A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen-disparate haematopoietic stem cells from parental donors

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)-disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA-matched donor. Success against major obstacles such as graft-versus-host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long-term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34(+) haematopoietic stem cells after rigorous T-cell depletion were prospectively monitored for their immune reconstitution during the first post-transplant year. Natural killer (NK) cells showed a marked increase on d +30. T and B cells began to reconstitute on d +72 and +68 respectively. During extended follow-up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T-cell receptor (TCR)-repertoire complexity. Naive T cells emerged 6 months post transplantation, paralleled by an increase in TCR-repertoire diversity. All patients self-maintained sufficient immunoglobulin levels after d +200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T-, B- and NK-cell compartments within the first post-transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA-matched donor.     

Identical twins discordant for multiple sclerosis have a shift in their T-cell receptor repertoires

CD4 T‐cells have an important role in the autoimmune response in multiple sclerosis (MS). We investigate the possibility that a shift occurs in the T‐cell receptor (TR) repertoire of identical twins discordant for MS. We compare the CDR3 spectratype distributions of 24 different TR V beta (TRBV) segments in naïve CD4 T‐cells from discordant MS twins and from healthy identical twins. We also compare the CDR3 spectratype distributions in unrelated healthy pairs, formed by combining members of different healthy twins, with the CDR3 spectratype distributions in unrelated pairs of MS patients and in unrelated pairs of their apparently healthy cotwins, formed by combining members of different discordant twins. We use the correlation coefficient (r‐value) as a measure of similarity of CDR3 spectratypes in each pair, and we test for the significance of the difference between r‐values from the different pairs. We observe that the r‐value for the CDR3 spectratype distributions among discordant twins differs significantly from the corresponding r‐value for the healthy twins for two TRBV segments. Further, the r‐values, for both the unrelated MS patient pairs and the unrelated pairs of their apparently healthy cotwins, differ significantly from the r‐values for healthy unrelated pairs of individuals. We conclude that both the MS patients and their apparently healthy cotwins have shifts in their CDR3 repertoires. Because we study naïve CD4 T‐cells, we postulate that CDR3 repertoire shifts precede MS and predispose to MS, but are unlikely to be sufficient to cause MS.     

Analysis of T cell repertoire in the liver of patients with chronic hepatitis C

Many T cells infiltrate into the liver of patients with chronic hepatitis C (CH-C). They are believed to play a crucial role in the immunopathogenesis of hepatic inflammation, but their clonality and specificity are unknown. The aim of this study was to clarify the characteristics of these T cells. We analysed the complementarity-determining region (CDR)3 size lengths of T cell receptor (TCR) beta-chains by size spectratyping, and determined the sequences of Vbeta CDR3 after subcloning Vbeta-specific polymerase chain reaction products. Spectratyping showed clonal expansions in all liver specimens, most of which showed more than two T cell clones. Moreover, many non-clonal T cells also accumulated in the liver. Clonality of the T cells suspected by spectratyping was confirmed by CDR3 sequencing. Although the sequences revealed no whole CDR3-shared clones among different patients, some common motif sequences were observed. Our data suggest that T cells are stimulated by several hepatitis C virus (HCV) epitopes, then accumulate in the liver of CH-C patients. Shared motifs of expanded T cell clones suggest that they might recognize the same regions of HCV peptides, but have differences due to HCV peptide mutational changes. These clones might also interact with non-clonal T cells and play a crucial role in the immunopathogenesis of CH-C.     

HLA-B27-restricted T cells from patients with ankylosing spondylitis recognize peptides from B*2705 that are similar to bacteria-derived peptides

Ankylosing spondylitis (AS) is an inflammatory systemic disease affecting the spine, sacroiliacal and peripheral joints. Although the aetiology of AS remains unknown, the strong association with the HLA-B27 allele might reflect directly a detrimental effect of the HLA-B27 molecule itself, resulting from its potential capability to present 'arthritogenic' peptides to CD8+ T cells. Because some forms of SpA are triggered by enterobacterial infection, such arthritogenic peptides might originate from autologous and/or bacterial proteins triggering cross-reactive CD8+ T cell clones. Intriguingly, two peptides from the second extracellular domain of HLA-B*2705 share sequence homologies with several enterobacterial antigens, exhibit the HLA-B27-binding-motif, and are presented by HLA-B*2705 itself. The objective of this study was to examine the clonal T cell reactivity against these peptides in patients with AS. To this end, we screened peripheral blood lymphocytes (PBL) of 26 patients with AS and 24 healthy donors for TNF-alpha-producing cells using ELISPOT assays. PBL and synovial fluid-derived lymphocytes (SFL) of peptide-responsive patients were then stimulated and cultured with the relevant peptide and control peptides in vitro. Antigen-specific T cell lines (TCL) were identified by standard chromium release assays. Clonal analysis was performed subsequently applying TCRB-CDR3 spectratyping. Among eight peptides tested, only the HLA-B27 168-176 peptide LRRYLENGK was recognized by PBL from B27+ AS patients but not from B27+ healthy controls (P=0.001). LRRYLENGK-specific T cell clones used preferentially the TCRBV5S1 and the BV14 segment. These results suggest that an HLA-B27-derived peptide with homology to bacterial peptides may play a role in AS.     

系统性红斑狼疮患者外周血T细胞TCRβ链CDR3谱系漂移和序列鉴定

目的 探讨系统性红斑狼疮（SLE）患者外周血T细胞TCRβ链CDR3谱系漂移，为SLE的免疫应答机制和个性化治疗研究提供基础。方法 采用免疫扫描谱型分析技术，分析5例正常献血员的CDR3分布特征及5例SLE患者PBMC中T细胞TCRβ链CDR3的优势利用情况，对克隆性增生T细胞的CDR3区进行序列分析。结果 5例正常献血员PBMC TCR BV CDR3谱型均呈高斯分布，5例活动型SLE患者24TCR BV CDR3家族均出现不同的优势表达。对单／寡克隆性增生T细胞B链CDR3区基因进行测序，证实存在不同的CDR3序列。结论 SLE活动期外周血T细胞TCRβ链CDR3谱系出现明显漂移，提示CDR3的选择性表达可能与SLE的免疫发病机理有关。特异应答的T细胞TCRCDR3序列的确定，将为SLE的发病机制研究和个性化治疗提供新的方法与手段。     

1月龄Balb/c小鼠胸腺、脾脏、肝脏、小肠和血液TRBV-TRBJ1-1 CDR3谱型分析

目的初步分析1月龄Balb/c小鼠胸腺、脾脏、肝脏、小肠和血液中TRBV-TRBJ1-1 CDR3谱型特征。方法提取1月龄Balb/c小鼠胸腺、脾脏、肝脏、小肠和血液样本的总RNA,逆转录成cDNA,以22条TRBV基因家族为上游引物,共同的TRBJ1-1基因为下游引物(荧光标记),PCR扩增包含完整CDR3区片断,毛细管电泳激光DNA扫描技术分析CDR3谱型的长度和多态性。结果胸腺样本毛细管电泳激光扫描图呈现标准的中间高两端低的多态性分布;各家族CDR3区最长和最短比较,多数为相差8～15个氨基酸的谱型分布。脾脏、肝脏、小肠、血液样本扫描图显示多数家族呈多态性分布,但均出现数量不等的单峰、寡峰、偏峰、低峰;多数家族表现为相差3～13个氨基酸的谱型分布,部分家族在预测大小位置无对应峰型。结论 1月龄Balb/c小鼠胸腺中基本包含TCR基因随机重排的多种克隆性T细胞;脾脏、肝脏、小肠、血液表现为单、寡、多克隆形式的T细胞分布,部分家族T细胞缺失,本实验结果可为进一步研究Balb/c小鼠的T细胞发育、分布、应答及CDR3谱型与疾病关系提供方法和基础。     

Analysis of the CDR3 region of alpha/beta T‐cell receptors (TCRs) and TCR BD gene double‐stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T‐cell receptor (TCR) beta chain in T‐cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo‐clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia (T‐ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T‐ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T‐ALL patients had the recombination signal sequence (RSS) 5′‐ and 3′‐breaks end in the TCR BD2 gene using a specialized ligation‐mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T‐ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T‐ALL vaccine, as well as in improving our understanding of healthy human T‐cell development.     

Analysis of T cell receptor Vβ diversity in peripheral CD4+ and CD8+ T lymphocytes in patients with autoimmune thyroid diseases

Autoimmune thyroid diseases are characterized by intrathyroidal infiltration of CD4⁺ and CD8⁺ T lymphocytes reactive to self‐thyroid antigens. Early studies analysing T cell receptor (TCR) Vα gene usage have shown oligoclonal expansion of intrathyroidal T lymphocytes but not peripheral blood T cells. However, TCR Vβ diversity of the isolated CD4⁺ and CD8⁺ T cell compartments in the peripheral blood has not been characterized fully in these patients. We performed complementarity‐determining region 3 (CDR3) spectratyping as well as flow cytometric analysis for the TCR Vβ repertoire in peripheral CD4⁺ and CD8⁺ T cells from 13 patients with Graves' disease and 17 patients with Hashimoto's thyroiditis. Polyclonal TCR Vβ repertoire was demonstrated by flow cytometry in both diseases. In contrast, CDR3 spectratyping showed significantly higher skewing of TCR Vβ in peripheral CD8⁺ T cells but not CD4⁺ T cells among patients with Hashimoto's thyroiditis compared with healthy adults. We found trends towards a more skewed CDR3 size distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves' disease exhibited no skewing both in CD4⁺ and CD8⁺ T cells. These findings indicate that clonal expansion of CD8⁺ T cells in Hashimoto's thyroiditis can be detected in peripheral blood and may support the role of CD8⁺ T cells in cell‐mediated autoimmune attacks on the thyroid gland in Hashimoto's thyroiditis.     

Glomerular T cells in Heymann nephritis.

Active Heymann nephritis (HN) is a rat model of human membranous nephropathy. The appearance of T cells within the glomeruli of HN rats suggests a role for these cells in the pathogenesis of the disease. The aims of this study were to investigate T cells infiltrating the glomerulus in HN in Lewis rats by polymerase chain reaction (PCR) of their Vbeta chains, CDR3 spectratyping and sequencing. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in CFA. Kidneys were collected between 4 and 10 weeks. The glomeruli were separated, homogenized and RNA extracted. RT-PCR, CDR3 spectratyping and sequencing were used to further characterize the infiltrating T cells. Multiple Vbeta families showed restriction of their CDR3 spectratypes in each animal. Several TCR Vbeta families had identical-sized restricted spectratypes across several different animals. Four Vbeta families were sequenced. In three of those four families, the dominant clones showed identical sized CDR3 regions and a striking over-expression of Jbeta2.6. Further analysis of the CDR3 regions of the Jbeta2.6 clones showed a significant restriction of the amino acids at four of the six CDR3 positions. Glomerular T cells bearing similar CDR3 sequences, using Jbeta2.6 and expressing at least two, and possibly more, Vbeta genes are involved in the pathogenesis of HN.     

T CELL REPERTOIRE COMPLEXITY IN SEVER COMBINED IMMUNODEFICIENCY PATIENTS AFTER BONE MARROW TRANSPLANTATION

INTRODUCTIONHumanSCIDiscausedbyseveralgeneticdefects,characterizedbyprofounddeficienciesinT,BandNKcellfunction(1-3).SCIDinfantsrarelysurvivebeyondoneyearofagewithouttherapeuticinterven-tion,suchashematopoieticstemcelltransplantationorgenetherapy(2,4).TcellreconstitutioninSCIDrecipientsofrigorouslyTcell-depletedallogeneicre-latedbonemarrowstemcells,isduetothedevelop-mentandmaturationofdonorstemcellsinthein-fant'svestigialthymus(3).Withinthethymus,TcellprecursorsundergoTCRgenerearra…