Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Pbx1 is required for adult subventricular zone neurogenesis.

TALE-homeodomain proteins function as components of heteromeric complexes that contain one member each of the PBC and MEIS/PREP subclasses. We recently showed that MEIS2 cooperates with the neurogenic transcription factor PAX6 in the control of adult subventricular zone (SVZ) neurogenesis in rodents. Expression of the PBC protein PBX1 in the SVZ has been reported, but its functional role(s) has not been investigated. Using a genetic loss-of-function mouse model, we now show that Pbx1 is an early regulator of SVZ neurogenesis. Targeted deletion of Pbx1 by retroviral transduction of Cre recombinase into Pbx2-deficient SVZ stem and progenitor cells carrying floxed alleles of Pbx1 significantly reduced the production of neurons and increased the generation of oligodendrocytes. Loss of Pbx1 expression in neuronally committed neuroblasts in the rostral migratory stream in a Pbx2 null background, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we further detected PBX1 binding to known regulatory regions of the neuron-specific genes Dcx and Th days or even weeks before the respective genes are expressed during the normal program of SVZ neurogenesis, suggesting that PBX1 might act as a priming factor to mark these genes for subsequent activation. Collectively, our results establish that PBX1 regulates adult neural cell fate determination in a manner beyond that of its heterodimerization partner MEIS2.

Congenital asplenia in mice and humans with mutations in a Pbx/Nkx2-5/p15 module.

The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.

Epigenetic regulation of early osteogenesis and mineralized tissue formation by a HOXA10-PBX1-associated complex.

Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

Spatio-temporal expression of Pbx3 during mouse organogenesis.

Pbx3 is a member of the Pbx family of TALE (three amino acid loop extension) class homeodomain transcription factors. These transcription factors are implicated in developmental and transcriptional gene regulation in numerous cell types through their abilities to form hetero-oligomeric DNA-binding complexes. Pbx3 was found to be expressed at high levels in the developing central nervous system (CNS), including a region of the medulla oblongata which is implicated in the control of respiration. Furthermore, as reported, Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure. In this study, we have characterized Pbx3 expression patterns during organogenesis in numerous tissues and organ systems other than the CNS, as a first step toward understanding the potentially overlapping functions of Pbx3 with other Pbx family members during vertebrate development. We have performed in situ hybridization on whole mount and sectioned mouse embryos from gestational day (E) 9 to E16.5. During early organogenesis, until E12.5, Pbx3 expression is found mostly in the embryonic head, forelimbs, and septum transversum, unlike Pbx1 and Pbx2 expression which is more widespread. Conversely, later in organogenesis, Pbx3 expression becomes more widely detectable throughout the developing embryo. Epithelial and mesenchymal tissues, as well as the CNS, represent major sites of Pbx3 expression. The enteric nervous system also expresses high levels of Pbx3, distinctively in the cells of the ganglia of Auerbach's myenteric nerve plexus, that also express Dlx2 and Notch1. Cartilage is also a site of Pbx3 expression. Interestingly, like Pbx1, Pbx3 is highly expressed in proliferating chondrocytes but is lost as chondrocytes become hypertrophic during endochondral ossification. Finally, Pbx3 is expressed only in the forelimb buds during early limb development, while the hindlimb bud is devoid of Pbx3. This finding leads us to add Pbx3 to the sparse list of early forelimb-specific molecular markers.