Abstract
Background. Graft versus host disease (GVHD) after allogeneic stem cell transplantation is associated with significant morbidity and mortality and targeted therapies are needed. The transcription factor nuclear factor of activated T-cells 1 (NFAT1) is known to interact with various other proteins, including AP-1 (fos/jun heterodimer), regulating both active immune responses and T-cell anergy. We have previously demonstrated lower expression of NFAT-associated transcripts via microarray, including IFN-γ, TNF-α, M-CSF, IL-3, 4, 5, and 13, IL-2R-α, CD40L, MIP1-α, as well as related transcription factors such as JunB, FOSL1, STAT4, T-bet, and c-maf in umbilical cord blood (UCB) CD4+ cells following primary stimulation when compared to cells obtained from adult blood (AB). We hypothesize a critical role for these NFAT1-dependent factors in the increased proliferation, decreased cytokine production seen in UCB, and lower incidence of GVHD following UCB transplantation. Here we present data more closely exploring the behavior of NFAT1 and associated genes in both UCB and AB CD4+ T-cells following primary stimulation.
Methods. siRNA targeting NFAT1 mRNA was utilized to study the effects of lowered NFAT1 in primary human T-cells. CD14neg/4+ T-cells were isolated from adult peripheral blood by ficoll density gradient and selection by MACS. These cells were then immediately transfected via Nucleofector electroporation (Amaxa Biosystems, Gaithersburg, MD) with siRNA duplexes targeting NFAT1 and Cyclophilin B as well as an eGFP-encoding plasmid along with appropriate controls. Cultures were stimulated by anti-CD3/CD28 for 16h. 24h following transfection. Whole cell protein was harvested from a portion of each culture, and the remaining cells washed with PBS and replaced in non-stimulating media. Protein was extracted from the remaining cells 24h later. Protein lysate was analyzed by western blot for NFAT1, Cyclophilin-B, and β-Actin. Separately, expression of 7 transcripts (FOS, JUN, JUNB, FOSL1, BACH2, NFAM, and NFAT1) was analyzed via real-time PCR (RT-PCR). mRNA was obtained from 4 UCB and 4 AB samples at baseline (0h) (n=2) and 16h (n=2) of stimulation, using β-2-microglobulin as an endogenous control.
Results. Transfection efficiency was measured to be approximately 70% via fluorescence microscopy of the eGFP-transfected culture. Our data indicate marginal reduction of NFAT1 protein at 24h. Notably after 48h approximately 65% knockdown is evident. Time course studies to determine the stability of siRNA-mediated NFAT1 knockdown are ongoing. No significant increase in the level of NFAT1 mRNA as measured by RT-PCR was detected. This suggests that the increase in NFAT1 protein levels post stimulation may not be a result of transcriptional regulation. Both UCB and AB samples exhibited a strong (15 fold) increase in FOSL1 transcription, and confirmed the enhanced up-regulation (about 3 fold) in the AB sample seen in microarray. Conclusions. Our data elucidate differences in the transcriptional program between UCB and AB T-cells, suggesting a crucial role for post-transcriptional events in the regulation of NFAT1 and its associated genes. Our findings identify differing regulation and expression of transcription factors in UCB vs. AB T-cells that may underlie the lower incidence and severity of GVHD seen in patients infused with UCB-derived grafts, and implicate additional elements which may modulate UCB T-cell alloreactivity. Studies to analyze the direct effects of decreased NFAT1 expression on relevant cytokines and other signaling molecules are currently ongoing.
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