Analysis of human T-cell receptor α-chain cDNAs isolated from peripheral blood mononuclear cells

T-cell receptor α-chain cDNA were generated from unstimulated peripheral blood mononuclear cells of a DR2,3,52a individual using a modified anchor PCR method. Fifty-six cDNA clones were identified representing 47 distinct T-cell receptor clonotypes and 26 VA loci. This analysis identified a new VA gene family VA30, and aew member of the VA6 gene family.     

Analysis of human T-cell receptor alpha-chain cDNAs isolated from peripheral blood mononuclear cells.

T-cell receptor alpha-chain cDNA were generated from unstimulated peripheral blood mononuclear cells of a DR2,3,52a individual using a modified anchor PCR method. Fifty-six cDNA clones were identified representing 47 distinct T-cell receptor clonotypes and 26 VA loci. This analysis identified a new VA gene family VA30, and a new member of the VA6 gene family.     

Expansion of clonotype-restricted HLA-identical maternal CD4+ T cells in a patient with severe combined immunodeficiency and a homozygous mutation in the Artemis gene

We have observed a male infant with severe combined immunodeficiency (SCID) responsible for Artemis gene mutation, in whom marked expansion of the transplacentally grafted maternal CD4(+) T cells was observed in various tissues. His class I and II major histocompatibility antigens (MHC) were identical to his mother's. We analyzed the T-cell populations within target tissues at a molecular level in order to determine whether different T-cell clonotypes are expanded in different types of tissue. Prior to T-cell expansion, the T-cell receptor variable beta (TCRBV) 5.1 subfamily predominated in peripheral blood (PB) lymphocytes. Third complementarity determining region (CDR3) size spectratyping and amino acid sequencing showed that the range of T-cell clonotypes was very restricted. After T-cell expansion, different TCRBV subfamilies were found to predominate in different target tissues; these included TCRBV 5.1 and 17 in the PB, TCRBV 13 and 21.3 in the bone marrow, and TCRBV 17 in lymph nodes. CDR3 size analysis showed that the expression of different proliferating T-cell clonotypes remained restricted after T-cell expansion. These results indicate that highly restricted maternal T-cell clonotypes can markedly expand, possibly in response to tissue-specific antigens, in a MHC-identical recipient.     

外周血淋巴细胞穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应的早期诊断价值

探讨外周血淋巴细胞(peripheral blood lymphocyte,PBL)穿孔素和颗粒酶B mRNA检测对肾移植急性排斥反应(AR)的早期诊断价值。采用RT-PCR方法动态检测67例肾移植AR患者外周血淋巴细胞穿孔素和颗粒酶B mRNA的表达水平,同时检测所有患者肌酐(Cr)表达水平情况,比较各时间点穿孔素、颗粒酶B mRNA及Cr表达水平,并探讨穿孔素、颗粒酶B与AR的关系。移植后1d与移植前1d比较,PBL穿孔素、颗粒酶B mRNA表达水平比较无明显差异性(P>0.05),Cr表达水平明显下降(P<0.05);AR前3d较之前各时间点比较,PBL穿孔素、颗粒酶B mRNA表达水平明显上升(P<0.05),而Cr表达水平明显下降(P<0.05);AR后1d时PBL穿孔素、颗粒酶B mRNA表达水平达到最高峰,随后逐渐下降(P<0.05),而AR后5d时Cr表达水平达到最高峰,随后逐渐下降(P<0.05);PBL穿孔素、颗粒酶B mRNA表达水平升高时间比Cr早4d,AR患者经MP/OKT3冲击治疗后,PBL穿孔素和颗粒酶B mRNA表达水平逐渐降低至原有基础水平。PBL穿孔素和颗粒酶B mRNA在AR早期诊断和抗排斥反应疗效评估等方面具有一定的临床价值。     

Correlation of clonal T cell expansion with disease activity in systemic lupus erythematosus

It has been proposed that T cell activation may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In order to examine this hypothesis, we assessed the whole degree of clonal accumulation of T cells using RT-PCR and subsequent single-strand conformation polymorphism (SSCP) analysis. The analysis of the beta chain of the TCR revealed little clonotypic T cell expansion in the peripheral blood of lupus patients in remission, whereas in patients with active disease many dominant T cell clonal expansions without any distinct V beta bias were observed. The alteration in the number of T cell clones correlated well with disease activities, since these T cell expansions decreased as patients had improved. Furthermore, similar but more intense accumulations of T cell clones were found in pleural and pericardial effusions of patients with lupus serositis. Some of these identical expanded clonotypes were observed irrespective of the lesions and the times of sampling, and some of them were identical to those observed in the peripheral blood. These results suggest that the T cell clonal expansions correlate with the disease activities and that the expansion might contribute to the pathogenic lesion formation in SLE.      

Clonally Expanded CD8+ T cells in Allogeneic Bone Marrow Transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with haematologic malignancies. Several lines of evidence demonstrate that donor T cells are involved in the antitumour effects observed after BMT. Thus, patients receiving T-cell-depleted BMT have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated BMT, and patients experiencing graft-versus-host disease (GVHD) have a lower risk of disease relapse than patients who do not experience GVHD. Although the importance of donor T cells for the curative action of BMT has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. In a recently initiated project, we have conducted a longitudinal study of T-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. Peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (MNCs) were isolated and cryopreserved. CD8⁺ T cells were isolated from the MNCs by use of immunomagnetic beads or FACS and analysed for the presence of clonally expanded cells by T-cell receptor clonotype mapping based on RT-PCR and denaturing gradient gel electrophoresis (DGGE). Using this gel-based methodology, clonally expanded T cells were monitored after transplant and compared to the clinical data of the patients. The preliminary results demonstrates the presence of clonally expanded CD8⁺ T cells at all time points analysed. Furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. The appearance of newly emerged clonotypes which coincided with clinical GVHD could indicate a role for these T cells in the pathogenesis of GVHD.     

Theophylline and the immune response: in vitro and in vivo effects

We analyzed the in vitro and in vivo effects of theophylline on various immunological parameters including proliferation of peripheral mononuclear cells (PMNC) in response to phytohemagglutinin (PHA), anti-T3 and anti-T11 monoclonal antibodies (MAb), PHA-induced interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by PMNC, interleukin-1 (IL-1) production by accessory cells, PHA-induced IL-2 production by T-cell clones, and PHA- and anti-T3 MAb-induced DNA synthesis by T cell clones. Results showed that theophylline inhibited PHA- and anti-T3-induced proliferation of both PMNC and T-cell clones, whereas the PMNC proliferation induced by MAb anti-T11 was not affected. The inhibition appeared to be dose-dependent and strictly related to the presence of the drug in the culture. Moreover, PHA-induced IL-2 production by both PMNC and T-cell clones also appeared to be reduced by theophylline. IL-1 production by accessory cells was not affected. These data suggest that the immunological inhibition exerted by theophylline is confined to the T-cell compartment, mainly by acting on structure(s) related to the T3/Ti complex, the primary site for T-cell activation. The alternative pathway of T-cell activation (i.e., via T11 site) seems unaffected. In addition, these results suggest possible clinical relations between the inhibition of the immune response and the plasma levels of the drug reached after a "once daily" or "twice daily" oral ingestion of slow-release theophylline products.     

胶原诱导的关节炎大鼠关节中浸润的T细胞TCR Vβ克隆型分析

目的：研究胶原诱导的关节炎（CIA）大鼠关节中局部浸润的T细胞TCR Vβ克隆型。方法：用Ⅱ型胶原（CⅡ）诱导Lewis大鼠发生关节炎后，经流式细胞术分析大鼠外周血CD4^＋和CD8^＋细胞的变化。提取CIA大鼠关节组织的总RNA，通过RT-PCR／SSCP方法分析大鼠两后肢足关节中TCR Vβ克隆型的一致率。将CIA大鼠脾淋巴细胞转移至同系正常大鼠，观察发病情况。结果：从注射CⅡ的第30天起，大鼠后爪出现红、肿等症状。流式细胞术分析显示，与正常大鼠相比较，CIA大鼠外周血CD8^＋T细胞的降低有显著性意义（P〈0．01），CD4^＋T／CD8^＋T的比值大于2（P〈0．01），提示CIA大鼠体内存在T淋巴细胞亚群的比例失调。CIA大鼠两后肢足关节中聚集的TCR Vβ克隆型的一致率，以TCR Vβ5．2和8．2最高，分别为78．89％和72．23％。脾淋巴细胞转移试验显示，受者发病的大鼠关节中，出现与供者CIA大鼠一致的TCR Vβ克隆型条带，表明关节局部聚集的TCR Vβ克隆型与关节炎（RA）的发病有关。结论：CIA大鼠关节局部存在以TCR Vβ5．2和8．2为主的致病性的T细胞TCR Vβ克隆型，可将其作为治疗CIA的靶物质，进行实验动物RA治疗的研究。     

The repertoires of circulating human CD8(+) central and effector memory T cell subsets are largely distinct

Memory T cells are divided into central and effector subsets with distinct functions and homing capabilities. We analyzed the composition and dynamics of the CD8(+) T cell repertoire of these subsets within the peripheral blood of four healthy individuals. Both subsets had largely distinct and autonomous TCRbeta repertoires. Their composition remained stable over a 9 month period, during which no cell passage between these subsets was detected despite important size variation of several clones. In one donor, four out of six TCRbeta clonotypes specific for the influenza A virus were detected in the central subset only, while the two others were shared. Altogether, these observations suggest that most effector memory T cells may not have derived from the central memory subset.     

Heterogeneity of the TCR Repertoire in Synovial Fluid T Lymphocytes Responding to BCG in a Patient with Early Rheumatoid Arthritis

T lymphocytes have been implicated in the pathogenesis of rheumatoid arthritis. Interestingly, many of the activated T cells isolated from the synovial fluid of individuals with rheumatoid arthritis react with antigens from Mycobacterium tuberculosis or BCG. This response is seen to a much lesser extent in the peripheral blood of these patients. To investigate the nature of the T-cell response to BCG in RA, we isolated T cells from the synovial fluid of a patient with early-stage rheumatoid arthritis, stimulated them with BCG and cloned by limiting dilution. Staining with monoclonal antibodies specific for different Vβ gene families revealed a statistically significant greater proportion of synovial-derived T-cell clones expressing the Vβ8 gene family product compared with peripheral blood clones. While the antigen specificity of some of the clones could not be determined, several of the clones displayed distinct antigen reactivities. Sequencing the TCR P chain genes of these T cells suggested that although the Vβ8 gene products appeared to be over-represented in these BCG-specific clones, each clone utilized distinct Jβ gene segments and used N segment addition to different extents. In addition, no common motifs were identified in the β chain CDR3s of the clones sequenced. Analysis of bulk cultured BCG-specific SF T cells and unstimulated peripheral blood T cells for Vβ8 gene expression also revealed a large amount of diversity within the CDR3 region. Thus, the T-lymphocyte response to BCG in this patient with early rheumatoid arthritis appears to be quite heterogeneous.     

Quantitative Assessment of T Cell Clonotypes in Human Acute Graft-versus-Host Disease Tissues

Owing to the difficulty in isolating T cells from human biopsy samples, the characteristics of T cells that are infiltratinghuman acute graft-versus-host disease (GVHD) tissues remain largely uninvestigated. In the present study, TCR-β deep sequencing of various GVHD tissue samples and concurrent peripheral blood obtained from transplant recipients was performed in combination with functional assays of tissue-infiltrating T cell clones. The T cell repertoire was more skewed in GVHD tissues than in the peripheral blood. The frequent clonotypes differed from tissue to tissue in the same patient, and the frequent clonotypes in the same tissue differed from patient to patient. Two T cell clones were successfully isolated from GVHD skin of a patient. In a cytotoxicity assay, both Tcell clones lysed patient peripheral blood mononuclear cells, but not donor-derived Epstein-Barr virus-transformed lymphoblastoid cells. Their clonotypes were identical to the most and second most frequent T cell clonotypes in the original GVHD skin and accounted for almost all of the skin-infiltrating T cells. These results suggest that human acute GVHD may result from only a few different alloreactive cytotoxic T cell clones, which differ from tissue to tissue and from patient to patient. The characterization of T cells infiltrating human GVHD tissues should be further investigated.     

T-cell receptor heterogeneity of gamma delta T-cell clones from human female reproductive tissues.

gamma delta T cells were isolated from human decidua parietalis, decidua basalis and cervix and cloned in the presence of interleukin-2 (IL-2). T-cell receptor (TcR) expression was then analysed and compared with that of a panel of gamma delta T-cell clones from peripheral blood. Only 17/40 (42.5%) clones from decidua parietalis were V gamma 9+/V delta 2+ as compared to 68/94 (72%) of peripheral blood clones (P < 0.005). Conversely, 50% of clones from decidua parietalis but only 15% of clones from peripheral blood were V delta 1+ (P < 0.001). At least seven distinct TcR types were identified among the panel of clones from decidua parietalis and at least six different types were expressed by the panel of 17 clones from cervix. This receptor heterogeneity was not a result of interdonor variation as in all instances where more than one clone was obtained from a single sample, individual clones having between two and five receptor types were identified. However, 23/24 (95.8%) of clones from decidua basalis were V gamma 9+/V delta 2+. Most clones from decidua parietalis and cervix, whether V gamma 9+/V delta 2+ or V delta 1+, were positive for the mucosal lymphocyte marker, HML-1, but expression was often heterogeneous within a single clone. In contrast, almost all gamma delta T-cell clones from peripheral blood were HML-1-. Thus, unlike the mouse, gamma delta T cells within these human female reproductive tissues have a diverse TcR repertoire which, in decidua parietalis, is distinct from that of peripheral blood.     

Functional heterogeneity among CD4+ T-cell clones from blood and skin lesions of leprosy patients. Identification of T-cell clones distinct from Th0, Th1 and Th2.

In the present study we examined the functional properties of T-cell clones reactive with Mycobacterium leprae and other mycobacterial antigens. Clones isolated from the skin lesions and blood of leprosy patients across the spectrum were exclusively CD4+CD8- and expressed the alpha beta T-cell receptor. Substantial heterogeneity in the production of cytokines, in particular interleukin-4 (IL-4), was observed, although no striking correlation with clinical status was apparent. A variety of patterns of cytokine secretion distinct from those of T-helper type-1 (Th1) Th2 or Th0, as defined in murine studies, was evident. Most noteworthy was a large number of clones from skin which secreted neither IL-2 nor IL-4, but large amounts of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Clones isolated from the blood of leprosy patients had a more restricted cytokine secretion profile, and appeared to resemble more closely previously described patterns, including those of high level production of IL-2 and/or IL-4. Virtually all clones, from either skin or blood, produced high levels of IFN-gamma, and thus many clones were IL-4 and IFN-gamma co-producers. The pattern of cytokine production by skin-derived T-cell clones was significantly affected by the in vitro activation status of the cells. Cells enriched in activated blasts tended to produce more IL-4 than small resting cells. In addition, the production of IFN-gamma by skin T-cell clones after < or = 10 weeks of culture was strikingly distinct from that of these clones after 5 months of culture. IL-4 and IFN-gamma co-producing clones shifted to a Th2-like pattern with much less IFN-gamma secretion, whereas non-IL-4-producing clones secreted much higher levels of IFN-gamma after prolonged culture, and became much more Th1-like. However, there was still no correlation between clinical status and pattern of cytokines produced. These results imply that a high fraction of T cells exists in leprosy lesions that is distinct from or that has not yet fully matured into Th1 or Th2 cells.     

Down-regulated donor-specific T-cell reactivity during successful tapering of immunosuppression after kidney transplantation

Stable cadaveric renal transplant patients were routinely converted from cyclosporin A (CsA) to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation to reduce the side effects of long-term immunosuppressive therapy. Thereafter, the AZA and MMF dose was gradually tapered to 50% at 2 years after transplantation. We questioned whether a reduction of immunosuppressive treatment results in a rise of donor-specific T-cell reactivity. Before transplantation (no immunosuppression), 1 year (high dose immunosuppression) and 2 years (low dose immunosuppression) after transplantation, the T-cell reactivity of peripheral blood mononuclear cells (PBMC) against donor and third-party spleen cells was tested in mixed lymphocyte cultures (MLC) and against tetanus toxoid (TET) to test the general immune response. We also measured the frequency of donor and third-party reactive helper (HTLpf) and cytotoxic (CTLpf) T-lymphocyte precursors in a limiting dilution assay. Donor-specific responses, calculated by relative responses (RR = donor/third-party reactivity), were determined. Comparing responses after transplantation during high dose immunosuppression with responses before transplantation (no immmunosuppression), the donor-specific MLC-RR (P = 0.04), HTLp-RR (P = 0.04) and CTLp-RR (P = 0.09) decreased, while the TET-reactivity did not change. Comparing the responses during low dose with high dose immunosuppression, no donor- specific differences were found in the MLC-RR, HTLp-RR and CTLp-RR, although TET-reactivity increased considerably (P = 0.0005). We observed a reduction in donor-specific T-cell reactivity in stable patients after renal transplantation during in vivo high dose immunosuppression. Tapering of the immunosuppressive load had no rebound effect on the donor-specific reactivity, while it allowed recovery of the response to nominal antigens.     

Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell clonality.

Single-stranded conformational polymorphism (SSCP) is often used for the diagnosis of T-cell clonality in lymphoproliferative disorders. We introduce a semireannealing SSCP (SR-SSCP) protocol that is rapid, reproducible, and effective. By denaturing and reannealing the polymerase chain reaction (PCR) product before high-resolution polyacrylamide gel electrophoresis, it is possible to generate a diagnostic fingerprint for each case with clonal T-cell receptor-gamma (TCR-gamma) gene rearrangement detected after PCR with TCR-gamma specific consensus primers. Discrete and distinct denatured single-stranded DNA band profiles characterize the rearranged TCR-gamma clones. In the same gel, the clone size may be estimated in the reannealed double-stranded PCR DNA and can be assessed down to the 2% clonal T-cell population level. Eighty-four cases, including 37 T-cell neoplasms, 29 B-cell neoplasms, and 18 reactive lymph node samples were analyzed by SR-SSCP. Clonal TCR-gamma rearrangement was diagnosed in 32 out of 37 T-cell neoplasms but in none of the B-cell tumors or reactive lymph node samples corresponding to sensitivity and specificity of 86.5% and 100%, respectively. We compare the results of SR-SSCP to those obtained by capillary electrophoresis and direct sequence analysis with 100% correlation. This novel method is applicable to any system for identification and quantitation of microheterogeneity in PCR products.