Idiopathic aplastic anemia (AA) has two key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predict response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified two mechanisms that lead to skewed Treg composition in AA. Firstly, FasL mediated apoptosis on ligand interaction and, secondly, relative IL-2 deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T cell-mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

MicroRNAs are short endogenous noncoding RNAs that play pivotal roles in a diverse range of cellular processes. The miR-181 family is important in T cell development, proliferation, and activation. In this study, we have identified BRK1 as a potential target of miR-181c using a dual selection functional assay and have showed that miR-181c regulates BRK1 by translational inhibition. Given the importance of miR-181 in T cell function and the potential role of BRK1 in the involvement of WAVE2 complex and actin polymerization in T cells, we therefore investigated the influence of miR-181c-BRK1 axis in T cell function. Stimulation of PBMC derived CD3 T cells resulted in reduced miR-181c expression and up-regulation of BRK1 protein expression, suggesting that miR-181c-BRK1 axis is important in T cell activation. We further showed that overexpression of miR-181c or suppression of BRK1 resulted in inhibition of T cell activation and actin polymerization coupled with defective lamellipodia generation and immunological synapse formation. Additionally, we found that BRK1 silencing led to reduced expressions of other proteins in the WAVE2 complex, suggesting that the impairment of T cell actin dynamics was a result of the instability of the WAVE2 complex following BRK1 depletion. Collectively, we demonstrated that miR-181c reduces BRK1 protein expression level and highlighted the important role of miR-181c-BRK1 axis in T cell activation and actin polymerization-mediated T cell functions.

Introduction MicroRNAs (miRNAs) are short endogenous non-coding RNAs consisting of 18-25 nucleotides in length which influence gene expression and play pivotal roles in a diverse range of cellular processes. Aberrant miRNA expression has been implicated in a variety of cancers, including haematological malignancies. The miR-181 family plays a crucial role in haematopoiesis, including megakaryocytic, erythroid and myeloid differentiation and both B and T cell development and differentiation. We therefore focused our study on validating novel downstream targets of miR-181. Methods A novel functional assay utilising an optimised 3'UTR enriched library and a dual selection strategy (Gäken et al., 2012) was performed to identify biologically relevant targets of miR-181c. BRK1 (BRICK1, SCAR/WAVE Actin Nucleating Complex Subunit) was identified as a potential target and validation was performed by quantitative real time PCR and western blot analysis. Given the potential role of BRK1 in the Wiskott-Aldrich Syndrome Protein Family Verprolin-Homologous Protein-2 (WAVE2) complex and actin polymerisation in T cells, we investigated the influence of the miR-181c-BRK1 axis on T cell function. Knockdown of BRK1, using short hairpin RNA (shRNA) lentiviral vectors, and overexpression of miR-181c, via transfection with miR-181c expression vectors, were performed in Jurkat and primary T cells. T cell activation was examined by measurement of CD69 and CD154 expression and actin polymerisation was quantified by total cellular F-actin content. Immune synapse formation was studied by conjugate formation between T cells and antigen-pulsed B cells. Lastly, lamellipodia formation was investigated by assessing the ability of T cells to spread on anti-CD3 coated slides. Results Target genes downregulated by miR-181c were identified. One such target was BRK1, a component of the WAVE2 complex that has been shown to play a pivotal role in actin polymerisation. Validation experiments showed that overexpression and inhibition of miR-181c had no impact on BRK1 mRNA expression but did in fact modulate protein expression, suggesting that miR-181c regulates BRK1 at the translational level. We demonstrated that primary T cell activation resulted in downregulation of miR-181c and upregulation of BRK1 protein expression, further strengthening our hypothesis that the miR-181c-BRK1 axis may play an important role in T cell activation. Next, we found that loss of BRK1 resulted in reduced T cell activation as shown by decreased expression of CD69 and CD154. Furthermore, we showed that downregulation of BRK1 expression by shRNA resulted in reduced actin polymerisation after T cell stimulation. Reduced expression of BRK1 led to a marked reduction in the total area (in square micrometers) of F-actin accumulation at T cell contact sites and synapses with B cells indicating defective immune synapse formation. Moreover, reduced BRK1 expression resulted in defect in lamellipodia formation in response to T cell receptor stimulation. Similarly, ectopic expression of miR-181c in Jurkat T cells also led to a reduction in T cell activation and actin polymerisation coupled with defects in immune synapse and lamellipodia formation, hence confirming the important role of the miR-181c-BRK1 axis in T cell activation. Lastly, we demonstrated that suppression of BRK1 induced reduced expression of other pivotal proteins in the WAVE2 complex including WAVE2, Abi1 and Sra1. This suggests that impairment of actin polymerisation-dependent T cell functions were a result of instability of the WAVE2 complex following BRK1 suppression. Conclusion For the first time, we hereby demonstrate that BRK1 is a target of miR-181c. Moreover, we have highlighted the potential role of the miR-181c-BRK1 axis in impaired actin polymerisation-dependent T cell function and immune synapse formation. Deregulation of the miR-181c-BRK1 axis requires further evaluation in haematological malignancies. Disclosures No relevant conflicts of interest to declare.

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