Irradiated Syngeneic Mice Research Articles

Potent Cooperation Between NUP98-NSD1 and FLT3-ITD In AML Induction

The NUP98-NSD1 fusion protein, product of the t(5;11)(q35;p15.5) chromosomal translocation, is an AML-associated cytogenetically silent genetic aberration, recently identified as the most frequent fusion in pediatric AML, generally associated with aggressive disease and poor prognosis. Interestingly, the vast majority (>70%) of the reported NUP98-NSD1-positive cases also carried an activating FLT3-ITD mutation suggesting functional cooperation. The purpose of this study was to search for experimental evidence of a functional cooperation between NUP98-NSD1 and FLT3-ITD in the transformation of murine hematopoietic cells in vitro and in vivo.Lineage surface marker-depleted murine bone marrow cells were transduced with either pMSCV-NUP98-NSD1-neo or pMSCV-FLT3-ITD-GFP or both expression constructs on fibronectin-coated plates. Serial colony formation assays in myeloid favoring medium and immunophenotypic analysis by flow cytometry indicated that retroviral expression of NUP98-NSD1 provided increased self-renewal capacity and impaired differentiation of murine bone marrow stem and progenitor cells.NUP98–NSD1 expressing cells displayed a typical myeloblastic morphology and co-expressed myeloid and early stem cell surface markers (CD34low/c-kit+/FcgR+/Gr-1+/ Mac-I+/B220-). Co-expression of FLT3-ITD resulted in high rates of cell proliferation, showed a more differentiated phenotype and concomitantly impaired the in vitro clonogenic capacity in methylcellulose cultures.Bone marrow cells expressing NUP98-NSD1 with or without FLT3-ITD were harvested from methylcellulose cultures and transplanted into sub-lethally irradiated syngeneic mice. All mice receiving cells co-expressing NUP98-NSD1 and FLT3-ITD developed AML that was transplantable into all secondary recipients. Myeloid leukemic blasts that co-expressed NUP98-NSD1 and FLT3-ITD were present in abundance both in BM preparations and in blood smears, and histopathological analysis showed widespread infiltration into solid organs. By contrast, no AML ever developed in mice receiving cells expressing only NUP98-NSD1. These mice, similar to mice receiving cells expressing FLT3-ITD only, developed signs of a chronic myeloproliferative disorder, characterized by expansion of Mac-1+/Gr-1+ BM cells with granulocytic/monocytic differentiation that in some cases caused severe distress after a latency period of more than one year. Intriguingly, upon injection with double transduced NUP98-NSD1 and FLT3-ITD progenitors rather different latency periods of the AML development were observed between different experiments. Interestingly, the latency periods could be correlated to the ratio of expression levels of FLT3-ITD to wildtype FLT3, with higher FLT3-ITD levels associated with a shorter latency. To further investigate the significance of aberrant FLT3 signaling, in vitro and in vivo transformed NUP98-NSD1 and NUP98-NSD1/FLT3-ITD cells were treated with a selective FLT3 tyrosine kinase inhibitor (PKC412). The higher sensitivity of cells co-expressing NUP98-NSD1 and FLT3-ITD to PKC412, compared to cells expressing NUP98-NSD1 only, indicated that proliferation and survival were dependent on FLT3-derived signals.Taken together, these observations demonstrate a potent cooperation between NUP98-NSD1 fusion and FLT3-ITD in leukemic transformation. However, neither the NUP98-NSD1 fusion protein nor the FLT3-ITD mutation alone was sufficient to induce AML. Moreover, the high sensitivity of NUP98-NSD1 and FLT3-ITD co-expressing leukemic blasts to FLT3 signaling inhibition suggests a possible therapeutic strategy to be further explored in this AML subgroup. Disclosures:No relevant conflicts of interest to declare.

In Vivo RNAi Screening Identifies HDAC4 As a Mediator Of Chemoresistance In Acute Myeloid Leukemia

The majority of AML patients still relapse, despite intensive chemotherapy. A subset of more chemoresistant leukemia cells might be responsible for this phenomenon. These relapse-initiating cells might differ epigenetically from the more chemosensitive leukemia bulk. Since interaction of leukemic cells with the niche might contribute to chemoresistance, we decided to perform an in vivo RNAi screen for modulators of chemoresistance. We constructed a focussed shRNA library with ∼400 constructs that target 142 genes involved in stem cell functions, leukemogenesis and epigenetic regulation. Myc/Bcl2 induced murine leukemias were retrovirally transduced and injected into sublethally irradiated syngeneic mice. Mice (n=24) were either treated with Cytarabine, Azacytidine, Decitabine, or vehicle. Upon signs of leukemia, DNA was extracted and the shRNA pool was PCR-amplified from bone marrow, spleen and liver of each individual mouse. Overall, 61 libraries were sequenced with more than 500-fold coverage of each construct. Suppression of HDAC4, Pik3r1, Atr, Ccna2 and HDAC1 significantly enhanced sensitivity towards Cytarabine treatment. Histone deacetylase 4 (HDAC4) was identified as a top candidate since it enhanced the efficacy of all three drugs. It was depleted more than sixteen-fold upon Cytarabine treatment. HDAC4 suppression by the specific shRNAs was confirmed by real-time RT-PCR and western blot analysis. In a competitive re-transplantation assay in vivo, HDAC4 suppression inhibited leukemic growth. Further, we analyzed inhibition of HDAC4 by LMK235, a novel and specific inhibitor of HDAC4 and 5 in human HL60 leukemia cells. Synergistic inhibition was demonstrated for LMK235 and Cytarabine in proliferation assays and in colony formation assays. These findings demonstrate that in vivo RNAi screening for therapeutic efficacy is feasible. HDAC4 might be an important target to enhance efficacy of anti-leukemic drugs. Disclosures:No relevant conflicts of interest to declare.

Role for notch/RBPj in preserving hematopoietic stem cell activity

Notch and Wnt signals are required for Hematopoietic Stem Cells (HSCs) generation, however, their putative role for maintenance of HSC in physiological conditions remains unclear. For example specific deletion of RBPj or β-catenin in the hematopoietic system does not compromise the viability of the mice and HSC maintain some repopulation capacity. However, limiting dilution demonstrated that β-catenin-deficient HSCs have decreased self-renewal capacity. In contrast, no evidence for Notch signalling on HSC function has been described. We here analyzed the reconstitution capacity of vav-cre;RBPjlox bone marrow cells by competitive transplant assays into irradiated syngenic mice. We found a significant decrease in the capacity of RBP mutant cells to engraft in primary recipients when compared to wildtype cells, similar to the β-catenin-deficient phenotype. Since both pathways function in an intimate collaboration in other tissues, we generated double RBPj and β-catenin deletion mutants of the hematopoietic system (by using vav1-cre). Interestingly, double knockout animals were viable and born at Mendelian ratios. We will here describe and discuss the phenotype of these double mutants including a detailed analysis of HSC potential and differentiation of the different hematopoietic lineages. Our preliminary data reveals a new role for Notch in maintaining the functionality of adult HSCs, which may involve Wnt/β-catenin. This research is funded by by Ministerio Ciencia e Innovación (PLE2009-0111, SAF2010-15450, RD12/0036/0054), AGAUR (2009SGR-23 and CONES2010-0006). Notch and Wnt signals are required for Hematopoietic Stem Cells (HSCs) generation, however, their putative role for maintenance of HSC in physiological conditions remains unclear. For example specific deletion of RBPj or β-catenin in the hematopoietic system does not compromise the viability of the mice and HSC maintain some repopulation capacity. However, limiting dilution demonstrated that β-catenin-deficient HSCs have decreased self-renewal capacity. In contrast, no evidence for Notch signalling on HSC function has been described. We here analyzed the reconstitution capacity of vav-cre;RBPjlox bone marrow cells by competitive transplant assays into irradiated syngenic mice. We found a significant decrease in the capacity of RBP mutant cells to engraft in primary recipients when compared to wildtype cells, similar to the β-catenin-deficient phenotype. Since both pathways function in an intimate collaboration in other tissues, we generated double RBPj and β-catenin deletion mutants of the hematopoietic system (by using vav1-cre). Interestingly, double knockout animals were viable and born at Mendelian ratios. We will here describe and discuss the phenotype of these double mutants including a detailed analysis of HSC potential and differentiation of the different hematopoietic lineages. Our preliminary data reveals a new role for Notch in maintaining the functionality of adult HSCs, which may involve Wnt/β-catenin. This research is funded by by Ministerio Ciencia e Innovación (PLE2009-0111, SAF2010-15450, RD12/0036/0054), AGAUR (2009SGR-23 and CONES2010-0006).

Enhanced and aberrant T cell trafficking following total body irradiation: a gateway to graft‐versus‐host disease?

Pre-transplant conditioning regimens play a major role in triggering graft-versus-host disease (GVHD). This study investigated the effect of irradiation on donor T cell trafficking to lymphoid and non-lymphoid tissues by comparing the migration of carboxy-fluorescein diacetate succinimidyl ester-labelled, naïve donor T lymphocytes in vivo in irradiated and non-irradiated syngeneic mice recipients. Recruitment of adoptively transferred naïve T cells to secondary lymphoid organs was increased in irradiated mice and naïve T cells also aberrantly localized to non-lymphoid tissues. Irradiation also induced aberrant effector memory T cell migration into lymph nodes and their localization to homing-privileged non-lymphoid sites, such as the gut. The presence of a minor histocompatibility mismatch further enhanced the aberrant accumulation of T cells in both lymphoid and non-lymphoid tissue, whilst their migratory pattern was not modified as compared to fully matched irradiated recipients. These effects correlated with decreased permeability of, and the secretion of chemotactic factors by the endothelium. Our findings are consistent with the possibility that excessive, dysregulated extravasation of T cells induced by irradiation promotes the development of GVHD.

Transcription Factor Hes1 Functions As a Tumor Suppressor in MLL-Associated Acute Leukemia

AbstractAbstract 118 Background:The transcription factor Hairy enhancer of split1 (Hes1) is well characterised as a downstream target of Notch signaling. Hes1 is a basic helix-loop-helix-type protein, and represses target gene expression. Notch signaling has been proposed to play bivalent roles in tumorigenesis, depending on the cell types. Notch/Hes1 pathway promotes leukemogenesis in T-cell acute lymphoblastic leukemia, and Hes1 also plays an oncogenic role in a mouse model of blastic transformation of chronic myelogenous leukemia. Recent studies, however, demonstrated that Notch signaling serves as a tumor suppressor for chronic myelomonocytic leukemia. Meanwhile, the role of Notch signaling in acute myeloid leukemia (AML) remains to be elucidated. To explore whether Hes1 could possibly play oncongenic or tumor suppressive roles in AML, we focused on a mouse leukemia model established by retroviral transduction with MLL-AF9. Methods:Common myeloid progenitors (CMPs), purified from Hes1−/− or Hes1+/+mouse fetal liver, were retrovirally transduced with MLL-AF9 cDNA. MLL-AF9-transduced cells were subjected to a colony-forming assay, with multiple rounds of replating. These cells were transplanted into lethally irradiated syngenic mice. CMPs from wild-type mouse bone marrow (BM-CMPs) were transduced with MLL-AF9 and dominant negative Hes1 (dnHes1), which lacks WRPW domain, and these cells were transplanted into irradiated mice. Secondary transplantation was performed by infusing leukemic bone marrow cells obtained from the primary recipients. Expression levels of downstream targets were evaluated by cDNA array and quantitative RT-PCR. Result:MLL-AF9-transduced Hes1−/− CMPs formed higher number of colonies at third replating compared to MLL-AF9-transduced Hes1+/+ CMPs, while colony numbers were not different in first and second plating. When infused to irradiated syngenic mice, MLL-AF9/Hes1−/− cells developed leukemia at shorter latencies than MLL-AF9/Hes1+/+ cells (MLL-AF9/Hes1−/−, 7–10 weeks, n=18 vs MLL-AF9/Hes1+/+, 10–14 weeks, n=18; p<0.001). Both MLL-AF9/Hes1−/−- and MLL-AF9/Hes1+/+- derived leukemia cells prepared from bone marrow of the first recipients were transplantable and caused leukemia in the secondary recipients with shorter and similar latencies (MLL-AF9/Hes1−/−, 4 weeks, n=10 vs MLL-AF9/Hes1+/+, 4 weeks, n=5). Mice receiving BM-CMPs transduced with MLL-AF9 and dnHes1 also developed leukemia at shorter latencies than mice receiving BM-CMPs transduced with MLL-AF9 and mock (dnHes1/MLL-AF9 8–10 weeks, n=10 vs mock/MLL-AF9 14–20 weeks, n=10, p<0.01). Leukemia cells induced by both MLL-AF9 and dnHes1 were transplantable and caused leukemia in the secondary recipients with latencies shorter than those induced by MLL-AF9 alone (dnHes1/MLL-AF9 3–4 weeks, n=10 vs mock/MLL-AF9 4–5 weeks, n=5, p<0.05). MLL-AF9/Hes1−/− and MLL-AF9/Hes1+/+ leukemia cells were then compared for mRNA expression by cDNA microarray. Among the genes with different expression levels between MLL-AF9/Hes1−/− and MLL-AF9/Hes1+/+leukemia cells, Meis1 was expressed at significantly higher levels in MLL-AF9/Hes1−/− leukemia cells. Discussion:These results imply that Hes1 serves as a tumor suppressor in MLL-AF9-induced AML. In most of the MLL-associated leukemias, dysregulation of HOX genes is thought to be a mainstay of leukemogenesis. Meis1 is a key cooperative factor for the function of each HOX protein, and thus, presumed to play an important role in development of MLL-associated leukemias. Repression of Meis1 might be among mechanisms how Hes1 functions as a tumor suppressor in MLL-associated leukemia. Disclosures:No relevant conflicts of interest to declare.

Stress Response Gene Egr-1 As Tumor Suppressor in BCR-ABL Mediated Leukemia

Abstract 33Chronic Myelogenous Leukemia (CML) is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. In the late 1990s a Tyrosine Kinase Inhibitor (TKI), imatinib mesylate (Gleevec, Novartis) started to be effectively used on CML patients. However, imatinib, therapy suppresses rather than eliminates the disease, and resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. The transcription factor early growth response 1 (Egr-1) was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There's evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). Therefore, we chose to investigate if Egr-1 can act as a suppressor of BCR-ABL driven leukemia. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, we investigated the stem cell compartment of both Egr-1 WT and Egr-1−/− BM, by determining the percentage of stem cells (Lin−Sca+c-Kit+, LSK), before infection with BCR-ABL; no statistically significant difference in the percentage of LSK cells was observed between Egr-1 WT and Egr-1−/− BM. Thus, the BM stem cell compartment of the Egr-1−/− mice does not offer a quantitiative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. Furthermore, when BM of transplanted mice was analyzed we observed an increased population of lineage negative cells in Egr-1−/− BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1−/− BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Additional data from our animal model, as well as analysis of human leukemia samples will be presented, further corroborating that Egr-1 functions as a suppressor of BCR-ABL driven CML. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics for CML, as well as for other leukemic diseases. Disclosures:No relevant conflicts of interest to declare.

Implication of the Distinct Differentiation Pathway of Megakaryocytes From Hematopoietic Stem Cells in the Mouse Bone Marrow

AbstractAbstract 1204 Background.Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist. Purpose.To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice. Results and Discussion.GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis.In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC.We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC.We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1.Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them. Disclosures:No relevant conflicts of interest to declare.

The in Vitro and In Vivo Oncogenic Activity of Homodimeric Mutant of Interleukin-7 Receptor a Chain (IL7Rα) Highlights the Significance of the IL7Rα/Jak1 Pathway in T-Cell Acute Lymphoblastic Leukemia

Abstract 73Interleukin-7 (IL7) is essential for T cell development and homeostasis. Dysregulation of signals that control normal T-cell development has been implicated in the onset of T-cell acute lymphoblastic leukemia (T-ALL). By analogy to activating mutations in the Notch pathways, we hypothesized that any mutations in the IL7 signaling axis might also contribute to T-ALL. Direct sequencing of human IL7 receptor a chain (hIL7RA) gene in a panel of 16 T-ALL cell lines identified two types of mutations in two different cell lines. One was an insertion mutation of 4 amino acids (LSRC) in the transmembrane region (INS, Fig.1A) from DND-41, a gamma-delta TCR+ T-ALL cell line, and the other was a truncated, loss-of-function, mutation in the cytoplasmic region from MOLT-4. We demonstrated that hIL7RA-INS mutant spontaneously formed a homodimer and constitutively activated downstream signals including Stat family members (1, 3 and 5), Akt and Erk via Jak1, but not Jak3.Next, we investigated oncogenic activity of hIL7RA-INS in primary hematopoietic progenitor cells. To this aim, lin− E.14 Balb/c fetal liver (FL) cells were retrovirally transduced with hIL7RA-INS in parallel with hIL7RA-wild type (WT), and then tested for their cytokine dependence in vitro. As expected, only hIL7RA-INS-transduced lin−FL cells showed abrogation of cytokine dependence. hIL7RA-transduced lin−FL cells were also transplanted into lethally irradiated syngeneic mice. Within 7–9 weeks after transplantation of lin−FL cells transduced with hIL7RA-INS, but not with hIL7RA-WT, recipient mice developed well-tolerated myelo- and lymphoproliferative disorders, characterized by marked leukocytosis, systemic lymphadenopathy and splenomegaly (Fig.1B). Notably, concomitant increase in hIL7RA+gamma-delta TCR+ T cells and decrease in B cells were observed in peripheral blood (Fig.1C). Histological examination of bone marrow, spleen and liver specimens from diseased mice revealed moderate to severe myeloid hyperplasia, disrupted splenic architecture by disseminated mature myeloid cells and infiltration of both myeloid and mononuclear cells into hepatic parenchyma, respectively. In addition, recipient mice for hIL7RA-INS-transduced lin−FL cells frequently manifested ruffled fur as well as mononuclear cell infiltration into salivary gland and pericardium, suggesting an autoimmune-like disorder. However, during median follow-up of 11 weeks, these recipient mice did not develop either overt leukemia or lymphoma, indicating that additional transforming events are required for evolution to aggressive hematological malignancies. These in vivo findings highlighted the possibility that aberrant signals via IL7RA in hematopoietic stem/progenitor cells might preferentially stimulate myelopoiesis over lymphopoiesis, and also confirmed the essential role of IL7RA in gamma-delta TCR+ T cell development, previously shown by IL7RA-knockout mice. Taken together, we speculated that dysregulated IL7RA signaling axis might be involved in the onset of T-ALL, especially with gamma-delta TCR+ phenotype. Finally, the present study, together with the recent report (JEM 208:901, 2011), emphasizes the significance of the sequential Notch-IL7RA pathways in the pathogenesis of T-ALL as well as the dominant role of the IL7RA/Jak1 axis in IL7 proliferative signal. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 3098: Myc preferentially transforms hematopoietic stem cells into Precursor B cell lymphoblastic leukemia/lymphoma

Abstract Overexpression of Myc oncogenes (c-Myc, N-Myc and L-Myc) is frequently found in a wide range of tumors. Although many reports have shown that Myc can directly induce tumors in mice models, the cell-of-origin of the Myc-induced tumor is still unclear. In this study, we attempted to determine the cell-of-origin in the Myc-induced hematopoietic tumor by using bone marrow transplantation model. Bone marrow (BM) cells were harvested from 5FU-treated mice (6 to 8-week-old C57BL/6 mice) and then infected with retroviral vectors to express one of Myc genes, N-Myc. The infected cells were intravenously transplanted into irradiated syngenic mice. As a result, all mice transplanted with N-Myc-transduced BM cells died around 2 months. Those mice showed enlarged lymph nodes, splenomegaly and invaded livers. Abnormally increased WBCs in those mice showed B220+CD19+CD43+/−IgM−, indicating that enhanced expression of N-Myc in BM cells caused Precursor B cell lymphoblastic leukemia/lymphoma (Pre-B LBL). To determine the cell-of-origin of this tumor, the N-Myc-transduced BM cells were fractionated into hematopoietic stem cells (HSCs), committed lymphoid progenitors (CLPs) or myeloid progenitors (MPs) by cell sorter, and those fractions were transplanted into irradiated recipients. Limiting dilution analysis revealed that mice transplanted with HSCs had Pre-B LBL at significant higher frequencies (1:184) than mice transplanted with CLPs (1:558) or MPs (1:9286). Furthermore, to determine whether more lineage-committed cells can be directly the cell of origin, we sorted immature-B cells transduced with N-Myc from non-treated BM cells and transplanted those cells into recipients. While we could not obtain any tumors from those transplanted mice, mice transplanted with immature-B (B220+CD19+IgD−IgM−) cells derived from p16Ink4a−/−p19Arf−/− mice had Pre-B LBL, suggesting that committed B cells could be the direct cell-of-origin in the absence of p16Ink4a and p19Arf genes which are known as tumor suppressor genes. Lastly, we confirmed that another Myc family gene c-Myc also could induce Pre-B LBL, showing similar histopathology and immunophenotype to the N-Myc-induced pre-B LBL. Collectively, our findings indicate that Myc genes can preferentially transform HSCs into Pre-B LBL and, that p16Ink4a and p19Arf genes can be crucial barriers to block Myc-induced oncogenic stress in more committed cells than HSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3098.

Pre-Transplant Integration Site Diagnostics in Hematopoietic Stem Cell Gene Transfer.

Abstract 3581Poster Board III-518We and others have previously shown that insertional activation of cellular genes caused by integrated retroviral vectors can lead to clonal dominance and malignant transformation. Pre-transplant diagnostics of vector flanking sequences and subsequent elimination of those clones that carry potentially dangerous integration sites prior to transplantation would dramatically improve the safety of clinical gene therapy regimens. Such a strategy requires efficient transduction of few or individual stem cells, their in vitro amplification and highly sensitive integration site determination before transplantation.To define optimal time points for transduction and ascertain the transplantability of ex vivo expanded murine stem cell clones, single CD45+Lin−Rho+SP cells isolated from bone marrow of male C57BL/6J (B6J) mice were cultivated for 8-10 days in the presence of IL11, SCF and Flt3-L. 10% of the sorted cells formed clones in vitro. In 28% ± 5% of these clones, the first division occurred during the first 48 hours after sorting, another 32% ± 8% divided up to 72 hours after sorting and additional 33% ± 7% up to 96 hours after sorting. 7% ± 4% had undergone their first division at a later time point. To examine the transplantability after ex vivo expansion, individual cell clones (containing 12 to >600 cells) were transplanted together with 105 carrier cells into lethally irradiated sex-mismatched syngeneic mice. The presence of donor-derived cells in peripheral blood of 20 transplanted mice was analyzed by Y-chromosome specific PCR. 55% of the ex vivo expanded clones contributed to post-transplant hematopoiesis. 25% of these clones exhibited long-term activity for >6 months after transplantation. Interestingly, only cell clones that had undergone their first division 48-96 hours after cell sorting contributed to long-term post-transplant hematopoiesis.For transduction, individual stem cell clones were spinoculated for 60 minutes with a GFP encoding lentiviral vector (MOI 100-5000). 5 days after transduction, 50% of cells generated by each clone were harvested, lysed and analyzed by LAM-PCR and integration site sequencing. After an additional 3 days, single clones were transplanted together with 105 carrier cells into lethally irradiated congeneic B6.SJL-PtprcaPepcb/BoyJ mice. Four weeks after transplantation, in 30% of these mice ≥0.4% CD45.1+ cells derived from single cell clones were detected in the peripheral blood. In 50% of these mice, the transduced clones contributed to myelopoiesis as well as lymphopoiesis for more than 24 weeks after transplantation, demonstrating that the longterm hematopoietic stem cell potential was retained after single cell marking and expansion.These results demonstrate that single stem cell gene transfer and subsequent expansion is possible to allow integration site determination. Long-term stem cells with defined lentiviral integration sites can be selected for transplantation. In summary, we provide proof of concept that pre-transplant diagnostics of integration sites is feasible to increase the safety of gene therapy by eliminating stem cell clones from transplants that carry unwanted integration sites. Disclosures:No relevant conflicts of interest to declare.

The Differential Role of Stromal Derived Factor-1 (SDF-1) and Monocyte Chemoattractant Protein-1 (MCP-1) in the Recruitment of Angiogenic Cells to Ischemic Tissue.

There is compelling evidence that circulating angiogenic cells exist in humans that are able to home to sites of vascular injury and stimulate angiogenesis. This observation has led to tremendous interest in the translational potential of these cells to mediate therapeutic angiogenesis. However, the number of angiogenic cells in the blood at baseline is low, limiting their delivery to sites of ischemia. To circumvent this limitation angiogenic cells can be mobilized into the blood. Using a murine model of acute hindlimb ischemia, we previously showed that treatment with G-CSF and/or AMD3100 potently enhanced revascularization. Adoptive transfer studies showed that mobilized monocytes mediate this response. Finally, the monocytes, once recruited to the ischemic tissue, stimulate angiogenesis in a paracrine fashion. The signals that recruit monocytes to sites of ischemia remain poorly defined. A better understanding of these signals may provide important insights into strategies to enhance or inhibit angiogenic cell recruitment. Herein, we summarize our recent findings suggesting that MCP-1 and SDF-1 play key but distinct roles in this process.SDF-1, through its major receptor CXCR4, is thought to play an important role in angiogenic cell recruitment and/or retention at sites of ischemia. To test this hypothesis, we studied the angiogenic capacity of CXCR4−/− cells. Since CXCR4 deficiency is embryonic lethal, we first generated bone marrow chimeras by transplanting CXCR4−/− fetal liver cells into irradiated syngeneic mice. Hematopoietic reconstitution with CXCR4−/− cells was confirmed by flow cytometry. Endogenous revascularization following the induction of hindlimb ischemia in these CXCR4−/− chimeric mice was comparable to wild type chimeric mice. To further assess the angiogenic capacity of CXCR4−/− cells, adoptive transfer experiments into wild type recipient mice were performed. As reported previously, adoptive transfer of wild type bone marrow cells markedly improved blood flow following induction of hindlimb ischemia [ratio of ischemic to nonischemic limb perfusion on day 14 after surgery: 0.54 ± 0.04 (saline control) vs. 0.81 ± 0.06 (WT bone marrow); p<0.001]. Surprisingly, adoptive transfer of CXCR4−/− bone marrow cells resulted in a comparable revascularization (0.81 ± 0.16; p<0.001 compared with saline treated controls). Since there is data that T lymphocytes may modulate the angiogenic response to ischemia, we repeated these experiments using RAG-1 deficient recipient mice, which lack B and T lymphocytes. Adoptive transfer of CXCR4−/− bone marrow cells into RAG-1−/− mice failed to stimulate angiogenesis [ratio of ischemic to nonischemic limb perfusion on day 14 after surgery: 0.61 ± 0.04 (saline control) vs. 0.68 ± 0.07(CXCR4−/− bone marrow) vs. 0.82 ± 0.07 (WT bone marrow)]. Collectively, these data show that the contribution of CXCR4 signals to angiogenesis in this vascular injury model is dependant upon the presence of inflammatory cells at the site of injury.MCP-1 is a major chemokine regulating monocyte chemotaxis and activation. Thus, we repeated the adoptive transfer experiments using MCP-1 deficient recipient mice. Adoptive transfer of wild type bone marrow cells into these mice had no effect on revascularization. Moreover, treatment of MCP-1−/− mice with G-CSF and AMD3100, though inducing the expected level of monocyte mobilization, failed to stimulate angiogenesis. These data highlight the importance of MCP-1 to the recruitment, retention, and/or activation of angiogenic cells at sites of ischemia.

Expression of Jak2V617F causes a polycythemia vera–like disease with associated myelofibrosis in a murine bone marrow transplant model

AbstractAn acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

Expression of activated M-Ras in hemopoietic stem cells initiates leukemogenic transformation, immortalization and preferential generation of mast cells

Cultures of purified hemopoietic stem cells transduced with an activated mutant of M-Ras contained abnormal cells that, despite the presence of only low levels of growth factors, generated large, dense colonies of macrophages and blast cells. Cells from these colonies survived and grew continuously in the absence of growth factors and generated clonal cell-lines that were mainly composed of well-differentiated mast cells, with a low frequency of undifferentiated cells. When transplanted into sublethally irradiated syngeneic mice, four out of four such clones gave rise to a systemic mastocytosis and mast-cell leukemia. However, the donor clones also generated low percentages of cells with the morphological and cell-surface characteristics of erythrocytes, granulocytes, monocytes and T- and B-lymphocytes. These data indicate that signals downstream of activated M-Ras are sufficient to transform hemopoietic stem cells, and while preserving their capacity to generate other cell-lineages in vivo, result in preferential generation of mast cells.

Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vector Serotypes 1 Through 5

Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

A Murine Marrow-Derived Multipotent Stem Cell Line Could Reconstitute Hematopoiesis after Transplantation into Lethallly Irradiated Syngeneic Mice.

Objectives In previous study, we have successfully established a murine marrow-derived multipotent stem cell line (mMMSC) from male C57BL/6 mice with highly proliferation and multipotency, which could differentiate into cells of three germ layers in vitro, such as adipocytes, osteoblasts, endothelium cells, hepatocytes and neutral cells, but not hematopoietic cells. The mMMSC expressed high levels of CD29, CD44 and Sca-1, low levels of CD13, CD90, Flk-1 and MHC-I, while negative for CD117, CD45 and MHC-II. RT-PCR showed that two transcription factors important in maintaining undifferentiated embryonic stem cell (ES), Oct-4 and Rex-1, were expressed. The present study was aimed at developing a mMMSC transplantation animal model to evaluate its role in hematopoietic reconstitution. Methods mMMSC and bone marrow cells (BM) freshly harvested from male C57BL/6 mice were used as donor cells. Female C57BL/6 mice lethally irradiated for 8Gy (25cGy/min) with 60Co were used as recipients, which were randomly defined into 4 groups: Group A (Control, n=5) were injected with 0.2ml PBS/per mouse within 4–6 hours after irradiation (injection timepoint was similar thereafter); Group B (BM, n=5): 0.2ml of 6×106 donor BM cells/per mouse; Group C (mMMSC, n=10): 0.2ml of 2×106 mMMSC/per mouse; Group D(mMMSC+BM, n=5): 0.2ml of 2×106mMMSC+6×106 BM cells /per mouse. The fluctuations of WBC and PLT in peripheral blood (PB) were evaluated every week until day 42 after transplantation. At day 42 post-transplantation, all alive mice were sacrificed for histological examination, chromosome analysis with R-banding, FISH using murine chromosome Y painting probe and sex-determining region of Y chromosome(sry) gene analysis with PCR. Results After irradiation, all mice in control group died of bone marrow failure between day 14 and 17. WBC and Hb recovered to (4.03±0.61)×109/L and (127.33±24.00)g/L at day 21 and PLT recovered to (812.33±67.38)×109/L at day 28 post-transplantation in mMMSC group. WBC and PLT in mMMSC group were lower than that in BM and mMMSC+BM groups on days 14 and 21 (P<0.05), and recovered to the same level on day 28, without significant difference among the above 3 groups thereafter. The recovery of WBC and PLT count in mMMSC+BM group was faster than that of BM group (P<0.05). In groups B,C and D, sry gene was detectable in BM, PB and spleen. Y chromosome was found in 100±0%, 50±5% and 100±0% of BM nucleated cells by conventional chromosomal analysis, and Y-positive signals were detected by FISH in 90.67±1.46%, 45.03±1.11% and 94.82±0.90% of interphase nuclei and metaphases, respectively. Highly purified CD45+cells sorted with MACS from BM and PB of mMMSC group showed Y chromosome in 33.3±11.55% of CD45+ BM cells by karyotyping. FISH ascertained Y chromosome in 31.18±1.83% and 24.65±2.77% of CD45+ cells from BM and PB, respectively. Conclusions 1.mMMSC transplantation alone can successfully reconstitute hematopoiesis in lethally irradiated mice. mMMSC can differentiate into hematopoietic cells in vivo and thus might be used as a novel substitute for hematopoietic stem cells (HSC). 2. Co-transplantation of mMMSC and HSC can enhancing hematopoietic reconstitution, which could be one of the potential strategies to use mMMSC for transplantation purposes.

Bone Marrow-Derived Cells Contribute to Pulmonary Vascular Remodeling in Hypoxia-Induced Pulmonary Hypertension

In these days, it was reported that bone marrow (BM) cells might take part in the remodeling of some systemic vascular diseases; however, it remains unknown whether the BM cells were involved in the vascular remodeling of pulmonary arteries and the progression of pulmonary hypertension (PH). The purpose of this study was to investigate whether BM-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced PH. To investigate the role of BM-derived cells, we transplanted the whole BM of enhanced green fluorescent protein (GFP)-transgenic mice to the lethally irradiated syngeneic mice (n = 30). After 8 weeks, chimera mice were exposed to consistent hypoxia using a hypoxic chamber (10% O(2)) for up to 4 or 8 weeks (10 mice per group). After hemodynamics and the ratio of right ventricular (RV) weight to left ventricle (LV) weight, RV/(LV + septum [S]), were measured, histologic and immunofluorescent staining were performed. BM-transplanted mice showed a high chimerism (mean [+/- SEM], 91 +/- 2.3%). RV systolic pressure and the RV/(LV + S) ratio increased significantly with time in PH mice, indicating RV hypertrophy. Marked vascular remodeling including medial hypertrophy and adventitial proliferation was observed in the pulmonary arteries of PH mice. Strikingly, a number of GFP(+) cells were observed at the pulmonary arterial wall, including the adventitia, in hypoxia-induced PH mice, while very few cells were observed in the control mice. Metaspectrometer measurements using confocal laser scanning microscopy confirmed that this green fluorescence was produced by GFP, suggesting that these GFP(+) cells were mobilized from the BM. Most of them expressed alpha-smooth muscle actin, a smooth muscle cell, or myofibroblast phenotype, and contributed to the pulmonary vascular remodeling. A semiquantitative polymerase chain reaction of the GFP gene revealed that the BM-derived GFP-positive cells in the PH group were observed more than eightfold as often compared with the control mice. The BM-derived cells mobilize to the hypertensive pulmonary arteries and contribute to the pulmonary vascular remodeling in hypoxia-induced PH mice.

FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.

Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL).Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated.To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation.Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days).The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.

Dynamic Patterns of Migration and Expansion of Hematopoiesis during MGMT Mediated Drug Selection.

While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.

P53 Silencing by Lentiviral Vector Mediated RNA Interference in Murine Hematopoietic Cells and the Development of a Myelogenous Proliferative Disease.

The ability for siRNA containing lentiviral vectors to specifically downregulate gene expression in the hematopoietic system following hematopoietic stem cell transplantation provides a new tool for developing disease models of leukemia and other hematological disorders. The tumor suppressor gene, p53, has been implicated in the genesis or progression of hematological malignancies, such as AML, MDS, and CML, and disorders such as myeloproliferative disease (MPD). Following transduction of Ly5.1 hematopoietic stem cells using a lentiviral vector expressing small interfering RNAs specific for mouse p53, cells were transplanted into lethally irradiated syngeneic recipient mice. Peripheral blood analyses by FACS at 7 weeks posttransplant showed significantly increased numbers of Gr1 Mac1 double positive progenitor like cells in 4 of 4 mice (73±14% compared to control siGFP transplanted mice 5±0.5%). Interestingly, 3 of 4 transplanted mice showed signs of morbidity by 16 weeks posttransplant due to a myelogenous proliferative disease (MPD) phenotype characterized by high peripheral blood neutrophil counts (>70% of white blood cells) and anemia (<106 RBC/μL), and extensive neutrophil infiltration into spleen and liver. Furthermore, peripheral blood smears show an expansion of megakaryocytes. Bone marrow mononuclear cells from transplanted mice were analyzed for p53 protein expression following p53 stabilization by irradiation and significantly lower levels of p53 were detected in sip53-transduced mice compared to siGFP lentiviral vector control mice. No altering of normal hematopoietic cell differentiation was observed in control mice from nonspecific RNAi interactions, as determined by FACS or blood cell count analyses. These results demonstrate that downregulated expression of p53 in mice hematopoietic cells yields a myelogenous proliferative disease and creates a model for the study of p53 involvement in the genesis and progression of hematological disorders. Furthermore, these results demonstrate the ability of lentiviral vector mediated siRNA to efficiently downregulate endogenous gene expression in vivo and model human disease.

Loss of Ly‐6A.2 expression on immature developing T cells in the thymus is necessary for their normal growth and generation of the Vβ T‐cell repertoire

Stage-specific expression of a number of cell-surface and signaling proteins is critical for normal development of T cells in the thymus. Equally important may be the loss of expression/signaling of developmentally regulated proteins for proper transitioning of developing T cells into thymic subsets. Ly-6A.2 exhibits a regulated pattern of expression on T cells maturing in the thymus, and dysregulating its expression results in arrest of developing T cells within the CD3-CD4-CD8- triple negative (TN) stage where the normal expression of Ly-6A.2 is extinguished. To further characterize the mechanisms underlying this block, we examined whether cell signaling and/or cell adhesion properties of the Ly-6A.2 molecule influenced the block in T-cell development. Analysis of bone marrow chimeras generated by injecting CFSE-labeled Ly-6A.2 transgenic bone marrow cells into irradiated syngeneic non-transgenic mice revealed normal trafficking of developing T cells from the cortex into the medulla. Production of LAT but not p56lck was diminished in CD4-CD8- DN cells from Ly-6A.2 dysregulated mice when compared with control littermates. Dysregulated expression of Ly-6A.2 did not suppress endogenous TCR-Vbeta expression. Finally, dysregulated expression of Ly-6A.2 enhanced apoptosis of an immature CD4+CD8+ (DP) subset of developing cells and altered the selected TCR-Vbeta repertoire. Taken together, these observations indicate that the termination of Ly-6A.2 expression and signaling within the CD4-CD8-CD3- subset of developing T cells is an important checkpoint during normal thymic development.