Change of the Notch1 Expression Level in Lymphoid Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4425-4425
Author(s):  
Haitao Zheng ◽  
Lijian Yang ◽  
Shao-Hua Chen ◽  
Liye Zhong ◽  
Yangqiu Li
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
B Cell ◽  
Natural Science ◽  
Research Funding ◽  
Gene Expression Level ◽  
Expression Level ◽  
Lymphoid Malignancies ◽  
Healthy Control ◽  
Cell Commitment

Abstract Abstract 4425 Notch gene family encodes single pass transmembrane receptors able to transducer intercellular signals involved in cell-fate determination. Notch signaling is the leading causes to the development of malignant tumors. Notch pathway plays a role in haematopoiesis affecting both stem cells and committed progenitors. The expression of Notch1 in thymic stromal cells and in developing thymocytes is consistent with Notch function. Notch1 is turn on before T-cell commitment along the lymphoid development; a variety of model systems indicates that Notch1 signaling is required for the earliest stages of T-cell commitment in the thymus and that, in its absence, cells develop toward the B-lineage. Overexpression of Notch1 gene was found in T-ALL, however, little is known the expression pattern of Notch1 gene in the different lymphoid malignancies. In the present study, the expression level of Notch1 gene was analyzed in patients with lymphoid malignancies. Real-time PCR with SYBR Green I technique was used for detecting Notch1 gene expression level in peripheral blood mononuclear cells of 43 patients with lymphoid malignancies [including 7 cases with T-ALL, 6 cases with T-NHL, 9 cases with B-ALL, 5 cases with B-CLL, 16 cases with B-NHL] and 20 healthy individuals. β2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in Notch1 gene expression level were used by the 2-ΔCt×100% ×100% method. The expression level of Notch1 in both T-cell lymphoid malignancies group (Median: 0.897%) and B-cell lymphoid malignancies group (Median: 0.726%) was significant higher than those in the healthy control (Median: 0.288%) (P<0.01, P<0.01). Moreover, the expression level of Notch1 was different in the five types of lymphoid malignancies. That is, the highest in the B-CLL group, secondary in the T-NHL group, the lowest in the B-NHL group. And, it is not significant different between the B-NHL and healthy control groups. In conclusions, Overexpression of Notch1 gene was found not only in T-ALL, but also in T-NHL and B-cell leukemia and suggests that the abnormal overexpression pattern of Notch1 might play an important role in the pathogenesis of lymphoid malignancies. Down-regulation of Notch1 might be considered as target therapeutic strategy for lymphocytic malignancies. Disclosures: Zheng: The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Zhong:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding.

The Change of Elf-1 Gene Expression Level in Hematological Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4704-4704
Author(s):  
Qi Shen ◽  
Haitao Zheng ◽  
Shao hua Chen ◽  
Lijian Yang ◽  
Yangqiu Li
Keyword(s):  
T Cell ◽  
B Cell ◽  
Natural Science ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Hematologic Malignancies ◽  
Expression Level ◽  
Lymphoid Malignancies ◽  
Non Hodgkin Lymphoma ◽  
Healthy Control

Abstract Abstract 4704 Elf-1 (E-74-like factor 1), a special transcription factor of T cells, is a member of the Ets (E-26-specific) family. Elf-1 plays a critical role in the transcription of the TCR ζ-chain. Our previous studies showed that the low expression level of TCR ζ-chain in hematologic malignancies, in the present study, we farther analyzed the expression of Elf-1 in patients with hematologic malignancies. Real-time PCR with SYBR Green I technique was used for detecting Elf-1 expression level in peripheral blood mononuclear cells from 33 patients with acute myeloid leukemia (AML) [including 17 cases with AML-M2 subtype (M2), 6 cases with (M3), 10 cases with (M5)], 49 patients with lymphoid malignancies [including T-cell acute lymphoblastic leukemia (T-ALL) 7 cases, T-cell non-Hodgkin lymphoma (T-NHL) 6 cases, B-cell acute lymphoblastic leukemia (B-ALL) 10 cases, B-cell chronic lymphoblastic leukemia (B-CLL) 7 cases, B-cell non-Hodgkin lymphoma (B-NHL) 19 cases] and 20 healthy volunteers. β2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in Elf-1 expression level were used by the 2-ΔCt×100% method. We found that the expression level of Elf-1 in 33 cases with AML (13.518±19.197%) was significant higher than those in the healthy control (2.044±1.321%) (P<0.01). However, the expression level of Elf-1 had no significant difference among the three AML subtypes (P>0.05). The expression level of Elf-1 in both T-cell lymphoid malignancies group (12.050±10.334%) and B-cell lymphoid malignancies group (14.144±21.576%) was significant higher than those in the healthy control (P<0.01, P<0.01). Moreover, the expression level of Elf-1 was different in AML and the lymphoid malignancies groups (P<0.01). That was, the highest in the B-ALL group, the lowest in the B-NHL group. These results indicated that overexpression of Elf-1 is a common feature in peripheral blood from patients with both AML and lymphoid malignancies, as TCR ζ-chain was down regulation in AML and lymphoid malignancies, the increased expression of Elf-1 gene, which an upstream factor of the TCRζ-chain might indicate a feedback up regulation situation in patients with hematologic malignancies. Disclosures: Shen: The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Zheng:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding.

scholarly journals Delineation of Novel Subtypes Based on Microenvironment Immune Signature Provides Prognostic Stratification Strategy in Peripheral T-Cell Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4108-4108
Author(s):  
Takeshi Sugio ◽  
Kohta Miyawaki ◽  
Koji Kato ◽  
Hiroaki Miyoshi ◽  
Koichi Ohshima ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
M2 Macrophage ◽  
Cell Type ◽  
Peripheral T Cell Lymphoma ◽  
Immune Signature ◽  
Prognostic Stratification

Abstract Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is the largest subgroup with dismal prognosis in peripheral T-cell lymphomas (PTCL) that do not fit into any of the specifically defined categories in the World Health Organization classification, and it comprises over 25% of PTCL. PTCL-NOS is sometimes referred to as the gwaste-basketh category because of the lack of specific features, and therefore contains heterogeneous disease entities. Its heterogeneity represents a major obstacle to elucidation of pathogenesis, development of novel therapeutic targets and establishment of standardized therapeutic strategies and results in its poor prognosis. The stratification strategy of PTCL-NOS still remains controversial, although previous studies tried to reclassify PTCL-NOS from the viewpoint of gCell-of-Originh of tumor cells (e.g. T helper 1/2 cells, regulatory T cells) by utilizing histopathologic methods or gene expression profiling. These situations prompted us to investigate microenvironment profile of PTCL-NOS and to develop the novel stratification strategy. For this purpose, we analyzed the expression levels of 800 of tumor microenvironment-related genes including the phenotypic markers, transcription factors and functional molecules of microenvironment cells in formalin fixed paraffin embedded (FFPE) samples derived from 33 PTCL-NOS patients utilizing nCounter system, which is novel RNA counting system based on a digital molecular barcoding technology and enables accurate quantification of genes with low expression levels. Thirty-three patients who were diagnosed as PTCL-NOS between 1993 and 2011 were included in this study. The median follow-up time was 796 days (range, 15-2973). The overall survival rate at 1 year after diagnosis (1yr OS) was 40%, and it was comparable to previous reports. Unsupervised hierarchical clustering analysis revealed three distinct clusters based on the gene expression pattern of the microenvironment-related genes: B-cell type (42.4 %), M2 macrophage type (24.2 %), and others (33.3 %) (Figure 1). On the other hand, the expression patterns of the major T-cell subset-associated genes were complicated and diverse depending on patients, and failed to form any distinct clusters. B-cell-related genes, such as CD19, CD79B and PAX5 were highly expressed in the B-cell type, and the M2 macrophage-related genes, such as CD68, CD163, CD14 and MARCO were highly expressed in the M2 macrophage type. More importantly, these microenvironment-based subgroups, B-cell type and M2 macrophage type, represent prognostically favorable and unfavorable subgroups, respectively (1yr OS: B-cell type, 84.8%; M2 macrophage type, 16.7%; others, 56.2%; log rank test, p = 0.01) (Figure 2). Interestingly, genes associated with immune checkpoint, such as PD-L1, TIM-3 and IDO-1, were highly expressed in the M2 macrophage type than the others (PD-L1, p = 0.02; TIM-3, p = 0.05; IDO-1, p < 0.01), indicating the existence of specific subgroup of PTCL-NOS patients as anticipated good-responder for immunotherapeutic agents including immune checkpoint inhibitors. Collectively, our findings strongly suggest that microenvironment immune signature, not gCell-of-Originh of tumor cells, could provide novel prognostic stratification method and therapeutic strategy of PTCL-NOS. Disclosures Akashi: Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Astellas Pharma: Research Funding; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Sunitomo Dainippon Pharma: Consultancy; Bristol Meyers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Persistent Clonal Expansion of Vδ3 and Vδ4 T Cells in T-ALL Case with Minimal Residual Disease after HSCT

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5885-5885
Author(s):  
Ling Xu ◽  
Jianyu Weng ◽  
Xin Huang ◽  
Shaohua Chen ◽  
Suxia Geng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Natural Science ◽  
Research Funding ◽  
Cell Clone ◽  
Science And Technology ◽  
Expression Level ◽  
Time Points ◽  
Technology Project ◽  
T Cell Clone

Abstract Analysis of diversity and clonality is an important tool in monitoring reconstitution of the immune system and identificating specific immune reaction clones involved in hematological malignancy individuals who accepted hematological stem cell transplantation (HSCT). We have previously reported the evolution of malignant and reactive γδ+ T cell clones in a case with relapse T-ALL before and after allogeneic stem cell transplantation (allo-SCT) (from 4 weeks to 108 weeks), relapse was discovered with the same TCR Vδ5+ T cell clone at 100 weeks after transplantation, and the patient underwent chemotherapy over the next two months and achieved again remission with minimal residual disease (MRD). We sequentially monitored the clinical and laboratorial features of this T-ALL case. The patient was received three times donor lymphocytes infusion (DLI) at 117, 120 and 124 weeks after HSCT. In 178 weeks after transplantation, the patient underwent relapse again, however, in this time the patient underwent III grade chronic graft versus host disease (cGvHD). We continued to analyze the samples at different time points after HSCT and DLI, the leukemic Vδ5+ T cell clone was detected in most samples, while this clone could not be detected in donor sample (Figure 1). Moreover, we found that a monoclonally expanded TCR Vδ4 which we reported before and it remained detectable in all samples post transplantation, interesting same monoclonal Vδ4 clone was identified in sample from donor which was collected at time for DLI. The sequence of Vδ4 was confirmed as same TCR rearrangement: Vδ4Dδ3Jδ1, and the expression level of Vδ4 was significantly increased at the time of disease relapse, the Vδ4 expression level in samples from PBMC and bone marrow at different time point was showed in figure 2. The monoclone Vδ4 from donor was thought as specifically expanded for unknown antigen (maybe for virus or leukemia) and may possess cytotoxicity in donor, however, the persistent expansion of Vδ4 in receptor was thought that might be responsible for leukemic cells, virus infection or cGvHD, it remains an opne question. Because patient underwent III grade cGvHD, and whether this Vδ4+ T cell clone was mainly associated with cGvHD, it is needed further investigation. In addition, the clonally expanded Vδ3 with same size was also detected at time before HSCT, and most time points after HSCT, while it could not be identified in donor sample (Figure 3). Obviously, this expanded Vδ3+ T cell clone originated from patient, and it is possible that this T cell clone possesssed a specific anti-leukemia function, because it appeared at the time with minimal residual disease, however, the function of clonally expanded Vδ3 is needed further confirmation. And it is needed also to analyze the clonality of TCR Vα and Vβ subfamilies. In conclusion, dynamically monitoring TCR repertoire and combining the characteristic of clinical status might benefit for characterizing the malignant clonal evolution, monitoring the specific T cell clone for graft-versus-leukemia (GvL) or GvHD effect and designing specific therapeutic strategies in T-ALL. Competing interests The authors declare that they have no competing interests. Grants: This study was supported by grants from the National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023), the Fundamental Research Funds for the Central Universities (No. 21610603, 21612116). Figure 1. The CDR3 spectratyping of the TCR Vγ and Vδ subfamily T cells in peripheral blood from donor and recipient at different time points after allo-HSCT. Figure 1. The CDR3 spectratyping of the TCR Vγ and Vδ subfamily T cells in peripheral blood from donor and recipient at different time points after allo-HSCT. Figure 2. The expression level of the TCR Vδ4 gene in recipient at different time points post-HSCT: Red dots showing the expression level of TCRVδ4 (TCDV4) gene in PBMC at time points of 117 W, 120 W, 135 W, 142 W, 146 W, 178 W, 190 W and 200 W post-HSCT. Blue triangles showing the expression level of TCRDV4 genes in bone marrow (BM) at time points of 129.5 W, 178 W, 190 W and 200 W post-HSCT. Figure 2. The expression level of the TCR Vδ4 gene in recipient at different time points post-HSCT: Red dots showing the expression level of TCRVδ4 (TCDV4) gene in PBMC at time points of 117 W, 120 W, 135 W, 142 W, 146 W, 178 W, 190 W and 200 W post-HSCT. Blue triangles showing the expression level of TCRDV4 genes in bone marrow (BM) at time points of 129.5 W, 178 W, 190 W and 200 W post-HSCT. Figure 3. The The CDR3 spectratyping of Vδ3 T cells in the samples of donor and recipient at different time points post-HSCT. The red boxes indicated the clonally expanded Vδ3 T cell clone with 526 bp which contained the same sequence confirmed by PCR product direct nucleotide sequencing. Figure 3. The The CDR3 spectratyping of Vδ3 T cells in the samples of donor and recipient at different time points post-HSCT. The red boxes indicated the clonally expanded Vδ3 T cell clone with 526 bp which contained the same sequence confirmed by PCR product direct nucleotide sequencing. Disclosures Xu: National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023): Research Funding; Fundamental Research Funds for the Central Universities (No. 21610603, 21612116): Research Funding. Li:Fundamental Research Funds for the Central Universities (No. 21610603, 21612116). : Research Funding; National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023): Research Funding.

scholarly journals The Characteristic of TCR Signaling Pathway in T Cell from Patients with Aplastic Anemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2226-2226
Author(s):  
Bo Li ◽  
Lixing Guo ◽  
Lingling Zhou ◽  
Mingjuan Wu ◽  
Yuping Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Natural Science ◽  
Research Funding ◽  
Cell Activation ◽  
Guangdong Province ◽  
Expression Level ◽  
Healthy Individuals ◽  
Healthy Group ◽  
Expression Levels

Abstract A number of abnormalities of T cell immune function leading to immune dysregulation have been reported in aplastic anemia (AA). Our previous study showed not only the abnotmal distribution and clonal expansion of TCR repertoire,but also significantly higher expression of CD3ζ in T cells from AA patients. Alternative CD3ζ gene expression level may directly represent a characteristic of abnormal T cell activation. Thus, we further investigated the expression level of CD28 and CTLA-4 which involved in mediating T cell activation; CD3ζ-regulating factors, the expression levels of Cbl-b and distribution of the CD3ζ 3' untranslated region (3'-UTR) splice variant. The splice variant of CD3ζ 3'-UTR were identified in peripheral blood mononuclear cells (PBMCs) from 55 healthy individuals and 68 patients with AA (36 cases with SAA and 32 cases with AA) respecitively by RT-PCR. The expression levels of the Cbl-b, CD28, CTLA4 and CD3ζ genes were analyzed by real-time quantitative PCR. The results showed that increased expression level of CD28 (0.91±0.67) and CD3ζ (1.72±2.02) were found in AA group compared with healthy group (CD28: 0.71±0.58, P = 0.08; CD3ζ:1.07±0.76, P<0.05), While significantly decreased expression level of CTLA-4 (0.16±0.17) and Cbl-b (0.34±0.31) were found in AA group compared with healthy group (CTLA-4: 0.22±0.14, P<0.05; Cbl-b: 0.62±0.35, P<0.01). There are not significantly differences of the expression levels from all detected genes between SAA and AA group. Two types of splicing variants forms, wild type CD3ζ 3'-UTR (WT) and alternatively spliced CD3ζ 3'-UTR (AS) which has significantly lower stability and translation of CD3ζ, were detected in all of the healthy individuals. However, 64.7% AA patients contained WT+ AS+ CD3ζ 3′-UTR, 22.1% AA patients contained WT+ AS- CD3ζ 3′-UTR and 13.2% AA patients WT- AS+ CD3ζ 3′-UTR. The frequencies of three types of CD3ζ 3′-UTR also have no significantly differences between SAA and AA patients. Based on the CD3ζ 3'-UTR isoform expression characteristic, we divided the AA into three subgroups: WT+ AS+, WT+ AS- and WT- AS+ group. Significantly higher expression level of CD3ζ gene was found in WT- AS+ group than that in WT+ AS+ groups. In conclusion, to our knowledge, this is the first study on mechanism of increased CD3ζ gene in AA. The increased expression level of CD3ζ gene may be associated with decreased expression level of Cbl-b gene in AA. Additionally, the expression feature of CD3ζ 3'-UTR isoform may be affect the expression of CD3ζ gene in some AA patient. Increased expression level of CD3ζ in AA patients contain WT- AS+ CD3ζ 3'-UTR may be feedback regulation. Meanwhile, aberrant of T cell activation in AA may be related to changed expression of CD28 and CTLA-4. Disclosures Li: the National Natural Science Foundation of China (#81370605 and #81460026), a China Post-doctoral Science Foundation funded project (#20070410840), the Natural Science Foundation of Guangdong Province (#S2012010008794 and #S20130200-12863),: Research Funding; the Technology Programme of Guangdong Province (#2014A020212209), the Foundation for High-level Talents in Higher Education of Guangdong, China (#[2013]246-54),: Research Funding; Science and Information Technology of Guangzhou funded basic research for application project (#2011J4100028), and the Fundamental Research Funds for the Central Universities (#21612425).: Research Funding.

scholarly journals Immune Profiling of Diagnostic DLBCL Biopsies Dramatically Improves upon Cell-of-Origin Risk Stratification

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 719-719
Author(s):  
Michael Nissen ◽  
Xuehai Wang ◽  
Clementine Sarkozy ◽  
Aixiang Jiang ◽  
Daisuke Ennishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Patient Outcomes ◽  
Expression Profiling ◽  
Research Funding ◽  
Cell Of Origin ◽  
Discovery Cohort

Abstract Background: Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy of mature B cells. The disease has traditionally been subdivided into cell-of-origin (COO) subtypes - germinal centre B cell-like (GCB) or activated B cell-like (ABC) - as determined by expression profiling or immunohistochemistry of the tumor cells. However the role of the immune microenvironment, and how the tumor and immune system interact to influence patient outcomes, remains to be fully investigated. Methods: In this project, we used mass cytometry (CyTOF) to deeply profile both tumor cell phenotypes and the immune microenvironments, alongside ABC/GCB classification and mutation profiling, in a discovery cohort of 54 DLBCL cases. As well, a validation cohort of 129 DLBCL patients were immunologically profiled by high-dimensional conventional flow cytometry, and their immune profiles alongside ABC/GCB classification, mutation profiling, and RNAseq data, were correlated with patient outcomes as measured by progression-free survival (PFS). Results: Analysis of the CyTOF/discovery cohort demonstrated that DLBCL tumor cells are phenotypically unique to each patient, with a small number of samples displaying distinct sub-clonal structure, often distinguished by differential expression of immune-related proteins like MHC-II. ABC/GCB classifications could be recapitulated based on tumor cell phenotypes, demonstrating that while COO was a robust feature, a great deal of heterogeneity exists within these established subtypes. Immunological profiling of the CyTOF/discovery cohort revealed that DLBCL samples could be divided into three distinct groups which roughly correlated with abundances of naïve, activated, or terminally differentiated T cells, respectively. This profiling schema was extended to the validation cohort of 129 patients which in turn led to identification of a subset of patients with a very high risk of disease progression (5-year PFS; 30% high risk vs. 80% low risk, p&lt;0.0001). This final classifier was based on a combination of ABC-DLBCL designation, combined with the presence of an immune microenvironment dominated by terminally differentiated (CD57+) T cells. We performed a limited series of functional studies using primary DLBCL biopsy samples to characterize further these CD57+ T cells as clonally restricted and incapable of responding to antigenic challenge. Interestingly, traditional immune markers of T cell exhaustion such as PD-1, TIM3, LAG3 and TIGIT were not correlated with patient outcomes. Conclusions: Overall, this study demonstrates the utility of immune profiling in risk stratification based on initial diagnostic biopsy material and highlights a subset of DLBCL patients who may benefit from immune-based therapies to rejuvenate the anti-tumor T cell response. We conclude that T cell senescence, rather than exhaustion, is the more relevant feature in DLBCL disease biology and highlights an alternate target for immunomodulatory therapy. Figure 1 Figure 1. Disclosures Craig: Bayer: Consultancy. Slack: Seagen: Consultancy, Honoraria. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; Rich/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Incyte: Consultancy. Steidl: Epizyme: Research Funding; Bayer: Consultancy; Curis Inc.: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Trillium Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding.

IL7 Counteraction with Notch Signaling Followed by EBF1 Expression Marks the B-Cell Commitment in CLP Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2452-2452
Author(s):  
Sasan Zandi ◽  
Panagiotis Tsapogas ◽  
Robert Månsson ◽  
Mikael Sigvardsson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cell ◽  
Single Cell ◽  
Reporter Gene ◽  
B Cell Development ◽  
Expression Level ◽  
Single Cell Pcr ◽  
Cell Commitment

Abstract Development of B-cell lineage from hematopoietic Stem cells in bone marrow is a stepwise process associated with a gradual loss of myeloid and T cell potential. This process involves a complex interaction of transcription factors like EBF1 and E2A, and extrinsic signals including IL7. It has been suggested that IL7 plays an inductive role in B-cell commitment through EBF activation in early B-Cell development. Mice deficient in Il-7 signaling show a dramatic reduction in the number of B-cell progenitors and reduced expression of EBF1 in the common lymphoid progenitor (CLP) compartment and ectopic expression of EBF can partially rescue the B-cell phenotype. However, the rather limited ability of EBF1 to rescue the phenotype as well as the powerful function of Il-7 in the expansion of committed cells creates a complex situation with an inherent difficulty to separate instructive and permissive actions of Il-7. Using transgenic mice carrying a reporter gene under the control of the EBF1 dependent Igll1 promoter, we were able to identify a B220−CD19− committed B-cell progenitor likely to represent the earliest committed population in the mouse bone marrow. This has opened the possibility to investigate lineage commitment in cells not expressing classical surface markers creating increased possibilities to study lineage choices. In order to investigate the inductive role of Il-7 we crossed the Igll1 reporter mice to Il-7 deficient mice. Analysis of reporter gene expression, gene expression by multiplex single cell PCR as well as functional analysis by in vitro differentiation assays, all supported that the committed lineage negative population was dramatically decreased in the absence of Il-7. These data all support the idea that Il-7 is critical not only for expansion of B-lineage progenitors but also for commitment per se. Investigation of the expression of EBF-1 by Real time and single cell PCR suggested that the expression level of EBF was on an average 50% lower in Il-7 deficient progenitors as compared to wild type cells. This expression level was recapitulated in mice heterozygote for a mutation in the EBF1 gene but since the formation of the early committed cells was not as dramatically effected in these mice, we found a need to look for a further function of Il-7 in its instructive role in B-cell development. This prompted us to investigate a potential function of Il-7 in the modulation of Notch signals known to counteract B-cell commitment and EBF function. This revealed that the addition of Il-7 largely inhibits the Notch response in pro-B cells in vitro. Therefore we suggest that Il-7 is directly involved in B-cell commitment and that this function is achieved by modulation of EBF1 both at the transcriptional and functional level.

Effects of Down-Regulating BCL11B Expression On the Proliferation, Apoptosis and Global Gene Expression Profiling of Molt-4 Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4505-4505
Author(s):  
Xin Huang ◽  
Si Chen ◽  
Lijian Yang ◽  
Shao-Hua Chen ◽  
Yubing Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Gene Expression Profiling ◽  
Natural Science ◽  
Expression Profiling ◽  
Research Funding ◽  
Science And Technology ◽  
Guangdong Province ◽  
Global Gene Expression ◽  
Global Gene Expression Profiling

Abstract Abstract 4505 The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a crucial role in T-cell development, differentiation, and proliferation. BCL11B disturbed expression, mutation, disruption, or rearrangement has been associated with T-cell malignancies. Here, we described the effects of BCL11B target-specific small interfering RNA (BCL11B-siRNA) on the proliferation, apoptosis, and global gene expression profiling of Molt-4 cells. The BCL11B-siRNA beginning with the 924bp of mRNA sequence, and the scrambled non-silencing siRNA control (sc) were obtained by chemosynthesis. Non-treated, mock-transfected, and sc-treated cells were used as controls. BCL11B expression in mRNA levels were analyzed at 24 h, 48 h and 72 h after siRNA delivered by nucleofection using the real-time quantitative PCR with Taqman technique. At the same time points, cell proliferations were assayed by the cell count kit-8 method. The morphology and the percentage of apoptosis were revealed by Hoechst33258 stain and flow cytometry at 72 h, respectively. An obvious reduction in mRNA level was observed in BCL11B-siRNA treated cells at 24 h, compared to the controls (P< 0.05). The proliferation rates of BCL11B–siRNA treated cells were significantly decreased from 24 h to 72 h (P< 0.05). BCL11B–siRNA treated cells showed a large increase in PI-positive cells, reaching to 84.6%. However, the percentage of each cell phases did not reveal significantly difference. Thus, the apoptosis could be induced in all phases of the cell cycle. The Hoechst33258 stain was used to verify the apoptosis. To elucidate the molecular mechanisms accounting for BCL11B-siRNA-mediated cell death, global gene expression profiling were performed on the BCL11B–siRNA treated cells and non-treated control by the Affymetrix HG U133 Plus 2.0 GeneChip at 24 h after nucleofection. The BCL11B showed 10.1 fold reductions in mRNA levels. Upregulated genes was found in 1982 probe sets, while 1884 genes down-regulated, at least two folds upon Bcl11b suppression. Involvement of the T-cell activation or differentiation as TRAT1, CD3G, CD247, LCK, PTGER4, or CD1D, all revealed at least 7 folds down-regulated, which may contribute to the inhibition of proliferation. Six genes of the TNF receptor family were up-regulated at least 2 folds as well as the BCL-2 gene down-regulated 3.5 folds. The apoptosis was most likely affected by the simultaneous activation of TNF receptor family genes and suppression of the Bcl-2 gene. Those related to the autophagy and the cell cycle did not find significant change. In conclusion, we demonstrated that the survival of Molt-4 cells was critically dependent on BCL11B gene. Suppression of BCL11B by RNA interference selectively inhibited the proliferation and induced the apoptosis effectively in transformed Molt-4 cells. Therefore, down-regulation of Bcl11b might be considered as a new target therapeutic strategy in T-cell malignancies. Disclosures: Huang: The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Zhou:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30771980) and Key Project Foundation of the Science and Technology of Guangdong province (No. 2007B030703008): Research Funding.

Expression Feature of CD3 and FcεRIγ Genes in Patients with Aplastics Anemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4372-4372
Author(s):  
Bo Li ◽  
Yu Zhe Niu ◽  
Si Chu Liu ◽  
Shao Hua Chen ◽  
Yang Li Jian ◽  
...  
Keyword(s):  
T Cell ◽  
Natural Science ◽  
Research Funding ◽  
Expression Level ◽  
Technology Program ◽  
Expression Levels ◽  
Science Technology ◽  
Tcr Signal Transduction ◽  
Funded Project ◽  
Fundamental Research

Abstract Abstract 4372 Aplastic anemia (AA) is characterized by pancytopenia and bone marrow hypoplasia, resulting from immune-mediated suppression of hematopoiesis. Understanding of the immune pathophysiology, especially T-cell immune of the disease over the past 3 decades have led to improvement the treatment of AA. However, primary and secondary failures after immunosuppressive therapy remain frequent. Thereby, more knowledge of the immune mechanisms leading to the aplastic anemia is crucial to more understanding the mechanism of the disease. In order to elucidate the feature of TCR signal transduction in AA, the expression levels of CD3γ, δ, ε and ζ chain and FcεRIγ genes which involed in TCR signal transduction and negative correlation of the expression levels between CD3ζ and FcεRIγ genes in actived T-cell in peripheral blood mononuclear cells (PBMCs) were analyzed. Real-time RT-PCR with SYBR Green technique was used for detecting the gene expression level in PBMCs from 27 patients with AA and 9 healthy individuals. The β2-microglobulin gene (β2M) was used as an endogenous reference. The expression levels of CD3γ, CD3ε and CD3ζ gene in AA patients (19.96±21.46, 17.50±17.53 and 5.50±3.53, respectively) were significant increased compared with healthy group (4.00±1.70, 4.48±2.37 and 1.95±1.77, respectively) (P< 0.01), whereas the expression level of FcεRIγ gene was significant decreased in AA patients (17.58±10.23) in comparison with healthy group (40.20±25.46) (P< 0.01), while no significant difference of the CD3δ expression level was detected between AA and healthy groups. Moreover, there was lost the negative correlation of the expression levels between CD3ζ and FcεRIγ genes (r=0.19,p=0.33). In conclusions, this is, to our knowledge, the first description of the feature of CD3γ, CD3δ, CD3ε, CD3ζ and FcεRIγ gene expression in AA patients. The abnormality expression of CD3γ, CD3ε, CD3ζand FcεRIγ gene might related to the T-cell dysfunction in AA. Further research is needed to investigate the different expression pattern of these genes in different subset of T cells. Disclosures: Li: China Postdoctoral Science Foundation funded project (No. 20070410840): Research Funding; Natural Science Foundation of Guangdong province (No. 07301046): Research Funding; The Fundamental Research Funds for the Central Universities (No. 21610408): Research Funding; Medical Science Technology Program of Guangzhou (2009-ZDI-18): Research Funding; National Natural Science Foundation of China (No. 30972455): Research Funding. Li:Medical Science Technology Program of Guangzhou (2009-ZDI-18): Research Funding; The Fundamental Research Funds for the Central Universities (No. 21610408): Research Funding; Natural Science Foundation of Guangdong province (No. 07301046): Research Funding; China Postdoctoral Science Foundation funded project (No. 20070410840): Research Funding; National Natural Science Foundation of China (No. 30972455): Research Funding.

scholarly journals Genetic Perturbation of CD70/CD27 Co-Stimulation Promotes the Development of Bcl6-Driven Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 713-713
Author(s):  
Elisa Mandato ◽  
Yanbo Sun ◽  
Vignesh Shanmugam ◽  
Il-Kyu Choi ◽  
Kyle T. Wright ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Nature Medicine ◽  
Immune Surveillance ◽  
B Cell Lymphoma ◽  
Genetic Alterations ◽  
Lymphoid Malignancies

Abstract Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation. CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway. In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL. To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. Disclosures Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

Gene Expression Profiling of Pulmonary Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Identifies New Diagnostic Markers, Biological Heterogeneity and Therapeutic Targets.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 560-560
Author(s):  
Wee J. Chng ◽  
Rafael Fonseca ◽  
Peter Leif Bergsagel ◽  
Julia A. Vrana ◽  
Paul J. Kurtin ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
B Cell ◽  
Malt Lymphoma ◽  
Lymphoid Tissue ◽  
Cell Lymphoma ◽  
Therapeutic Targets ◽  
Unsupervised Clustering ◽  
T Cell Lymphoma ◽  
Lymphoid Malignancies

Abstract To further understand the molecular biology of mucosa-associated lymphoid tissue (MALT) lymphoma, we conducted a comprehensive gene expression study on the Affymetrix platform using total RNA extracted from biopsy specimen of 35 well-characterized pulmonary MALT lymphoma samples and compared them to gene expression profiles of a range of B and T cell lymphoma, such as Burkitt’s lymphoma, diffuse large B-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, multiple myeloma, and peripheral T-cell lymphoma, and normal cellular counterparts, such as different B-cell and T-cell populations, normal plasma cells and lung tussue. Analytical strategies were adopted to negate tissue specific effect such that our results can be generalized to MALT lymphoma from other anatomical sites. We further analyzed the data using functional tools such as gene-set enrichment analysis and gene ontology. Network/pathway analysis was performed using Metacore. Unsupervised clustering of whole dataset (including other lymphoid malignancies and normal cellular counterpart) using genes with variable expression, showed that compared to other B-cell lymphoid malignancies, MALT lymphoma have a prominent T-cell signature. Using established gene expression of different B-cell compartment, we confirmed that MALT has a marginal zone B-cell and memory B-cell cell-of-origin signature. Using ANOVA with post-hoc pair-wise comparison, we identified 4 novel transcripts over-expressed in MALT but not other B-cell tumors, and validated one of these using immunohistochemistry. MALT lymphoma with t(11;18) or t(14;18) over-expressed genes enriched for pathways other than the NFKB pathway such as JAK-STAT and SRC signaling pathways, which may represent novel therapeutic targets. We identified a number of genes with ‘outlier’ expression including RARA and RGS13. However, translocation involving RARA and the immunoglobulin heavy chain locus was not detected by FISH, suggesting other mechanisms of deregulating gene expression were involved. Using unsupervised clustering of only the MALT samples, we found that the molecular heterogeneity within MALT is composed of a group characterized by MALT1 translocations and another group with plasmacytic differentiation. Interestingly, further refinement of this grouping can be achieved by clustering the samples based on ‘outlier’ gene expression. Samples with MALT1 translocations have high expression of RARA, samples with plasmacytic differentiation have high FKBP11 expression and samples with high RGS13 expression tend to have trisomy 3 and reactive follicles. Our study therefore identified novel molecular markers, deregulated pathways and genetic defects in MALT. Furthermore, subgroups with distinct pathological features and anchored by unique pattern of ‘outlier’ gene expression were identified.

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