N-Myc Is Overexpressed In Both Murine and Human Early T-Cell Precursor Leukemia and Is Sufficient To Initiate this Leukemia In Multipotent Primitive Arf-/- thymocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 348-348
Author(s):  
Louise M. Treanor ◽  
Sheng Zhou ◽  
Yu Fukuda ◽  
Nandakumar Satish ◽  
David Finkelstein ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Murine Model ◽  
Leukemic Cells ◽  
Control Element ◽  
Mrna Levels ◽  
Cell Precursor ◽  
Double Positive ◽  
Fluorescent Intensity ◽  
Cell Counts

Abstract Early T-cell precursor–ALL (ETP-ALL) is a subtype of T-cell acute lymphoblastic leukemia that displays both myeloid and lymphoid phenotypic features and has a relatively poor prognosis. Our previous work has shown that activating mutations in the Il7 receptor (Il7r) and Lmo2 overexpression in CD4-CD8- thymocytes cause ETP-ALL in mice via DN2 thymocyte differentiation block. To further understand the molecular mechanisms responsible for initiation and maintenance of ETP-ALL, we compared expression profiles of seven murine ETP leukemias induced by Lmo2 and Il7r mutant vectors and four preleukemic blocked DN2 Arf-/- thymocyte populations to normal murine DN2 Arf-/- thymocytes. This analysis and qRT-PCR identified N-Myc as one of the top upregulated genes in either Lmo2 or Il7r mutant samples. Importantly in pediatric ETP-ALL N-Myc RNA is expressed at high levels in 11/13 human cases compared to 26/72 T-ALL cases corroborating our murine model with the human disease. To investigate if N-Myc itself was sufficient to induce ETP leukemia, immature murine Arf-/- thymocytes were transduced with a retroviral vector expressing the N-Myc cDNA and YFP. After 20 days in culture a block at the DN2 stage of thymocyte development was observed that recapitulated the Lmo2 and the Il7r receptor mutant phenotype. When sublethally irradiated Rag2-/-, γc-/- recipient mice were transplanted with N-Myc transduced Arf-/- thymocytes 5/5 of the primary recipients developed ETP leukemia originating from the thymocyte graft with a median latency of 60 days (Figure 1). These mice had elevated white blood cell counts between 17-85x103/µl and enlarged spleens ranging in weight from 0.42g-0.67g. Flow cytometry analysis showed that their spleen, thymus and peripheral blood contained greater than 70% YFP+ Gr1+ cells. These N-Myc induced leukemias expressed both T-cell (cytoplasmic CD3) and myeloid (Gr1 and Mac1) markers and displayed other features typical of ETP disease. Having established that N-Myc is sufficient to initiate ETP leukemia in the murine model, we next tested if N-Myc is critical in the maintenance of ETP leukemia induced by Il7r mutants. ETP leukemic cells arising from the Il7r 241-242TC mutant were transduced with a mir30 lentiviral vector that contains a short hairpin N-Myc inhibiting sequence and mCherry as a reporter gene (shN-Myc). This vector has been shown to reduce N-Myc mRNA levels by five fold in N-Myc overexpressing cells. The transduced cells were analyzed and sorted for leukemic+ (GFP) scrambled+ (mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells. Analysis of the cells showed the mCherry mean fluorescence intensity (MFI) of the leukemic cells transduced with shN-Myc was approximately double that of the scrambled transduced cells, demonstrating at least equal transduction efficiency of the two vectors. Sorted double positive leukemic cells were transplanted into sublethally irradiated WT recipients. All animals succumbed to leukemia and were subsequently analyzed by flow cytometry for expression of scrambled and shN-Myc vector expression. Analysis of tumor cells showed a significant decrease in the mCherry expression of the cells containing the shN-Myc compared to the scrambled control indicating that sustained N-Myc expression is selected for during the maintenance of ETP leukemia and in the future a tetracycline control element will be incorporated into the shN-Myc to confirm this (Figure 2). Furthermore we plan to test therapeutics against N-Myc such as bromodomain and Aurora kinase A inhibitors in vitro and plan to expand this in vivo. This demonstrates the utility of this novel mouse model combined with gene expression profiling to elucidate the signaling networks in ETP leukemia to develop targeted therapy for this aggressive disease.Figure 1Survival curve of recipient mice transplanted with CD4-CD8- thymocytes transduced with N-Myc and Il7r mutants.Figure 1. Survival curve of recipient mice transplanted with CD4-CD8- thymocytes transduced with N-Myc and Il7r mutants.Figure 2Mean fluorescent intensity (MFI) of mCherry in mice transplanted with leukemic+ (GFP) Scrambled+(mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells.Figure 2. Mean fluorescent intensity (MFI) of mCherry in mice transplanted with leukemic+ (GFP) Scrambled+(mCherry) or leukemic+(GFP) shN-Myc+(mCherry) double positive cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD7+ and CD56+ Myeloid/Natural Killer Cell Precursor Acute Leukemia: A Distinct Hematolymphoid Disease Entity

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2417-2428 ◽  
Author(s):  
Ritsuro Suzuki ◽  
Kazuhito Yamamoto ◽  
Masao Seto ◽  
Yoshitoyo Kagami ◽  
Michinori Ogura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Acute Leukemia ◽  
Natural Killer ◽  
Nk Cell ◽  
Bone Marrow Involvement ◽  
Leukemic Cells ◽  
Allogeneic Bone ◽  
Disease Entity ◽  
Cell Precursor

Abstract The disease spectrum of natural killer (NK) cell leukemias and lymphomas has recently been expanding with the continuing evolution in diagnostic concepts. We describe here seven cases of acute leukemia of conceivable myeloid and NK cell precursor phenotype in six men and one woman varying from 19 to 59 years of age (median, 46 years). Striking extramedullary involvement was evident at initial presentation, with peripheral lymphadenopathy and/or mediastinal masses. Two lacked any leukemic cells in the bone marrow at diagnosis. Using cytochemical myeloperoxidase staining, less than 3% of the leukemic cells showed positive reactivity. However, expression of CD7, CD33, CD34, CD56, and frequently HLA-DR, but not other NK, T-cell, and B-cell markers was observed. Cytoplasmic CD3 was detected in three of the cases by flow cytometry and in six by Northern blotting, suggesting an origin from common progenitors between the NK cell and myeloid lineages. All but one presented germline configurations of the T-cell receptor β and γ chain genes and Ig heavy chain gene. With regard to morphology, the cells were generally L2-shaped, with variation in cell size, round to moderately irregular nuclei and prominent nucleoli, pale cytoplasm, and a lack of azurophilic granules. Histopathologic examination of biopsied specimens of extramedullary tumors showed a lymphoblast-like morphology, implying the differential diagnostic problem from lymphoblastic lymphomas, especially in cases lacking bone marrow involvement. Three patients were successfully treated with chemotherapy for acute myeloid leukemia (AML), whereas three other patients proved refractory to chemotherapeutic regimens for lymphoid malignancies, although two responded to subsequent AML chemotherapy. However, despite intensive chemotherapy, including allogeneic bone marrow transplantation, most persued fatal courses within 41 months. These data suggested that the CD7+ and CD56+ myeloid/NK cell precursor acute leukemia might constitute a distinct biologic and clinical disease entity. Its recognition appears to be particularly important for the clinicopathologic evaluation of CD56+ hematolymphoid malignancies and the development of therapeutic approaches to such disease.

CD26+CD4+T cell counts and attack risk in interferon-treated multiple sclerosis

2005 ◽  
Vol 11 (6) ◽  
pp. 641-645 ◽  
Author(s):  
F Sellebjerg ◽  
C Ross ◽  
N Koch-Henriksen ◽  
P Soelberg Sørensen ◽  
J L Frederiksen ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hazard Ratio ◽  
Cd4 T Cell ◽  
Immunomodulatory Treatment ◽  
Cell Counts ◽  
Increased Risk ◽  
Insufficient Response

Biomarkers that allow the identification of patients with multiple sclerosis (MS) with an insufficient response to immunomodulatory treatment would be desirable, as currently available treatments are only incompletely efficacious. Previous studies have shown that the expression of CD25, CD26 and CCR5 on T cells is altered in patients with active MS. We studied the expression of these molecules by flow cytometry in patients followed for six months during immunomodulatory treatment. In interferon (IFN)-β-treated patients, we found that the hazard ratio for developing an attack was 2.8 in patients with CD26+CD4+T cell counts above median, and this risk was independent of the risk conferred by neutralizing anti-IFN-β antibodies. CD26+CD4+T cell counts may identify patients with MS at increased risk of attack during treatment with IFN-β.

scholarly journals B7-H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xu Zheng ◽  
Qikai Wang ◽  
Xiang Yuan ◽  
Yingbo Zhou ◽  
Hui Chu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Salivary Glands ◽  
Inflammatory Cytokines ◽  
Mrna Levels ◽  
Lymphocyte Infiltration ◽  
Flow Cytometry Analysis ◽  
Treg Differentiation

Background. This study is aimed at exploring the role of B7-H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods. B7-H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti-B7-H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT-PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7-H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two-tailed Student’s t-tests were used to detect the statistical difference in various measures between the two groups. Results. B7-H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti-B7-H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL-12, IL-18, IL-1α, TNF-α, IFN-α, and BAFF were upregulated markedly in anti-B7-H4 mAb-treated mice compared to IgG isotype-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb-treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti-B7-H4 mAb-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7-H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7-H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7-H4Ig treatment revealed that the mRNA levels of IL-12, IL-6, IL-18, IL-1α, TNF-α, and IFN-α were significantly downregulated in B7-H4Ig-treated mice compared to control Ig treatment. B7-H4Ig-treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7-H4Ig treatment mouse were more than control Ig treatment. Conclusions. Our findings implicate that B7-H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.

A Longitudinal Analysis of the T-Cell Receptor Vβ Repertoire in Aplastic Anemia Patients at Initial Presentation, Remission, and Relapse.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2402-2402
Author(s):  
Yunfeng Cheng ◽  
Yong Tang ◽  
Spencer Green ◽  
Keyvan Keyvanfar ◽  
Tullia Bruno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Receptor ◽  
Immunosuppressive Treatment ◽  
Cell Receptor ◽  
Initial Presentation ◽  
Cell Counts ◽  
Therapeutic Decisions

Abstract Aplastic anemia is a bone-marrow-failure syndrome characterized by low blood-cell counts and a fatty bone marrow. In most cases, no obvious etiological factor can be identified, but clinical responses to immunosuppressive treatment (IST) strongly suggest an immune pathophysiology. Our previous study of T-cell receptor (TCR) Vβ (variable region of β-chain) repertoire usage by flow cytometry suggested that aplastic anemia results from antigen-specific lymphocyte attack on hematopoietic progenitors (Risitano et al. Lancet2004; 364:355). In the current work, 7 patients were investigated for Vβ pattern expression before first immunosuppresive treatment, at the remission, and again on relapse. The TCR Vβ repertoire was analyzed for CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 22 different Vβ chains. Most patients had very different patterns of Vβ usage from healthy individuals, and all but one showed expansion of at least one Vβ family before immunosuppressive treatment (Vβ family expansions were defined as 2 standard deviations (SD) from the means in controls). The median number of expanded Vβ families was 4 per patient among CD8CD28dim effector cells. At remission, almost all the initially expanded Vβ subfamilies decreased to less than 2SD of controls. At relapse, most of the expanded Vβ subsets were increased again. However, 5/7 patients showed new expanded Vβ subsets at recurrence of cytopenias, suggesting antigenic spread of new epitopes recognized by immune systems. Although no common pattern of specific expanded Vβ subsets could be identified among different patients, some Vβ subfamilies appeared to be more frequently involved (Vβ 5.1 and Vβ 5.2 were expanded in 4 of 7 patients both at initial presentation and relapse ). These data suggest that monitoring Vβ subsets in aplastic anemia, and potentially in other immune-mediated human diseases of a similar pathophysiology could be used to guide individual therapeutic decisions and in the development of new treatments.

scholarly journals Affordable CD4+-T-Cell Counting by Flow Cytometry: CD45 Gating for Volumetric Analysis

2002 ◽  
Vol 9 (5) ◽  
pp. 1085-1094 ◽  
Author(s):  
George Janossy ◽  
Ilesh V. Jani ◽  
Nicholas J. Bradley ◽  
Arsene Bikoue ◽  
Tim Pitfield ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
High Efficiency ◽  
Cell Counting ◽  
Limits Of Agreement ◽  
Cd4 Counts ◽  
Single Tube ◽  
Cell Counts ◽  
Industry Standards ◽  
Resource Poor

ABSTRACT The flow cytometers that are currently supported by industry provide accurate CD4+-T-cell counts for monitoring human immunodeficiency virus disease but remain unaffordable for routine service work under resource-poor conditions. We therefore combined volumetric flow cytometry (measuring absolute lymphocyte counts in unit volumes of blood) and simpler protocols with generic monoclonal antibodies (MAbs) to increase cost efficiency. Volumetric absolute counts were generated using CD45/CD4 and CD45/CD8 MAb combinations in two parallel tubes. The percentage values for the various subsets were also determined within the leukocyte and lymphocyte populations utilizing a fully automated protocol. The levels of agreement between the newly developed method and the present industry standards, including both volumetric and bead-based systems using a full MAb panel for subset analysis, were tested by Bland-Altman analyses. The limits of agreement for CD4 counts generated by the volumetric methods using either CD45/CD4 (in a single tube) or the full Trio MAb panel (in three tubes) on the CytoronAbsolute flow cytometer were between −29 and +46 cells/mm3 with very little bias for CD4 counts (in favor of the Trio method: +8 CD4+ lymphocytes/mm3; 0.38% of lymphocytes). The limits of agreement for absolute CD4 counts yielded by the volumetric CD45/CD4 method and the bead-based method were between −118 and +98 cells/mm3, again with a negligible bias (−10 CD4+ lymphocytes/mm3). In the volumetric method using CD45/CD8, the strongly CD8+ cells were gated and the levels of agreement with the full Trio showed a minor bias (in favor of the Trio; +40 CD8+ cells/mm3; 5.2% of lymphocytes) without a significant influence on CD4/CD8 ratios. One trained flow cytometrist was able to process 300 to 400 stained tubes per day. This workload extrapolates to a throughput of >30,000 samples per year if both CD45/CD4 and CD45/CD8 stainings are performed for each patient or a throughput of >60,000 samples if only CD45/CD4 counts are tested in a single tube. Thus, on the basis of the high efficiency and excellent agreement with the present industry standards, volumetric flow cytometers with automated gating protocols and autobiosamplers, complemented by generic CD45, CD4, and CD8 MAbs used in two-color immunofluorescence, represent the most suitable arrangements for large regional laboratories in resource-poor settings.

scholarly journals CD7+ and CD56+ Myeloid/Natural Killer Cell Precursor Acute Leukemia: A Distinct Hematolymphoid Disease Entity

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2417-2428 ◽  
Author(s):  
Ritsuro Suzuki ◽  
Kazuhito Yamamoto ◽  
Masao Seto ◽  
Yoshitoyo Kagami ◽  
Michinori Ogura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Acute Leukemia ◽  
Natural Killer ◽  
Nk Cell ◽  
Bone Marrow Involvement ◽  
Leukemic Cells ◽  
Allogeneic Bone ◽  
Disease Entity ◽  
Cell Precursor

The disease spectrum of natural killer (NK) cell leukemias and lymphomas has recently been expanding with the continuing evolution in diagnostic concepts. We describe here seven cases of acute leukemia of conceivable myeloid and NK cell precursor phenotype in six men and one woman varying from 19 to 59 years of age (median, 46 years). Striking extramedullary involvement was evident at initial presentation, with peripheral lymphadenopathy and/or mediastinal masses. Two lacked any leukemic cells in the bone marrow at diagnosis. Using cytochemical myeloperoxidase staining, less than 3% of the leukemic cells showed positive reactivity. However, expression of CD7, CD33, CD34, CD56, and frequently HLA-DR, but not other NK, T-cell, and B-cell markers was observed. Cytoplasmic CD3 was detected in three of the cases by flow cytometry and in six by Northern blotting, suggesting an origin from common progenitors between the NK cell and myeloid lineages. All but one presented germline configurations of the T-cell receptor β and γ chain genes and Ig heavy chain gene. With regard to morphology, the cells were generally L2-shaped, with variation in cell size, round to moderately irregular nuclei and prominent nucleoli, pale cytoplasm, and a lack of azurophilic granules. Histopathologic examination of biopsied specimens of extramedullary tumors showed a lymphoblast-like morphology, implying the differential diagnostic problem from lymphoblastic lymphomas, especially in cases lacking bone marrow involvement. Three patients were successfully treated with chemotherapy for acute myeloid leukemia (AML), whereas three other patients proved refractory to chemotherapeutic regimens for lymphoid malignancies, although two responded to subsequent AML chemotherapy. However, despite intensive chemotherapy, including allogeneic bone marrow transplantation, most persued fatal courses within 41 months. These data suggested that the CD7+ and CD56+ myeloid/NK cell precursor acute leukemia might constitute a distinct biologic and clinical disease entity. Its recognition appears to be particularly important for the clinicopathologic evaluation of CD56+ hematolymphoid malignancies and the development of therapeutic approaches to such disease.

scholarly journals Inhibitory effect and mechanism of action (MOA) of hirsutine on the proliferation of T-cell leukemia Jurkat clone E6-1 cells

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10692
Author(s):  
Jie Meng ◽  
Rui Su ◽  
Luping Wang ◽  
Bo Yuan ◽  
Ling Li
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Death ◽  
T Cell ◽  
Mechanism Of Action ◽  
Mrna Levels ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Antitumor Effects

Background The bark of Uncaria rhynchophylla has been traditionally used to treat convulsion, bleeding, hypertension, auto-immune conditions, cancer, and other diseases. The main focus of this research is done for the purpose of exploring the antitumor activity and mechanism of action (MOA) for hirsutine isolated from U. rhynchophylla. Methods Jurkat clone E6-1 cells were treated using 10, 25 and 50 μM for 48 h. Inhibition of cell proliferation due to hirsutine treatment was evaluated by CCK8 assay. Flow cytometry was applied to ascertain Jurkat cell cycle progression and apoptosis after treatment with 10, 25 and 50 μM hirsutine for 48 h. The expression and level of the apoptosis-related genes and proteins was analyzed by Real-time Quantitative polymerase chain reaction (qPCR) and Western blotting method, respectively. Results CCK8 analyses revealed that hirsutine could significantly inhibit the proliferation of Jurkat clone E6-1 cells, in a concentration and time-dependent fashion. Flow cytometry assays revealed that hirsutine could drive apoptotic death and G0/G1 phase arrest in Jurkat cells. Apoptotic cells frequencies were 4.99 ± 0.51%, 13.69 ± 2.00% and 40.21 ± 15.19%, and respective cell cycle arrest in G0/G1 accounted for 34.85 ± 1.81%, 42.83 ± 0.70% and 49.12 ± 4.07%. Simultaneously, compared with the control group, Western blot assays indicated that the up-regulation of pro-apoptotic Bax, cleaved-caspase3, cleaved-caspase9 and Cyto c proteins, as well as the down-regulation of Bcl-2 protein which guards against cell death, might be correlated with cell death induction and inhibition of cell proliferation. QPCR analyses indicated that hirsutine could diminish BCL2 expression and, at the same time, improve Bax, caspase-3 and caspase-9 mRNA levels, thus reiterating a putative correlation of hirsutine treatment in vitro with apoptosis induction and inhibition of cell proliferation (p-value < 0.05). Excessive hirsutine damages the ultrastructure in mitochondria, leading to the release of Cyt c from the mitochondria to cytoplasm in Jurkat clone E6-1 cells, thereby inducing the activated caspase cascade apoptosis process through a mitochondria-mediated pathway. Conclusion An important bioactive constituent—hirsutine—appears to have antitumor effects in human T-cell leukemia, thus enlightening the use of phytomedicines as a novel source for tumor therapy. It is speculated that hirsutine may induce apoptosis of Jurkat Clone E6-1 cells through the mitochondrial apoptotic pathway.

scholarly journals Combination immunotherapy induces distinct T-cell repertoire responses when administered to patients with different malignancies

2020 ◽  
Vol 8 (1) ◽  
pp. e000368
Author(s):  
Jason Cham ◽  
Li Zhang ◽  
Serena Kwek ◽  
Alan Paciorek ◽  
Tao He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Metastatic Melanoma ◽  
Flow Cytometry Analysis ◽  
Cell Repertoire ◽  
Gm Csf ◽  
Cell Counts ◽  
Melanoma Patients ◽  
Circulating T Cells

BackgroundCTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.MethodsWe used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman’s rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.ResultsWhile melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=−0.81, p<0.001 vs r=−0.59, p=0.02).Trial registration numbersNCT00064129;NCT01363206.

Heme Metabolic Gene Expression In FLVCR-Knockout Mice During T Cell Development

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2787-2787
Author(s):  
Mary Philip ◽  
Alexandra R. Zaballa ◽  
Blake T. Hovde ◽  
Janis L. Abkowitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Double Positive ◽  
Metabolic Gene Expression ◽  
Free Heme ◽  
Single Positive

Abstract Abstract 2787 Heme is essential for nearly every organism and cell. However, free heme can induce free radical formation and cellular damage, therefore cells must carefully regulate heme levels. The feline leukemia virus subgroup C receptor (FLVCR) exports heme from cells. Conditional deletion of Flvcr has been shown to cause progressive anemia in neonatal and adult mice (Science 319:825-8, 2008). Recently, we developed a transplant model in which developing lymphocytes lacked FLVCR while erythroid cells expressed FLVCR, preventing anemia, and found that CD4 and CD8 peripheral T cells were severely decreased while B cell numbers were normal. We further demonstrated that FLVCR-knockout thymocytes were blocked at the CD4CD8 double-positive (DP) stage (Blood [ASH Annual Meeting Abstracts] 114: 913, 2009). We hypothesized that developing T cells lacking FLVCR are arrested at the DP stage because of increased intracellular free heme (IFH). While heme is required for erythroid function, little is known about the role of heme in T cell development. Real-time dynamic quantification of IFH in vivo or from ex vivo tissue is a major challenge in heme biology. We reasoned that by measuring the expression of genes transcriptionally-regulated by heme, we could indirectly assess IFH. Three proteins are key regulators of IFH in non-erythroid cells: aminolevulinic acid synthase-1 (ALAS1) is the rate-limiting enzyme in heme synthesis, FLVCR exports heme, and heme oxygenase-1 (HMOX1) degrades heme. Normal thymic T cell development proceeds from the CD4CD8 double-negative (DN) to the CD4CD8 double-positive (DP) stage, which then go on to either the CD4 single-positive (CD4SP) or CD8 single-positive (CD8SP) stage. We flow-sorted cells from each stage and used multiplex quantitative PCR (qPCR) to determine that all three genes were expressed at higher levels early in normal T cell development during the DN and DP stages and then at lower levels in the CD4SP and CD8SP. Heme binding to the negative regulatory protein BACH1 causes dissociation of BACH1 from the Hmox1 promoter and increased Hmox1 transcription, while expression and stability of Alas1 mRNA is under negative feedback control by heme. Therefore, we predicted that increased IFH in FLVCR-knockout thymocytes would lead to an increase in Hmox1 mRNA and a decrease in Alas1 mRNA levels. We compared expression of heme metabolic genes in FLVCR-knockout and control thymocytes. Flvcr expression was nearly absent in FLVCR-knockout DN and DP cells, however, there was a slight increase in Flvcr expression by the few CD4SP and CD8SP present. To understand this result, we analyzed the extent of genomic Flvcr deletion in FLVCR-knockout thymocytes and peripheral B and T cells by genomic qPCR. DN and DP thymocytes had near complete deletion of Flvcr while CD4SP and CD8SP had slightly less-efficient deletion, likely accounting for the increased Flvcr mRNA levels. Strikingly, Flvcr deletion in the few peripheral T cells present was 50–60% in contrast to peripheral B cells (>90%): only those T cells with incomplete Flvcr deletion survived, further underscoring the absolute requirement for FLVCR in developing T cells. We next examined Hmox1 mRNA expression and found that Hmox1 expression was higher in FLVCR-knockout DP, CD4SP, and CD8SP compared to wild-type FLVCR controls. This supports our hypothesis that FLVCR loss leads to increased IFH during T cell development. Alas1 expression was similar in FLVCR-knockout and control thymocytes, a finding that could be explained because heme regulates ALAS1 activity not only at the transcriptional level but also at the post-transcriptional level. Thus Alas1 expression may not be a good indicator of IFH. In summary, we developed a method to quantify relative free heme levels in developing thymocytes through the measurement of heme metabolic gene expression and found that IFH levels were increased in FLVCR-knockout thymocytes compared to controls. Whether and how excess free heme derails the T cell developmental program, remains to be discovered. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia

2008 ◽  
Vol 205 (4) ◽  
pp. 751-758 ◽  
Author(s):  
Elisabetta Flex ◽  
Valentina Petrangeli ◽  
Lorenzo Stella ◽  
Sabina Chiaretti ◽  
Tekla Hornakova ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Signature ◽  
Poor Response ◽  
Response To Therapy ◽  
Janus Kinase ◽  
Leukemic Cells ◽  
Cell Precursor ◽  
Induced Apoptosis

Aberrant signal transduction contributes substantially to leukemogenesis. The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors and plays a nonredundant role in lymphoid cell precursor proliferation, survival, and differentiation. We report that somatic mutations in JAK1 occur in individuals with acute lymphoblastic leukemia (ALL). JAK1 mutations were more prevalent among adult subjects with the T cell precursor ALL, where they accounted for 18% of cases, and were associated with advanced age at diagnosis, poor response to therapy, and overall prognosis. All mutations were missense, and some were predicted to destabilize interdomain interactions controlling the activity of the kinase. Three mutations that were studied promoted JAK1 gain of function and conferred interleukin (IL)-3–independent growth in Ba/F3 cells and/or IL-9–independent resistance to dexamethasone-induced apoptosis in T cell lymphoma BW5147 cells. Such effects were associated with variably enhanced activation of multiple downstream signaling pathways. Leukemic cells with mutated JAK1 alleles shared a gene expression signature characterized by transcriptional up-regulation of genes positively controlled by JAK signaling. Our findings implicate dysregulated JAK1 function in ALL, particularly of T cell origin, and point to this kinase as a target for the development of novel antileukemic drugs.

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