'Humanized mice' are anticipated to be a valuable tool for studying the human immune system, but the reconstituted human immune cells have not yet been well characterized. Here, we extensively investigated the differentiation and functions of human B and T cells in a supra-immunodeficient mouse strain, NOD/shi-scid/gammac(null) (NOG) reconstituted with CD34(+) hematopoietic stem cells obtained from umbilical cord blood. In these hu-HSC NOG mice, the development of human B cells was partially blocked, and a significant number of B-cell progenitors accumulated in the spleen. The mature CD19(+)IgM(+)IgD(+) human B cells of the hu-HSC NOG mice could produce IgG in vivo and in vitro by antigenic stimulation. In contrast, although human T cells with an apparently normal phenotype developed, most of them could neither proliferate nor produce IL-2 in response to antigenic stimulation by anti-CD3 and anti-CD28 antibodies in vitro. The positive selection of human T cells in the thymus was sufficiently functional, if not complete, and mainly mediated by mouse class II, suggesting that the human T cells lost their function in the periphery. We found that multiple mechanisms were involved in the T-cell abnormalities. Collectively, our results demonstrate that further improvements are necessary before humanized mice with a functional human immune system are achieved.

SummaryI.ntegrin/hgand binding evokes tyrosine phosphorylation of various proteins. We reported previously that a 105 kD protein (ppl05) was tyrosine phosphorylated by the engagement of [31 integrins in T lymphocytes. We show here that ppl05 is a novel p130Cas (Crk-associated substrate)-related protein. Deduced amino acid sequence revealed that ppl05 contains conserved motifs with p130Cas, and both ppl05 and p130Cas bind to focal adhesion kinase (pp125FAK) and Crk. However, ppl05 has a clearly distinct structure from p130Cas, and ppl05 is preferentially expressed in lymphocytes, whereas p130Cas is expressed in adherent cells. With these findings, we designate ppl05 as Cas-L, lymphocyte-type Cas. Furthermore, we demonstrate that integrin/hgand binding results in the recruitment of Crk, Nck, and SHPTP2 to ppl05. These findings further define the roles of ppl05/Cas-L and pp125FAK in the integrin-mediated signaling pathways. ever, a 105-kD tyrosine-phosphorylated protein (ppl05) has not been identified.ppl05 is a protein that we first identified in T lymphoblastoid H9 cells as well as peripheral T cells (11). ppl05 is a 105-kD protein that is tyrosine phosphorylated by the engagement of 0L4131 integrin. Our previous studies demonstrated that ppl05 is distinct from pp125FAK, although both ppl05 and pp125FAK are tyrosine phosphorylated via similar kinetics by the engagement of oL4 [~l integrin in H9 ceils (12). Given the fact that ppl05 is one of the major proteins that is tyrosine phosphorylated by [31 integrin stimulation in peripheral T cells, ppl05 may play an important role in [31 integfin-mediated signaling in T cells.Recently, Sakai et al. (13) cloned the cDNA ofpl30Cas (Crk-associated substrate), p130Cas was originally identified as a tyrosine-phosphorylated 130 kD protein in the p60 v-src-or p47V-Crk-transformed fibroblasts. The deduced amino acid sequence of pl30Cas revealed that p130Cas contained one Src homology (SH) 3 domain in the NH zterminal region and multiple putative binding sites of SH2 domains. Tyrosine phosphorylated pl30Cas bound to Crk, and the deduced amino acid sequence of p130Cas contained multiple binding motifs of the Crk SH2 domain. Crk can bind to guanine nucleotide-releasing factors son of sevenless (SOS) and C3G by the SH3 domain of Crk (14,15). SOS and C3G are involved in the activation of Kas

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Five monoclonal antibodies detected a surface antigen expressed exclusively on T-cell acute lymphoblastic leukemia (T-ALL) in a panel of 45 human hematopoietic cell lines, including T-cell lines derived from adult T-cell leukemia and those established by immortalization with human T-cell leukemia virus type 1 or Herpesvirus saimiri. Peripheral blood mononuclear cells, including fresh and activated T cells, were also completely devoid of this antigen. We designated this antigen as TALLA-1 (from T-ALL-associated antigen 1). By expression cloning, a cDNA clone encoding TALLA-1 was isolated from T-ALL cell line Molt-4. TALLA-1 was found to be a member of the transmembrane 4 superfamily (TM4SF). The cDNA was also essentially identical to A15, which was isolated from another T-ALL cell line, HPB-ALL, by differential hybridization with normal peripheral blood lymphocytes, and to CCG-B7, which was isolated from a brain cDNA library using CCG repeat as a probe. The gene product was now characterized in detail at the protein level. Northern blot analysis showed that the gene was expressed most strongly in brain, skeletal muscle and spleen. In a panel of 52 non-hematopoietic human cell lines, the majority of neuroblastoma cell lines were found to be positive for TALLA-1. Like ME491, CO-029 and L6, TALLA-1 may be another TM4SF member behaving as a potential tumor-associated antigen.

Transcription factor GATA-2 is essential for the proper function of hematopoietic stem cells and progenitors. Two first exons/promoters have been found in the mouse GATA-2 gene, and a distal IS promoter shows activity specific to hematopoietic progenitors and neural tissues. To ascertain whether the two-promoter system is also utilized in the human GATA-2 gene, we isolated and analyzed a P1 phage clone containing this gene. The nucleotide sequence of the human GATA-2 gene 5' flanking region was determined over 10 kbp, and a human IS exon was identified in the locus through sequence comparison analysis with that of the mouse GATA-2 IS exon. RNA blotting and reverse-transcribed PCR analyses identified a transcript that starts from the IS exon in human leukemia-derived cell lines. The IS-originated transcript was also identified in CD34-positive bone marrow and cord blood mononuclear cells, which are recognized as clinically important hematopoietic stem cell-enriched fractions. Phylogenic comparison of the human and mouse GATA-2 gene sequences revealed several regions in the locus that exhibit high sequence similarity. These results demonstrate that the GATA-2 gene regulatory machinery is conserved among vertebrates. The fact that the human IS promoter is active in the hematopoietic stem cell/progenitor fraction may be an important clue for the design of a vector system that can specifically express various genes in hematopoietic stem cells and progenitors.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental pollutant causing public concern.Its toxic effects include disruption of the immune, endocrine, and reproductive systems, impairment of fetal development, carcinogenicity, and lethality in rodents. Here, we report that TCDD induces apoptosis in two cultured human leukemic lymphoblastic T cell lines. This cell death was found not to be dependent on an aryl hydrocarbon receptor and to be inhibited by the inhibitor of tyrosine kinases and caspases. Apoptosislinked c-Jun N-terminal kinase is rapidly activated in these cells by the treatment with TCDD. A dominantnegative mutant of c-Jun N-terminal kinase prevented cell death in the treatment with TCDD. Furthermore, TCDD decreases the Bcl-2 protein level in these cell lines. These findings will help in the understanding of the molecular mechanism underlying TCDD-mediated immunotoxicity. 2,3,7, 1 is a prototype halogenated aromatic hydrocarbon, and it is considered to be one of the most potent toxicants studied (1). The adverse biological effects of TCDD seen in experimental animals include immune, reproductive, and developmental toxicity, carcinogenicity, wasting syndrome, chloracne, and lethality (2). While the immunotoxic effects of TCDD have been well characterized in the rodent model, little data are available for humans. The thymic atrophy caused by TCDD in rodents is mediated by a selective killing of immature thymocytes, although the exact mechanism remains unknown. In the rat (3) and mouse (4) immature thymocytes treated with TCDD (in vitro) have been reported to die due to apoptosis, although other studies have produced contradictory results (5). However, the demonstration of this effect in vivo has met with only limited success. The lack of a cell culture system in which toxicity can be readily detected and shown in a regulated manner has hindered efforts to understand the intracellular and molecular events by which TCDD induces apoptosis. It has been assumed that all the biological effects of TCDD are mediated through the Ah receptor (1), a ligand-activated member of the bHLH-PAS family of transcription factors (6, 7). However, not all the biological effects of TCDD can be explained using this receptor-based model.Thus while it is clear that TCDD is highly toxic to mammals, the cellular mechanisms by which TCDD exerts its toxic effects are obscure. We now describe a cell culture system derived from human T cell lymphomas that provides unique advantages for studying the molecular mechanisms underlying the action of TCDD. The idea for this culture system came to us when we observed, during our recent studies of TCDD, that, when the human leukemic lymphoblastic T cell line L-MAT (8) was exposed to TCDD, the cells underwent morphological changes and eventual apoptosis. Using this cell culture system, we can now more fully investigate the role of the Ah receptor in the mechanism of TCDD-mediated apoptosis, which has hitherto been difficult with the use of human tissues. MATERIALS AND METHODSCel...

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remains unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, while ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. ASNS CpG island is largely unmethylated in normal hematopoietic cells but is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knock-in mice. In three childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL with favorable karyotypes but is mostly unmethylated in BCP-ALL with poor prognostic karyotypes. Higher ASNS methylation is associated with higher l-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene due to aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.