared to 27+ cells. This result was validated by quantitative RT-PCR (qPCR) analysis of miR-146a mRNA levels in peripheral 27+ and27- T cell samples. miR-146a showed an approximately 4-fold higher expression levels in peripheral 27- T cells when compared to their 27+ T cells counterparts. Contrary to miR-146a, qPCR analysis of miR-146b revealed very low levels and a tendency but no significant changes between 27- and 27+ T cells. Of note, miR-146a expression levels were also high in CD4+ T cell subsets, whereas miR-146b was low abundant in all T cell popula-tions analysed. We thus pursued our studies with miR-146a, the only abundant miRNA enriched in 27- T cells.
Interestingly, the analysis of thymic subsets allowed us to establish that miR-146a is already differentially expressed during  T cell development as miR-146a relative expression levels is very low in thymic DN2, DN3, pre- T cells (CD25+CD27+) and 27+ T cells when compared to 27- thymocytes. miR-146a is, thus, the first miRNA identified with a differential expression in thymic and peripheral T cell subsets associated with IL-17 and IFN- cytokine pro-duction. This prompted us to investigate whether miR-146a is involved in the regulation of the functional properties of  T cells.

Aim 2: Functional impact of specific miRNAs on Teff differentiation in vitro
To gain insight into the function of miR-146a in effector  T cell differentiation, we performed gain-of-function studies either by retroviral overexpression of a miR-146a construct or by electroporation of miR-146a mimics in T cells. When using retroviral transduction to express the native stem loop of miR-146a in in vitro differentiated  T cells, the mRNA levels of mature miR-146a in-creased approximately 10-fold when compared to those observed in cells retrovirally transduced with GFP alone. miR-146a overexpression led to a significant reduction of IFN-+  T cell frequency when compared to the GFP control indicating that miR-146a restricts IFN- production in  T cells. This effect of miR-146a on IFN- production by  T cells was confirmed by an alternative gain-of-function approach based on the electroporation of  T cells with synthetic miR-146a oligonucleotides. Thus, miR-146a “mimics” significant-ly decreased the frequency of IFN--producing  T cells compared to neutral control oligonucleotides. These results are consistent with the low abundance of miR-146a, that shows a low abundance in IFN- producing 27+ T cells; and lead us to hypothesize that miR-146a high expression in 27- T cells suppressed to prevent IFN- expression.
To further explore the functional role of miR-146a i

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

MiR-146a is, to our knowledge, the first miRNA to play a non-redundant role in T cell differentiation. Its impact on IFN- production may have important implications in inflammatory diseases where this cytokine promotes disease progression, such as ulcerative colitis/ Crohn’s disease or type I diabetes. Thus, we can envisage that the manipulation of miRNA, for example through synthet-ic mimics, may reduce IFN- production and thus attenuate disease pathology.
Our sub-project on CD4+ T cell subsets is at an earlier stage of development but will likely identify novel miRNA targets that can also impact on inflammatory diseases, especially through modulation of the balance between effector and regulatory CD4+ T cell subsets.
In a different context, our work on cancer opens new avenues for the manipula-tion of T cells in hematological malignancies through CK2-based targeted therapies (Ribeiro et al. 2016).

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