Acetaldehyde-induced mutation at the hprt locus in human lymphocytes in vitro

Acetaldehyde (Aa) induces chromosomal aberration and sister chromatid exchange in a variety of test systems, but has not previously been evaluated for its ability to induce gene mutation in mammalian cells. We have studied the mutagenic effect of Aa at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human lymphocytes in vitro by using the T-cell cloning technique and selection of mutant cell clones in medium containing thioguanine. Cells treated with 1.2-2.4 mM Aa for 24 hr or 0.2-0.6 mM Aa for 48 hr showed a dose-dependent decrease of cell survival and a 3- to 16-fold increase of the mutant frequency. The inverse relationship between cell survival and mutant frequency was linear down to a relative survival of 15%, and showed a similar slope in the 24-hr and 48-hr treatment experiments. Forty-one mutant T-cell clones derived from cultures treated with 1.2 or 2.4 mM Aa and 15 from untreated controls were expanded for DNA extraction and Southern blot analysis to study deletion mutation using a full length hprt cDNA probe, and clonal identity on the basis of T-cell receptor rearrangements. In the culture with a 16-fold increase of mutant frequency, 4 out of 10 independent mutants (40%) showed partial deletions extending beyond the 3' coding sequences of the hprt gene. Two of 22 independent mutants derived from the other treated cultures with at most a 6-fold increase of mutant frequency, and 1 of 11 independent control clones showed rearrangement of the hprt gene, none of which affected the 3'-end of the hprt gene. These results show that Aa is capable of inducing gene mutation at the hprt locus in human cells, and suggest that deletion mutation affecting the 3'-end of the gene may be a major type of Aa-induced mutation of this locus.     

Diagnostic and prognostic significance of clonal T-cell receptor beta gene rearrangements in lymph nodes of patients with mycosis fungoides

In this study, 25 involved and uninvolved lymph nodes from 22 patients with mycosis fungoides (MF) and seven dermatopathic lymph nodes from patients with benign skin disorders were studied for the presence of clonal T-cell receptor beta (TCRβ) gene rearrangements by Southern blot analysis. These results were correlated with the histological classification, follow-up data, and survival. The results of the histological classification and Southern blot analysis were concordant in 26 of 32 cases. Clonal TCRβ gene rearrangements were found in all six MF lymph nodes showing (partial) effacement of the normal lymph node architecture, but in none of the eight uninvolved dermatopathic MF lymph nodes and in none of the seven dermatopathic control lymph nodes. In addition, in 5 of 11 dermatopathic MF lymph nodes that were considered to have early involvement by MF at histological examination, clonal TCRβ gene rearrangements were detected. In the group of MF patients with dermatopathic lymphadenopathy, patients with detectable clonal T-cell populations had a significantly shorter survival than patients without such a population (P<0.01). The results of this study indicate that within the group of dermatopathic MF lymph nodes, prognostically different groups can be distinguished and that TCRβ gene rearrangement analysis may be an important adjunct in the early diagnosis of lymph node involvement by MF.     

T细胞淋巴瘤伴黏液间质1例及文献复习

目的 报道1例罕见的臀部伴黏液间质的T细胞淋巴瘤并文献复习,探讨其诊断要点。方法 应用组织学、免疫组化、基因重排检测技术进行诊断与鉴别诊断。结果经多项免疫组化排除了软组织肿瘤（纤维、平滑肌、横纹肌、脂肪、滑膜肉瘤及肌纤维母细胞瘤）及恶性黑色素瘤等以后,进行了淋巴瘤标记：CD45、CD45RO、CD3、CD20、CD79α、CD68、和TCRγ基因重排检测。显示CD45、CD45RO、CD3阳性,TCRγ克隆性基因重排。结论 本例与文献报道4例资料提示：伴黏液间质的非霍奇金淋巴瘤好发于淋巴结外软组织,由弥漫性淋巴瘤样片状区与黏液间质区组成,必需应用免疫组化排除间叶源性肿瘤,加用淋巴瘤标记和基因重排检测阳性确诊。     

分化早期小儿急性白血病细胞免疫球蛋白和T细胞受体基因结构的变化

从29例免疫分型的小儿急性白血病中选出髓过氧化物酶染色阴性和不与细胞系相关或成熟细胞系相关的单克隆抗体反应的6例白血病细胞,进一步作免疫球蛋白(包括重链和κ轻链)和T细胞受体(包括δ、γ、β)基因结构的分析。所有6例均有IgH基因的重排,提示这些白血病细胞是β细胞来源。两例CD_(10)阴性和白病细胞.κ链基因没有重排,其中一例的TCRδ、γ、β基因也都处于胚系,另一例的TCRβ处于胚系。在CD_(10)阳性白血病细胞中,其中两例κ链基因丢失,TCR基因结构都有不同程度的变化(重排或丢失)。这些结果可能提示在CD_(10)阳性白血病细胞中能产生功能性IgH的重排,并可导致IgL和TCR基因结构的变化。     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

Development and selection of γδ T cells

Summary:  Two main lineages of T cells develop in the thymus: those that express the αβ T-cell receptor (TCR) and those that express the γδ TCR. Whereas the development, selection, and peripheral localization of newly differentiated αβ T cells are understood in some detail, these processes are less well characterized in γδ T cells. This review describes research carried out in this laboratory and others, which addresses several key aspects of γδ T-cell development, including the decision of precursor cells to differentiate into the γδ versus αβ lineage, the ordered differentiation over the course of ontogeny of functional γδ T-cell subsets expressing distinct TCR structures, programming of ordered Vγ gene rearrangement in the thymus, including a molecular switch that ensures appropriate Vγ rearrangements at the appropriate stage of development, positive selection in the thymus of γδ T cells destined for the epidermis, and the acquisition by developing γδ T cells of cues that determine their correct localization in the periphery. This research suggests a coordination of molecularly programmed events and cellular selection, which enables specialization of the thymus for production of distinct T-cell subsets at different stages of development.     

Molecular analyses of in vivo hprt mutant T cells from atomic bomb survivors

In vivo-derived hprt-deficient mutant T cells isolated from three nonirradiated controls and two atomic bomb survivors were studied by Southern blot analysis to investigate the molecular spectra of the mutations. Mutant frequencies for the three controls were 1.8, 2.3, and 7.3 x 10(-6), and those for the two survivors (who had received radiation doses of 2.46 and 2.15 Gy, based upon the revised atomic bomb shielded kerma estimates) were 9.3 and 14.4 x 10(-6), respectively. Fourteen (13%) of 105 mutant T-cell colonies from the controls showed various structural changes in the hprt gene. The frequency of mutants with hprt gene structural changes in one atomic bomb survivor, who exhibited a mutant frequency of 9.3 x 10(-6), was 26% (16/61), which was significantly higher than that of the controls. However, the frequency of structural changes in the other survivor (14%, 8/59) was not higher than that of the controls. Two sets of mutants (in total, eight mutants) from the survivor, who showed a significantly higher frequency of mutants with hprt gross alterations than did the controls, had the same hprt changes and the same rearrangements of T-cell receptor (TcR) beta- and gamma-chain genes, indicating a clonal expansion from one progenitor mutant. This phenomenon may reflect an in vivo recovery process of T cells in the periphery after exposure to atomic bomb radiation. However, when comparing the frequency of mutations, these two sets of mutants should be reduced. After reducing the total number of mutants from the number of gross hprt changes, the frequency was not significantly higher than that of the controls.     

Intestinal T-cell lymphoma: a reassessment of cytomorphological and phenotypic features in relation to patterns of small bowel remodelling

Intestinal T-cell lymphoma (ITCL) is an uncommon entity among primary gastrointestinal lymphomas. In this study we evaluated tumours from 20 patients presenting with (n=8) or without (n=12) a history of coeliac disease (CD). Neoplastic lesions were composed of predominantly small (n=4), small-to-medium (n=2), medium/mixed-to-large (n=7) or large and anaplastic (n=7) cells. Different patterns of tumour growth and remodelling of the small bowel wall were observed. Pattern a (n=4) was characterized by an intramucosal spread of small tumour cells with a small growth fraction. This pattern resembles mucosal inflammation in CD. In pattern b (n=2), ulcerated solitary or multiple tumours composed of small to medium-sized cells were observed. The adjacent or distant mucosa showed a nearly normal architecture. In pattern c (n=7), ulcerated lesions were composed of medium-sized to large cells. Mucosal flattening occurred in all segments infiltrated by lymphoma. In pattern d (n=7), bowel remodelling was observed along the small intestine even at sites not affected by lymphoma. The main neoplastic lesions were composed of pleomorphic large or anaplastic cells frequently expressing the CD30 molecule. Intramucosal spread of a small epitheliotropic T-cell population was observed in the vicinity or even at distant segments of the small bowel. The demonstration of clonal rearrangements of T-cell receptor genes helped to trace widespread occurrence of this small intraepithelial neoplastic component. We suggest that different features of tumour cells such as the expression of activation antigens may contribute to the remodelling of small bowel mucosa. The addition of immunophenotyping data to macroscopic and microscopic features of specimens provided evidence that this uncommon lymphoma exhibits a spectrum in cytological composition and growth patterns. However, despite the considerable heterogeneity of the cases analysed, most of them shared a characteristic immunohistochemical profile (CD3⁺, CD8^+/–, CD103⁺), further substantiating the view that ITCL is the neoplastic equivalent of an intraepithelial T-cell subset of the small intestine. This phenotype and the intraepithelial accumulation of lymphoma cells observed in the surviving mucosa are clues to the diagnosis of this clinicopathological lymphoma entity characterized by a broad range of morphological expressions.     

Each of the two productive T cell receptor alpha-gene rearrangements found in both the A10 and BM 3.3 T cell clones give rise to an alpha chain which can contribute to the constitution of a surface-expressed alpha beta dimer.

The H-2 Kb specific cytotoxic T cell clone BM3.3 carries two productive TCR alpha gene rearrangements but expresses a single detectable TCR alpha beta chain combination on its surface. However, when separately analyzed by gene transfer, both productive alpha gene rearrangements are translated into alpha chains capable of pairing with the BM3.3 beta chain and of cell surface expression. Similar observations have been made with the products of the two productive alpha gene rearrangements previously identified in the A10 T cell clone. These observations contrast with those made for the two TCR alpha chains expressed in the MS202 T cell clone. In the latter instance, when analyzed by gene transfer, one of the alpha chains was unable to make a pair with the beta chain. Consequently, when considered with respect to the mechanisms of TCR allelic exclusion, our data suggest that the mere expression of a TCR alpha beta dimer on the surface of immature CD4+ CD8+ thymocytes may be necessary but not sufficient to shutdown further alpha gene rearrangements. Rather, the ability of a TCR alpha beta dimer to be positively selected may be needed to trigger the maturation of thymocytes to a stage devoid of recombinase activity. Alternatively, if non-dividing CD4+ CD8+ CD3+ small thymocytes do not experience secondary alpha-gene rearrangement, our data suggest that TCR alpha gene rearrangements are attempted quasi-simultaneously on both alpha alleles.     

The polymerase chain reaction in the diagnosis of early mycosis fungoides

The earliest or patch stage of mycosis fungoides may present diagnostic difficulty both clinically and pathologically. The present study of the polymerase chain reaction (PCR) as a diagnostic tool in early mycosis fungoides was therefore undertaken, using a rapid PCR method for the detection of γ- and β-chain T-cell receptor (TCR) gene rearrangements in routine formalin-fixed, paraffin-embedded histological sections. Forty-two biopsies were studied from 26 patients with mycosis fungoides. Twenty-three skin biopsies with a clinicopathological diagnosis of early, or patch stage, mycosis fungoides were investigated. Of these, 18 (78 per cent) showed TCR-γ or both β- and γ-chain TCR gene rearrangements. TCR gene rearrangements were shown in seven of the 14 plaque stage lesions (50 per cent) and also in the single case of tumour stage disease. Where gene rearrangements were identified, these were identical in all biopsies from an individual patient, irrespective of the site of the lesion, the disease stage, or the time lapse between the biopsies. The PCR is therefore a highly sensitive technique, which can be performed on routine pathological material, in cases where the diagnosis of early mycosis fungoides cannot be made with certainty on conventional histopathological and immunohistochemical grounds. © 1997 John Wiley & Sons, Ltd.     

TCR基因重排检测在T细胞淋巴瘤病理诊断中的作用

基因重排现象是每个淋巴细胞在早期经历的一个正常的过程,由于机体每个淋巴细胞的重排基因都是特有的,使每个淋巴细胞都有独特的重排形式。如若机体在某些致瘤因素的作用下失去对某个基因重排细胞的控制,则该细胞就会出现无控制的、单克隆性增生,最终导致淋巴瘤的形成。所以淋巴瘤所具有的是单克隆性重排。因此通过聚合酶链反应（polymerase chain reaction,PCR）技术进行T细胞抗原受体（T cell receptor,TCR）基因克隆性重排分析,尤其在常规HE形态学与免疫组化结果都不能确诊时,显得尤为重要。该文对其在T细胞淋巴瘤病理诊断中的作用作一综述。     

Bohemian T cell receptors: sketching the repertoires of unconventional lymphocytes

Over the last several decades, novel populations of unconventional T cells have been identified; defined by an invariant (or nearly invariant) T cell receptor (TCR) with a fixed specificity to non-canonical antigens and major histocompatibility (MHC) molecules, they form large, functionally monoclonal populations tasked with surveying for their specific antigens. With residence in both lymphoid and non-lymphoid tissues coupled with their ability to rapidly produce a spectrum of cytokines and effector molecules, the unconventional T cells are poised as some of the first responders to infection/damage and are thought to provide critical coverage before more focused, conventional T cell responses are mobilized. However, new technologies for the measurement and characterization of TCR repertoires have identified an underappreciated amount of TCR diversity in the unconventional T cells. In many cases, the specificities of these diverse TCRs converge on the same or similar antigens as their invariant counterparts, while others have yet to be defined. Here, we will review the current knowledge of the TCR repertoires of unconventional T cells and discuss how repertoires might be used as a framework for their organization, and further our understanding of their role not only during an immune response, but also their contribution in maintaining homeostasis.     

非特指外周T细胞淋巴瘤中PTEN的改变

目的观察非特指外周T细胞淋巴瘤(peripheral T cell lymphoma,not otherwise specified,PTCL-NOS)中抑癌基因(phos-phatase and tensin homolog deleted on chromosome ten,PTEN)的改变情况,探讨其与肿瘤生物学行为的关系,为阐明PTCL-NOS的发生、发展机制提供科学依据。方法应用间期双色荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测36例PTCL-NOS石蜡包埋组织中PTEN基因的改变情况,分析其改变与各临床参数的关系。结果 36例PTCL-NOS中8例出现PTEN杂合性缺失(loss of heterozygosity,LOH);Kaplan-Meier生存分析显示该基因异常组较正常组生存期明显缩短(P<0.05);PTEN基因改变与病理分期、发生部位、年龄、性别、乳酸脱氢酶(LDH)水平均无相关性(P>0.05)。结论 PTCL-NOS存在的抑癌基因PTEN杂合性缺失,在PTCL-NOS发生、发展中可能起重要作用,是评估该肿瘤预后的重要指标。     

急性单核细胞白血病病人外周血TCR Vα基因谱系和T细胞克隆性增殖特点

目的：了解急性单核细胞白血病（AML-M5）病人TCR Vα29个亚家族的分布及其克隆性。方法: 利用RT-PCR分别扩增8例M5病人外周血单核细胞的TCR Vα29个亚家族基因的CDR3。阳性的PCR产物进一步经荧光素标记和基因扫描分析产物的CDR3长度，了解T细胞克隆性。9例健康人作为对照。结果: RT-PCR分析显示正常人表达绝大多数Vα亚家族基因，而8例M5病人外周血仅可以检测到1-10个Vα亚家族基因，出现频率最高的是Vα3（6/8例，75%），其次为Vα12（5/8例，62.5%），有15个Vα亚家族表达缺失（Vα1、4、5、7、9、14-18、20、21、26、28和Vα29）。基因扫描分析显示：8例AML-M5病人中有6例存在克隆性增殖T细胞，其中，以Vα12亚家族出现克隆性增殖T细胞频率（3/5例）最高，有2例仅存在单一的克隆性增殖Vα3亚家族T细胞。正常人外周血各Vα亚家族T细胞主要呈多克隆性。结论: M5病人外周血Vα亚家族T细胞存在明显的选择性选用及克隆性增殖特点，这可能是机体T细胞受M5细胞刺激而引起机体产生相应的特异性免疫反应，同时T细胞TCR Vα亚家族分布及克隆性增殖情况具有个体特异性。     

正常人T细胞和胸腺细胞中多种新的TCRδRec基因重排

利用巢式或半巢式PCR扩增10例正常人外周血单个核细胞(PBMC)、4例分选CD3~+细胞和7例正常胸腺细胞DNA中TCR δRec区与Jδ1、Dδ3和Ja重排的基因片段,分析正常人外周血T细胞和胸腺细胞中TCR δRec基因重排情况。克隆性PCR产物进一步进行核苷酸序列分析确定其重排位置。结果发现了4种新的TCR δRec重排,包括TCR δRec_(149321)-Jδ1、TCRδRec_(149820)-Jδ1、TCR δRec_(151657)(Nx)-Ja和TCR δRec_(153199)-Jδ1等,其中以TCR δNx的重排最多见,通过利用不同模板DNA的PCR分析发现δRec重排在外周血和胸腺细胞中有所不同。结果显示TCR δNx-Jδ1重排在成熟和不成熟T细胞发生频率均较高,而TCR δNx-Dδ3重排在不成熟T细胞中发生率较高。但所有重排均不表达于mRNA中。本研究结果为TCR δ基因重排的研究补充了一些新的数据。     

Variation in the histological pattern of nodal involvement by gamma/delta T-cell lymphoma

AIMS: Gamma-delta (gammadelta) T-cell non-Hodgkin's lymphomas (NHLs) usually present with liver, spleen and marrow infiltration. Lymph node involvement by gammadelta T-cell NHL has been rarely documented so far; its histological pattern needs to be further defined. METHODS AND RESULTS: Two cases of nodal gammadelta T-cell NHL are reported: case 1, a 44-year-old man, presented with cytomegalovirus retinitis and superficial lymphadenopathies. Histological analysis of an inguinal lymph node showed complete destruction by a diffuse pleomorphic lymphoid proliferation, which was positive for CD2, CD3, CD43, CD45, TIA-1 and granzyme B, and displayed a gammadelta phenotype (deltaTCR1+, Vdelta1+, Vdelta2-, Vdelta3-, betaF1-). Bone marrow was normal. Case 2, a male 24-year-old patient with a history of renal transplantation, presented with hepatosplenomegaly and supraclavicular lymph node enlargement. Lymph node architecture was globally preserved. Peripheral sinuses contained scattered nests of medium-sized irregular lymphoid cells. Bone-marrow was infiltrated. Phenotype showed positivity for CD2, CD3, CD45 and TIA1 and expression of gammadelta TCR (deltaTCR1+, deltaV1+, deltaV2-, deltaV3-, betaF1-). Both patients died a short time after diagnosis. CONCLUSIONS: These observations suggest that at least two forms of nodal gammadelta T-cell NHL may be encountered: one mimicking classical alphabeta T-cell NHL, with diffuse pleomorphic cell proliferation, and one displaying sinusoidal neoplastic infiltration suggesting a close relationship with hepatosplenic gammadelta T-cell NHL.     

A helper T-cell antigen enhances generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro

A T-cell help for generation of hepatitis C virus-specific cytotoxic T lymphocytes was studied in three patients with chronic hepatitis C. In all three, human leukocyte antigen B44-restricted cytotoxic T lymphocytes recognizing an epitope in hepatitis C virus nucleocapsid protein residues 81–100 were generated from the peripheral blood lymphocytes by repeated stimulation with a synthetic hepatitis C virus nucleocapsid pep-tide. The proliferative response of peripheral blood lymphocytes to hepatitis C virus nucleocapsid protein residues 1–120 was observed in one patient, and was ascribed to CD4⁺ T cells. The helper T cells recognized a major epitope in residues 21–40 and a minor epitope(s) in residues 81–110. They produced interferon γ, but interleukin 4 was not detectable in the T-helper cell culture supernatants. The hepatitis C virus nucleocapsid protein residues 1–120 and the major helper T-cell epitope enhanced generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro, although the protein alone did not generate them. In the other two patients, the protein did not enhance generation of hepatitis C virus-specific cytotoxic T lymphocytes in vitro. The results suggest that a hepatitis C virus-specific helper T-cell epitope is helpful for inducing a strong specific cytotoxic T-lymphocyte response. © 1995 Wiley-Liss, Inc.