A Human SIL-SCL Fusion Gene Displays a Development Block at the CD4− CD8− to CD4+CD8+ Transition in Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2833-2833
Author(s):  
Yue Cheng ◽  
Zhenhua Zhang ◽  
Chris Slape ◽  
Peter D. Aplan
Keyword(s):  
T Cell ◽  
Fusion Gene ◽  
Chromosomal Rearrangements ◽  
Regulatory Elements ◽  
Interstitial Deletion ◽  
Lymphocytic Leukemia ◽  
Bac Clone ◽  
Cd8 Cells ◽  
Human T Cell ◽  
Bac Transgene

Abstract Chromosomal rearrangements leading to dysregulation of the SCL gene, located at chromosome 1p32, is a common event in the development of human T-cell acute lymphocytic leukemia (T-ALL). In the most common form of SCL gene rearrangement, an interstitial deletion of approximately 90 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SIL (SCL interrupting locus) gene, which is located directly upstream of SCL. To investigate the consequences of this event, we reproduced this gene alteration by using cre-mediated recombination. A BAC clone containing both human SIL and SCL genes was isolated and loxP sites were cloned into the intron 1 region of both the SIL and SCL genes, corresponding to the sites at which recombination leading to an interstitial deletion occurs in human T-ALL patients. This BAC clone was used to generate transgenic SILloxloxSCL mice. The SILloxloxSCL mice were bred to mice that express the cre recombinase in the thymus (Lck-cre mice). The BAC transgene was recombined between the two loxP sites in over 50% of the thymocytes from double transgenic Lck-cre/SILloxloxSCL mice, faithfully recapitulating the event seen in human T-ALL patients, and bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression was verified by RT-PCR. Using FACS analysis, we found that mice carrying both the SILloxloxSCL transgene and the Lck-cre transgene have decreased CD4+/CD8+, CD4+/CD8−, CD4−/CD8+ and increased CD4−/CD8− thymocytes compared to transgene-negative mice or mice that carried the SILloxloxSCL transgene but not the Lck-cre transgene. These findings were detected in mice from 6 to 15 months of age. Interestingly, increased numbers of CD44+ thymocytes can also been identified in SILloxloxSCL/Lck-cre mice. In the spleen, the SILloxloxSCL/Lck-cre mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results indicate an accumulation of immature T-cells in mice transgenic for both the SILloxloxSCL transgene and the Lck-cre transgene, and demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

Juxtaposition of the BCL11B Gene to a Novel Region at 17q by a t(14;17)(q32;Q21) in Childhood T-Cell Lymphoblastic Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4130-4130 ◽  
Author(s):  
Sabine Strehl ◽  
Margit König ◽  
Katharina Spath ◽  
Markus Pisecker ◽  
Georg Mann
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Peripheral Blood ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Regulatory Elements ◽  
Lymphoblastic Lymphoma ◽  
Fish Analysis ◽  
Bac Clone ◽  
Expression Levels

Abstract T-cell acute lymphoblastic lymphoma/leukemia is frequently associated with recurrent genetic aberrations that result in the deregulation of transcription factors. In this respect, BCL11B plays a key role in the differentiation and survival during T-cell development. The 3′-located regulatory elements of BCL11B are juxtaposed to TLX3 by a cryptic t(5;14)(q35;q32) in approximately 20% of childhood T-ALL, which leads to inappropriate expression of TLX3. BCL11B can also fuse to TRDC through an inv(14)(q11.2q32.31) resulting in the expression of a BCL11B-TRDC fusion transcript in the absence of wild-type BCL11B. Moreover, a t(6;14) involving BCL11B and the 6q26 region has been described. We have identified a novel BCL11B rearrangement in a case of childhood T-cell lymphoblastic lymphoma. Cytogenetics detected a t(14;17)(q32;q21) and subsequent FISH analysis using BCL11B-spanning and BCL11B 3′-breakpoint-cluster-region flanking BAC clones revealed that BCL11B itself was not disrupted. However, a translocation breakpoint downstream of the BCL11B was observed suggesting the activation of a juxtaposed gene usually residing at 17q by the transcriptional regulatory elements of BCL11B. To narrow down the breakpoint at 17q a FISH-based chromosome-walking strategy using a set of chromosome 17q-specific BACs was employed. A BAC clone encompassing - from centromere to telomere - the genes RAB5C (a member of the RAS oncogene family), KCNH4 (potassium voltage-gated channel, subfamily H (eag-related), member 4), HCRT (hypocretin (orexin) neuropeptide precursor), GHDC (GH3 domain containing; LGP1), STAT5B (signal transducer and activator of transcription 5B), and the 5′-end of STAT5A showed a split signal indicating that one of these genes was juxataposed to the BCL11B enhancer. RAB5C, KCNH4, GHDC, and STAT5B are transcribed in a telomere-centromere orientation, whereas STAT5A shows the opposite transcriptional direction. Together with the FISH pattern observed these data suggested that STAT5A was the most likely candidate gene that might be inappropriately expressed via the regulatory elements of BCL11B. However, semi-quantitative expression analysis showed that neither STAT5A nor STAT5B were significantly upregulated in the affected lymph node as compared to normal bone marrow, peripheral blood, and thymus. In fact, compared to the expression levels in the other tissues STAT5A seemed to be expressed at lower levels. Thus, also the expression levels of RAB5C, KCNH4, and GHDC were analyzed. KCNH4 expression was almost undetectable in bone marrow, peripheral blood, and thymus and for all three genes no elevated expression was observed in the T-cell lymphoma. Owing to the unchanged expression of these genes also the transcription level of STAT3, which is localized further distal to the breakpoint determined by FISH was analyzed, and similar to STAT5A showed lower expression. However, depletion of STATs usually results in reduced cell viability and apoptosis. Together, our data suggest several scenarios: rearrangements of the region containing the remote enhancer of BCL11B are not necessarily accompanied by high expression of a gene juxtaposed into the close vicinity, expression levels of the juxtaposed gene may be just modulated rather than strongly enhanced, the presence of a more complex translocation undetectable by cytogenetics that results in the overexpression of a gene not obviously affected by the translocation or the generation of a fusion gene.

The CALM-AF10 Fusion Gene Expressed in Transgenic Mice Produces Acute Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 358-358
Author(s):  
David L. Caudell ◽  
Zhenhau Zhang ◽  
Yangjo Chung ◽  
Peter D. Aplan
Keyword(s):  
T Cell ◽  
Acute Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
U937 Cell ◽  
Fusion Gene ◽  
Regulatory Elements ◽  
Recent Analysis ◽  
Incomplete Penetrance ◽  
Cd8 Cells

Abstract The rare but recurring translocation [t(10;11)(p13;q21)] occurs in patients with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML); a more recent analysis of clinical cases showed that of the T-ALL cases, t(10;11) are restricted to the TCRγδ lineage. This translocation was initially described in immature T-cells; subsequently the molecular features were characterized in the U937 cell line and shown to result in the production of a CALM-AF10 fusion gene. In order to study the leukemic effects of a CALM-AF10 fusion in mice as a model of human disease, a CALM-AF10 fusion cDNA was cloned from the U937 cell line and inserted into an expression vector that utilized Vav 5′ and 3′ regulatory elements. The Vav gene is expressed in all hematopoietic tissues and is essential for lymphocyte development; Vav regulatory elements have been used by a number of groups to direct transgene expression to the hematopoietic compartment. We generated transgenic Vav-CALM-AF10 mice by pronuclear injection, and followed offspring from two founder mice that were shown to express the CALM-AF10 transgene in hematopoietic tissues for 18 months. Mice were clinically healthy for the first 9 months of life, but had T and B cell developmental abnormalities, including decreased CD4+/CD8+ cells and increased CD4−/CD8− cells in the thymus. In addition, CALM-AF10 mice had decreased B220+/IgM+ cells in the spleen, as well as an unusual Mac1+/B220+ population of cells in the spleen. Eight of 20 offspring from founder C10, and 7 of 16 from founder E6 developed acute leukemia between 9 and 18 months-of-age. Additionally, 5 of 20 mice from the C10 founder and 3 of 16 mice from the E6 founder were found dead unexpectedly but were too necrotic for analysis. Animals that developed acute leukemia were either found dead without premonitory symptoms or presented with ruffled fur, hunched posture and dyspnea. Gross findings included splenomegaly and hepatomegaly. CBC analysis indicated a leukocytosis and anemia and peripheral blood and bone marrow contained increased numbers of blasts and immature forms. Spleens from leukemic mice were effaced by leukemic blasts, which also invaded peripheral tissues including liver, lung, and kidney. Tissue sections from most of the leukemias were myeloperoxidase (MPO) positive and negative for CD3, F4/80, and B220, and immunophenotype was typically Mac1+, Gr+/−, CD4−, CD8−, and B220−. Spleen and/or liver from animals with leukemia were evaluated for clonal T-cell receptor (TCR) β or δ rearrangements. Two of 11 had clonal TCR β rearrangements, one of which also had a clonal TCR δ rearrangement. These findings suggest that CALM-AF10 produces a predominately myeloid leukemia in mice, some of which also have T-cell features. The long latency period and incomplete penetrance suggests that collaborative events may be needed to initiate leukemogenesis.

scholarly journals Gamma (immune) interferon production by leukocytes from a patient with a TG cell proliferative disease

Blood ◽  
1982 ◽  
Vol 59 (1) ◽  
pp. 198-201 ◽  
Author(s):  
JJ Hooks ◽  
BF Haynes ◽  
B Detrick-Hooks ◽  
LF Diehl ◽  
TL Gerrard ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Activity ◽  
Morphological Characteristics ◽  
Lymphocytic Leukemia ◽  
Lymphoid Cells ◽  
T Cell Subsets ◽  
Ifn Gamma ◽  
Killer Activity ◽  
Human T Cell

Abstract We report a patient with a disease characterized by proliferation of T cells with Fc receptors for IgG (TG). However, unlike lymphoid cells from normal individuals or from patients with other lymphoid malignancies, the patient's lymphocytes spontaneously produced gamma interferon (IFN-gamma) in vitro. The peripheral lymphocytes consisted of 95% TG cells, which exhibited the morphological characteristics of T- cell chronic lymphocytic leukemia (CLL) and were normal on cytochemical and chromosome analysis. The majority of TG cells were OKT3+, OKT8+, and OKT4-, 3A1-. These cells failed to express suppressor cell activity and displayed depressed levels of natural killer activity, but mediated antibody-dependent cell-mediated cytotoxicity. The spontaneous production of IFN-gamma by human peripheral lymphoid cells as demonstrated in this study may serve as a probe for studying the relationship between IFN-gamma and the proliferation of human T-cell subsets.

scholarly journals Modulated expression of notch1 during thymocyte development

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 970-976 ◽  
Author(s):  
RP Hasserjian ◽  
JC Aster ◽  
F Davi ◽  
DS Weinberg ◽  
J Sklar
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
T Cell Differentiation ◽  
Thymocyte Development ◽  
Cd8 Cells ◽  
Flow Cytometric ◽  
Human T Cell ◽  
Weak Staining ◽  
Notch Gene

Abstract The Notch gene family encodes transmembrane proteins that have been implicated in control of diverse cellular differentiation events in the fly, frog, and mouse. Mammalian Notch1 is expressed at high levels in thymus and is mutated in a subset of human T-cell acute lymphoblastic neoplasms, suggesting a role in T-cell differentiation. To investigate the patterns of expression of NOTCH1 protein in thymocytes of the developing and mature thymus, antibodies raised against NOTCH1 were used to perform immunohistochemical and flow cytometric analyses. Strong staining for NOTCH1 within the fetal murine thymus was observed as early as 13.5 days postcoitum. By 17.5 days postcoitum, preferential staining of superficial cortical thymocytes was observed, with weak staining of developing medulla. Flow cytometric analysis and immunohistochemical staining of flow-sorted cells confirmed that the highest levels of NOTCH1 expression in adult murine thymus were present in immature cortical thymocytes (CD24high, CD4-CD8-). In contrast, NOTCH1 expression was low or absent in more mature cortical thymocytes (CD24low, CD4+CD8+), whereas intermediate levels of expression were observed in CD4+CD8- and CD4-CD8+ cells. These data indicate a dynamic pattern of NOTCH1 expression during T-cell differentiation and suggest that downregulation of NOTCH1 may be required for maturation of cortical thymocytes.

scholarly journals T-cell activation and subset patterns are altered in B-CLL and correlate with the stage of the disease

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 786-792 ◽  
Author(s):  
TH Totterman ◽  
M Carlsson ◽  
B Simonsson ◽  
M Bengtsson ◽  
K Nilsson
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Healthy Subjects ◽  
Lymphocytic Leukemia ◽  
Stage Ii ◽  
T Cell Subsets ◽  
Cd8 Cells ◽  
Hla Dr ◽  
The Absolute

Abstract Two-color FACS analysis was used to study activated and “functional” T and natural killer (NK) cell subsets of circulating lymphocytes in 23 patients with B-type chronic lymphocytic leukemia (B-CLL) and in 30 healthy subjects. As compared with controls, B-CLL patients had increased absolute numbers of phenotypically activated, HLA-DR+ CD4+ and CD8+ cells and T suppressor/effector (CD11b+CD8+) cells. When patients in Rai stages II through IV (n = 11) were compared with cases in Rai stages O through I (n = 12), the former group of patients had higher numbers of activated CD4+ and CD8+ T cells and decreased levels of suppressor/effector T cells. The absolute numbers of T suppressor/inducer (CD45R+CD4+) cells were elevated in patients with stage O through I disease but within normal range in stage II through IV leukemia. We further showed that the absolute numbers of NK-like (CD16+) cells and their activated counterparts (DR+CD16+) are elevated in B-CLL patients as compared with healthy subjects. The comparison of relative T and NK subsets in the blood of patients and controls showed that the proportions of CD4+, CD8+, and CD16+ cells expressing the activation marker HLA-DR were increased in B-CLL. Furthermore, the percentage of T-suppressor/inducer (CD45R+) cells within the CD4+ population was decreased in the patients. The proportion of T- suppressor/effector (CD11b+) cells within the CD8+ subset was reduced in subjects with stage II-IV disease only. When stimulated in vitro with the T-cell-dependent inducer TPA, B-CLL cells from patients in Rai stages II through IV secreted larger amounts of IgM as compared with cells from stage O through I patients. A positive correlation was observed between the degree of phenotypic activation of CD4+ T-helper cells and their functional capacity to augment IgM secretion by autologous B-CLL cells. Our findings indicate a tumor cell-directed regulatory role of T cells (and possibly NK cells as well) in B-CLL. Furthermore, monitoring of phenotypically activated and functional T- cell subsets may be helpful in the prediction of disease progression and timing of therapy in B-CLL.

scholarly journals Blueprint of human thymopoiesis reveals molecular mechanisms of stage-specific TCR enhancer activation

2020 ◽  
Vol 217 (9) ◽  
Author(s):  
Agata Cieslak ◽  
Guillaume Charbonnier ◽  
Melania Tesio ◽  
Eve-Lyne Mathieu ◽  
Mohamed Belhocine ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Regulatory Elements ◽  
T Cell Differentiation ◽  
Epigenetic Changes ◽  
Human T Cell ◽  
Cell Commitment

Cell differentiation is accompanied by epigenetic changes leading to precise lineage definition and cell identity. Here we present a comprehensive resource of epigenomic data of human T cell precursors along with an integrative analysis of other hematopoietic populations. Although T cell commitment is accompanied by large scale epigenetic changes, we observed that the majority of distal regulatory elements are constitutively unmethylated throughout T cell differentiation, irrespective of their activation status. Among these, the TCRA gene enhancer (Eα) is in an open and unmethylated chromatin structure well before activation. Integrative analyses revealed that the HOXA5-9 transcription factors repress the Eα enhancer at early stages of T cell differentiation, while their decommission is required for TCRA locus activation and enforced αβ T lineage differentiation. Remarkably, the HOXA-mediated repression of Eα is paralleled by the ectopic expression of homeodomain-related oncogenes in T cell acute lymphoblastic leukemia. These results highlight an analogous enhancer repression mechanism at play in normal and cancer conditions, but imposing distinct developmental constraints.

Analysis of the Human T Cell Receptor (TCR) Repertoire from Birth to Old Age Suggests That TCRVβfrequencies Are Established Independent of HLA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3313-3313
Author(s):  
J. Joseph Melenhorst ◽  
Josette Zeilah ◽  
Edgardo Sosa ◽  
Dean Follmann ◽  
Nancy F. Hensel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Expression ◽  
Rank Order ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Rank Test ◽  
Cell Repertoire ◽  
Cd8 Cells ◽  
Human T Cell

Abstract Human T cell development occurs in two waves of development and proliferation: first, early T cells expressing the TCRb chain but not the α-chain are selected for functional TCRβ protein independent of HLA recognition, a process called β-selection; second, thymocytes expressing both the α- and β-TCR are selected for intermediate affinity for self-MHC/ self-peptide complex. This latter process is referred to as positive selection. We sought to determine whether the peripheral TCRVβ frequencies in the naïve T cell repertoire start off at a fixed rank order with minimal skewing as would be expected from a predominantly β-selected repertoire. A total of 22 TCRVβ proteins was quantitated by flow cytometry in a group of 20 unselected umbilical cord blood (UCB) samples (a kind gift from Dr. P. Rubinstein, NY Blood Center, NY), consisting of >80% naïve T cells as defined by CD27+CD45RA+ staining in CD4+ and CD8+ cells. A common rank order of TCRVβ protein frequencies was found in both CD4 and CD8 T cell subsets (figure 1). Median TCRVβ frequencies in CD4 and in CD8 cells of UCB were statistically not significantly different from the frequencies in adult donor CD4 and CD8 cells (Wilcoxon signed rank test; p > 0.2). Furthermore, the percentages of CD4 cells expressing a particular Vβ correlated significantly in CD8 cells (figure 2) with some Vβ proteins being predominantly expressed by either CD4 (Vβ2, Vβ5.1) or CD8 (Vβ14, Vβ7) cells. Our data therefore conform to the prediction that the TCRVβ frequencies are dominantly shaped by β-selection, and not by interactions of the αβTCR/ co-receptor with MHC/ antigen complexes during thymic selection. Figure 1. TCRBV in UCB CD4+ (top) and CD8+ (bottom) T cells Figure 1. TCRBV in UCB CD4+ (top) and CD8+ (bottom) T cells Figure 2. Comparison of TCRBV protein expression frequencies in CD4 and CD8 cells of UCB Figure 2. Comparison of TCRBV protein expression frequencies in CD4 and CD8 cells of UCB

scholarly journals Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

2020 ◽  
Author(s):  
Emilie Coppin ◽  
Bala Sai Sundarasetty ◽  
Susann Rahmig ◽  
Jonas Blume ◽  
Nikita A. Verheyden ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Regulatory Elements ◽  
T Cell Differentiation ◽  
Human Immune System ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Human T Cell ◽  
Human T Cells

AbstractHumanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T cell differentiation remains inefficient. We generated mice expressing human interleukin (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

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