scholarly journals Epigenetic Disruption of ZEB1 Binding Causes Constitutive Activation of IL-15 in Cutaneous T-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3635-3635
Author(s):  
Anjali Mishra ◽  
Sonya Kwiatkowski ◽  
Laura Sullivan ◽  
Leah Grinshpun ◽  
Giandomenico Russo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Binding Sites ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Luciferase Activity ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Normal Donor ◽  
Relative Luciferase Activity

Abstract Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin-homing T-cells that represents 70-80% of all cutaneous lymphomas. Studies have shown that interleukin-15 (IL-15) plays a key role in T-cell oncogenesis by increasing T-cell survival, proliferation, migration and invasion in patient-derived CTCL cell lines. Previously, we have demonstrated that IL-15 is overexpressed in lesional biopsies and peripheral blood of CTCL patients (Mishra et al., Blood, 122:1826, 2013). However, the underlying mechanisms of IL-15 deregulation are largely unknown. Since epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T-cells from the blood of CTCL patients and performed a pyro-sequencing analysis to determine methylation of the IL-15 gene promoter. We observed significantly higher methylation in the IL-15 promoter of CD4+ T-cells from CTCL patients vs. normal donors (mean ± SEM of relative % methylation= 232.0 ± 49.18, n=9 vs 100.1 ± 7.619, n=6, P =0.03). Furthermore, methylation at the IL-15 promoter was higher in malignant CD4+ T-cells compared to non-malignant neutrophils from the same patient (mean ± SEM of relative % methylation = 232.0 ± 49.18 vs 52.31 ± 6.28%, N=9 each, paired t-test, P =0.0025). Using this approach, we also analyzed methylation of each 'CpG dinucleotide' in the IL-15 promoter. Once again, methylation was higher in CD4+ T-cells of patient versus normal donors and, within each patient, in CD4+ T-cells versus neutrophils. The CpG rich region of the human IL-15 promoter contains three putative binding sites for the known transcriptional repressor Zeb1, which has been reported to be mutated or deleted in CTCL. In order to examine the effect of CpG methylation at the Zeb1 binding region on IL-15 transcription, the CpG rich 5' regulatory region of the IL-15 promoter was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, the region of the IL-15 promoter containing the 3 ZEB1-binding sites is transcriptionally active in its native form (mean ± SEM of relative luciferase activity of promoter-less vector vs. IL-15 promoter vector = 77.00 ± 3.29 vs. 626.8 ± 25.05 respectively, N=3 each, P<0.0001). Deletion of the putative Zeb1 binding sites (BS#1, BS#2, BS#3 and BS#1-3) or the entire Zeb-binding region (BR) in the IL-15 promoter led to a significant increase in IL-15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL-15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1-3 vs. BR vectors = 99.56 ± 0.33 vs. 7135 ± 108.5 vs. 3940 ± 62.36 vs. 2372 ± 24.65 vs. 1099 ± 9.72 vs. 1525 ± 32.00, n=4 each; P <0.0001 each). Since CpG methylation of DNA can physically prevent transcription factor binding to regulatory regions of DNA, we hypothesized that CD4+ cells from CTCL patients might display reduced binding of Zeb1 to the IL-15 promoter thereby increasing IL-15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)-PCR, we observed a substantial loss of Zeb1 binding at the IL-15 promoter in the CD4+ cells of CTCL patients compared to those of normal donors (mean ± SEM of relative Zeb1 binding to IL-15 promoter in normal donor vs. CTCL patient CD4+ T-cells = 101.3 ± 1.48 vs. 25.31 ± 3.78, n=3 each; P <0.0001). Similarly, silencing Zeb1 in normal donor CD4+ T-cells caused an increase in IL-15 transcription. Finally, constitutive IL-15 overexpression was sufficient to cause a high penetrance lymphoproliferative disease in transgenic mice, with typical dermatologic features of human CTCL, including alopecia, scaly erythematous plaques/patches, and ulcerations, caused by an epidermotropic T-cell infiltrate. Thus, we defined Zeb1 as a novel repressor for IL-15 transcription in normal T-cells and show that epigenetic interference with its repressive function at the IL-15 promoter is associated with T-cell lymphoma in vivo, highlighting the role of epigenetic processes in inflammation-induced cancers and further supporting the role of Zeb1 as a putative tumor suppressor gene in CTCL Disclosures Porcu: Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding.

HTLV-1 Dysregulates IL-6 and IL-10-JAK/STAT Signaling and Induces Leukemia/Lymphoma of Mature CD4+ T Cells with Regulatory T-Cell-like Signatures

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1516-1516
Author(s):  
Yusuke Higuchi ◽  
Jun-ichirou Yasunaga ◽  
Yu Mitagami ◽  
Koichi Ohshima ◽  
Masao Matsuoka
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Cell Leukemia ◽  
Chugai Pharmaceutical ◽  
Flow Cytometric ◽  
Stat Signaling

Human T-cell leukemia virus, type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases (e.g., HTLV-1 associated myelopathy, uveitis). Among viral genes encoded in the HTLV-1 provirus, HTLV-1 bZIP factor (HBZ) is the most important gene for its pathogenesis, since HBZ is constantly expressed in all ATL cases, and has a potential to enhance T-cell proliferation. We generated HBZ transgenic (Tg) mice and found that almost all mice developed systemic inflammation, such as dermatitis, and about 40% of mice suffered from T-cell lymphoma. In addition, this mouse demonstrates the similar immunophenotypes to HTLV-1-infected subjects, such as increase of effector/memory and Foxp3-expressing regulatory T cells (Tregs), indicating that it is a good animal model to analyze the molecular mechanisms of pathogenesis by HTLV-1. There is a clear correlation between the severity of inflammation and the incidence of T-cell lymphoma, suggesting that some immune factors are closely involved in oncogenesis by HBZ. Recent studies showed that several pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6), participate in not only inflammation but also tumor development. Especially, IL-6 has been reported to play important roles in oncogenesis, tumor progression, angiogenesis and migration. To clarify the roles of IL-6 in HBZ-mediated lymphomagenesis, we crossed HBZ-Tg mice with IL-6 knockout (KO) mice. Contrary to our hypothesis, inflammation was exacerbated, and incidence of T-cell lymphomas was markedly increased in HBZ-Tg/IL-6KO mice. In the pathological analysis of the lymphoma tissues, the percentage of Foxp3+ cells were much higher in HBZ-Tg/IL-6KO mice than in HBZ-Tg mice. Moreover, flow cytometric analysis also showed that CD4+Foxp3+ T cells were increased in spleens from HBZ-Tg/IL-6KO mice compared with HBZ-Tg mice or WT mice. Since IL-6 inhibits TGF-beta-induced Foxp3 expression and subsequently suppresses differentiation to Tregs, our results suggest that depletion of IL-6 induces aberrant differentiation of CD4+ T cells toward Treg-like cells in HBZ-Tg mice and accelerates lymphomagenesis of this subset. RNA-seq and flow cytometric analyses revealed that expression of IL-10 was significantly higher in CD4+ T cells of HBZ-Tg/IL-6KO than HBZ-Tg. Importantly, IL-10 accelerated the proliferation of CD4+ T cells of HBZ-Tg whereas it did not influence control T cells. We also found that HBZ interacted with both STAT1 and STAT3, which are critical transcription factors in IL-10-JAK/STAT signaling pathways, and enhanced their transcriptional activities. Foxp3-expressing T cells and IL-10 are known to be immune-suppressive, but these results indicate that increased Foxp3-expressing T cells and activation of IL-10-JAK/STAT signaling are associated with inflammation and lymphomagenesis by HBZ in vivo. Our results suggest that IL-6 and IL-10 have suppressive and promoting effects on HBZ-induced pathogenesis, respectively. It has been reported that progression of HTLV-1-associated diseases was observed in several HTLV-1 carriers after administration of anti-IL-6R antibody, Tocilizumab. Inhibition of IL-6/IL-6R signaling and consequent activation of Foxp3/IL-10/JAK/STAT axis seem to be important for HTLV-1 pathogenesis. Disclosures Ohshima: Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding; SRL, Inc.: Consultancy. Matsuoka:Kyowa Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb Corp.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Combined Effect of Mtor Inhibitors and FTI for the Growth Inhibition of T Cell Lymphoma - Involvement of Different Molecular Mechanisms According to the Lymphoma Subtype

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4870-4870
Author(s):  
Leonidas Zierock ◽  
Wolfgang Melchinger ◽  
Bettina Wehrle ◽  
Juergen Finke ◽  
Reinhard Marks
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Molecular Mechanisms ◽  
Cell Lymphoma ◽  
Mtor Inhibitors ◽  
T Cell Lymphoma ◽  
Ampk Phosphorylation ◽  
Lymphoma Subtype

Abstract Abstract 4870 Since the succesful treatment of T cell lymphoma remains to be problematic, identification of new pharmacological targets in this malignancies are desperately needed. The AMPK-Rheb-mTOR signaling pathway plays an important role in regulating processes such as proliferation and proteinsynthesis according to energy and nutrient levels in normal and malignant T cells. Inhibitors of mTOR have shown promising results in clinical trials in several lymphoma types. Similarly, recent data could prove inhibitors of farnesyltransferase (FTI) to be effective as a single agent in certain subtypes of T cell lymphoma. Despite divergent data regarding the molecular target of FTI action, recently published work suggest inhibition of prenylation of the GTPase Rheb as putative mechanism for the antineoplastic effects of FTI (Basso et al., J Biol Chem, 2005). Therefore, combining inhibition of mTOR and Rheb might result in increased inhibition of T cell lymphoma proliferation. To investigate this hypothesis, human T cell lymphoma cell lines DERL-2 (originated from hepatosplenic gamma-delta T cell lymphoma), Karpas-299 (originated from anaplastic large cell T cell lymphoma) and normal human CD4+ T cells were incubated with a combination of everolimus as mTOR inhibitor and FTI (lonafarnib, SCH-66336) or the single agents. While both substances showed an additive combined anti-proliferative effect in DERL-2 cells, proliferation of Karpas cells were more susceptible to inhibition by FTI. On a molecular level, despite substantial growth inhibition in both cell lines by everolimus alone, phosphorylation of 4EBP1 and p70S6K remained unaffected, while FTI mediated reduction of Karpas cell proliferation was associated with a substantial decrease in AMPK phosphorylation together with an overexpression of p27kip, which could not be observed in DERL-2 cells. In contrast, incubation of stimulated human CD4+ T cells with the drugs alone or in combination did not result in changes in the phosporylation status of AMPK. Nevertheless, in contrast to everolimus, FTI induced a reduction of total protein expression of AMPK and other proteins, e.g. AKT. In addition, contrary to the observations in the malignant T cells, FTI treatment of unstimulated human CD4+ T cells resulted even in an increase of AMPK-phosphorylation. A hint for the explanation of these conflicting data came from analyses of Rheb expression in the examined cell types. While Rheb was easily detectable in the malignant T cell lines and the stimulated CD4+ T cells, it was almost absent in unstimulated CD4+ T cells. A model derived from this findings is that FTI effects depend on different targets available for inhibition of prenylation according to the activation or differentiation status of the T cells. While Rheb might be the target in malignant or activated T cells, another target, e.g. phosphatases, might be responsible for the FTI effect in resting T cells where Rheb is not available. In Karpas cells a particular connection between Rheb and AMPK might exist, as described for other cell lines (Lacher et al., Oncogene, 2010). Inhibition of this Rheb-AMPK axis might explain the particular gowth inhibiting effect of FTI in this model of anaplastic large T cell lymphoma. Nevertherless, the presented data show a combined effect of mTOR inhibitors and FTI for the potent treatment of T cell lymphoma involving different molecular mechanisms according to the lymphoma subtype. Disclosures: Finke: Fresenius Biotech GmbH: Honoraria, Research Funding.

Molecular Heterogeneity in Peripheral T-Cell Lymphoma Not Otherwise Specified Revealed By Comprehensive Mutational Profiling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2927-2927 ◽  
Author(s):  
Yosaku Watatani ◽  
Yasuharu Sato ◽  
Kenji Nishida ◽  
Hiroaki Miyoshi ◽  
Yuichi Shiraishi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Genetic Alterations ◽  
T Cell Lymphoma ◽  
Molecular Heterogeneity ◽  
Not Otherwise Specified ◽  
T Cell Lymphomas ◽  
Somatic Alterations

Abstract Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoproliferative disorders arising from mature T-cells. Among them, PTCL-not otherwise specified (PTCL-NOS) is a diagnosis of exclusion, comprising the largest fraction of PTCL with a diverse underlying pathogenesis. Recently, the concept of nodal T-cell lymphomas with T-follicular helper (TFH) phenotype, including angioimmunoblastic T-cell lymphoma (AITL) and PTCL-NOS that manifests a TFH phenotype, has been proposed, a distinguishing feature of which is the high frequency of TET2, IDH2, DNMT3A, and RHOA(G17V) mutations. Although recent large-scale genetic studies have uncovered mutational landscapes of several other subtypes of PTCLs, such as cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma (ATL), the entire picture of somatic alterations in PTCL-NOS still remains elusive. In addition, their similarities and differences among various histological subtypes in PTCLs have not been fully elucidated. To address this issue, we initially analyzed our and publicly available whole-exome/genome as well as transcriptome sequencing data from PTCL-NOS and other related PTCLs. Then, we carried out an extensive investigation of somatic mutations and structural variations (SVs) in PTCL-NOS using targeted-capture sequencing of 118 PTCL-NOS samples. Consistent with previous reports, TET2 (35%) was the most frequently mutated gene in PTCL-NOS with the majority (78%) affected by multiple mutations, followed by RHOA (25%), TP53 (16%), KMT2C (12%), PLCG1 (12%), and HLA-B (11%). Besides them, a considerable proportion of patients harbored mutations in components of T-cell receptor (TCR) /NF-κB pathway (such as PRKCB, CARD11, IRF4, and PRDM1), other signal transduction molecules (STAT3, NOTCH1, and SOCS1), chemokine receptors (CCR4 and CCR7), epigenetic modifiers (CREBBP, KDM6A, IDH2, and DNMT3A), transcriptional regulators (GATA3 and TBL1XR1), and molecules associated with immune evasion (HLA-A, HLA-B, FAS, B2M, and CD58). In addition to deteriorating SVs involving frequently affected genes (TP53, FAS, GATA3, and TBL1XR1), we discovered several genes almost exclusively affected by SVs, including TP73, IKZF2, and NFKB2, and CD274. Novel targets of recurrent mutation were also identified, including PDCD1, YTHDF2, and LRP1B, which were frequently targeted by nonsense and frameshift mutations distributed throughout the entire genes. Among them, PDCD1encodes PD-1 receptor transmitting an inhibitory signal from PD-L1 and PD-L2 ligands in T cells, and its loss of function seems to enable tumor cells to escape from the suppression by this negative signal. Although the roles of YTHDF2, a reader protein of N6-methyladenosine, and LRP1B, a member of the low density lipoprotein receptor family, in T cells are not immediately apparent, these findings shed light on a new biological function of these genes. Next, we investigated the co-existence relationship between frequently altered genes in PTCL-NOS. Interestingly, mutations characteristic of TFH lymphomas (TET2, RHOA, IDH2, and DNMT3A) tended to co-occur in a subset of PTCL-NOS cases, whereas they were almost mutually exclusive with mutations in TP53 and TCR/NF-κB pathway genes. This observation reveals the molecular distinction between TFH and non-TFH lymphomas in PTCL-NOS: the former is similar to AITL, although TET2 mutations did not show higher allelic burden than RHOA and IDH2mutations. In contrast, the latter is at least partly characterized by the genetic alterations shared with ATL. In summary, our findings illuminate the landscape of somatic alterations in PTCL-NOS and provide a novel insight into their genetic and molecular heterogeneity, which would help us to exploit a new therapeutic strategy to combat this disease. Disclosures Ohshima: CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria.

scholarly journals Immune cell topography predicts response to PD-1 blockade in cutaneous T cell lymphoma

2021 ◽  
Author(s):  
Garry Nolan ◽  
Darci Phillips ◽  
Magdalena Matusiak ◽  
Belén Gutierrez ◽  
Salil Bhate ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Clinical Response ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
Post Treatment ◽  
T Cell Lymphoma ◽  
T Cell Subsets ◽  
Cutaneous T Cell Lymphoma

Abstract Anti-PD-1 immunotherapies have transformed cancer treatment, yet the determinants of clinical response are largely unknown. We performed CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced cutaneous T cell lymphoma (CTCL) patients enrolled in a clinical trial of pembrolizumab therapy. Clinical response was not associated with the frequency of tumor-infiltrating T cell subsets, but rather with striking differences in the spatial organization and functional immune state of the tumor microenvironment (TME). After treatment, pembrolizumab responders had a localized enrichment of tumor and CD4+ T cells, which coincided with immune activation and cytotoxic PD-1+ CD4+ T cells. In contrast, non-responders had a localized enrichment of Tregs pre- and post-treatment, consistent with a persistently immunosuppressed TME and exhausted PD-1+ CD4+ T cells. Integrating these findings by computing the physical distances between PD-1+ CD4+ T cells, tumor cells, and Tregs revealed a spatial biomarker predictive of pembrolizumab response. Finally, the chemokine CXCL13 was upregulated in tumor cells in responders post-treatment, suggesting that chemoattraction of PD-1+ CD4+ T cells towards tumor cells facilitates a positive outcome. Together, these data show that T cell topography reflects the balance of effector and suppressive activity within the TME and predicts clinical response to PD-1 blockade in CTCL.

scholarly journals Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2486-2492
Author(s):  
Y Fukunaga ◽  
SS Evans ◽  
M Yamamoto ◽  
Y Ueda ◽  
K Tamura ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
T Cell Subsets ◽  
Cutaneous T Cell Lymphoma ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Adult T Cell Leukemia

Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP- 40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5′ nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5′NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5′NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL.

Hypermethylation of Bcl6 Is a Potential Cause of Development of Lymphoma with Tfh Features in Tet2 Knockdown Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2490-2490 ◽  
Author(s):  
Hideharu Muto ◽  
Mamiko Sakata-Yanagimoto ◽  
Yasuyuki Miyake ◽  
Genta Nagae ◽  
Terukazu Enami ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
Mrna Level ◽  
T Cell Lymphoma ◽  
Lymphoma Cells ◽  
Cpg Sites ◽  
Intron 1 ◽  
Bcl6 Expression

Abstract Background TET2 is known as an enzyme which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss-of-function mutations in TET2 are frequent in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Normal counterpart of AITL is thought to be follicular helper T cells (Tfh). PTCL-NOS is likely to consist of heterogeneous groups. Some of the PTCL-NOS cases also have features of Tfh. In the last annual meeting, we reported that aged homozygous Tet2 gene-trap mice (Tet2gt/gt), which showed 80% reduction in the Tet2 mRNA level in various hematopoietic cells, developed T-cell lymphoma. To further investigate the mechanism of T-lymphomagenesis, we analyzed methylome, hydroxymethylome, and transcriptome in the lymphoma cells. Material and Method Tet2 gt mice have a trapping vector inserted into the second intron of the Tet2 locus. In all the analyses, we used CD4+ T cells prepared from lymphoma cells developed in these mice, as well as CD4+ T cells prepared from spleen of wild-type mice as a control. To investigate genome-wide methylation and hydroxymethylation statuses, we performed MeDIP and hMeDIP sequencing, and bisulfite sequencing. To examine comprehensive gene expression, we performed microarray-based analysis, followed by Gene Set Enrichment Analysis (GSEA). Result After observation over a year (median, 67 weeks), 5 out of 7 Tet2gt/gt mice developed T-cell lymphomas with Tfh-like immunostaining pattern. No differences were found in the average levels of 5mC between lymphoma and control CD4+ cells throughout the regions around transcription start sites (TSS) +/- 5 kb. In contrast, when the same regions were analyzed for 5hmC levels, those in the lymphoma cells were significantly lower at the regions around TSS +/- 1 kb. When focused on regions having high 5mC contents (MACS score>5.0), lymphoma cells demonstrated a significant enrichment at regions around TSS +/- 1 kb, intragenic regions, and CpG islands (p=0.013, 0.006, 0.022 respectively). On the other hand, 5hmC was significantly decreased at regions around TSS +/- 1 kb in lymphoma cells than control cells (p=0.018). In a set of genes whose expression was higher in lymphoma cells than control cells, 5hmC levels were significantly lower in lymphoma cells. GSEA analysis revealed upregulation of Tfh-associated genes such as Bcl6 and cMaf (FDR q value=0.0004), key transcription factors for Tfh differentiation, in lymphoma cells compared with control cells. The expression of upregulated Tfh-associated genes was validated by real-time PCR. We focused on the epigenetic change of Bcl6 because it is among the most important transcription factors for Tfh development. It was reported that hypermethylation at intron 1 of Bcl6 upregulated its transcriptional activity in B cell lymphomas. Bisulfite sequencing revealed that the CpG sites in the intron 1 of Bcl6 were massively methylated/hydroxymathylated in lymphoma cells, whereas those in control cells were mostly at an unmodified status. MeDIP sequencing indicated that intron 1 of Bcl6 had more methylated status in lymphoma cells than control cells. We also found that CpG sites in the same region were densely methylated/hydroxymathylated in EL4, mouse T-cell lymphoma cell line, and that the decitabine treatment converted them into unmodified CpG along with the decrease in the Bcl6 expression levels. Discussion and Conclusion Tet2 gt/gt mice developed T-cell lymphoma with both Tfh-like immunohistological character and gene expression pattern. We found distinct changes in methylome, hydroxymethylome, and transcriptome. We also found a tight linkage between the increased methylation of intron 1 of Bcl6 and increased expression of its mRNA level in lymphoma cells developed in Tet2 knockdown mice. The same scenario is indicated in a T-cell lymphoma cell line. These observations imply that, in normal CD4+ T cells, reduced Tet2 function might increase the methylation status of the CpG sites in intron 1 of Bcl6, which may result in upregulation of Bcl6 expression and deviated Tfh generation. These processes might be an initiating event for the development of T-cell lymphoma with the Tfh features. Because of the long latency before lymphoma development in Tet2gt/gtmice, it is likely that additional hits are necessary. Disclosures: No relevant conflicts of interest to declare.

Peripheral blood sCD3−CD4+T cells: a useful diagnostic tool in angioimmunoblastic T cell lymphoma

2013 ◽  
Vol 32 (1) ◽  
pp. 16-21 ◽  
Author(s):  
Anju Singh ◽  
Richard Schabath ◽  
Richard Ratei ◽  
Andrea Stroux ◽  
Claus-Detlev Klemke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Diagnostic Tool ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Angioimmunoblastic T Cell Lymphoma

scholarly journals Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma

Blood ◽  
1989 ◽  
Vol 74 (7) ◽  
pp. 2486-2492 ◽  
Author(s):  
Y Fukunaga ◽  
SS Evans ◽  
M Yamamoto ◽  
Y Ueda ◽  
K Tamura ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
T Cell Subsets ◽  
Cutaneous T Cell Lymphoma ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Adult T Cell Leukemia

Abstract Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP- 40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5′ nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5′NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5′NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL.

scholarly journals Expression of Vav1-Myo1F Fusion Affects T-Cell Differentiation and Induces T-Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Jose Rodriguez Cortes ◽  
Robert Albero Gallego ◽  
Ioan Filip ◽  
Juan Angel Patino ◽  
Anisha Cooke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Gene Fusions ◽  
Cell Specification ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T cell lymphomas not otherwise specified (PTCL, NOS) account for &gt;45% diagnoses, show limited response to intensified chemotherapy treatment and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knockin mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with up-regulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T-cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histological examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Disclosures Palomero: Kura Onclology: Research Funding.

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