SummaryMADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic a-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.

As the threshold nucleated cell dose for single unit umbilical cord blood (UCB) in adults has not to date been firmly established, we prospectively compared single vs. 2-unit UCB transplantation after reduced intensity conditioning (RIC) in adult patients with hematologic malignancies. Study design specified one UCB unit if the cryopreserved total nucleated cell (TNC) dose was ≥2.5×10 7 /kg recipient weight, otherwise 2-units matched at minimum 4/6 HLA loci to the patient and 3/6 to each other were infused. Twenty-seven patients received 1 unit; 23 patients received 2 units. Median time to absolute neutrophil count (ANC) >500/μL was 24 days (95% CI 22–28 days), 25 days for 1-unit and 23 days for 2-units (p=0.99). At day 100, ANC >500/μL was 88.4% and 91.3% in the 1 and 2-unit groups (p=0.99), respectively. Three-year event free survival (EFS) was 28.6% and 39.1% in the 1 and 2-unit groups (p=0.71), respectively. Infusion of 2 units was associated with significantly lower relapse risk, 30.4% vs. 59.3% (p=0.045). Infused cell doses (TNC, CD3 + , CD34 + , CD56 + CD3 neg ) did not impact engraftment, overall survival (OS), or EFS. Taken together, single unit UCB transplantation with threshold cell dose ≥2.5×10 7 /kg recipient weight after RIC is a viable option for adults, although infusion of 2 units confers a lower relapse incidence.

+ HLADR+ CD38 + (P = 0AE108) cell dose and event-free survival (EFS). Highresolution matching for HLA-class II (DRB1) resulted in improved EFS (P = 0AE02) and decreased risk for acute graft-versus-host disease (GVHD) (P = 0AE004). Early mortality (prior to post-transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34 + , committed CD34 + progenitors and CD3 + T cells favourably influence UCB allogeneic engraftment.

On activation, umbilical cord blood (UCB) CD4þ T cells demonstrate reduced expression of tumor necrosis factor-a (TNF-a) and interferon-c (IFN-c), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4 þ T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-a and IFN-c, is reduced in resting and activated UCB CD4 þ T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4 þ T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4 þ T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4þ T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4þ T cells at levels equivalent to adult PB CD4 þ T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4þ T-cell allogeneic responses.

UCB graft lymphocyte immune naivety and observed early tolerance induction may contribute to the observed favorable GVHD incidence, despite infusion of HLA mismatch grafts in the unrelated allogeneic setting.