BACH2 Regulates Foxp3 Expression in Umbilical Cord Blood CD4⁺/45RA⁺ T-Cells.

Abstract 2677

Poster Board II-653

There are clear differences in the response of umbilical cord blood (UCB) T-cells with regards to improved immune tolerance after allogeneic stem cell transplant and lower incidence of acute graft versus host disease (aGvHD) which have not been determined at a cellular level. Activated, UCB CD4⁺ T-cells express less TNF-α and IFN-γ then their adult complement. At the protein level, NFAT1, a transcription factor known to regulate the expression of FoxP3, TNF-α, and IFN-γ is attenuated in resting UCB CD4⁺ T-cells. In addition, activated NFAT1 temporal response to stimulation is delayed by ∼6 hours. However our analysis by gene array did not see any attenuation of FoxP3 mRNA compared to normal naïve adult peripherial CD4⁺/CD45RA⁺ T-cells. The absence of a key transcription factor activity having little or no effect on subsequent downstream gene expression is normally a strong indicator of compensatory regulatory factor(s). Previous observation demonstrated that the transcription factor BACH2 plays a compensatory regulatory role in the up-regulation of IL-2 in UCB CD4⁺/CD45RA⁺ T-cells. Studies of the effect of BACH2 inhibition on FoxP3 expression in UCB CD4⁺/CD45RA⁺ T-cells were conducted. Loss of BACH2 expression correlated with the loss of FoxP3 mRNA and protein expression in transient siRNA knockdown experiments as observed by qRT-PCR, and validated by Western Blot. Using cross-linking chromatin immunoprecipitation (ChIP), we show that BACH2 binds to the proximal promoter and within intron 1 of FoxP3 gene in naïve UCB CD4⁺/CD45RA⁺ T-cells and within the intron 1 of adult CD4⁺/CD45RA⁺ T-cells. This report summarizes studies demonstrating a functional role for BACH2 in UCB CD4⁺/CD45RA⁺ T-cells. Transient knockdown of BACH2 by siRNA results in decreased FoxP3 expression at both at mRNA and protein level prior to CD3/CD28 stimulation. There were several putative BACH2 DNA binds sites both associated and unassociated with NFAT1 sites. ChIP analysis demonstrated that BACH2 co-precipitated with 2 separate sites surrounding exon 1 of the FoxP3 gene in unstimulated UCB CD4⁺/CD45RA⁺ T-cells, but with only one site in unstimulated AB CD4⁺/CD45RA⁺ T-cells. In addition, the bZip protein sMafK was observed co-immunoprecipitated with the same DNA regions in UCB and AB CD4⁺/CD45RA⁺ T-cells.