EXPRESSION OF VASCULAR CELL ADHESION MOLECULE-1 IN MURINE HEARTS WITH ACUTE MYOCARDITIS CAUSED BY COXSACKIEVIRUS B3

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. Using a murine model of acute myocarditis caused by Coxsackievirus B3 (CVB3), perforin-expressing killer cells have been shown to infiltrate the heart, and intercellular adhesion molecular-1 (ICAM-1) together with major histocompatibility complex (MHC) antigen was induced on myocardial cells in acute viral myocarditis. To clarify the immunological mechanisms in more detail, the expression of vascular cell adhesion molecular-1 (VCAM-1) has been examined in the heart of acute myocarditis and on cultured cardiac myocytes treated with interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). The effects of in vivo antibody treatment to VCAM-1 on myocardial damage involved in acute myocarditis were also analysed. CVB3-induced myocarditis resulted in enhanced expression of VCAM-1 on myocardial cells. VCAM-1 expression was also induced on cultured cardiac myocytes by treatment with IFN-γ and TNF-α. The in vivo antibody treatment to VCAM-1 decreased the myocardial damage to some extent, but the effects were not statistically significant. These data suggest that the expression of VCAM-1 on myocardial cells may play at least a partial role in the myocardial damage involved in acute viral myocarditis.     

T-cell receptor Vα region usage in the cerebrospinal fluid of patients with mumps meningitis

To investigate clonality of local T cells in viral infections of the central nervous system, the T-cell receptor (TCR) repertoire was evaluated in T cells from the cerebrospinal fluid (CSF) of nine patients with mumps meningitis, using the polymerase chain reaction (PCR) assay. The usage of the variable region of TCR α chain gene (Vα gene) in the CSF was widespread, and an average of 13 out of the 18 Vα families were expressed. Quantitative PCR analyses showed that the Vα gene expression was biased toward three or less Vα families in the CSF of each patient. When compared with peripheral blood T cells, the average percentages of Vα 11 and Vα 12 gene expression were significantly higher in the CSF than in the peripheral blood. These results suggested that mumps-specific T lymphocytes with a restricted TCR repertoire are selectively recruited to the central nervous system in mumps meningitis, although polyclonal, probably nonspecific, T-cell populations are present in the CSF.     

Expression of cytokine mRNAs in murine hearts with acute myocarditis caused by coxsackievirus B3

In murine acute viral myocarditis, natural killer (NK) cells infiltrate the heart first, followed by activated T-cells, which play an important role in the pathogenesis of the myocardial damage. Because of their multipotential effects, cytokines are thought to play a role in the induction and development of these immune processes. To clarify in more detail the precise mechanism of the cytokine networks involved, the expression of various cytokine mRNAs has been investigated in myocardial cells infected with Coxsackievirus B3 (CVB3) in vivo and in vitro by a semiquantitative polymerase chain reaction (PCR) method. Interleukin (IL)-1α, IL-1β, IL-6, tumour necrosis factor (TNF)-α, and TNF-β were expressed almost throughout the early phase of virus infection with some variations. IL-2, IL-3, IL-4, IL-10, interferon (IFN)-γ, granulocyte/macrophage colony stimulating factor (GM-CSF), and IL-2 receptor (IL-2R) were mainly expressed by the infiltrating cells. TNF-α, TNF-β, and IL-1β were also expressed partly by the infiltrating cells. T-helper (Th)1-related cytokines (IL-2, IFN-γ, and TNF-β) were more strongly expressed than Th2-related cytokines (IL-4 and IL-10) in vivo, indicating that the Th cells which infiltrated the heart and mediated the immune responses in the early phase of acute myocarditis were mainly of Th1-type. © 1997 by John Wiley & Sons, Ltd.     

原发性血小板减少性紫癜患者T细胞受体Vβ亚家族基因的表达特点

目的：了解原发性血小板减少性紫癜（ITP）患者T细胞受体 Vβ亚家族基因表达特点。 方法： 采用反转录酶－多聚酶链反应（RT-PCR）方法检测5例ITP患者外周血T细胞TCR Vβ 24个亚家族基因表达情况，10例正常人作为对照。 结果： 正常人外周血T细胞表达全部24个TCR Vβ亚家族，而5例ITP患者外周血T细胞仅表达4-11个Vβ亚家族，主要为Vβ2（100％）和Vβ3（100％），其次为Vβ19（80％）和Vβ21（80％）部分。全部样本未检测出Vβ4、Vβ6、Vβ17、Vβ20、Vβ24亚家族表达。 结论： ITP患者外周血T细胞TCR Vβ基因谱系呈限制性表达，与其存在细胞免疫功能异常有关。     

High‐throughput single‐cell sequencing of paired TCRα and TCRβ genes for the direct expression‐cloning and functional analysis of murine T‐cell receptors

Precise clonal and functional assessments of the T cell receptor (TCR) repertoire diversity require paired TCRα and TCRβ gene sequence information at monoclonal level. However, available single‐cell strategies are typically limited in throughput and often do not provide full‐length DNA templates for direct gene cloning. Here, we describe a high‐throughput strategy for the unbiased amplification and automated sequence analysis of paired TCRα and TCRβ genes from primary mouse T cells. The platform links cell phenotype and TCR gene sequence information at single‐cell level. Furthermore, it enables direct functional analyses through the efficient cloning of both genes and the generation of stable TCR expressing cell lines. This highly efficient workflow is a powerful tool to determine the diversity and quality of the murine T‐cell repertoire in various settings, for example in vaccine development, infectious diseases, autoimmunity, or cancer.     

Quantitative analysis of T cell receptor diversity in clinical samples of human peripheral blood

The analysis of T cell receptor diversity provides a clinically relevant and sensitive marker of repertoire loss, gain, or skewing. Spectratyping is a broadly utilized technique to measure global TCR diversity by the analysis of the lengths of CDR3 fragments in each Vβ family. However the common use of large numbers of T cells to obtain a global view of TCR Vβ CDR3 diversity has restricted spectratyping analyses when limited T-cell numbers are available in clinical setting, such as following transplant regimens. We here demonstrate that one hundred thousand T cells are sufficient to obtain a robust, highly reproducible measure of the global TCR Vβ repertoire diversity among twenty Vβ families in human peripheral blood. We also show that use of lower cell number results not in a dwindling of observed diversity but rather in non-reproducible patterns in replicate spectratypes. Finally, we report here a simple to use but sensitive method to quantify repertoire divergence in patient samples by comparison to a standard repertoire profile we generated from fifteen normal donors. We provide examples using this method to statistically evaluate the changes in the global TCR Vβ repertoire diversity that may take place during T subset immune reconstitution after hematopoietic stem cell transplantation or after immune modulating therapies.     

Effects of interferon-α on cytokine profile,T cell receptor repertoire and peptide reactivity of human allergen-specific T cells

A large panel of T cell clones (TCC) specific for the recombinant form of Poa pratensis allergen (rKBG7.2 or Poa p9) were established from the peripheral blood of a grass pollen-sensitive donor in the absence or presence of recombinant interferon-α (IFN-α) in bulk culture and their pattern of cytokine secretion, peptide reactivity and TCR Vβ repertoire was examined. The majority of allergen-specific TCC derived in absence of IFN-α produced high amounts of interleukin-4 (IL-4) and IL-5 but not IFN-γ (Th2 cells), while most of TCC derived in presence of IFN-α produced IFN-γ but not, or limited amounts of, IL-4 and IL-5 (Th1 or Th0 cells). Of 24 TCC established in the presence of IFN-α, 22 were able to recognize a single allergen peptide, p26, while none of the clones established in the absence of IFN-α showed a similar specificity. The majority of both clones expressed the Vβ2 element regardless of whether they were established in the presence of IFN-α, but the presence of IFN-α favored the expansion of Vβ2⁺, Vβ17⁺ and Vβ22⁺ Poa p9-specific T cells, whereas in the absence of IFN-α, other TCR Vβ-bearing T cells (Vβ5, Vβ6.7 and Vβ14) were expanded in addition to Vβ2⁺ T cells. None of Vβ2⁺ clones established in the absence of IFN-α reacted with p26, whereas all the Vβ2⁺ clones established in its presence responded to this peptide. IFN-α also shifted the TCR Vβ repertoire of both Poa p9- and Lolium perenne group 1 (Lol p1)-specific T cell lines generated from the same patient and from a different grass-sensitive individual. These data demonstrate that IFN-α modulates the development of allergen-specific T cells in vitro, and suggest that IFN-α may represent an useful tool for novel immunotherapeutic approaches in allergic disorders.     

T cell receptor diversity in alloreactive responses against HLA-B27 (B*2705) is limited by multiple-level restrictions in both α and β chains

The T cell receptors (TCR) in HLA-B27 (B*2705) alloreactivity were analyzed in cytotoxic T lymphocytes (CTL) from two individuals. Non-random usage was found in Vβ, N+Dβ, Vα, and Jα, but not in Jβ segments or Nα-regions. Vβ segments from homology subgroup 4 were predominant and not associated to a particular donor or fine specificity, suggesting involvement in recognizing the HLA-B27 molecule. In contrast, preferential Vα usage was associated with particular individuals and fine specificities, indicating distinct Vβ and Vα recruitment and contribution to allorecognition. Recurrent N+Dβ motifs and Jα segments, even from different donors, limited junctional diversity, suggesting that CDR3 usage was determined by the alloantigenic epitope independently of individuals. TCR were selected differently at various levels, as indicated by the following findings. Four clonotypes with similar fine specificity had identical β and unrelated α chains. Similar α were associated with unrelated β chains, and vice versa. CTL using Vβ subgroup 4 did not globally show concomitant predominance of other TCR elements. Vα7, one of the preferred Vα segments, was always associated with Vβ subgroups other than 4. Sometimes, a TCR showed homology in elements of one chain to a second TCR or group of TCR, and to another in the other chain. These results are best explained by differential selection of TCR elements by different epitopes, providing a key to the inner structure of allospecific TCR repertoires.     

Lack of directed Vα14-Jα281 rearrangements in NK1+ T cells

NK1.1⁺ T cells are an unusual subset of TCRαβ cells distinguished by their highly restricted Vβ repertoire and predominant usage of an invariant Vα14-Jα281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant α chain, we have analyzed Vα14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1⁺ T cells. As expected a high proportion (17/20) of the hybrids had rearranged Vα14 to Jα281. However, Vα14-Jα281 rearrangements always occurred on only one chromosome and were accompanied by other Vα-Ja rearrangements (not involving Vα14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of Vα14-Jα281 rearrangements in NK1.1⁺ T cells.     

Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-α-reactive CD4 T cells in A/J mice

Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IA(k) dextramers for cardiac myosin heavy chain (Myhc)-α 334-352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in na?ve mice as measured by proliferative response to Myhc-α 334-352 and IA(k) dextramer staining. We also detected Myhc-α 334-352 dextramer(+) cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in na?ve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology.     

Evidence for the persistence of monoclonal expansions of CD8+ T cells following primary simian immunodeficiency virus infection

A longitudinal study of the CD8⁺ TCR variable (Vβ) chain repertoire was performed in rhesus macaques experimentally infected with simian immunodeficiency virus (SIV) using both TCR Vβ chain-specific monoclonal antibodies and TCR β chain CDR3 length analysis. Expansions of subpopulations of CD8⁺ T cells were detected during the acute phase of SIV infection. In all monkeys studied, monoclonal expansions persisted for at least 18 months and increasingly dominated the repertoire of CD8⁺ T cells expressing the relevant Vβ chain. This study shows that persistent CD8⁺ T cell expansions develop in response to a virus infection. This is important not only for our understanding of the T cell response to viruses but also for understanding the factors that determine the normal CD8⁺ TCR repertoire.     

High-throughput sequencing reveals restricted TCR Vβ usage and public TCRβ clonotypes among pancreatic lymph node memory CD4+ T cells and their involvement in autoimmune diabetes

Islet-reactive memory CD4⁺ T cells are an essential feature of type 1 diabetes (T1D) as they are involved in both spontaneous disease and in its recurrence after islet transplantation. Expansion and enrichment of memory T cells have also been shown in the peripheral blood of diabetic patients. Here, using high-throughput sequencing, we investigated the clonal diversity of the TCRβ repertoire of memory CD4⁺ T cells in the pancreatic lymph nodes (PaLN) of non-obese diabetic (NOD) mice and examined their clonal overlap with islet-infiltrating memory CD4 T cells. Both prediabetic and diabetic NOD mice exhibited a restricted TCRβ repertoire dominated by clones expressing TRBV13-2, TRBV13-1 or TRBV5 gene segments. There is a limited degree of TCRβ overlap between the memory CD4 repertoire of PaLN and pancreas as well as between the prediabetic and diabetic group. However, public TCRβ clonotypes were identified across several individual animals, some of them with sequences similar to the TCRs from the islet-reactive T cells suggesting their antigen-driven expansion. Moreover, the majority of the public clonotypes expressed TRBV13-2 (Vβ8.2) gene segment. Nasal vaccination with an immunodominat peptide derived from the TCR Vβ8.2 chain led to protection from diabetes, suggesting a critical role for Vβ8.2⁺ CD4⁺ memory T cells in T1D. These results suggest that memory CD4⁺ T cells bearing limited dominant TRBV genes contribute to the autoimmune diabetes and can be potentially targeted for intervention in diabetes. Furthermore, our results have important implications for the identification of public T cell clonotypes as potential novel targets for immune manipulation in human T1D.     

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。     

Natural killer-like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin-4 production

An unusual subset of mature T cells expresses natural killer (NK) cell-related surface markers such as interleukin-2 receptor β (IL-2Rβ CD122) and the polymorphic antigen NK1.1. These “NK-like” T cells are distinguished by their highly skewed Vα and Vβ repertoire and by their ability to rapidly produce large amounts of IL-4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1⁺ T cells and consequently to be deficient in IL-4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL-2Rβ⁺ T cells with a skewed Vβ repertoire characteristic of “NK-like” T cells. Furthermore lack of NK1.1 expression on IL-2Rβ⁺ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR-P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL-2Rβ⁺ T cells segregated with NKR-P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL-4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR-P1. Collectively, our data indicate that “NK-like” T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL-4 production.     

职业铅接触工人外周血T细胞Vβ基因谱系明显缺失

目的:分析职业铅接触工人外周血中T细胞受体Vβ基因(TCR Vβ)谱系分布和克隆性特点,进一步了解铅接触后对T细胞免疫的影响。方法:利用RT-PCR分别检测6例职业铅接触工人和6例健康体检者的外周血中各TCR Vβ亚家族的表达情况;阳性产物经荧光素标记和基因扫描分析其CDR3长度以了解T细胞克隆性。结果:所有健康体检者均可检测到24个Vβ亚家族并呈多克隆,而6例职业铅接触工人外周血TCR Vβ亚家族表现明显的限制性利用,仅能检测到1～7个,并且多数所检测到的Vβ亚家族T细胞呈现为寡克隆或双克隆增殖,发生克隆性改变的亚家族主要集中在Vβ1和Vβ16。结论:职业铅接触可能在不同程度上影响机体TCR Vβ谱系的利用和克隆性改变,可能与铅的免疫毒性有关。     

肿瘤患者胸/腹水和外周血中TCR Vβ亚家族表达及调节性T细胞亚群的研究

目的:探讨肿瘤患者胸/腹水中肿瘤浸润性淋巴细胞(TIL)和外周血T细胞中T细胞受体(TCR)Vβ亚家族的表达变化情况、CD4/CD8细胞比例变化和Treg细胞的分布情况,分析肿瘤患者的免疫状态。方法:分离11例肿瘤患者胸/腹水和外周血以及4例健康人外周血的T细胞,采用流式方法检测Vβ24个亚家族及CD3、CD4、CD8、CD25、CD127的表达情况,统计分析T细胞中Vβ24个亚家族、Treg细胞的特点。结果:TCR Vβ亚家族的表达在肿瘤患者胸/腹水TIL中与血液淋巴细胞中存在着显著差异,其中肺癌患者(4/5)TIL中TCR Vβ8,结肠癌患者(3/4)TIL中TCR Vβ2等亚家族显著高表达;11例肿瘤患者胸/腹水样本中Treg的比例均比外周血高(P<0.05),其中5例肺癌患者胸腹水中CD8+T细胞比例降低。结论:肿瘤患者胸/腹水中的淋巴细胞TCR Vβ亚家族表达与外周血存在差异,表明肿瘤患者体内淋巴细胞发生了明显的优势取用和定向趋化。此外肿瘤微环境可能影响了TIL中CD4+细胞的分化导致胸腹水中Treg细胞的比例升高,同时伴随着CD8+T比例的下降,并可能因此影响到TIL中TCR Vβ亚家族的优势取用情况,且导致免疫耐受。     

Preferential Positive Selection of T Lymphocytes which Express Two Different TCRα Chains, an Endogenous and a Transgenic

A hallmark of positive selection in T-cell receptor (TCR)-transgenic mice is a strong skewing towards the CD4⁺ or the CD8⁺ subset, depending on the class II or I restriction of the TCR, respectively. However, previous experiments in TCR transgenic mice specific for an Ig light chain (λ2³¹⁵)/I-E^d class II molecule did not fit into this scheme because the authors observed an anomalous skewing towards CD8. In this paper, the authors show that endogenous TCRα chains are expressed on > 90% of CD4⁺ and CDS⁺ cells in this particular transgenic strain, even on a selecting H-2^d haplotype. Endogenous TCRQ chains are first detected when double-positive thymocytes down-regulate either CD4 or CD8. Endogenous Vα seems to influence generation of T-cell subsets because CD4⁺ and CD8⁺ cells express different frequencies of endogenous Vα2 and Vα8. In the absence of endogenous TCRα chains in recombination-deficient TCR-transgenic severe combined immunodeficiency (SCID) mice, a strong skewing towards CD4⁺ T cells is seen, but such mice are severely T-cell deficient. As an explanation for these results, the authors suggest that the transgenic TCR has a too low affinity for efficient positive selection, therefore, TCRa gene rearrangements proceed. Endogenous TCRa paired with transgenic TCRβ could bind to class I or class II molecules, enhance positive selection and thereby production of CD4⁺ or CD8⁺ cells. Most of the ‘mismatched’ CD8⁺ cells are λ2³¹⁵-specific and I-E^d class II restricted, and may function as idiotype-specific suppressors of B cells. These results may help explain the origin of dual TCRα T cells. Furthermore, the authors suggest that T cells ‘mismatched’ for co-receptor/TCR MHC-specificity may be enriched among dual TCRa T cells.     

Effects of in vivo administration of anti-B7-1/B7-2 monoclonal antibodies on the survival of mice with chronic ongoing myocarditis caused by Coxsackievirus B3

In acute myocarditis and dilated cardiomyopathy, it has previously been reported that antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved. For antigen-specific T-cell activation to occur, it is necessary for the T-cell to receive co-stimulatory signals provided by co-stimulatory molecules expressed on the antigen-presenting cell (APC), as well as the main signal provided by binding of the T-cell receptor (TCR) to the antigen. To investigate the roles for the co-stimulatory molecules B7-1 and B7-2 in the development of chronic ongoing viral myocarditis, firstly the expression of B7-1/B7-2 was analysed in the hearts of A/J mice with myocarditis induced by Coxsackievirus B3 (CVB3). Secondly the induction of B7-1/B7-2 on cultured cardiac myocytes treated with interferon (IFN)-γ was evaluated. Thirdly the effects of the in vivo administration of anti-B7-1/B7-2 monoclonal antibodies (MAbs) on the survival of mice with viral myocarditis were examined. CVB3-induced myocarditis resulted in enhanced expression of B7-1/B7-2 on cardiac myocytes. The expression of B7-1/B7-2 on cardiac myocytes could be induced by IFN-γ in vitro. In vivo anti-B7-1 MAb treatment significantly prolonged the survival of mice with myocarditis, whereas anti-B7-2 MAb treatment abrogated the protective effect of anti-B7-1. These findings indicate that distinct roles for B7-1 and B7-2 antigens are involved in the development of viral myocarditis and raise the possibility of immunotherapy with anti-B7-1 MAb to prevent T-cell-mediated cardiac myocyte injury and to improve the prognosis of viral myocarditis. Copyright © 1999 John Wiley & Sons, Ltd.