BACH2 Directly Regulates Expression of Foxp3 in UCB CD4+ T-Cells
Authors: Lesniewski Mathew L. , Haviernik Peter , Weitzel R. Patrick , Fanning Laura , Finney Marcie , Yang Yu-Chung , Ratajczak Mariusz Z , Greco Nicholas , Paul Paul , Maciejewski Jaroslaw P. , Laughlin Mary 
Published: April 2020

Abstract
Introduction: We have previously reported low constitutive nuclear factor of activated T cells (NFAT1) protein expression in unstimulated umbilical cord blood (UCB) CD4+ T-cells, and delayed up-regulation of NFAT1 expression during primary stimulation compared to adult primary CD4+ T-cells. Recent data has shown NFAT1 regulating Foxp3, an observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, and our work demonstrating BACH2 directly regulates IL2 expression in the absence of NFAT1 protein. We sought to determine if BACH2 plays a role in regulating Foxp3 expression in UCB CD4+ T-cells. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein mafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. We hypothesized that BACH2 may directly regulate Foxp3 transcription by binding to putative NFAT1/AP1 binding sites within the Foxp3 proximal promoter. These sites may be vacant due to an absence of NFAT1 protein in resting UCB CD4+ T-cells. This hypothesis was tested by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells and then examining the level of Foxp3 expression at both the mRNA and protein level. In addition chromatin immunoprecipitation (ChIP) was performed in order to identify if BACH2 was binding to regions proximal to the Foxp3 gene. Finally Foxp3 driven luciferase plasmids were constructed and transiently transfected into primary UCB CD4+ T-cells with and without BACH2 siRNA and the relative expression determined.
Methods: UCB T-cells were purified using autoMACs system. After overnight culture, T-cells were transfected with BACH2 siRNA using Amaxa nucleofector system. Both siRNA treated and control cells were incubated in media for 24 hours, and then stimulated using anti-CD3 and anti-CD28 antibodies. Aliquots of cells were collected at 24 hours and 30 hours post-stimulation for protein and total RNA isolation. The relative changes in mRNA levels for BACH2, FoxP3, were determined by Applied Biosystems Taqman real time RT-PCR system. Western blots were run to confirm results seen in the qRT-PCR. ChIP using BACH2 antibody, and PCR was used to determine putative BACH2 binding in DNA region surround exon 1 of Foxp3.
Results: Loss of BACH2 expression correlates with reduced expression of (40-fold) FoxP3, mRNA in CD4+ UCB T-cells. Western blot performed on BACH2 siRNA treated, and anti-CD3/CD28 antibody stimulated UCB CD4+ T-cells; confirmed the observed loss of Foxp3 in unstimulated UCB CD4+ T-cells. Analysis of ChIP data from unstimulated UCB CD4+ T-cells showed that BACH2 and smafK bound to 2 regions flanking exon 1 of the Foxp3 gene, while results from unstimulated adult PB CD4+ T-cells showed BACH2 and smafK co-precipitated with only one region flanking exon 1 of Foxp3.
Conclusions: NFAT1 protein has recently been shown to be required for the activation of Foxp3 in CD4+ T-cells, yet in UCB CD4+ T-cells NFAT1 protein expression is significantly attenuated. However, Foxp3 expression is unchanged in UCB CD4+ T-cells compared to adult PB CD4+ T-cells. UCB CD4+ T-cells BACH2 binds to two site surrounding exon 1 of Foxp3 gene, promoting the expression of Foxp3 mRNA in the absence of NFAT1 protein expression. These results suggest that expression of BACH2 in UCB CD4+ T-cells may underlie UCB immune tolerance in the unrelated allogeneic setting via regulation of FoxP3 impacting Treg development.