Null mutation of the Lmo4 gene or a combined null mutation of the Lmo1/Lmo3 genes causes perinatal lethality, and Lmo4 controls neural tube development in mice

The LIM-only family of proteins comprises four members; two of these (LMO1 and LMO2) are involved in human T-cell leukemia via chromosomal translocations, and LMO2 is a master regulator of hematopoiesis. We have carried out gene targeting of the other members of the LIM-only family, viz., genes Lmo1, Lmo3 and Lmo4, to investigate their role in mouse development. None of these genes has an obligatory role in lymphopoiesis. In addition, while null mutations of Lmo1 or Lmo3 have no discernible phenotype, null mutation of Lmo4 alone causes perinatal lethality due to a severe neural tube defect which occurs in the form of anencephaly or exencephaly. Since the Lmo1 and Lmo3 gene sequences are highly related and have partly overlapping expression domains, we assessed the effect of compound Lmo1/Lmo3 null mutations. Although no anatomical defects were apparent in compound null pups, these animals also die within 24 h of birth, suggesting that a compensation between the related Lmo1 and 3 proteins can occur during embryogenesis to negate the individual loss of these genes. Our results complete the gene targeting of the LIM-only family in mice and suggest that all four members of this family are important in regulators of distinct developmental pathways.     

The oncogenic T cell LIM-protein Lmo2 forms part of a DNA-binding complex specifically in immature T cells.

The LIM-only protein LMO2 is expressed aberrantly in acute T-cell leukaemias as a result of the chromosomal translocations t(11;14) (p13;q11) or t(7;11) (q35;p13). In a transgenic model of tumorigenesis by Lmo2, T-cell acute leukaemias arise after an asymptomatic phase in which an accumulation of immature CD4(-) CD8(-) double negative thymocytes occurs. Possible molecular mechanisms underlying these effects have been investigated in T cells from Lmo2 transgenic mice. Isolation of DNA-binding sites by CASTing and band shift assays demonstrates the presence of an oligomeric complex involving Lmo2 which can bind to a bipartite DNA motif comprising two E-box sequences approximately 10 bp apart, which is distinct from that found in erythroid cells. This complex occurs in T-cell tumours and it is restricted to the immature CD4(- )CD8(-) thymocyte subset in asymptomatic transgenic mice. Thus, ectopic expression of Lmo2 by transgenesis, or by chromosomal translocations in humans, may result in the aberrant protein interactions causing abnormal regulation of gene expression, resulting in a blockage of T-cell differentiation and providing precursor cells for overt tumour formation.     

The LIM domain protein Lmo4 is highly expressed in proliferating mouse epithelial tissues.

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.     

Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy

Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy.     

Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development

Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.     

LMO2与T细胞白血病

Lmo2基因是LMO(LIM-only)家族的成员之一。作为一个原癌基因,Lmo2的染色体异位t(11;14)(p13;q11)或t(7;11)(q35;p13)与T细胞急性淋巴细胞白血病密切相关。LMO2是细胞中介导转录因子复合物形成的重要接头分子。现对LMO2的分子结构及其在正常和白血病细胞中的调控作用机制的差异作重点介绍。在此基础上还讨论了LMO2成为逆转录病毒介导的基因治疗X染色体连锁的严重联合免疫缺陷综合征过程中成为病毒插入靶位点的可能原因。     

Retroviral insertional mutagenesis can contribute to immortalization of mature T lymphocytes

Several cases of T-cell leukemia caused by gammaretroviral insertional mutagenesis in children treated for x-linked severe combined immunodeficiency (SCID) by transplantation of autologous gene-modified stem cells were reported. In a comparative analysis, we recently showed that mature T cells, on the contrary, are highly resistant to transformation by gammaretroviral gene transfer. In the present study, we observed immortalization of a single T-cell clone in vitro after gammaretroviral transduction of the T-cell protooncogene LMO2. This clone was CD4/CD8 double-negative, but expressed a single rearranged T-cell receptor. The clone was able to overgrow nonmanipulated competitor T-cell populations in vitro, but no tumor formation was observed after transplantation into Rag-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptors were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. This study demonstrates that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results show that signaling of the IL-2 receptor and the protooncogene LMO2 can act synergistically in maligniant transformation of mature T lymphocytes.     

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell-T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell-mediated immunity.     

Disordered T-Cell Development and T-Cell Malignancies in SCL LMO1 Double-Transgenic Mice: Parallels with E2A-Deficient Mice

The gene most commonly activated by chromosomal rearrangements in patients with T-cell acute lymphoblastic leukemia (T-ALL) is SCL/tal. In collaboration with LMO1 or LMO2, the thymic expression of SCL/tal leads to T-ALL at a young age with a high degree of penetrance in transgenic mice. We now show that SCL LMO1 double-transgenic mice display thymocyte developmental abnormalities in terms of proliferation, apoptosis, clonality, and immunophenotype prior to the onset of a frank malignancy. At 4 weeks of age, thymocytes from SCL LMO1 mice show 70% fewer total thymocytes, with increased rates of both proliferation and apoptosis, than control thymocytes. At this age, a clonal population of thymocytes begins to populate the thymus, as evidenced by oligoclonal T-cell-receptor gene rearrangements. Also, there is a dramatic increase in immature CD44(+) CD25(-) cells, a decrease in the more mature CD4(+) CD8(+) cells, and development of an abnormal CD44(+) CD8(+) population. An identical pattern of premalignant changes is seen with either a full-length SCL protein or an amino-terminal truncated protein which lacks the SCL transactivation domain, demonstrating that the amino-terminal portion of SCL is not important for leukemogenesis. Lastly, we show that the T-ALL which develop in the SCL LMO1 mice are strikingly similar to those which develop in E2A null mice, supporting the hypothesis that SCL exerts its oncogenic action through a functional inactivation of E proteins.     

Single domain intracellular antibodies from diverse libraries: emphasizing dual functions of LMO2 protein interactions using a single VH domain

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC(3)) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Co-crystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC(3) offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.     

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.     

A novel POU domain protein which binds to the T-cell receptor beta enhancer.

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.