Cell clonality in hypereosinophilic syndrome: what pathogenetic role?

OBJECTIVE: Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous disorder, including either a myeloproliferative or a lymphoproliferative variant (l-HES). In l-HES, T-lymphocytes could be involved in the pathogenesis through several cytokines, including IL5. METHODS: We assayed both TCR Beta- and delta-rearrangements by fluorescent PCR, characterizing 14 patients affected by HES. Lyn activation (a src-kinase involved in the IL5 pathway) was also tested in 6 cases. RESULTS: FIP1L1-PDGFRa was detected in 4 cases (28.6%); a clonal TCR was found in 10 cases (71.4%), including cases FIP1L1-PDGFRalpha-positive; four cases did not show any molecular marker. In this series, levels of IL5, IL4, IL2 and gammaIFN were measured, without any significant difference among different subgroups. All pathological samples tested did not show Lyn activation. Immunophenotype was also characterized: only one case showed an atypical CD3-/CD4+ population in the bone marrow. CONCLUSION: This study would suggest that a real distinction between m- and l-HES is not wholly convincing and that clonal T-cell expansion could not be the "primum movens" but an epiphenomenon in HES.     

Is TCRgamma clonality assay useful to detect early celiac disease?

BACKGROUND: Although histology is considered the gold standard for the diagnosis of celiac disease, the early stages (latent or potential) of this disease are difficult to diagnose, because of the negativity of laboratory tests and the lack of villous atrophy. Thus, markers of early disease are needed. AIMS: We investigated the possibility to detect latent or potential celiac disease by means of TCRgamma clonality assay in intraepithelial T cells in patients with suspected disease, negative laboratory tests, and an increased number of intraepithelial lymphocytes. PATIENTS AND METHODS: Duodenal biopsies were obtained from 35 patients with nonspecific duodenitis (controls), 13 latent or potential celiac disease subjects, 28 well-defined celiac patients, and 8 celiac patients in gluten-free diet. Histologic and immunohistochemical quantification of intraepithelial lymphocytes, as well as TCRgamma clonality assay, were carried out in all subjects by means of standard techniques. RESULTS: Intraepithelial lymphocytes and TCRgamma clonality were significantly increased in potential and defined celiac patients with respect to the controls, even though the increase in TCRgamma clonality was lesser with respect to that of intraepithelial lymphocytes. No significant differences were found concerning this variable between the potential and defined celiac subjects. CONCLUSIONS: TCRgamma clonality does not represent a marker of early disease. However, it might be useful to help in distinguishing celiac disease from other causes of nonspecific duodenitis.     

应用半重叠TCRγ引物扩增TCRγ基因重排检测淋巴细胞白血病的研究

采用更加敏感的半重叠式T细胞受体（TCR）γ链特异性引物的多聚酶链反应（PCR）法,对28例急性淋巴细胞白血病（ALL）初治、完全缓解（CR）及骨髓移植（BMT）患儿的骨髓标本进行检测,用消化煮沸及酚抽提法分别对所有骨髓标本进行DNA提取,将PCR结果进行比较。结果表明,消化煮沸法用于微量标本的DNA提取优于酚抽提法。28例ALL患儿中16例检出TCRγ特异性条带,其中微小残留病（MRD）组18例;阳性检出12例,其中免疫分型标记为B细胞型的4例（25.0%）,免疫标记为T细胞型者全部出现TCRγ阳性条带。表明TCRγ基因重排并非克隆性T细胞增生所特有,部分ALL患儿存在双克隆重排。     

应用半重叠TCRγ引物扩增TCRγ基因重排检测淋巴细胞白血病的…

采用更加敏感的半重叠式Ｔ细胞受体（ＴＣＲ）γ链特异性引物的多聚酶链反应（ＰＣＲ）法，对２８例急性淋巴细胞白血病（ＡＬＬ）初治，完全缓解（ＣＲ）及骨髓移植（ＢＭＴ）患儿的骨髓标本进行检测，用消化煮沸及酚抽提法分别对所有骨髓标本进行ＤＮＡ提取，将ＰＣＲ结果进行比较，结果表明，消化煮沸法用于微量标本的ＤＮＡ提取优于酚抽提法，２８例ＡＬＬ患儿１６例检出ＴＣＲγ特异性条带，其中微小残留病（ＭＲＤ）组１８例，     

Analysis of B-cell clonality in the hepatic tissue of patients with Sjögren's syndrome

OBJECTIVE: We investigated the incidence of B-cell clonality in the minor salivary gland and liver (extra-glandular lesion) of patients with Sj?gren's syndrome (SS). We also compared B-cell clonality in the minor salivary gland and liver in the same individuals, and compared its incidence among patients with various liver diseases, such as primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH). METHODS: A minor salivary gland biopsy was performed on 35 patients with SS (30 patients with primary SS, and five patients with secondary SS). A liver biopsy was performed on nine patients with SS associated with bile duct lesions, two patients with PBC, one patient with AIH, one patient with drug-induced liver dysfunction, and three patients with viral hepatitis. DNA was extracted from each tissue sample and then subjected to Polymerase Chain Reaction (PCR). B-cell clonality was analysed by assessing the rearrangement of the immunoglobulin heavy chain (IgH) gene by PCR. RESULTS: B-cell clonality was confirmed in the minor salivary gland biopsy sample in 23 of the 35 patients (65.7%), and in the liver biopsy sample (non-exocrine organ involvement) in seven of the nine patients (77.8%). The presence or absence of B-cell clonality was investigated in both the minor salivary gland and liver in seven patients, but B-cell clonality was confirmed in both tissues in only one patient, and the pattern of clonality in the minor salivary gland differed from that in the liver. B-cell clonality was detected in the liver of the PBC and AIH patients. CONCLUSION: B-cell clonality is a phenomenon that is observed frequently in SS lesions in the salivary glands and liver. The appearance of B-cell clonality was shown to be attributable to antigen-driven clonal expansion.     

Differential utilization of T cell receptor TCRα/TCRδ locus variable region gene segments is mediated by accessibility

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRδ locus is contained within the TCRα locus; TCRα variable region exons are encoded by TRAV and TRAJ segments and those of TCRδ by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5′TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.     

Evidence for a functional sidedness to the αβTCR

The T cell receptor (TCR) and associated CD3γε, δε, and ζζ signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the αβ and γδTCR-CD3 complexes. We also report that the other side of the αβTCR mediates homotypic αβTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3ε chains of two CD3 heterodimers are arranged side-by-side in both the αβ and γδTCR-CD3 complexes. This system was also used to show that αβTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Cα domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair αβTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a “functional-sidedness” to the αβTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located.     

How αβ T cells deal with induced TCRα ablation

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)alpha chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7--10 days, but minute amounts of surface-bound TCR beta chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naive T cells decay over time, the decay rates being faster for CD8(+) cells (t(1/2) approximately 16 days) than for CD4(+) cells (t(1/2) approximately 46 days). TCR(+) na?ve cells are either maintained (CD8(+)) or decay more slowly (CD4(+); t(1/2) approximately 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8(+) cells; t(1/2) approximately 52 days) or not at all (CD4(+) cells), but at the population level, these cells fail to expand as their TCR(+) counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the alpha beta TCR contributes significantly to T-cell homeostasis.     

SLE中的反抑制细胞与γδTCR阳性T细胞

对60例SLE细胞亚群表型分析发现,大部分SLE患者外周血中CD_8阳性T细胞亚群数量未见降低,研究表明,尤以CD_8~+VV~+、CD_8~+γδ-TCR~+和CD_8~+VV~+γδ~+三类T细胞亚群高表达最为明显,由于CD_8~+VV~+、CD_8~+γδ-TCR~+和CD8_8~+VV~+γδ~+T细胞亚群均具有反抑制活性。因此在活动性SLE患者中更为明显。可作为临床从免疫学角度分析SLE活动与否,评估稳定期SLE患者免疫重建的参考指标之一。     

SLE中的反抑制细胞与γδTCR阳性T细胞

对６０例ＳＬＥ细胞亚群表型分析发现，大部分ＳＬＥ患者处周血ＣＤ８阳性Ｔ细胞亚群数量未见降低，研究表明，尤以ＣＤ８＾＋ＶＶ＾＋、ＣＤ８＾＋γδ－ＴＣＲ＾＋和ＣＤ８＾＋ＶＶ＾＋γδ＾＋三类Ｔ细胞亚群高表达最明显，由于ＣＤ８＾＋ＶＶ＾＋、ＣＤ８＾＋γδ－ＴＣＲ＾＋和ＣＤ８＾＋ＶＶ＾＋γδ＾＋Ｔ细胞亚群均具有反抑制活性。因此在生ＳＬＥ患者中更为明显。可作为临床从免疫学角度分析ＳＬＥ活动与否，评估稳定期ＳＬ     

Leukemia Associated Clonal Expansion of TCR Vβ Subfamily T Cells

The analysis of the T cell receptor (TCR) Vβ repertoire is one of the most sensitive methods to identify the clonal expansion T cells which respond to tumor associated antigens. Recently, studies have focused on clonally expanded T cells from patients or normal donors induced by leukemia associated antigens in vivo or in vitro. Understanding such clonality and restricted usage of TCR Vβ repertoire of leukemia-associated expanded T cells may be useful for the design of new immunotherapeutic strategy for leukemia.     

HBsAg HBeAg作用PBLs对TCR Vβ基因表达的影响

目的探讨乙肝病毒（ＨＢＶ）与肝癌的相关性．方法取用乙肝疫苗注射３次前后外周血淋巴细胞（ＰＢＬ）；ＨＢＶＤＮＡ转染的ＨｅｐＧ２２２１５株培养上清（ＨＢｓＡｇ，ＨＢｅＡｇ阳性）感染健康人ＰＢＬ后进行ＴＣＲＶβ基因１－２０亚家族表达水平的分析．结果乙肝疫苗注射后及ＨｅｐＧ２２２１５株培养上清感染健康人ＰＢＬ后ＴＣＲＶβ６，１４；Ｖβ６，１５特异性扩增而表达水平显著增高．结论Ｖβ６可能为识别ＨＢＶ抗原或引发免疫应答的基因片段，但Ｖβ１４，１５各自在限制和杀伤功能方面起着重要的作用，这从分子水平上又证明了ＨＢＶ与肝癌的关系     

Absence of clonality of Campylobacter jejuni in serotypes other than HS:19 associated with Guillain-Barré syndrome and gastroenteritis

Guillain-Barré syndrome (GBS) is recognized as a complication that occurs after Campylobacter infection. Certain Penner serotypes, such as HS:19, are linked particularly to GBS in some parts of the world, and there is good evidence for restricted genetic diversity in these isolates. However, GBS also occurs after Campylobacter infection due to other serotypes. Therefore, we asked whether Campylobacter jejuni non-HS:19 serotypes associated with GBS have a clonal structure and differ from strains isolated from patients with Campylobacter gastroenteritis. A worldwide selected population of C. jejuni non-HS:19 strains associated with GBS and gastroenteritis was analyzed by use of multilocus enzyme electrophoresis, automated ribotyping, pulsed-field gel electrophoresis, and flagellin gene typing. The results show that these isolates represent a heterogenic population and do not constitute a unique population across serotypes. No epidemiologic marker for GBS-associated strains was identified.     

不明原因发热患者检测淋巴细胞IgH和TCR基因重排的意义

目的:探讨不明原因发热(FUO)患者淋巴细胞免疫球蛋白重链(Ig H)和T细胞受体-γ(TCR-γ)基因重排的临床意义。方法:采用半巢式PCR方法检测110例FUO患者骨髓淋巴细胞Ig H和TCR-γ基因重排的阳性率。结果:84例淋巴瘤发热患者中,Ig H基因重排阳性34例,TCR-r基因重排阳性29例,两者阳性率达75%;26例非淋巴瘤发热患者中,Ig H基因重排阳性0例,TCR-r基因重排阳性1例,基因重排阳性率3.8%,差异有统计学意义(P0.05)。结论:对于FUO患者行基因重排有助于淋巴瘤的早期诊断,特别是对无法取得病理组织的患者意义更大。     

共刺激PBLs作用肝癌细胞TCR Vβ基因表达与PTK活性

目的探讨在Ｔ细胞活化信号传递中特异性识别和杀伤肝癌抗原的Ｔ细胞受体（ＴＣＲ）Ｖβ基因亚家族的表达特征,及与蛋白质酪氨酸激酶（ＰＴＫ）活性的关系．方法用单克隆抗体ＣＤ２８＋Ｂ７１（ＣＤ８０）共刺激正常人外周血淋巴细胞（ＰＢＬｓ）后作用于肝癌细胞（ＢＥＬ７４０２）,用ＲＴＰＣＲ,Ｓｏｕｔｈｅｒｎ印迹、Ｗｅｓｔｅｒｎ印迹分析ＴＣＲＶβ基因表达特征及ＰＴＫ含量．结果ＴＣＲＶβ７基因在体外选择性扩增,ｄ４表达水平最高,为２４％,对照组为６％,相应地ＰＴＫ的含量也较高,为５８％,对照组为１１％．结论Ｖβ７基因特异性识别肝癌抗原,并介导ＰＴＫ信号转导途径的激活     

How structural adaptability exists alongside HLA-A2 bias in the human αβ TCR repertoire

How T-cell receptors (TCRs) can be intrinsically biased toward MHC proteins while simultaneously display the structural adaptability required to engage diverse ligands remains a controversial puzzle. We addressed this by examining αβ TCR sequences and structures for evidence of physicochemical compatibility with MHC proteins. We found that human TCRs are enriched in the capacity to engage a polymorphic, positively charged “hot-spot” region that is almost exclusive to the α1-helix of the common human class I MHC protein, HLA-A*0201 (HLA-A2). TCR binding necessitates hot-spot burial, yielding high energetic penalties that must be offset via complementary electrostatic interactions. Enrichment of negative charges in TCR binding loops, particularly the germ-line loops encoded by the TCR Vα and Vβ genes, provides this capacity and is correlated with restricted positioning of TCRs over HLA-A2. Notably, this enrichment is absent from antibody genes. The data suggest a built-in TCR compatibility with HLA-A2 that biases receptors toward, but does not compel, particular binding modes. Our findings provide an instructional example for how structurally pliant MHC biases can be encoded within TCRs.MHC restriction is a hallmark of T-cell recognition (1). The first crystallographic structures of T-cell receptors (TCRs) in complex with antigen revealed the structural basis for MHC restriction, illustrating how TCRs bind a composite ligand consisting of the MHC protein and its presented peptide (pMHC) (2, 3). Around the same time, evidence began to mount that T cells, and by extension their TCRs, maintain a physical bias toward MHC proteins (4). The mechanism underlying this bias, and indeed its very existence, has remained controversial. On one hand, structural evidence has been presented that germ-line complementarity-determining region (CDR) loops incorporate evolved regions with intrinsic affinities toward MHC proteins (5). On the other hand, the requirement for coreceptor during thymic selection provides a mechanism for selecting TCRs that bind MHC (6–9).Efforts to reconcile these positions have emphasized that they are not mutually exclusive (9–11). However, it is clear that any intrinsic bias TCRs have toward MHC proteins must exist alongside the need for TCRs to structurally adapt to different ligands or alterations in the TCR–pMHC interface (12). For example, changes to a peptide alone are sufficient to alter how the same TCR sits over the same MHC protein, altering TCR–MHC atomic contacts (13, 14). Similar results are seen with changes to TCR variable domains or hypervariable loops, or even MHC mutations (15–18). Perhaps not surprisingly then, there are no germ-line–based TCR–MHC contacts that are fully conserved at the atomic level when structures with TCRs sharing the same variable domain are compared. For example, TCRs sharing Vβ 8.2 make similar contacts with the helices of the class II MHC protein I-A, but actual contacts are modulated by different peptides, hypervariable loops, and Vα domains (15, 19–21). Although such variances have been noted and even predicted (5, 22), they nonetheless make it difficult to clearly identify interactions encoding a TCR–MHC bias (12).Furthermore, in addition to class I and class II MHC proteins, TCRs can bind a variety of nonclassical MHC molecules, non-MHC ligands (6, 23, 24), and as demonstrated very recently can even bind MHC molecules in orientations reverse from the “canonical” diagonal binding mode (25). Thus, whatever MHC biases actually do exist do not exempt a receptor from identifying alternate binding solutions or targets.A possibility often overlooked in discussions of TCR binding is that an evolutionarily encoded MHC bias does not need to specify attractive (i.e., energetically favorable) interactions. A more fundamental feature is structural or physicochemical complementarity, or simply “compatibility.” A set of structurally/chemically compatible interactions whose formation is energetically weak, neutral, or even slightly unfavorable is better than a set of noncomplementary, unfavorable interactions. A well-understood example in protein chemistry is interactions between opposing charges in protein structures. Formation of such interactions require removal of the charges from bulk water, which comes with a substantial, unfavorable “desolvation” penalty (26, 27). The coulombic energy from the interaction between the charges is often insufficient to offset the desolvation penalty (28). However, a weak (or even unfavorable) charge–charge interaction is still energetically better than two uncompensated charges buried in a protein. In this way, charges can influence structural specificity even if they do not drive binding. Similar arguments can be made for shape complementarity: an energetically neutral but complementary set of interactions is better than a forced set of interactions that might result in steric hindrance or require costly conformational changes. This concept of compatibility is well understood in antibody–antigen interactions (29).Considering the above arguments, evolution could have specified a TCR bias toward MHC by facilitating structural/chemical compatibility. Because they need not be energetic drivers, regions of compatibility might be expected to be adaptable and even overridden when sufficient “glue” can be found elsewhere in an interface. To examine this possibility, we studied human αβ TCRs that recognize the highly prevalent human class I MHC protein, HLA-A*0201 (HLA-A2 or simply A2). Given the high occurrence of A2 in human populations (30), we reasoned that, should it exist, a signature of evolutionarily encoded compatibility should be present within the large amount of structural data available for A2-binding TCRs. We quantified sequence variability and used this to guide queries of electrostatic compatibility. In doing so, we identified a set of interactions between TCRs and a polymorphic region on A2 that are strongly conserved in TCR–A2 structures. These interactions, which involve a positively charged polymorphic “hot-spot” region that is almost exclusive to the A2 α1-helix (31–33), are correlated not only with A2 binding but also restricted positioning over the MHC. Different TCRs engage this hot spot differently, in some cases using hypervariable rather than (or in addition to) germ-line loops. Nonetheless, we show that TCR germ-line loops are enriched in the negatively charged amino acids needed to engage the hot spot and that this enrichment is absent from the corresponding loops in antibodies. Thus, the capacity for achieving structural and chemical complementarity with A2 is not only built-in to the human TCR repertoire but present in a way that facilitates the structural adaptability needed to engage diverse pMHC ligands. Although examples of other forms of evolved compatibility remain to be identified, our findings support the conclusion that TCRs have an evolutionarily encoded, inherent compatibility with MHC proteins that facilitates recognition but does not compel particular binding solutions. This can explain how TCRs can show MHC bias in functional experiments, while still displaying considerable structural adaptability, and sometimes violate expectations.