Repression of NFAT1 Protein Expression by MicroRNA-184 in Umbilical Cord Blood-Derived CD4⁺ T-Cells as a Mechanism Underlying Lower Incidence of aGVHD

Clinical benefits of umbilical cord blood (UCB) grafts include lower incidence of acute graft-versus-host disease (aGVHD) following allogeneic transplantation. The dramatically lower expression of the transcription factor Nuclear Factor of Activated T-cells 1 (NFAT1) in UCB-derived CD4⁺ T-cells compared to adult blood (AB) results in reduced production of inflammatory cytokines following stimulation, in part underlying this advantage. Here we present a novel mechanism by which NFAT1 protein expression is restrained in these cells. Importantly, NFAT1 mRNA (NFATc2) is not significantly reduced in UCB CD4⁺ T-cells. Gradient separation and cellular fractionation reveal that NFAT1 is indeed translationally repressed in UCB through early stimulation timepoints, which we hypothesized might be due to the activity of microRNA. A search of known microRNA species reveals that miR- 184 is strongly predicted to bind to the NFATc2 3′ untranslated region (UTR). We have confirmed this interaction through luciferase expression and Western Blot analysis. First, luciferase expression vectors containing the short miR-184 binding sequence or the cloned 3′ UTR were constructed and transfected into UCB and AB CD4⁺ T-cells. In UCB, insertion of the miR-184 binding sequence alone reduced luciferase expression to 38% of control. The cloned 3′ UTR vector exhibited 60% expression compared to control. Both these negative effects were fully reversible by cotransfection with an excess of blocking antisense to miR- 184. Likewise, the miR-184 site did not impact luciferase expression in AB CD4⁺ T-cells, but expression was reduced by 61% in AB upon cotransfection with miR-184 mimic. miR-184 mimic produced a 23% decrease in luciferase activity from vector containing the NFATc2 3′ UTR. Both these effects were attenuated by cotransfection with an excess of blocking antisense to miR-184. Blocking antisense increased endogenous NFAT1 protein expression in UCB CD4⁺ T-cells by 86% as measured by quantified Western Blot, without producing a significant change in NFATc2 mRNA quantity. Conversely, miR-184 mimic produced a 31% decrease in NFAT1 protein expression by AB CD4⁺ T-cells. We confirmed by quantitative RT-PCR (qRT-PCR) that miR-184 exhibited an average of 58.4-times greater expression in UCB than in AB CD4⁺ T-cells (p=0.005). We have additionally demonstrated via gain-of-function and loss-of-function analyses that the effects on NFAT1 expression by miR-184 are sufficient to affect transcription of the NFAT-dependent pro-inflammatory cytokine IL-2 through 16 hours of in vitro stimulation. This study comprises the first identification of miR-184 activity in primary human lymphocytes and reveals its function in repressing NFAT1 protein expression uniquely in UCB CD4⁺ T-cells. These findings hold important implications for our further understanding of GVHD, autoimmunity, and neonatal lymphocyte development, and suggest miR-184 as a useful biomarker or target of ex vivo graft analysis.