Semiquantitative and qualitative assessment of B-lymphocyte V H repertoire by a fluorescent multiplex PCR

We established a new tool to perform semiquantitative and qualitative screening for V(H) gene usage frequency during IgH rearrangements in human B-lymphocytes. In two separate multiplex PCRs, the rearranged VDJ regions were amplified with V(H) family-specific primers labeled with different fluorescent dyes (FAM, HEX, NED, or ROX). The relative amount of each of the particular V(H) family products and their ratios were determined by fragment analysis on a ABI PRISM 377 sequencer. We verified that the fluorescent multiplex PCR (FMPCR) shows high specificity and sensitivity, acceptable reproducibility and reliability. Data obtained were well in agreement with results revealed by sequencing following single-cell PCR. Ten healthy volunteers showed a comparable semiquantitative V(H) family distribution. The FMPCR also correctly detected a monoclonal peak in a CLL patient. Thus, labeling primers with various fluorescent dyes allows for an assessment of V(H) family usage and an immediate determination of the involved V(H) gene family if any clonal peaks are present. This method provides a quick, easy, and reliable tool for V(H) repertoire screening of larger populations of patients suffering from diseases with changes in the V(H) repertoire allowing for selection of cases worth a more detailed and cumbersome sequence analysis later on.     

The germline repertoire of T-cell receptor beta-chain genes in patients with multiple sclerosis

The T-cell receptor (TCR) beta-chain gene repertoire of 40 multiple sclerosis (MS) patients was compared to that of 100 normal individuals. V-beta probes that represent 14 different V-beta subfamilies plus a C-beta probe were used to identify 53 separate beta-chain gene segments. No duplication or deletion of any of these 53 gene segments was found in the MS patients. Restriction fragment length polymorphism (RFLP) alleles detected by V-beta 8, V-beta 11 and C-beta probes defined 8 different beta-chain haplotypes. The distribution of these haplotypes in Caucasian MS patients and normal individuals was significantly different (p = 0.012). Comparison of the DR2+ subset of MS patients (n = 32) to a second group of 43 Caucasian DR2+ normal individuals revealed that the distribution of these beta-chain haplotypes was significantly different in these two populations (p = 0.015). These results suggest that an MS susceptibility gene(s) may be located in the region of the TCR/beta-chain gene complex.     

Germline repertoire of T-cell receptor beta-chain genes in patients with insulin-dependent diabetes mellitus.

We have investigated the genotype and allelic distribution of germline restriction fragment length polymorphisms of the T-cell receptor beta chain, segment C beta, and two variable segments which are in linkage disequilibrium, V beta 8 and V beta 11, in 42 insulin-dependent diabetes mellitus (IDDM) patients and in 51 healthy blood donors used as controls. Recently, several works have reported contradictory results showing or not showing an association between polymorphic alleles of the C beta gene and diabetes type I. We found no significant differences in the allele, genotype, and haplotype distribution of the gene segments studied, between IDDM patients and control populations.     

A comparison of multiplex and monoplex T-cell receptor gamma PCR.

T-cell receptor gamma (TCRgamma) PCR is often used to detect clonal T-cell populations. Because TCRgamma contains a limited number of variable (Vgamma) and joining (Jgamma) regions, a small number of PCR primers can be used to assess T-cell clonality. The seven primers used in the current study were described previously and were split into 2 or 3 multiplex primer sets. In this study, a single 7-primer multiplex (7-plex) PCR reaction was compared with all 12 possible monoplex primer combinations on 18 samples previously analyzed for T-cell receptor rearrangements by TCRbeta Southern blot and/or TCRgamma PCR followed by temporal temperature gradient gel electrophoresis. Using fluorescent Vgamma-region primers, unlabeled Jgamma-region primers, and capillary electrophoresis, we show all TCRgamma rearrangements seen by 7-plex PCR on known positive samples were seen following monoplex PCR. However, additional TCRgamma gene rearrangements were seen in monoplex PCR reactions that were not seen in the 7-plex PCR reactions. Monoplex but not 7-plex PCR of known negative samples occasionally showed TCRgamma gene rearrangements, often with less frequently used Vgamma and Jgamma-region primers, and may have represented false positive results. In summary, the single 7-plex PCR reaction correctly identified specimens with TCRgamma clonal populations and represents an improvement over existing assays that use these same primers split into several smaller multiplex reactions. Monoplex PCR has no advantage over multiplex PCR and has the potential to lead to false positive results.     

Discrimination of T-cell subsets and T-cell receptor repertoire distribution

Flow cytometry-based analysis of T-cell receptor (TCR) repertoires is an essential tool for the detection of clonal T-cell expansions in physiologic and pathologic conditions. Individual T-cell subsets can be investigated based on their surface properties. The aims of our study were to provide reference values for various disease settings and delineate the contribution of individual TCR repertoires to the human T-cell differentiation pathway. We analyzed blood of 66 healthy subjects aged 0 (cord blood) to 72 years. Lymphocyte subpopulations and TCR repertoires were simultaneously explored using antibodies specific to CD3, CD4, CD8, CD45RA, CCR7, CD27, CD57 and a set of 25 antibodies detecting human TCR-Vβ chains. Statistical analysis included Wilcoxon, paired t and ANOVA tests. Initially, TCR expansion values were calculated based on the analysis of TCR-Vβ distribution on CD4+ and CD8+ T cells. We then established gating strategies and an algorithm for data analysis allowing for discrimination of T-cell subsets and TCR distribution. Dominant TCR expansions were present within effector as opposed to central/effector memory or naive cells, e.g., median TCR-Vβ expansion rate was highest on CD45RA+/CCR7? effector CD4+/8+ cells (eight and 11-fold, respectively). Remarkably, TCR expansions were missing (0) or very low (0.5) on CD4+ and CD8+ central memory population, respectively. No significant gender-related variability of TCR repertoires was identified, and significant impact of chronic cytomegalovirus infection was shown. Our results serve as reference for future studies elucidating clonal TCR dominance of T-cell subsets.     

Evolution of antibody and T-cell receptor V genes--the antibody repertoire might have evolved abruptly

Structural homology present between antibody genes and T-cell antigen receptor (TCR) genes include not only V genes but also D and J segments. This may indicate that the gene duplication occurred involving a large chromosomal region containing all these genes and segments. It is conceivable, however, that the TCR gene complex is the ancestor, as effective selective forces to diversity V gene sequences are provided by MHC polymorphism only for TCR V genes. Consequently, we argue that the antibody repertoire may have emerged rather abruptly in evolution.     

Thymic stromal cell specialization and the T-cell receptor repertoire

Ten years ago, we proposed a model for thymus function in which thymic epithelial cells are primarily responsible for imprinting major histocompatibility complex (MHC)-restricted specificity, and bone marrow-derived macrophages or dendritic cells are responsible for the induction of self-tolerance. Since then, transgenic and knockout models have allowed for a dissection of thymic stromal components in vivo, leading to a new understanding of their specialized functions. We have determined that with regard to class II-restricted CD4 T-cell development, two distinct subsets of thymic epithelium help shape the repertoire: Cortical epithelium appears solely responsible for positive selection, whereas a fucose-bearing subset of medullary epithelium is specialized for negative selection. This absolute separation of positive and negative selection into two distinct spatial and temporal compartments leads to a much simpler view of the process of repertoire selection. Finally, a novel view of the function of the thymic medulla is discussed.     

Proliferation of thymocytes in relation to T-cell receptor beta-chain expression.

During proliferation and differentiation of maturing thymocytes, T-cell receptor beta-chain products are first expressed in the cytoplasm. Only subsequently are they expressed on the cell surface, presumably as part of the alpha beta/CD3 receptor complex. This study uses double immunofluorescence labelling to identify these cytoplasmic and surface phases separately in relationship to cell-cycle parameters. The use of a mitotic arrest agent and tritiated thymidine autoradiography both show that cells with cytoplasmic beta-chains are in cell cycle, whereas cells with surface beta-chains are cycling slowly, if at all.     

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

The profile of T-cell receptor beta-chain variable （TRBV） genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern （GMSP） can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection （AHI）, peripheral blood mononuclear cells （PBMCs） were separated and further sorted into CD4^＋ and CD8^＋ T-cell subsets. The molecular features of the TRBV complementary determining region 3 （CDR3） motifs were determined using GMSP analysis. When a GMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4^＋ and CD8^＋ T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8^＋ T-cell subset was significantly higher than that of the CD4^＋ T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4^＋ and CD8^＋ T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8＋ T-cell subset, may be relevant to the pathogenesis of subjects with AHh The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.     

中线T细胞淋巴瘤的TCR—γ基因重排研究

目的对中线Ｔ细胞淋巴瘤的克隆性进行研究。方法用一组针对Ｔ细胞受体γ链基因Ｖ区片段的家族特异性引物和ＰＣＲ方法,对１１例（２２个标本）中线Ｔ细胞淋巴瘤病例进行了Ｔ细胞受体γ基因重排的检测。结果２２个标本中２１个有Ｔ细胞受体γ链基因的克隆性重排（９４４５％）。在９例原发灶的连续活检和１例原发灶及其转移灶的标本中均未发现增生的克隆家族的改变。结论中线Ｔ细胞淋巴瘤的病变组织中存在Ｔ细胞的单克隆性增生,为该肿瘤的Ｔ细胞起源提供了分子生物学的证据。在中线Ｔ细胞淋巴瘤的疾病过程中（包括转移灶）增生Ｔ细胞的家族未发生变化,支持肿瘤的单克隆起源学说     

T-ALL及T细胞株相关TCR Vβ基因谱系和克隆性分析

目的了解T细胞-急性淋巴细胞白血病(T-ALL)患者外周血中的T细胞及T细胞株的TCR Vβ基因谱系及其克隆性增殖情况。方法利用RT-PCR方法扩增6个不同的T细胞株和6例初发未治T-ALL病人外周血单个核细胞中24个TCR Vβ基因的互补决定区3(CDR3)，PCR产物进一步经荧光标记和基因扫描分析CDR3长度而确定T细胞的克隆性，部分T细胞株的单克隆PCR产物进一步进行序列分析。结果与正常人外周血表达全部24个Vβ亚家族不同，6例T-ALL病人分别表达5～12个Vβ亚家族。6例病人均存在1个或多个Vβ亚家族的寡克隆或双克隆增殖T细胞，另外，还有一些Vβ亚家族多克隆模式发生改变，呈现寡克隆性增殖的趋势。T细胞株多显示为表达一个Vβ亚家族的单克隆T细胞，不同T细胞株的CDR3长度和序列不尽相同。结论T-ALL患者外周血T细胞的TCR Vβ谱系出现限制性改变，均可检测到克隆性增殖T细胞，尚需进一步鉴定其性质(肿瘤性或抗原特异性增殖)，对于检测微小残留病变和设计抗白血病独特型疫苗均有一定的意义。     

Microbial colonization influences composition and T-cell receptor V beta repertoire of intraepithelial lymphocytes in rat intestine.

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ-free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T-cell receptor (TCR) alpha/beta + IEL in that the proportions of CD4+ and CD8 alpha beta + TCR alpha/beta + cells were increased, while the double negative (CD4- CD8-) fraction was reduced. microbial colonization also influenced the TCR V beta repertoire of CD8+ IEL in that the proportions of V beta 8.2+ and V beta 10+ cells were increased, whereas V beta 8.5+ and V beta 16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same V beta subsets. Three-colour flow-cytometric analysis demonstrated that skewing of the V beta repertoire was most pronounced in the CD8 alpha alpha + subset, although the numerical increase of IEL mainly included the CD8 alpha beta + subset. In contrast to IEL, the TCR V beta repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCR alpha/beta + IEL subpopulations respond dynamically to the microbial gut flora and suggest that their V beta repertoire can be shaped by luminal microbial antigens.     

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.     

Rearrangement of kappa-chain and T-cell receptor beta-chain genes in malignant lymphomas of "T-cell" phenotype

Detection of immunoglobulin gene rearrangements by molecular hybridization is considered to be a highly sensitive approach to the evaluation of clonality of B-cell-derived neoplasms. Like monoclonal surface immunoglobulin in B cells, which serves as a reliable immunophenotypic marker for clonality, rearrangement of the genes encoding immunoglobulin light chains has been accepted as a reliable genotypic marker for the presence of a clonal expansion of B lymphocytes. The authors now report 3 cases of non-Hodgkin's lymphoma that were immunologically classified as having a T-cell phenotype and in which, in addition to rearrangements of the T-cell receptor beta-chain gene, a rearrangement of an immunoglobulin light-chain gene was clearly identified by Southern blot hybridization. The results demonstrate that the data obtained by molecular hybridization should be interpreted with caution when the immunogenetic findings do not correlate with immunophenotypic expression, and that the results of molecular genetics studies should be interpreted in conjunction with morphologic and immunologic findings.     

Dominant T-cell receptor beta-chain gene rearrangements indicate clonal expansion in the rheumatoid joint

T-cell receptor (TcR) beta and delta gene rearrangements were studied in anti-CD3 expanded T-cell populations cultured from the synovial membrane (SM) (n = 5) or synovial fluid (SF) (n = 2) of rheumatoid arthritis (RA) patients. Dominant TcR beta-chain gene rearrangements to C beta 1 were demonstrated in all the patients tested and 1-3 expanded clones per patient were found. Clonal rearrangements to C beta 2 were detected in one SM sample (two clones) and one SF sample (one clone). The TcR delta gene was deleted in all the samples tested. We conclude that clonal dominance may be found in expanded T-cell populations from SM and SF of RA patients. Multiple clones may be present, either using the C beta 1 or C beta 2 gene segment.     

运用荧光定量多重PCR技术检测RB1基因突变

目的　建立一种半自动、简易、快速和不需放射性同位素技术,用以检测RB1 基因突变的方法。方法　应用包括扩增RB1 基因27 个外显子和启动区在内的荧光定量多重PCR 技术。将全基因分成若干组,每组3～7 对引物,同时含对照C4 引物并使用4 对外对照,分别为RB的缺体、单体、双倍体及三体。在测试标本中,一个片段的拷贝数根据比较对照与标本的荧光强度而获得。利用自动片段处理软件2.1 处理结果。结果　观察到小片段缺失、插入及外显子拷贝数缺失等突变。测序结果和RB瘤细胞杂合性丢失进一步证实荧光定量多重PCR 的筛查结果。结论　此法是筛查患者基因缺失、插入的一种快速、简易的方法。应用此法可查出约50% 的阳性病例。查出的突变不仅有小缺失、插入,也可发现用以往的方法所不能见到的外显子杂合性缺失病例。对临床基因缺陷的诊断有重要意义