Correlation of aberrant proliferation with T-cell growth factor in adult T-cell leukemia cells

Peripheral blood lymphocytes from seven patients with adult T-cell leukemia (ATL) were found to lack PHA-responsiveness. However, in most of the cases, minute but distinct proliferation could be induced and maintained by human spleen cell conditioned medium containing PHA or by a combination of PHA and conditioned medium of gibbon cell line, MLA-144 (MLA-144 CM). These results indicate that the lack of response to mitogens of ATL cells might be attributed not only to the failure of these cells to produce T-cell growth factor (TCGF) upon activation, but also to their poor responsiveness to TCGF. Furthermore, a direct proliferative response to mitogen-free MLA-144 CM was shown in two out of seven patients; these two patients experienced rapidly progressive clinical courses. This observation raises the possibility that TCGF promotes the growth of ATL cells in vivo, and is related to the clinical course of the disease.     

Adult T-cell leukemia cells expressing OKT 17 antigen in the activated state

The surface phenotype and the functional activities of leukemic cells from seven patients with adult T-cell leukemia (ATL) were studied using monoclonal antibodies OKT 3, 4 and 8, anti-Tac, and OKT 17. The latter defines the heterogeneity of the activated T4+ T cell subset. In all cases, ATL cells with the typical OKT3-T4+ T8- phenotype expressed OKT 17 antigen. In addition, in five out of the seven cases the fresh ATL cells possessed Tac antigen which is expressed on activated T cells in varying degree. After cultivation with PWM, most populations of ATL cells acquired Tac even in the cases expressing little antigen in uncultured preparations. However the PWM activated ATL cells did not lose OKT 17 antigen. Functional assays showed the suppressor activity of ATL cells on normal B cell differentiation in three out of six cases examined. These results suggest that ATL cells most probably arise from a particular subset characterized by OKT 17 antigen within the activated OKT4+ T cell subset.     

Arginine deiminase enhances dexamethasone-induced cytotoxicity in human T-lymphoblastic leukemia CCRF-CEM cells

Since arginine deiminase (ADI; EC 3.5.3.6) inhibits cell proliferation by arresting cells in the G1 phase, we tested its synergistic effect on cell death induced by dexamethasone (DEX), which also induces apoptosis by G1 cell cycle arrest. ADI inhibited cell proliferation and induced apoptosis in human leukemic CEM cells in a dose-dependent manner. Simultaneous treatment with ADI and DEX showed synergistic effects on DNA fragmentation and LDH release. In addition, ADI exerted its anti-proliferative activity against DEX-resistant CEM cells. ADI suppressed expression of c-myc, a potential key regulator of cell proliferation and apoptosis, and increased expression of p27Kip1 cyclin-dependent kinase inhibitor. These results suggest that ADI efficiently increases the anti-cancer effect of DEX on human leukemic CEM cells through G1 cell cycle arrest involving downregulation of c-myc and upregulation of p27Kip1.     

Clinical features of OKT4+/OKT8+ adult T-cell leukemia

Adult T-cell leukemia (ATL) has a range of clinical characteristics. Phenotypically the leukemic cells usually express the helper/inducer associated antigen OKT4 with lack of OKT8. We have observed three patients with acute ATL cytologically indistinguishable from OKT4+/OKT8- ATL but whose neoplastic cells had the unusual phenotype, OKT3+, OKT4+, OKT6-, OKT8+ OKT9+/-, OKT11+, Tac+/-, TdT-. All patients had abnormal karyotypes and antibodies against anti-ATL associated antigens as well as proviral DNA of human T-cell leukemia virus in the leukemic cells. The clinical course was complicated by skin eruptions, hypercalcemia, pulmonary infection and disseminated intravascular coagulopathy. All died of complications shortly after diagnosis. The clinical features of these patients were similar to those of OKT4+/OKT8- ATL. However, their acute course suggests that co-expression surface antigens OKT4 and OKT8 may be a sign of aggressive nature of the disease with poor prognosis.     

PD-1 在急性T淋巴细胞白血病细胞中的表达及其临床意义

[摘要] 目的：探讨PD-1 分子在急性T淋巴细胞白血病（T-ALL）患者来源的肿瘤细胞（T-ALL细胞）中的表达及其临床意义。方法：选用2015 年12 月江苏省中医院血液科提供的1 例T-ALL细胞、4 例健康志愿者提供的PBMC和博生吉医药科技（苏州）有限公司提供的人293T/PD-1 细胞,将T-ALL细胞经尾静脉注射到B-NDG小鼠构建T-ALL细胞异种移植瘤模型,用流式细胞术检测移植瘤小鼠脾中获得的细胞是否主要是由T-ALL细胞组成。用流式细胞术检测T-ALL细胞中PD-1 蛋白的表达,用RT-PCR进一步验证T-ALL细胞中PD-1 mRNA表达水平。对T-ALL细胞中PD-1 基因进行SNP测序,以检测PD-1 基因DNA序列是否发生改变。在体外使用PD-1 抑制剂研究其对T-ALL细胞增殖、凋亡以及相关因子mRNA表达水平的影响。结果：成功构建小鼠TALL细胞异种移植瘤模型,用流式细胞术确认了该例疾病是T-ALL。T-ALL 细胞中PD-1 mRNA和蛋白均高表达（均P<0.01）。PD-1 基因的第5 个外显子的一个碱基由胞嘧啶突变成胸腺嘧啶。在体外PD-1 抑制剂对T-ALL细胞的增殖和凋亡均无明显影响;PD-1 抑制剂上调抑癌蛋白IGFBP3 mRNA表达,降低促癌蛋白SULT1A3 mRNA表达（均P<0.01）。结论：PD-1 在T-ALL细胞中高表达,PD-1可作为临床上T-ALL诊断及治疗的靶点。     

大颗粒T淋巴细胞白血病误诊1例报告

大颗粒淋巴细胞(LGL)占外周血有核细胞的10%~15%,其中T淋巴细胞占85%,自然杀伤细胞(NK)占15%.而大颗粒淋巴细胞白血病1985年首次被描述,是一种涉及血液、骨髓、脾脏的克隆性疾病[1].1993年Lubomir Sokolet根据其免疫表型将其分为T、NK细胞型[2].尽管大颗粒T细胞淋巴细胞白血病(T-cell large granular lymphocytic leukemia,T-LGLL)有粒细胞缺乏及贫血等症状,但无临床特异性,且疾病发展较慢,呈潜隐过程,常致漏诊和误诊.现将我科收治的1例以初诊时误诊为粒细胞缺乏症的大颗粒T淋巴细胞白血病的诊疗过程报道如下.     

Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.     

成人早期前体T急性淋巴细胞白血病（ETP-ALL）与非ETP-ALL的临床特点对比

目的:对比分析成人早期前体T急性淋巴细胞白血病（ETP-ALL）与非早期前体T急性淋巴细胞白血病（non-ETP-ALL）的临床特点。方法:回顾性分析于我科系统诊治的成人T细胞急性淋巴细胞白血病（T-ALL）患者19例,其中ETP-ALL 6例,non-ETP-ALL 13例,对比两组患者临床基本状况、血液及骨髓检测结果、免疫分型结果及诱导治疗后缓解情况。结果:ETP-ALL组患者白细胞水平显著低于non-ETP-ALL组患者,血小板水平显著高于non-ETP-AL组患者,主要见于pro-T-ALL,首次诱导治疗后完全缓解或接近完全缓解（CR/CRi）率显著低于后者。结论:ETP-ALL患者具有较独特的临床特点,对常规诱导治疗反应差,有必要积极探索新的治疗方法和药物。     

T-细胞幼淋巴细胞白血病治疗的研究进展

幼淋巴细胞白血病（prolymphocytic leukemia,PLL）是一种罕见但恶性程度极高的成熟淋巴肿瘤,其中T-细胞幼淋巴细胞白血病（T-cell prolymphocytic leukemia,T-PLL）这种亚型被认为难以治愈,临床治疗目的主要是提高治疗有效率和延长缓解期。随着对白血病发生发展机制研究的不断深入,目前T-PLL的治疗手段取得了较大的进步,其中表观遗传学的干预治疗为治愈TPLL提供了新的研究方向,本文就T-PLL的治疗研究进展进行综述。      

Dissociation of interleukin-2-mediated cell proliferation and interleukin-2 receptor upregulation in adult T-cell leukemia cells

We studied cell proliferation and interleukin-2 (IL-2) receptor upregulation mediated by exogenous IL-2 in leukemic cells from adult T-cell leukemia (ATL) patients to characterize some aspects of abnormal IL-2 receptor expression in ATL. Leukemic cells from 7 ATL patients examined showed no or very poor proliferative response to IL-2 though they expressed IL-2 receptors without any stimulation. In contrast, ATL leukemic cells cultured with IL-2 for 2 days expressed about twice as many IL-2 receptors as those of cells cultured without IL-2. Thus, in ATL leukemic cells, there seems to be a dissociation between the signal(s) for two different effects mediated by IL-2, cell proliferation and IL-2 receptor upregulation.     

Role of microRNA-143 in Fas-mediated apoptosis in human T-cell leukemia Jurkat cells

Treatment of Jurkat T cells with Fas-activating antibody (CH-11) facilitated rapid cell death that was shown to be caspase-dependent apoptosis. The expression of miR-143 was up-regulated during the apoptosis with time. The increased expression of miR-143 emerged from 1 to 2 h after the treatment, at which time the caspases-8 and -3 were also activated; and this increase was almost canceled by the pretreatment with an inhibitor of caspase-3 or -8. Furthermore, the transfection of Jurkat cells with mature miR-143 induced a significant growth suppression and enhancement of CH-11-induced apoptosis. On the contrary, an extracellular signal-regulated protein kinase 5 (ERK5), which was determined to be a target of miR-143 in colon cancer DLD-1 cells, was time-dependently down-regulated at the translational level after the treatment. During the apoptosis, the expression level of FasL was maintained and the level of nuclear-Foxo3a was increased in the early phase. These data suggest that the up-regulation of miR-143 could be related to the apoptosis in part by targeting ERK5, which leads to promotion of Foxo3a/FasL positive feedback loop.     

RNA interference-mediated silencing of NANOG leads to reduced proliferation and self-renewal,cell cycle arrest and apoptosis in T-cell acute lymphoblastic leukemia cells via the p53 signaling pathway

NANOG is critical for maintaining the self-renewal and proliferative properties of embryonic stem cells. Here we found that cultured T-cell acute lymphoblastic leukemia (T-ALL) cells, as well as human primary T-ALL cells, express a functional variant of NANOG. NANOG mRNA is derived predominantly from a retrogene locus termed NANOGP8. Furthermore, we showed that RNA interference-mediated NANOG knockdown inhibited cell proliferation, reduced self-renewal, promoted apoptosis and arrested the cell cycle through a p53-mediated pathway in leukemic cells. These findings demonstrate the oncogenic potential of this pluripotent gene in human T-ALL cells.     

Comparison of the performance of anti-CD7 and anti-CD38 bispecific antibodies and immunotoxins for the delivery of saporin to a human T-cell acute lymphoblastic leukemia cell line

We have investigated the cytotoxic performance of two different anti-CD7/anti-saporin BsAb's (HB2 × DB7-18 and Q1.1), three anti-CD38/anti-saporin BsAb's (OKT10 × RabSap, OKT10 × DB7-18 and Q4.1) and an anti-CD7 (HB2-Sap) and anti-CD38-saporin (OKT10-Sap) immunotoxin for delivering the ribosome inactivating protein (rip) to the human T-cell acute lymphoblastic leukemia cell line HSB-2. In the case of CD7 as target molecule the immunotoxin outperformed both anti-CD7 BsAb's being six times more effective than HB2 × DB7-18 and 98 times more so than Q1.1 at effectively inhibiting protein synthesis in a dose dependent manner. The chemically constructed HB2 × DB7-18 BsAb was more effective at inhibiting protein synthesis and cell growth in target HSB-2 cells in a dose dependent manner than the quadroma produced BsAb Q1.1. Both BsAb demonstrated a prozone effect used at concentrations above 0.1 nM though this was more pronounced for Q1.1 than for HB2 × DB7-18. The prozone effect was partially though not completely reversed by increasing the concentration of saporin in the system. In the case of CD38 as target molecule the anti-CD38 IT OKT10-Sap performed poorly, never actually achieving its IC₅₀. Two BsAb's constructed with monoclonal anti-saporin Fab arms each recognizing a different epitope on the saporin molecule also performed poorly. IN contrast the BsAb OKT10 × RabSap constructed with Fab derived from a rabbit polyclonal anti-saporin antiserum performed in a dose dependent manner achieving its IC₅₀ at a concentration of 1.3 nM. This BsAb also exhibited a prozone effect. These results exemplify the importance of cross linking adjacent target molecules on the cell surface in order to achieve effective delivery of saporin to the cell interior.     

Adenosine deaminase in plasma of patients with adult T-cell leukemia (ATL): correlation between enzyme activity and the ATL subtypes

Adenosine deaminase (ADA) was assayed in plasma from 14 patients with adult T-cell leukemia (ATL) (eight with acute ATL and six with smoldering or chronic ATL), 20 male family members (ten were anti-ATLA antibody positive and the other ten negative), and 25 normal individuals. ADA activity was uniformly higher in plasma from patients with ATL than normal controls. This enzyme activity significantly increased in acute ATL in comparison to smoldering or chronic ATL. In families of ATL patients, no statistical difference in ADA activity between the anti-ATLA antibody-positive group and -negative group could be discerned. The enzyme activity in a patient with acute ATL, after a bone-marrow transplant, rapidly increased as leukemic cells increased in peripheral blood. These findings indicate that the levels of ADA activities in plasma from ATL patients reflect the condition of this disease. Thus, measurement of this enzyme activity offers a further parameter to distinguish subtypes of ATL, and is of prognostic and therapeutic value.     

Accessory role of autologous T lymphocytes and adherent cells for the in vitro proliferation of T-cell colony-forming cells from patients with T-cell acute lymphoblastic leukemia

Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies can proliferate in methylcellulose in the absence of added growth factors or mitogenic stimulation. Mononuclear cells (MNC) from 7 patients with T-cell acute lymphoblastic leukemia were separated into cells forming rosettes with sheep erythrocytes (E+) or not (E-). E- cells were further depleted by complement-mediated cytotoxicity with OKT3 monoclonal antibody (E-OKT3- cells). The study of their spontaneous T-cell colony-forming ability suggested that proliferation of T-CFC in the absence of added growth factors requires cellular cooperation because: (1) No colony growth was observed at low cell concentrations (up to 2 X 10(4) cells/ml) whereas at higher cell densities the number of colonies increased exponentially; (2) The plating efficiency from unfractionated MNC was higher than that from E-OKT3- or E+ cells. Irradiated autologous E+ cells enhanced the plating efficiency from blast-enriched cell fractions (E-OKT3-) when co-cultured either directly in methylcellulose or separately in a two layer assay (agar-methylcellulose), suggesting that their activity could be due to diffusible factors; (3) Adherent-cell depletion of MNC decreased colony formation. Autologous irradiated adherent cells were able to restore the plating efficiency from MNCA- cells when co-cultured directly in methylcellulose but not in separate layers; however, media conditioned by patients' A+ cells could enhance the colony growth from patients' MNCA- cells, indicating that their activity could also be mediated by constitutively released soluble factors.     

Urinary excretion of parathyroid hormone-related protein as a predictor of hypercalcemia in patients with adult T-cell leukemia.

Hypercalcemia with adult T-cell leukemia (ATL) is chiefly caused by an excessive production by tumor cells of parathyroid hormone-related protein (PTHrP). We have previously reported hypercalcemic patients with solid tumors to excrete a large amount of the C-terminal fragments of PTHrP (C-PTHrP) into their urine. To elucidate whether PTHrP production correlates with or predicts the development of hypercalcemia, we studied the urinary excretion of C-PTHrP in 36 ATL patients. The urinary excretion of C-PTHrP was in the normal range (< 0.40 nmol equivalent to PTHrP (109-141)/g creatinine) in HTLV-1-positive carriers (n 3), ATL patients in complete remission (n 2) and chronic type ATL patients (n 2). It was marginally increased in seven patients in partial remission, and gradually increased as the disease progressed. In 20 patients who died without or with hypercalcemia, it was increased to 1.98 +/- 0.69 (n 9) and 7.6 +/- 2.1 nmol/g creatinine (mean +/- SD, n 11, P < 0.01), respectively. Urinary C-PTHrP excretion was significantly correlated with serum calcium and LDH levels as well as with CD25-positive cells in the peripheral blood. In four patients whose urinary excretion had been serially determined, it increased prior to the development of hypercalcemia. The findings suggest the urinary excretion of C-PTHrP to be of use as a predictor of the development of hypercalcemia in ATL patients. In ATL patients whose urinary excretion of C-PTHrP is progressively increasing, the serum calcium concentration should be carefully monitored to prevent hypercalcemic crisis.     

成年人T细胞急性淋巴细胞白血病中Ikaros6表达及其临床意义

目的 检测成年T细胞急性淋巴细胞白血病（T-ALL）患者Ikaros6的表达,并探讨其临床意义.方法 提取成年T-ALL患者初发时骨髓单个核细胞的RNA,反转录为cDNA,采用反转录-聚合酶链反应（RT-PCR）扩增技术进行检测,测序进一步确定结果,并结合患者临床资料分析临床特点及其预后.结果 74例初发成年人T-ALL中,Ikaros6阳性率为21.6％（16例）,另外阳性患者较阴性患者更易发生髓外浸润（x2=4.084,P=0.043）,且外周血白细胞计数高（103.15×109/L比15.60×109/L）、贫血更严重（血红蛋白75.95 g/L比107.00 g/L）和血小板计数低（26.0×109/L比67.0×109/L）,但差异均无统计学意义（均P＞ 0.05）.Ikaros6阳性患者生存期短和具有高复发风险.结论 Ikaros6在成年人T-ALL中表达率较高,阳性患者具有外周血白细胞计数高和易出现髓外浸润等临床特点,预后差,具有高复发风险.Ikaros6可考虑作为成年人T-ALL分层治疗或预后判断的分子指标.     

HTLV-I infection of T and B cells of a patient with adult T-cell leukemia-lymphoma (ATLL) and transmission of HTLV-I from B cells to normal T cells

We analysed the DNA of different tissues of a patient (HS) with adult T-cell leukemia/lymphoma virus (HTLV-I). We detected viral sequences in fresh specimens from spleen, thymus, liver, skin and peripheral blood neoplastic lymphocytes. The pattern of HTLV-I intergration is identical in the leukemic cells and in all other tissues analysed, but the signal intensity is strongest in the leukemic cells, indicating the source of HTLV-I proviral sequences was the leukemic T-cells which had infiltrated these tissues. In fact, the cultured skin fibroblasts of the patient did not contain HTLV-I sequence. However, cultured lymphocytes of this patient was consistently an immortalized B-cell line containing HTLV-I sequences in a manner indicative of a polyclonal infection. This cell line was also infected with the Epstein-Barr virus (EBV). In order to determine whether HTLV-I alone was sufficient for B-cell immortalization, we obtained single cell clones by limiting dilution. The DNA of all the cell clones that we analysed contained both the HTLV-I and EBV genomes, suggesting that immortalization of the B-cell was more likely due to the EBV rather than HTLV-I. Infectious HTLV-I viruses produced by the B-cell line still had the propensity to infect and transform T-lymphocytes in normal human umbilical cord blood. Unlike the parental B cells, the transformed T lymphocytes were clonally selected. Our results indicate that although the predominant infected cell population of the patient was his leukemic T lymphocytes, some of his EBV-positive B-lymphocytes were also polyclonally infected. The latter had a growth advantage in culture over the T lymphocytes but the virus produced by these immortalized B cells has not been adapted and has maintained its tropism for T cells.     

Genetic profile of adult T-cell leukemia/lymphoma in Okinawa: Association with prognosis,ethnicity, and HTLV-1 strains

Genetic alterations in adult T-cell leukemia/lymphoma (ATLL), a T-cell malignancy associated with HTLV-1, and their clinical impacts, especially from the perspective of viral strains, are not fully elucidated. We employed targeted next-generation sequencing and single nucleotide polymorphism array for 89 patients with ATLL in Okinawa, the southernmost islands in Japan, where the frequency of HTLV-1 tax subgroup-A (HTLV-1-taxA) is notably higher than that in mainland Japan, where most ATLL cases have HTLV-1-taxB, and compared the results with previously reported genomic landscapes of ATLL in mainland Japan and the USA. Okinawan patients exhibited similar mutation profiles to mainland Japanese patients, with frequent alterations in TCR/NF-ĸB (eg, PRKCB, PLCG1, and CARD11) and T-cell trafficking pathways (CCR4 and CCR7), in contrast with North American patients who exhibited a predominance of epigenome-associated gene mutations. Some mutations, especially GATA3 and RHOA, were detected more frequently in Okinawan patients than in mainland Japanese patients. Compared to HTLV-1-taxB, HTLV-1-taxA was significantly dominant in Okinawan patients with these mutations (GATA3, 34.1% vs 14.6%, P = .044; RHOA, 24.4% vs 6.3%, P = .032), suggesting the contribution of viral strains to these mutation frequencies. From a clinical viewpoint, we identified a significant negative impact of biallelic inactivation of PRDM1 (P = .027) in addition to the previously reported PRKCB mutations, indicating the importance of integrated genetic analysis. This study suggests that heterogeneous genetic abnormalities in ATLL depend on the viral strain as well as on the ethnic background. This warrants the need to develop therapeutic interventions considering regional characteristics.     

A simple and reliable method for the detection and quantitation of the human T-cell leukemia virus type-I provirus in peripheral blood mononuclear cells of seropositive blood donors.

Human T-cell leukemia virus type I (HTLV-I) antibody detection has been widely used to screen HTLV-I carriers. Sometimes, however, it gives false positive or negative results. A demonstration of the HTLV-I provirus from patients' peripheral blood mononuclear cells (PBMC) should, therefore, give the crucial evidence for them being HTLV-I carriers. We established a simple and reliable method using the polymerase chain reaction (PCR) to detect one molecule of HTLV-I provirus in 100 x 10(3) PBMC, during which internal control primers for the human beta-globin gene were also employed in the same reaction tube to check the success of the amplification reaction. We can thus easily avoid any false negative judgement and quantitate the HTLV-I provirus in PBMC simply by diluting the sample before PCR. One ml blood was enough for ten or more determinations by PCR. Analysis of seropositive blood from donors demonstrated a wide range for the number of HTLV-I provirus in PBMC. The method could conveniently be used for quantitating HTLV-I proviruses and following up HTLV-I carriers to study the pathophysiology and mode of HTLV-I transmission.