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

Periodic Reporting for period 3 - DevoTed_miR (MicroRNA determinants of the balance between effector and regulatory T cells in vivo)

Reporting period: 2018-07-01 to 2019-12-31

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 cancer and autoinflam-matory 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 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.
As initially proposed, we have been analyzing the microRNA repertoires of CD4+, CD8+ and T cells. Here we describe the results obtained with the latter subset, since they have been published in Science Immunology (Schmolka et al.).
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 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 expressed 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, with miR-146a showing the highest levels of expression in peripheral 27- T cells.
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. 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. To further explore the functional role of miR-146a in T cell differentiation, we pursued a loss-of-function analysis using miR-146a-deficient mice. The number of 27+ and 27- as well as the production of IFN- and IL-17 ex vivo were not affected by the absence of miR-146a, but instead impacted the functional plasticity of 27- T cells. In particular, in mixed (1:1) bone marrow (BM) and mixed (1:1) neonatal thymus chimeras, 27- T cells derived from miR-146a-deficient progenitors had a higher frequency of IFN-+ 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- production by 27- T cells, thereby limiting their functional plasticity.
For an unbiased identification of the mRNAs targeted by miR-146a that contribute to IFN- regulation, we conducted differential AGO2 RNA immunoprecipitation experiments followed by deep sequencing (AGO2 RIP-seq) in total CD3+ T cells. To identify miR-146a targets we performed AGO2-RIP experiments in CD3+ T cells retrovirally transduced with either GFP-control or miR-146a-expressing vectors. We identified 225 mRNA targets after differential AGO2-RIP-seq, from which 96 (42,7%) had a predicted miR-146a binding site in their 3’ UTR region. We designed reporter constructs in a pmirGLO Dual-luciferase miRNA target expression vector for the five potential mRNA targets and transiently transfected them into 293 T cells together with an expression plasmid for either miR-146a or GFP-control. We observed a marked reduction in luciferase levels (sensitive to mutation of the miR-146a binding sites in the 3’ UTR) in various targets, most notably Nod1. Moreover, Nod1 (but not other candidates) expression negatively correlated with miR-146a expression in T cell subsets, suggesting that Nod1 could indeed be targeted by miR-146a in T cells. We have therefore analyzed the phenotype of Nod1-deficient mice, and obtained the opposite phenotype to miR-146a KO mice, i.e. a marked depletion of double producers (IFN+IL17+ 27- T cells). This established the miR-146a/ Nod1 pathway as a novel determinant of the functional plasticity of T cells – with miR-146a being the first miRNA to play a non-redundant role in the differentiation of pro-inflammatory T cell subsets.
MiR-146a is, to our knowledge, the first miRNA to play a non-redundant role in the differentiation of pro-inflammatory T cell subsets. Its impact on IFN- 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. Thus, we can envis-age that miRNA manipulation, for example through synthetic mimics or an-tagomiRs, may modulate IFN- levels and thus impact on disease outcome.
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