The Role of the bZip Protein Bach2 in Umbilical Cord Blood (UCB) T Lymphocytes.

Graft versus host disease (GVHD) occurring after allogeneic stem cell transplantation is mediated by donor T cell alloreactivity against major and minor histocompatibility antigens presented by recipient antigen presenting cells (APC). Clinical reports identify that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD due to reduced cytokine production. Nuclear factor of activated T cells (NFAT1) plays a pivotal role in the transcription of cytokine genes and other immune responses. NFAT1 regulates the expression of cytokine genes through direct interaction with AP-1 (Fos/Jun, basic-lucine zipper bZip proteins). NFAT1 and AP-1 cooperatively bind to a 15-bp DNA that contains both NFAT1 and AP-1 sites. We used Affymetrix oligonucleotide microarrays to compare gene expression of primary purified CD4+ UCB T-cells to adult peripheral blood CD4+ T-cells (AB) at baseline, 6, and 16 hours after primary stimulation with anti-CD3/anti-CD28. NFAT-regulated genes exhibited lower expression in UCB CD4+ T-cells including: GM-CSF, IFN- g, TNF-a, IL-3, IL-4, IL-5, IL-13, IL- 2R (CD 25). In addition, AP-1 (Fos/Jun) did not show a significant change. However, Bach2, an 841- amino-acid-residue containing bZip protein, did show a significant increase in UCB CD4+ T-cells. Previous reports identify from the crystal structure of the quaternary complex of NFAT1/AP-1/DNA (Chen et al., Nature 1998) that NFAT1 is in contact with the lucine zipper region of AP-1 while the basic region of AP-1 binds to the DNA site at TGTTTCA. The amino-acid residues that make contact with DNA in the bZip regions of Jun and Fos are highly conserved in the Jun and Fos families. These amino acid residues are also 100% conserved in the human and mouse Bach2. The high similarity of DNA-contacting amino-acid residues between Bach2 and AP-1 strongly suggests that Bach2 and AP-1 may bind to the same DNA site. We hypothesize that Bach2 may act as a transcriptional repressor of cytokine genes due to the fact that increased Bach2 in UCB may compete with AP-1 for same DNA binding site, which in turn may partly block the interaction of NFAT1 with AP-1 and the expression of cytokine genes in UCB. We are currently pursuing this hypothesis. Ongoing studies are to confirm whether the protein levels of Bach2 and AP-1 are different in UCB compared to with AB CD4+ T-cells. This includes using gel-shift assays in vitro to test Bach2 binding to the DNA site of AP-1 as well as to test potential competitive binding of Bach2 and AP-1 to the same DNA site.