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Dysregulated miR34a/diacylglycerol kinase ζ interaction enhances T-cell activation in acquired aplastic anemia

Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a−/− mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a−/− mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a−/− lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.


SUPPLEMENTARY METHODS
Quantitative RT-PCR analysis CD3 + CD45RA + CD45ROnaïve T cells and CD3 + CD45RA -CD45RO + non-naïve T cells were isolated from bone marrow mononuclear cells (BMMCs) by negative selection with immunomagnetic beads. In brief, BMMCs were incubated with a group of microbeads-conjugated antibodies, including anti-CD14, anti-CD16, anti-CD56, anti-CD19, anti-CD15, anti-CD123 and CD235a, together with anti-CD45RA or anti-CD45RO (Miltenyi). The purity of isolated CD3 + CD45RA + CD45ROand CD3 + CD45RA -CD45RO + T cells was generally 87% as determined by fluorescenceactivated cell sorter analysis. Total RNAs were extracted from the BMMCs of 41 patients and 20 healthy controls as well as naïve T cells and non-naïve T cells from 8 patients and 8 controls with Trizol reagent (Takara, Otsu, Japan). cDNA was prepared using a TaqMan microRNA reverse transcription kit (ABI, Foster City, USA) and a Prime Script reverse transcription reagent kit (Takara). TaqMan real-time RT-PCR was performed to confirm the expression level of miRNAs using TaqMan universal PCR master mix (ABI); U6 served as an internal control. The thermal cycling included 2 minutes at 50°C, 10 minutes at 95°C, then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute.
In order to verify the target genes that play a role in AA, the mRNA levels of Kruppel-like factor 4 (KLF4), lymphoid enhancer binding factor 1 (LEF1), SPI-1 protooncogene (SPI1), nuclear receptor subfamily 4 group A member 2 (NR4A2), sirtuin 1 (SIRT1), cyclin-dependent kinase 6 (CDK6), and DGKζ in BMMCs from patients and controls were measured using the SYBR Green RT-PCR Master Mix (Toyobo, Osaka, Japan). All these mRNA data were normalized to the beta-actin mRNA. The level of DGKζ mRNA in mice was examined by the same way. The thermal cycling conditions included an initial denaturation step at 95°C for 30s, followed by 40 cycles of 95°C for 5s, 62°C for 10s, and 72°C for 15s.
All reactions were performed in the light Cycler 480 II (Roche, Rotkreuz, Switzerland). Each sample was examined in triplicate. Relative transcripts were determined by the formula: 2 -(CTtarget-CTcontrol) .

T cell proliferation assays
LN cells and splenocytes were similarly stimulated as above mentioned for 72 h, and during the last 12 h were pulsed with 5-Bromo-2-deoxyuridine (Brdu, BD, San Jose, USA). To track cell division, cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) prior to stimulation. Brdu incorporation and progression of cell division were analyzed by flow cytometry after staining with anti-CD4 and anti-CD8 fluorescence-labeled mAbs.

Immunoblot analysis
To determine DGKζ expression, BMMC were extracted from 8 SAA patients and 8 healthy controls, and lymph node cells from miR34a -/mice and wild type mice were treated as method Part 'T cell activation' described.
To analyze ERK activation after TCR engagement, lymph node cells from 9 miR34a -/mice and 9 wild type mice were left unstimulated or were stimulated with anti-CD3ε and anti-CD28 mAbs for 5 and 15 min. Human BMMCs and mouse LN cells were lysed using a total protein extraction kit (BestBio, Shanghai, China). Proteins were resolved by SDS-PAGE, transferred to a trans-blot nitrocellulose membrane and probed with antibodies specific to DGKζ (Santa Cruz Biotechnology, Dallas, USA) and phosphorylated ERK1/2 (Cell Signal Technology, Danvers, MA, USA). β-actin or total ERK were determined as loading control.