Michelle Kushida

Dendritic Cells Genetically Engineered to Express Fas Ligand Induce Donor-Specific Hyporesponsiveness and Prolong Allograft Survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

Significance Glioblastoma is an incurable brain tumor. It is characterized by intratumoral phenotypic and genetic heterogeneity, but the functional significance of this heterogeneity is unclear. We devised an integrated functional and genomic strategy to obtain single cell-derived tumor clones directly from patient tumors to identify mechanisms of aggressive clone behavior and drug resistance. Genomic analysis of single clones identified genes associated with clonal phenotypes. We predict that integration of functional and genomic analysis at a clonal level will be essential for understanding evolution and therapeutic resistance of human cancer, and will lead to the discovery of novel driver mechanisms and clone-specific cancer treatment. Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.

Quiescent Sox2+ Cells Drive Hierarchical Growth and Relapse in Sonic Hedgehog Subgroup Medulloblastoma

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

ΔNp63 plays an anti-apoptotic role in ventral bladder development

The bladder, the largest smooth-muscle organ in the human body, is responsible for urine storage and micturition. P63, a homolog of the p53 tumor-suppressor gene, is essential for the development of all stratified epithelia, including the bladder urothelium. The N-terminal truncated isoform of p63, ΔNp63, is known to have anti-apoptotic characteristics. We have established that ΔNp63 is not only the predominant isoform expressed throughout the bladder, but is also preferentially expressed in the ventral bladder urothelium during early development. We observed a host of ventral defects in p63-/- embryos, including the absence of the abdominal and ventral bladder walls. This number of ventral defects is identical to bladder exstrophy, a congenital anomaly exhibited in human neonates. In the absence of p63, the ventral urothelium was neither committed nor differentiated, whereas the dorsal urothelium was both committed and differentiated. Furthermore, in p63-/- bladders, apoptosis in the ventral urothelium was significantly increased. This was accompanied by the upregulation of mitochondrial apoptotic mediators Bax and Apaf1, and concurrent upregulation of p53. Overexpression ofΔ Np63γ and ΔNp63β in p63-/- bladder primary cell cultures resulted in a rescue, evidenced by significantly reduced expressions of Bax and Apaf1. We conclude that ΔNp63 plays a crucial anti-apoptotic role in normal bladder development.

Regulation of Sox9 by Sonic Hedgehog (Shh) is Essential for Patterning and Formation of Tracheal Cartilage

We report that Sonic Hedgehog (Shh) regulates both formation and patterning of tracheal cartilage by controlling the expression pattern and level of the chondrogenic gene, Sox9. In Shh−/− tracheo‐esophageal tubes, Sox9 expression is transient and not restricted ventrally to the site of chondrogenesis, and is absent at the time of chondrogenesis, resulting in the failure of tracheal cartilage formation. Inhibition of Hedgehog signalling with cyclopamine in tracheal cultures prevents tracheal cartilage formation, while treatment of Shh−/− tracheal explant with exogenous Shh peptide rescues cartilage formation. Both exogenous Bmp4 and Noggin rescue cartilage phenotype in Shh−/− tracheal culture, while promoting excessive cartilage development in wild‐type trachea through induction of Sox9 expression. The ventral and segmented expression of Sox9 in tracheal primordia under Shh modulated by Bmp4 and Noggin thus determine where and when tracheal cartilage develops. These results indicate that Shh signalling is a critical determinant in tracheal cartilage development. Developmental Dynamics 239:514–526, 2010.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRβ, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare ‘outlier’ clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Murine xenogeneic immune responses to the human testis: a presumed immune-privileged tissue.

INTRODUCTION Immune privilege provides a natural paradigm for potentially down-regulating allogeneic and xenogeneic inflammatory immune responses. Fas ligand has been suggested as a general underlying mechanism of immune privilege; the human Fas ligand has been shown to ligate murine Fas in vitro. METHODS In this study, we examined whether the human testicular xenograft, a presumed immune-privileged tissue would have prolonged survival in mice. In addition, in vitro and in vivo murine xenogeneic immune responses to the human testicular xenografts were characterized using MHC class I, MHC class II, CD4, CD8, CD4/8 knockout mice. RESULTS Unlike in rodent testis, Fas ligand mRNA is not expressed and Fas is highly expressed in human testis. Human testicular xenografts are immunogenic, and do not induce any preferential pattern of recipient systemic Th1 or Th2 cytokine bias. Interestingly, an indefinite survival of the human testicular xenografts is observed in murine MHC class II knockout mice, whereas the human skin xenografts were rejected without a delay. In vivo murine immune responses to human testicular xenografts require a recipient MHC class II-dependent CD4 T cell-mediated process that appears to depend on B7-1/B7-2 costimulatory signals. CONCLUSIONS Our results demonstrate that the concept of immune privilege, as defined by the expression of Fas ligand and prolonged survival after transplantation, cannot be extended to human testis. The stringent restriction of murine xenogeneic immune responses to discordant human testicular xenografts to the indirect MHC class II-dependent CD4 T cell-mediated pathway suggests a potential venue for immune modulation to induce tolerance across a discordant species barrier.

T cells are necessary and critical for xenograft rejection in new concordant cardiac xenotransplant model.

BACKGROUND A new vascularized concordant xenotransplant model using the Chinese hamster as donor and mouse as recipient species is reported. This model takes advantage of the wealth of informative immune reagents and knockout and transgenic backgrounds available for the mouse. METHODS Heterotopic auxillary cardiac transplantation was performed. The mean survival time was assessed by daily palpation. Xenoreactive antibody production was measured by flow cytometry, and cardiac xenografts were examined by light microscopy. RESULTS The tempo of xenograft rejection in this model is consistent with concordant species combination. IgM and IgG3 responses were not critical for the concordant xenograft rejection. Long-term survival (>100 days) of the concordant cardiac xenografts was observed without any immunosuppression in nude mice. Reconstitution of nude mice with CD3+ T cells induced the xenograft rejection in 5.7 days (P<0.01). CONCLUSION This new concordant cardiac xenotransplant model demonstrates that T-dependent xenogeneic immune response is necessary and critical for the xenograft rejection.