Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting leukemia-propagating cell frequency.

NOTCH1 pathway activation contributes to the pathogenesis of over 60% of T-cell acute lymphoblastic leukemia (T-ALL). While Notch is thought to exert the majority of its effects through transcriptional activation of Myc, it also likely has independent roles in T-ALL malignancy. Here, we utilized a zebrafish transgenic model of T-ALL, where Notch does not induce Myc transcription, to identify a novel Notch gene expression signature that is also found in human T-ALL and is regulated independently of Myc. Cross-species microarray comparisons between zebrafish and mammalian disease identified a common T-ALL gene signature, suggesting that conserved genetic pathways underlie T-ALL development. Functionally, Notch expression induced a significant expansion of pre-leukemic clones; however, a majority of these clones were not fully transformed and could not induce leukemia when transplanted into recipient animals. Limiting-dilution cell transplantation revealed that Notch signaling does not increase the overall frequency of leukemia-propagating cells (LPCs), either alone or in collaboration with Myc. Taken together, these data indicate that a primary role of Notch signaling in T-ALL is to expand a population of pre-malignant thymocytes, of which a subset acquire the necessary mutations to become fully transformed LPCs.

Shared acquired genomic changes in zebrafish and human T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is a challenging clinical entity with high rates of induction failure and relapse. To discover the genetic changes occurring in T-ALL, and those contributing to relapse, we studied zebrafish (Danio rerio) T-ALL samples using array comparative genomic hybridization (aCGH). We performed aCGH on 17 T-ALLs from four zebrafish T-ALL models, and evaluated similarities between fish and humans by comparing all D. rerio genes with copy number aberrations (CNAs) with a cohort of 75 published human T-ALLs analyzed by aCGH. Within all D. rerio CNAs, we identified 893 genes with human homologues and found significant overlap (67%) with the human CNA dataset. In addition, when we restricted our analysis to primary T-ALLs (14 zebrafish and 61 human samples), 10 genes were recurrently altered in > 3 zebrafish cancers and ≥ 4 human cases, suggesting a conserved role for these loci in T-ALL transformation across species. We also conducted iterative allo-transplantation with three zebrafish malignancies. This technique selects for aggressive disease, resulting in shorter survival times in successive transplant rounds and modeling refractory and relapsed human T-ALL. Fifty-five percent of original CNAs were preserved after serial transplantation, demonstrating clonality between each primary and passaged leukemia. Cancers acquired an average of 34 new CNAs during passaging. Genes in these loci may underlie the enhanced malignant behavior of these neoplasias. We also compared genes from CNAs of passaged zebrafish malignancies with aCGH results from 50 human T-ALL patients who failed induction, relapsed or would eventually relapse. Again, many genes (88/164) were shared by both datasets. Further, nine recurrently altered genes in passaged D. rerio T-ALL were also found in multiple human T-ALL cases. These results suggest that zebrafish and human T-ALLs are similar at the genomic level, and are governed by factors that have persisted throughout evolution.

Heritable T-cell malignancy models established in a zebrafish phenotypic screen.

T-cell neoplasias are common in pediatric oncology, and include acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LBL). These cancers have worse prognoses than their B-cell counterparts, and their treatments carry significant morbidity. Although many pediatric malignancies have characteristic translocations, most T-lymphocyte-derived diseases lack cytogenetic hallmarks. Lacking these informative lesions, insight into their molecular pathogenesis is less complete. Although dysregulation of the NOTCH1 pathway occurs in a substantial fraction of cases, many other genetic lesions of T-cell malignancy have not yet been determined. To address this deficiency, we pioneered a phenotype-driven forward-genetic screen in zebrafish (Danio rerio). Using transgenic fish with T-lymphocyte-specific expression of enhanced green fluorescent protein (EGFP), we performed chemical mutagenesis, screened animals for GFP(+) tumors, and identified multiple lines with a heritable predisposition to T-cell malignancy. In each line, the patterns of infiltration and morphological appearance resembled human T-ALL and T-LBL. T-cell receptor analyses confirmed their clonality. Malignancies were transplantable and contained leukemia-initiating cells, like their human correlates. In summary, we have identified multiple zebrafish mutants that recapitulate human T-cell neoplasia and show heritable transmission. These vertebrate models provide new genetic platforms for the study of these important human cancers.

Fishing for lymphoid genes.

Thymic organogenesis and T-cell lymphopoiesis are crucial interdependent processes that establish a functional vertebrate immune system. The current understanding of vertebrate thymic development during embryogenesis remains incomplete and would benefit from novel approaches. The zebrafish Danio rerio is a powerful developmental and genetic system for the dissection of early events in the ontogeny of the immune system. Forward genetic screens have uncovered genes involved in hematopoiesis, and specific screens are being designed to examine the genes that regulate T-cell development and the origin of the thymus. Studies of the zebrafish should improve our understanding of lymphoid development in vertebrates.

Tacrolimus (FK506) in allogeneic bone marrow transplantation for severe aplastic anemia following orthotopic liver transplantation.

Severe aplastic anemia (SAA) is a frequent complication of orthotopic liver transplantation for non-typeable viral hepatitis. Allogeneic bone marrow transplantation (BMT) may successfully reconstitute hematopoiesis but the optimal conditioning regimen and graft-versus-host disease (GVHD) prophylaxis in such patients are unknown. Allogeneic BMT was undertaken in an 8-year-old male patient who developed SAA 6 weeks after cadaveric orthotopic liver transplantation for fulminant hepatic failure secondary to presumed non-typeable viral hepatitis. The preparative regimen for his HLA genotypically identical sibling BMT consisted of cytoxan and anti-thymocyte globulin. Tacrolimus (FK506) and prednisone, used to prevent liver graft rejection, were supplemented with methotrexate on post-BMT days, 1, 3, 6 and 11 for GVHD prophylaxis. Engraftment proceeded promptly and without complications. Transfusion dependence resolved 6 weeks after BMT. The patient is alive and well 1 year after his BMT on FK506 and prednisone without any signs of GVHD or liver allograft rejection. This case is the first demonstration of the feasibility of continuing FK506 used for prevention of liver graft rejection as GVHD prophylaxis for allogeneic BMT.

Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: role of NF-kappa B.

We have studied the transcriptional activation of the human TNF-alpha gene by the superantigen staphylococcal enterotoxin A (SEA) in the human premonocytic cell line THP-1. Nuclear proteins from SEA-stimulated THP-1 cells bound strongly to kappa 3, the most proximal of three putative NF-kappa B binding sites (kappa 1-kappa 3) found in the 5' regulatory region of the TNF-alpha gene, but only weakly to kappa 1, the most distal of the NF-kappa B binding sites, and showed no binding to kappa 2. The mobility of the kappa 3-nucleoprotein complex was identical to that of complexes formed between nuclear proteins and the consensus NF-kappa B seuqence. Moreover, both 5' and 3' mutants of kappa 3 were unable to displace kappa 3 binding, suggesting that the kappa 3 binding complex induced by SEA has the characteristics of NF-kappa B. Studies using Abs directed against the NF-kappa B subunits p50 and p65 suggested that both p50 and p65 bind to the kappa 3 sequence. Reporter gene assays showed that deletion of kappa 3 (-99 to -89 bp) and point mutation of the three 5' guanine bases in the kappa 3 sequence reduced the inducibility of the TNF-alpha promoter by SEA and LPS. These results indicate that superantigen induces NF-kappa B in human monocytic cells and suggest that binding of NF-kappa B to the kappa 3 site of the TNF-alpha promoter plays an important role in the transcriptional activation of the TNF-alpha gene by superantigen.

Early activation events induced by the staphylococcal superantigen toxic shock syndrome toxin-1 in human peripheral blood monocytes.

Staphylococcal exotoxins (SE) bind to MHC class II molecules and induce the production of IL-1 beta and TNF-alpha in human monocytic cells. Here we show that stimulation of peripheral blood monocytes with toxic shock syndrome toxin-1 (TSST-1) induced rapid increase in tyrosine phosphorylation of cytosolic protein substrates, accumulation of inositol phosphates, and de novo tyrosine phosphorylation of the PLC-gamma 1 isozyme. Accumulation of inositol phosphates was inhibited by preincubation of cells with inhibitors of protein tyrosine kinases (PTK). Stimulation of monocytes with TSST-1 furthermore led to activation of protein kinase C (PKC). PTK and PKC activation plays a role in the induction of monokine gene transcription by SE because inhibitors of PTK and PKC reduced TSST-1-stimulated IL-1 beta and TNF-alpha gene expression. We therefore propose a model in which the induction of monokine gene transcription by TSST-1 in monocytes necessitates activation of tyrosine kinase(s) and of PKC, the latter probably by way of PLC-gamma 1.

Activator protein-1 (AP-1) is stimulated by microbial superantigens in human monocytic cells.

Microbial superantigens bind to major histocompatibility complex (MHC) class II molecules and activate gene transcription in monocytes. In search of transcription factors that potentially mediate the effects of superantigens at the nuclear level, we examined the capacity of staphylococcal superantigens to stimulate the activity of the transcriptional promoter factor activator protein-1 (AP-1). Electrophoretic mobility shift assays showed an increase in nuclear proteins that bound to the consensus AP-1 motif within 5 min following the stimulation of the monocytic cell line THP-1 with toxic shock syndrome toxin-1 (TSST-1) or staphylococcal endotoxin A. We show that mRNA levels for the subunits that compose AP-1, the protooncogenes c-fos and c-jun, are upregulated by stimulation of THP-1 cells with TSST-1. The activated AP-1 complexes were functional, as evidenced by the capacity of TSST-1 to stimulate the expression of an AP-1-driven reporter gene construct transfected into THP-1 cells. These results establish that the engagement of MHC class II molecules by superantigens increases the activity of functional AP-1 complexes and that this may proceed in part by transcriptional activation of c-fos and c-jun protooncogenes.

Microbial superantigens induce NF-kappa B in the human monocytic cell line THP-1.

Staphylococcal superantigens bind to MHC class II molecules and induce transcriptional activation of IL-1 beta and TNF-alpha genes in human monocytic cells. The understanding of the mechanisms by which superantigens activate cytokine gene expression is incomplete. In this study, we demonstrate that toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A and B induce the activation of NF-kappa B, a transcriptional enhancer that binds to sequences found in both the IL-1 beta and TNF-alpha promoters. Electrophoretic mobility-shift assays showed a rapid induction of nuclear proteins that bound to the consensus kappa B motif. Furthermore, TSST-1 potently stimulated chloramphenicol acetyltransferase (CAT) expression by THP-1 cells transfected with a consensus NF-kappa B-promoter CAT construct, indicative of induction of NF-kappa B enhancer function. Induction of both NF-kappa B DNA-binding proteins and NF-kappa B enhancer function was down-regulated by inhibitors of protein kinase C and protein tyrosine kinase, indicating a role for these protein kinases in the induction of NF-kappa B by MHC class II ligands. Using neutralizing antibodies, we demonstrated that after the stimulation of cells with TSST-1, TNF-alpha, but not IL-1 beta, acted to up-regulate binding of NF-kappa B to DNA and the activation of the NF-kappa B-promoter CAT construct. These results indicate that induction of NF-kappa B by superantigens is up-regulated in part by an autocrine loop involving TNF-alpha.

Transcriptional activation of IL-1 beta and tumor necrosis factor-alpha genes by MHC class II ligands.

Ligands that bind MHC class II (Ia) molecules, including staphylococcal exotoxins (SE) and mAb induce IL-1 and TNF secretion in human monocytes and monocytic cell lines. In this study, we have analyzed the mechanisms by which SE induce IL-1 beta and TNF-alpha production. Treatment of human peripheral blood monocytes with staphylococcal exotoxin B and toxic shock syndrome toxin-1 resulted in a biphasic increase of IL-1 beta mRNA that lasted more than 12 h and in a more transient rise in TNF-alpha mRNA. A F(ab) preparation of the anti-HLA DR mAb L243 also caused a significant increase in monokine mRNA levels. Stimulation of a monocytic cell line, THP-1, with staphylococcal exotoxin B and toxic shock syndrome toxin-1 induced a rapid rise in IL-1 beta and TNF-alpha mRNA levels. This response peaked at 1 to 3 h poststimulation and remained detectable at 12 h. Nuclear run-on transcription assays demonstrated that SE cause transcriptional activation of the IL-1 beta and TNF-alpha genes. This transcriptional activation did not require de novo protein synthesis as it was not inhibited by the protein synthesis inhibitor cycloheximide. These results define an important function for Ia molecules as regulators of cytokine gene expression and add to the understanding of the changes in cellular function induced by SE.

Different large deletions of T cell receptor V beta genes in natural populations of mice.

A panel of geographically separate Mus m. domesticus and Swiss mice from several sources was screened for deletions in the T cell receptor variable (V) beta locus. Four out of forty-three strains tested show a deletion identical to or larger than the deletion previously described in SJL mice. To our knowledge, this is the first time that such important V beta deletions are described in inbred or partially inbred wild-derived strains of mice. On the other hand there seems to be very little polymorphism between the remaining V beta genes. Expression of V beta genes in peripheral and intra-thymic T cells was tested using antibodies specific for different V beta polypeptide chains. Flow cytometry analysis revealed a high expression of V beta 6 and V beta 17 genes in the Copacabana Swiss-derived strain and an absence of V beta 17 expression in the WLA wild-derived strain. The three Mus m. domesticus strains (WLA, DDO and WBG) having deleted two to three additional V beta subfamilies compared to SJL present no apparent immune deficiencies or autoimmune disorders. We conclude that relatively few V beta genes may suffice for unimpaired survival of wild mice and that there is a selective pressure for the structural conservation of the remaining V beta genes.

Combination of parenteral and oral immunotherapy in grass pollen-allergic children. A double-blind controlled study of clinical and immunological efficacy.

Twenty patients with a proven sensitization to grass pollens were treated with parenteral "priming" and subsequently with either oral "booster" (n = 10) or placebo (n = 10) extension course. The study was carried out in a double-blind manner. Cumulative preseasonal parenteral dosage was 3,100 NU (Noon Units), patients in the oral group subsequently received 123.9 mg of grass pollen extract during the pollen season. No side effects were noted after intake of the oral preparation. No significant difference (95% confidence interval) were noted comparing results of in vivo (skin prick test and conjunctival provocation test) and in vitro tests (specific serum IgE- and IgG-antibodies) between the two groups. Analysis of symptom and medication scores as well as subjective assessment of patients revealed no superiority of oral "booster" over placebo. Data obtained in this study does not support the concept of combined parenteral and oral treatment. This is in contrast to work reported previously.