T cell receptor diversity in the human thymus

A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRα and TCRβ gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3 × 10⁶ unique TCRβ and 0.5 × 10⁶ TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3 × 10⁶ unique TCRβ sequences and 3.7 × 10⁶ TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70 × 10⁶ unique TCRβ sequences and 60 to 100 × 10⁶ TCRα sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times.     

Isolation of functionally active T cell receptor gamma delta-bearing lymphocytes from human peripheral blood

We have developed a simple method for isolation of functionally active T cell receptor (TCR)-gamma delta positive cells from human peripheral blood, using immunomagnetic separation techniques. After culture with feeder cells and interleukin-2, cells thus isolated showed a CD3+ TCR-alpha beta- phenotype, and stained with antibodies against the TCR-gamma delta complex. The TCR-gamma delta+ cells were functionally active, responding with DNA synthesis when stimulated via their CD3 and CD2 molecules in concert, or when interleukin-2 was added.     

Methods for comparing the diversity of samples of the T cell receptor repertoire

Analysis of T cell receptor (TCR) data has become a crucial element in many studies aimed at better understanding the evolution of the T cell repertoire and the role of TCR diversity in immune responses. In this paper we focus on comparing the diversity between samples of the TCR repertoire. We discuss some of the limitations and potential problems inherent in some of the more popular approaches to comparing samples of the TCR repertoire and we suggest alternate methods that both avoid these problems and enrich the analysis of TCR data. Examples from published studies of the CD8(+) T cell responses to the influenza A virus D(b)NP(366) and D(b)PA(224) epitopes in mice are used to demonstrate the implementation of these methods. One example involves a comparison between the central and effector memory T cell subsets, defined on the basis of CD62L expression, and the other examines changes in the TCR repertoire over time.     

Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor gamma/delta

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) gamma/delta were analyzed. Cell preparations highly enriched for TcR gamma/delta+ cells were obtained by treatment of E rosette-forming lymphocytes with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64-82% TcR gamma/delta+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR gamma/delta+ cell subsets in the peripheral blood. TcR gamma/delta cells were able to form conjugates with the natural killer-sensitive K-562 and with the natural killer-resistant HL-60-R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%-98% of the cells in the TcR gamma/delta+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR gamma/delta+ cells had electron-dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3- or CD3+ granular lymphocyte subsets. Freshly isolated TcR gamma/delta+ cells were unable to lyse K-562 and natural killer-resistant targets, such as HL-60-R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti-CD3 TcR mAb or by lectin. After short-term culture with interleukin 2, TcR gamma/delta+ cells acquired a strong cytolytic activity against K-562 and HL-60-R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti-CD3/TcR mAb.     

Phenotypical heterogeneity among human T cell receptor gamma/delta-expressing clones derived from peripheral blood

Human T cell clones expressing the T cell receptor (TcR) gamma/delta were isolated from peripheral blood lymphocytes of two unrelated donors. The TcR gamma/delta+ clones derived from one of these donors were all of the Ti gamma A+, delta-TCS1-, BB3+ phenotype indicating the exclusive use of the V gamma 9 and V delta 3 gene segments. In contrast, the T cell clones derived from the second donor were either Ti gamma A+, delta-TCS1-, BB3+:Ti gamma A-, delta-TCS1+, BB3- or Ti gamma A-, delta-TCS1-, BB3-. The delta-TCS1 determinant was expressed on both nondisulfide- and disulfide-linked TcR gamma/delta. Northern blot and DNA sequence analysis indicated that the Ti gamma A-, delta-TCS1-, BB3- clones do use the V delta 1 gene segment demonstrating that the delta-TCS1 monoclonal antibody does not react with all TcR gamma/delta using this particular gene segment. In contrast to the delta-TCS1+ T cell clones, the V delta 1+ delta-TCS1- T cell clones were found to express V delta 1 in conjunction with the J delta 3 gene segment suggesting that this particular V delta 1-J delta 3 combination is not recognized by the delta-TCS1 monoclonal antibody. In T cell clones derived from one individual the V delta 1 gene segment was found to be expressed with either J delta 1, J delta 2 or J delta 3. Heterogeneity among the 18 clones was detected with respect to the expression of the CD4, CD5 and CD8 antigens: one clone was CD4+, nine clones were CD5+ and two clones were CD8+. Thus, in this panel of clones, heterogeneity exists both with regard to CD antigen expression and the TcR gamma/delta phenotype. Also, our results indicate that the delta-TCS1 monoclonal antibody does not react with all TcR gamma/delta using the V delta 1 gene segment.     

T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes.

In the present study we have characterized the gamma/delta T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial V delta 1+ cells have a high expression of activation markers CD45R0 ("memory cells") and HLA-DR as compared to PB, indicating a preactivated population of V delta 1-carrying T cells in vivo and (b) interleukin 2-induced expansion of synovial cells yields a high proportion of gamma/delta in most samples expressing predominantly the V delta 1 TcR. Junctional sequence analysis of the TcR delta chain from interleukin 2-expanded PB cell lines demonstrated a polyclonal V delta 1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed V delta 1 genes was found. Altogether, our data demonstrate the presence of preactivated V delta 1-expressing cells in the synovial compartment. This V delta 1 population is predominantly polyclonal, except in one patient where oligoclonally expanded V delta 1 cells were detected.     

Non-random expression of T cell receptor gamma and delta variable gene segments in functional T lymphocyte clones from human peripheral blood

Human T cell receptor (TcR) gamma delta displays a variety of protein forms. Disulfide-linked (type 1) or non disulfide-linked (type 2) receptors occur, with gamma chains encoded by the C gamma 1 or the C gamma 2 gene segment, respectively. Exon 2 of C gamma 2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR gamma chains differ in molecular mass and charge between type 1 and type 2 receptors. The delta chains as well as the gamma chains have different structural properties between receptor types. This cannot be due to the use of different C delta gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in gamma/delta combinations, rearrangement and expression of V gamma, J gamma, C gamma and V delta gene segments were determined in TcR gamma/delta+ clones derived randomly from peripheral blood of normal donors. Most clones used C gamma 1, a minority C gamma 2. The different protein properties of receptor types could be explained by the non-random expression of V gamma (J gamma) and V delta gene segments. Type 1 receptors preferentially used gamma chains encoded by the V gamma 9 and J gamma 1.2 gene segments together with delta chains encoded by V delta 2. In type 2a receptors, V gamma 9 was not predominant; often other V gamma gene segments were employed, but then in high frequency in coordination with V delta 1. Reactivity of the clones with monoclonal antibodies anti-Ti gamma A, BB3 and delta-TCS-1 correlated with the expression of the V gamma 9, V delta 2 and V delta 1 gene segments, respectively. Therefore, V gamma and V delta use in TcR gamma/delta+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the V gamma 9-V delta 2 combination was predominant, while the V gamma 9-V delta 1 and particularly the V gamma 9-"V delta other" combination was rare. These data indicate that the TcR gamma delta repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.     

人外周血T淋巴细胞胀亡现象的观察

目的观察人外周血T淋巴细胞的胀亡现象,探讨建立T细胞胀亡检测方法.方法密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及地塞米松组,培养后观察细胞光镜、荧光镜及电镜形态学,并用流式细胞仪检测胀亡细胞比例变化.结果①人外周血T淋巴细胞经96小时体外培养,可自然出现典型细胞胀亡形态学改变.②经72小时培养,不同浓度地塞米松组(1×10-6、1×10-5、1×10-4、1×10-3 mol/L)T细胞的胀亡率分别为(3.49±0.42)%、(5.17±0.48)%、(8.44±0.72)%、17.93±1.50)%.③在1×10-5mol/L地塞米松作用下,不同培养时间(48、72、96、120小时)T淋巴细胞的胀亡率分别为(0.53±0.10)%、(6.36±0.80)%、(20.60±1.59)%、25.56±1.76)%.结论人外周血T淋巴细胞存在胀亡现象,地塞米松可诱导人外周血T淋巴细胞胀亡.     

人外周血T淋巴细胞自噬现象的观察

目的：观察分离培养的人外周血T淋巴细胞的自噬现象。方法：密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞，分空白组及地塞米松（DXM）组，培养72小时后观察细胞光镜及电镜形态学、MDC荧光染色，并用流式细胞仪检测自噬细胞比例变化。结果：①通过自然培养后人外周血T淋巴细胞可出现典型自噬细胞形态学改变。②空白组及DXM组72小时自噬细胞发生率与0小时比较有显著性差异。③DXM组与空白组72小时自噬细胞发生率有显著性差异。结论：人外周血T淋巴细胞存在自噬现象，DXM可诱导人外周血T淋巴细胞自噬。     

Differential estrogen receptor gene expression in human peripheral blood mononuclear cell populations

Estrogens have been shown to modulate immune responses. Several studies have demonstrated the capacity of T cells, B cells, and monocytes to respond to estrogens and estrogen receptor (ER) expression in these cell types has been reported. However, little is known regarding the relative expression in these cells of ERalpha and the more recently identified ERbeta. In the present study, results of quantitative TaqMan RT-PCR analyses indicate that ERs are differentially expressed in PBMC subsets. CD4+ T cells express relatively high levels of ERalpha mRNA compared with ERbeta, whereas B cells express high levels of ERbeta mRNA but low levels of ERalpha. Peripheral blood CD8+ T cells and monocytes express low but comparable levels of both ERs. This quantitative analysis of ER expression in distinct PBMC subsets may provide a basis for dissecting the mechanisms of immune modulation by estrogens and identifying therapeutic targets for the treatment of inflammatory and immunologic disorders.     

Circannual variations in the B cell/T cell ratio in normal human peripheral blood

In previous studies we have shown that B cells and subpopulations of T cells can be identified in blood smears with bacteria used as markers that bind spontaneously to lymphocytes. We have also identified Ig-bearing cells by using an Escherichia coli coated with anti-human Ig antibody. Here we determined the absolute values and the percentage of B cells and of other lymphocyte subpopulations in the peripheral blood of six normal donors every 2 mo for 1 yr. We found that the total leukocyte counts and the total number of lymphocytes remained unchanged throughout the year, whereas the percentage of B cells in the coldest month was at almost twice the level observed in summer. The percentage of cells that bind Arizona hinshawii and Salmonella schottmülleri remained practically unchanged during the entire year. A variation was noted in the ratio between T₁ and T₂ cells, which also appears to be seasonally related. We speculate that hormonal factors, probably corticosteroids, are responsible for changes in the traffic of some lymphocyte subpopulations.     

非霍奇金淋巴瘤患者T细胞亚群、NK细胞检测的临床意义

目的：研究非霍奇金淋巴瘤（NHL）患者外周血T淋巴细胞亚群、NK细胞检测结果的变化与该病的关系及与慢性淋巴腺炎患者细胞免疫功能的不同变化。方法：采用流式细胞仪（FCM）检测非霍奇金淋巴瘤（NHL）患者、慢性淋巴腺炎及正常人外周血T淋巴细胞亚群比例、NK细胞的变化。结果：非雹奇金淋巴瘤患者与正常人比较总的T淋巴细胞、辅助性T淋巴细胞及CD4^＋／CD8^＋比值明显下降（P〈0．05），细胞毒性T淋巴细胞明显升高（P〈0．05），NK细胞则无明显变化（P〉0．05）。非霍奇金淋巴瘤患者与慢性淋巴腺炎患者比较，细胞毒性T淋巴细胞、NK细胞明显升高（P〈0.05），而总的T淋巴细胞、辅助性T淋巴细胞无明显改变（P〉0．05），CD4^＋／CD8^＋比值略有下降但无明显统计学意义。结论：非霍奇金淋巴瘤患者细胞免疫功能明显受到抑制，T细胞亚群及NK细胞的检测对NHL的诊断、治疗、预后判断有一定的临床价值。     

Characterization of the interstitial lung and peripheral blood T cell receptor repertoire in cigarette smokers

T lymphocytes modulate the pulmonary inflammatory response. The aim of this study was to evaluate the clonality within the interstitial lung and peripheral blood T cell receptor (TCR) repertoire in smokers. Interstitial T lymphocytes were isolated from surplus tissue of 16 patients (63 +/- 9 [+/- SD] yr old, 11 male) undergoing surgery due to lung cancer (n = 15) or emphysema. TCR clonality was assessed by PCR amplification followed by spectratyping. Nearly all TCR of interstitial lung lymphocytes showed oligoclonal bands (CD4(+) subset 13/16 patients, 81%; CD8(+) 100%) indicating a specific differentiation. Peripheral blood T lymphocytes (PBL) TCR (especially CD4(+)) had less oligoclonal bands (CD4(+) 31%, CD8(+) 88%). Likewise, more oligoclonal bands were seen in lung TCR (total of 168 bands; 37 CD4(+); 131 CD8(+)), compared with 59 bands in PBL TCR (13 CD4(+); 46 CD8(+)). Intraindividual comparison revealed a more prominent difference in TCR oligoclonality between lung and blood in CD8(+) T cells (median of difference lung minus blood 5; interquartile range 1-10; P = 0.002) compared with CD4(+) T cells (median 2, 0-3, P = 0.039). Thus, TCR oligoclonality is preferentially found in the CD8(+) T cell subset, most distinctive in the lung. These findings indicate a specific interstitial T cell differentiation in response to local stimuli.     

Measurement of absolute T cell receptor rearrangement diversity

Anti-drug antibodies are elicited by virtually all therapeutic proteins, and standardized assays are required for clinical monitoring of patients as well as for comparing antibody response to different therapeutic proteins in clinical trials. Velaglucerase alfa and imiglucerase are enzyme replacement therapies for the long-term treatment of type 1 Gaucher disease, a lysosomal storage disease resulting from an inherited deficiency of the enzyme glucocerebrosidase. We used state-of-the-art tools to develop a panel of assays for detection and characterization of antibody responses to velaglucerase alfa and imiglucerase. Highly-sensitive, direct bridging electrochemiluminescence screening assays were developed using samples from treatment-na?ve individuals with type 1 Gaucher disease to set cut points. A mouse anti-glucocerebrosidase monoclonal antibody used as a calibrator was shown to have similar affinity and binding kinetics for anti-velaglucerase alfa and anti-imiglucerase antibodies. A quantitative radioimmunoprecipitation assay for IgG antibodies was developed to eliminate false-positives from the highly sensitive screening assay. Using 59 samples from treatment-na?ve individuals with type 1 Gaucher disease, the confirmatory cut points were calculated to be 1.42 ng/mL for anti-velaglucerase alfa antibodies and 3.23 ng/mL for anti-imiglucerase antibodies. Isotype-specific indirect electrochemiluminescence assays were developed for IgE, IgA, and IgM subclasses. The IgE subclass assay was shown to be more sensitive than the confirmatory assay using sheep anti-glucocerebrosidase polyclonal antibody cross-linked with fragments specific to human IgE, with cut points for anti-velaglucerase alfa or anti-imiglucerase antibodies determined to be 0.53 and 0.55 ng/mL, respectively. An assay that detects inhibition in vitro of velaglucerase alfa and imiglucerase hydrolysis of a synthetic substrate in the presence of antibodies was developed to test for neutralizing antibodies. Using 52 individual healthy human donor samples and 35 samples from treatment-na?ve individuals with type 1 Gaucher disease, cut points for the velaglucerase alfa and imiglucerase neutralizing antibody assays were determined to be 20%, such that a sample with greater than 20% inhibition of enzyme activity in the presence of antibodies was considered positive for neutralizing antibodies. In conclusion, highly sensitive and equivalent methods were developed and validated to directly compare antibody response to velaglucerase alfa and imiglucerase treatments in patients with Gaucher disease, and may contribute to future internationally standardized assays for antibody detection in patients with Gaucher disease.     

The human T cell receptor V beta repertoire of normal peripheral blood lymphocytes before and after mitogen stimulation.

Mitogen stimulation of T cells in vitro has been employed in the analysis of the T cell antigen receptor (TCR) repertoire and as a method of generating T cell lines and clones. It has been suspected for some time that mitogen stimulation may bias the repertoire. We have addressed this problem employing a semi-quantitative technique utilizing the polymerase chain reaction (PCR) and flow cytometry. Using this PCR method and a panel of primers to 22 V beta subgroups, the V beta repertoire of both unstimulated and phytohaemagglutinin (PHA)-stimulated peripheral T cells from eight healthy individuals was investigated. The samples were also analysed by flow cytometry using anti-V beta 2, V beta 5 and V beta 8 MoAbs. A significant increase in the expression of V beta 6, V beta 7.2 and V beta 10.1 was found in all eight samples of PHA-stimulated T cells compared with unstimulated T cells using the PCR method. In contrast, no differences were found between unstimulated and PHA-stimulated T cells by flow cytometry. These results question the validity of using mitogen-stimulated T cells to investigate TCR gene usage.     

T cell development and receptor diversity during aging

The T cell system is a complex and highly dynamic system that is amazingly robust over many decades of human life. Its functional competence is determined not only by its size but also by its diversity. Homeostatic control mechanisms have to secure sufficient T cell replenishment while preventing loss of clonal diversity. Major homeostatic challenges include profound expansion and shrinkage of T cell clonotypes upon antigenic triggering and, more importantly, age-related changes in T cell regeneration. The ability of the thymus to rebuild a diverse repertoire ceases in the fifth decade of life. Emerging data suggest that the end of the 7th decade of life defines a critical time period when T cell homeostasis is no longer guaranteed and diversity of the na?ve T-cell repertoire collapses. Thus, failure of T cell homeostasis appears to result from cumulative defects of T cell generation. Elucidation of the underlying mechanisms will allow for extending this turning point to later in life; ultimately, interventions have to aim at restoring thymic function and complementary modes of T cell reconstitution.