A Broad Distribution of ZAP-70 Expression Is Demonstrated in Adult Acute Lymphoblastic Leukaemia by Quantitative PCR Analysis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4309-4309
Author(s):  
Geothy Kochethu ◽  
Andy Bell ◽  
Mike Griffiths ◽  
Gulnaz Begum ◽  
Farooq Wandroo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Cell Line ◽  
Quantitative Pcr ◽  
Microarray Analysis ◽  
Lymphoblastic Leukaemia ◽  
Prognostic Value ◽  
Wide Range

Abstract The ZAP-70 tyrosine kinase plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. However ZAP-70 is expressed in many cases of B cell chronic lymphocytic leukemia and correlates with a poor prognosis. Microarray analysis has recently identified ZAP-70 expression in a range of other B cell malignancies, including acute lymphoblastic leukaemia (ALL), and ZAP-70 expression may carry prognostic value in these diseases. Microarray analysis may not offer the most accurate method for quantitation of small changes in mRNA expression and this technology may therefore limit the potential information to be derived from ZAP-70 expression. We developed a real time quantitative PCR assay for ZAP-70 and used this to determine ZAP-70 expression in 76 adult patients with ALL (64 B lineage and 12 T lineage). RNA was extracted from diagnostic bone marrow specimens taken from 76 patients aged over 18 years presenting with ALL. Quantitative PCR was optimised using Jurkat T cells and utilized GAPDH as the internal control. ZAP-70 and GAPDH cDNA were co-amplified in a multiplex PCR reaction with five-fold serial dilutions of Jurkat cDNA ranging from 25ng to 40pg, and this data was used to construct the calibration curve. ZAP-70 expression within tumours was determined relative to expression within the T cell line Jurkat, defined as having an arbitrary value of 1.0. A wide distribution of ZAP-70 expression was seen in individual ALL cases with a range between 0.002 and 5.3. The average ZAP-70 expression level within ALL was below that of Jurkat with a mean of 0.332 and median of 0.185. Expression of ZAP-70 showed a relatively continuous pattern of expression across the cohort apart from six samples (8%) with a level of ZAP-70 expression above that of the Jurkat T cell line. Statistical analysis of a potential association between ZAP-70 expression and cytogenetic subgroup was performed by group comparison using either a two-sample test or ANOVA. No significant association between ZAP-70 expression and cytogenetic subgroup was found. Only 4 cases of t(1,19) translocation encoding the E2A/PBX1 gene fusion were seen in the cohort and all had ZAP-70 levels above the median level. Within the T-ALL subgroup, 11 of the 12 patients showed ZAP-70 expression above the median with a mean of 0.26. Low expression was observed in 4 of the 5 cases in the b3a2/b2a2 p210 BCR/ABL subgroup. Study of a potential correlation between ZAP-70 expression and clinical prognosis is continuing but the three patients with the highest levels of ZAP-70 failed to achieve remission with induction therapy and died of refractory disease. These data confirm that ZAP-70 expression is seen in the majority of cases of ALL and demonstrate the wide range of expression between individual cases. Large cohort studies are likely to be required to confirm an association between ZAP-70 expression and specific cytogenetic subgroup and to confirm the potential prognostic value of ZAP 70 expression.

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals Interleukin 2 induces antigen-reactive T cell lines to secrete BCGF-I.

1983 ◽  
Vol 158 (6) ◽  
pp. 2024-2039 ◽  
Author(s):  
M Howard ◽  
L Matis ◽  
T R Malek ◽  
E Shevach ◽  
W Kell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Interleukin 2 ◽  
Activated T Lymphocytes ◽  
T Cell Lines ◽  
Activated B Cells

Antigen-activated T lymphocytes produce within 24 h of stimulation a factor that is indistinguishable biochemically and functionally from the B cell co-stimulating growth factor, BCGF-I, originally identified in induced EL4 supernatants: Supernatants from antigen-stimulated T cell lines are not directly mitogenic for resting B cells, but synergize in an H-2-unrestricted manner with anti-Ig activated B cells to produce polyclonal proliferation but not antibody-forming-cell development; biochemical studies reveal the B cell co-stimulating factor present in antigen-stimulated T cell line supernatants is identical by phenyl Sepharose chromatography and isoelectric focusing (IEF) to EL4 supernatant BCGF-I. We thus conclude that normal T cells produce BCGF-I in response to antigenic stimulation. Analysis of the mechanism of BCGF-I production by antigen-stimulated T cells showed that optimum amounts of BCGF-I were obtained as quickly as 24 h post-stimulation, and that the factor producing cells in the T cell line investigated bore the Lyt-1+2- phenotype. As few as 10(4) T cells produced sufficient BCGF-I to support the proliferation of 5 X 10(4) purified anti-Ig activated B cells. Finally, the activation of normal T cell lines to produce BCGF-I required either antigen presented in the context of syngeneic antigen-presenting cells (APC) or interleukin 2 (IL-2).

Quantitative Proteomic Analysis Implicates An Altered B-Cell Differentiation State as a Mechanism Underlying GC-Resistance in An Acute Lymphoblastic Leukaemia (ALL) Cell Line Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1736-1736
Author(s):  
Lindsay Nicholson ◽  
Caroline Evans ◽  
Elizabeth Matheson ◽  
Lynne Minto ◽  
Christopher Keilty ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Cell Line ◽  
Mismatch Repair ◽  
Cell Lines ◽  
Lymphoblastic Leukaemia ◽  
B Cell Differentiation ◽  
Protein Levels ◽  
Resistant Phenotype

Abstract Abstract 1736 Poster Board I-762 Introduction Glucocorticoids (GC) are pivotal agents used in the treatment of childhood acute lymphoblastic leukaemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome in childhood ALL, but the underlying molecular basis remains unclear. Previous studies using cell lines have identified mutation/deletion of the glucocorticoid receptor (GR) as a mechanism of GC-resistance. However, genetic aberration of the GR is rare in clinical samples1. This disparity may be due to the mismatch repair deficient status of many ALL cell lines which consequently have a greater likelihood of acquiring mutations under GC-selection. We have used a discovery proteomics approach for hypothesis generation on potential mechanisms for resistance. To achieve this, we compared a well-characterized mismatch repair proficient GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9) both bearing wildtype GR, in a comparative proteomics experiment using 4-channel isobaric tagging for relative and absolute quantitation (the iTRAQ approach). Methods Cells were treated with either vehicle control or 0.1μM dexamethasone for 24 hours and subjected to subcellular fractionation to prepare a nuclear fraction. Each sample was labelled with a distinct isobaric tag for relative quantification and analysed by 2-dimensional liquid chromatography/ tandem mass spectrometry. The proteins were identified and relatively quantified using Protein Pilot software (Applied Biosystems). Ratios were calculated for dexamethasone-treated ‘versus’ control vehicle for each cell line and an ITRAQ ratio of greater than or equal to ± 1.2 or less than 0.8 fold change were considered to be differentially expressed. Results The comparative dataset highlighted two transcription factors which are involved in B-cell differentiation, PAX5 and IRF4, to be differentially expressed in the PreB 697 compared to the R3F9 cell line. The GC-resistant R3F9 cell line had reduced PAX5 and IRF4 protein expression compared to the parental cell line and this was further validated in other GC-resistant sub-clones derived from the PreB 697 cell line by western blot analysis. The reduced PAX5 level in the GC-resistant cell lines was not due to monoallelic loss, as measured by a QRT-PCR method or mutation as determined by DHPLC analysis of ‘hot-spot’ exons. In addition, PAX5 mRNA levels were not significantly altered, thus suggestive of a post-transcriptional mechanism for PAX5 protein reduction. To test the direct role of PAX5 in GC-resistance, we reduced PAX5 mRNA and protein levels using RNA interference in the parental GC-sensitive, PreB 697 cell line. PAX5 protein levels were reduced by at least 80% and were maintained for 48 hours post-transfection. The PreB 697 cell line was transfected with siRNA directed to PAX5 using electroporation, the cells were allowed to recover for 24 hours and the levels of cell kill were assessed in response to a 48 hour incubation with 1 μM dexamethasone by Annexin V staining and the MTS assay. Paradoxically, PAX5 knockdown increased GC-sensitivity (mean 60.4% apoptosis, S.D. 16.8, N=3) in comparison to a non-specific siRNA (mean 31.0% apoptosis, S.D. 5.2, N=3) but did not influence sensitivity to either vincristine or daunorubicin. Thus, this response was specific to glucocorticoids. Conclusion Using a proteomic approach we have shown alterations in PAX5 protein levels are associated with a GC-resistant phenotype which an mRNA-based technology would fail to detect. Modulation of PAX5 in ALL cells may influence the response to GC-therapy. It is known that GC-sensitivity alters during B-cell development, with early lymphoid precursors being highly sensitive and more mature B cells being highly resistant to GC-induced apoptosis. We propose that reduced PAX5 protein levels may reflect an altered differentiation state of the sub-clones of PreB 697 which are associated with a GC resistant phenotype. 1Irving et al, Cancer Res, 2005 2Schmidt et al, FASEB, 2006 Disclosures No relevant conflicts of interest to declare.

RUNX1 point mutations potentially identify a subset of early immature T-cell acute lymphoblastic leukaemia that may originate from differentiated T-cells

Gene ◽  
2014 ◽  
Vol 545 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Michelle Meng Huang Mok ◽  
Linsen Du ◽  
Chelsia Qiuxia Wang ◽  
Vinay Tergaonkar ◽  
Te Chih Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Point Mutations

Functional human T cell-B cell hybridomas established from fusion of normal T cells and an EBV-transformed B cell line

1990 ◽  
Vol 54 (1) ◽  
pp. 134-147
Author(s):  
Hajime Kimata ◽  
James Berenson ◽  
Jonathan Kagan ◽  
Andrew Saxon
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Human T Cell

scholarly journals Effect of thimerosal, methylmercury, and mercuric chloride in Jurkat T Cell Line

2012 ◽  
Vol 5 (3) ◽  
pp. 159-161 ◽  
Author(s):  
Gianpaolo Guzzi ◽  
Paolo D. Pigatto ◽  
Francesco Spadari ◽  
Caterina A.M. La Porta
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Mercuric Chloride ◽  
Methyl Mercury ◽  
Leukemia Cell Line ◽  
Cellular Viability ◽  
Jurkat T Cells ◽  
Jurkat T Cell ◽  
Wide Range

ABSTRACT Mercury is a ubiquitous environmental toxicant that causes a wide range of adverse health effects in humans. Three forms of mercury exist: elemental, inorganic and organic. Each of them has its own profile of toxicity. The aim of the present study was to determine the effect of thimerosal, a topical antiseptic and preservative in vaccines routinely given to children, methyl mercury, and mercuric chloride on cellular viability measured by MTT in Jurkat T cells, a human T leukemia cell line. The treatment of Jurkat T cells with thimerosal caused a significant decrease in cellular viability at 1 μM (25%, p<0.05; IC50: 10 μM). Methyl mercury exhibited a significant decrease in cellular viability at 50 μM (33%, p<0.01; IC50: 65 μM). Mercuric chloride (HgCl2) did not show any significant change in cellular survival. Our findings showed that contrary to thimerosal and methyl mercury, mercuric chloride did not modify Jurkat T cell viability.

The Role of CD4+ T Cells in a Murine Model of Acute Lymphoblastic Leukaemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1838-1838
Author(s):  
Ahmed N. Hegazy ◽  
Mathias Wolenski ◽  
Karl Welte ◽  
Christoph Klein
Keyword(s):  
T Cells ◽  
T Cell ◽  
Acute Lymphoblastic Leukaemia ◽  
Adoptive Transfer ◽  
Lymphoblastic Leukaemia ◽  
Tumor Antigen ◽  
T Cell Responses ◽  
Interferon Γ ◽  
Cell Responses

Abstract To assess CD4-mediated anti-tumor immunity in a murine acute lymphoblastic leukaemia model system, we have generated a series of BCR-ABL positive pre-B cell lines expressing the surrogate tumor antigen ovalbumine. Upon intravenous injection, PKH-26-labeled leukaemia cells were taken up by splenic CD8+ but not by CD8− dendritic cells (DC). In comparison to PBS-injected DCs, CD8+ DCs also showed increased expression of CD40, CD80, and CD86. Purified DCs from leukemic mice stimulated transgenic DO11.10 T cells recognizing OVA323–339 in the context of I-Ad, suggesting efficient presentation of the surrogate tumor antigen. Next, we utilized adoptive transfer of DO11.10 T cells to measure tumor-specific T cell responses in vivo. OVA-expressing BM185 cells were unable to directly stimulate DO11.10 T cells, as shown by 3H-thymidine incorporation. In contrast, DO11.10 T cells were activated in vivo in spleen and lymph nodes, as shown by upregulation of CD44 and CFSE staining, suggesting that DC effectively present tumor antigens to DO11.10 T cells in vivo. However, despite of detectable T cell activation and proliferative T cell responses, all animals succumbed to progressive leukemia. Furthermore, adoptive transfer of naïve DO11.10 cells did not induce protective anti-leukemia immunity. Interestingly, in vivo primed DO11.10 T cells did not express interferon-γ. We therefore hypothesized that inefficient in vivo priming of TH1 cells may contribute to immune evasion of ALL cells. To address this question, we primed DO11.10 T cells in vitro prior to adoptive transfer. In this setting, DO11.10 T cells expressed interferon-γ and induced regression of preestablished leukaemia. This effect was dependent on CD8 cells, as shown by in vivo depletion experiments. Our experimental system supports the concept of CD4-dependent antitumor immunity and provides a platform to assess immunological mechanisms of novel strategies to therapeutically enhance antileukaemic immune responses.

scholarly journals CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

2021 ◽  
Vol 94 ◽  
pp. 105570
Author(s):  
Odelaisy León-Triana ◽  
Soukaina Sabir ◽  
Gabriel F. Calvo ◽  
Juan Belmonte-Beitia ◽  
Salvador Chulián ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Therapy ◽  
Acute Lymphoblastic Leukaemia ◽  
Mathematical Models ◽  
B Cell ◽  
Lymphoblastic Leukaemia ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T
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