Decreasing the threshold for thymocyte activation biases CD4+ T cells toward a regulatory (CD4+CD25+) lineage

Thymus-derived CD4(+)CD25(+) regulatory T (T(r)) cells play a critical role in suppressing aberrant responses to self in vivo. The factors that influence a CD4(+) T cell's decision to commit to an immunoregulatory T(r) cell lineage are currently unknown. In the present study, we found that in mice, abundantly expressing a few or one peptide(s) bound to MHC class II molecules, a large portion of conventional CD4(+) T cells could be biased towards the commitment to a T(r) lineage by reducing the threshold required for thymocyte activation. This occurred in the presence of either an antisense glucocorticoid receptor transgene or a pharmacological inhibitor of glucocorticoid synthesis. These results demonstrate a novel in vivo pathway for the generation of T(r) cells, and raise the possibility that therapeutic enhancement of the T(r) cell repertoire through pharmacological manipulation of TCR signaling thresholds may provide a feasible means of ameliorating autoimmunity.     

Sox-2基因表达的变化在小鼠EST模型药物胚胎毒性评价中的应用

目的: 建立小鼠胚胎干细胞实验 (EST) 模型,验证该模型检测胚胎毒性的有效性,探讨未分化基因 Sox-2 表达水平的变化对药物胚胎毒性评价的作用。方法: 体外培养小鼠胚胎干细胞(ES),通过形态学观察、核型分析和碱性磷酸酶(AKP)染色等方法鉴定ES;MTT法检测5-氟尿嘧啶(5-FU)、苯妥英钠(DPH)和青霉素G(penicillin G)对ES的毒性作用,RT-PCR半定量分析法检测3种药物对未分化基因 Sox-2 表达的影响。结果: 成功建立EST 模型。5-FU、DPH和penicillin G细胞毒性检测结果表明,其胚胎毒性依次为强、弱和无,与临床药物毒性相一致。 Sox-2 基因表达结果显示,随药物浓度增高及毒性增加, Sox-2 基因表达量逐渐高于阴性对照组,但均低于未分化ES细胞。结论: 研究结果显示, Sox-2 基因的表达水平与药物毒性密切相关,可用于初步评价EST模型中药物的胚胎毒性。     

Human thymocyte development in mouse organ cultures

A novel system to study human thymocyte development is described in which embryonic mouse thymic rudiments are seeded with human precursor cells in vitro. In these cultures human thymocytes proliferate extensively (greater than 20-fold increase in cell number) and mature, as evidenced by the accumulation of double and single positive (CD4+ and/or CD8+) cells. Data presented here suggest that the survival and ordered development of the mature human thymocytes in chimeric thymuses is dependent on human stromal elements. Immature CD4-CD8- human thymocytes failed to colonize or minimally recolonized mouse thymic lobes unless provided with high density (greater than 1.077 g/ml) human thymic cell fractions. These fractions contain multicellular complexes of epithelial/nurse cells, thymocytes, and dendritic cells/macrophages which dramatically enhanced the recolonizing capacity of purified CD4-CD8- thymocytes. The chimeric organ culture system described here provides not only a new approach for studying human T cell ontogeny but also a direct means for the future dissection of stromal interactions necessary for successful transition of precursor cells (CD4-CD8-) to immature double positive (CD4+CD8+) and mature single positive cells (CD4+ or CD8+) in the thymus.     

Tumor necrosis factor (TNF) receptor type 2 mediates thymocyte proliferation independently of TNF receptor type 1

Tumor necrosis factor (TNF) mediates its biological effects by binding to two distinct but homologous receptor molecules. The type 1 receptor (TNF-R1) has been shown to be essential and sufficient for most cellular responses to soluble TNF. In contrast, only limited data exist concerning the role of the type 2 receptor (TNF-R2) in TNF responses, both in vitro and in vivo. Here, we demonstrate by the use of thymocytes from TNF-R-deficient mice that the TNF-R2-dependent enhancement of proliferation and secretion of granulocyte-macrophage colony-stimulating factor is in fact mediated by TNF-R2 on its own, independent of co-expression and/or stimulation of TNF-R1.     

Peptide antagonists that promote positive selection are inefficient at T cell activation and thymocyte deletion

We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I-restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen-driven deletion in single-cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the K^b-restricted peptide OVA_257-264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with K^b. OVA_257-264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double-positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse. In the absence of additional antigen-presenting cells, single-cell thymocyte suspensions showed that the specificity for double-positive thymocyte deletion and mature T cell activation was virtually identical, demonstrating a limited cross-reactivity with a number of variants having conservative substitutions at these exposed residues. These peptides were considerably more efficient at both thymic deletion and mature T cell activation than a number of non-conservative substitution analogs known to act as antagonists of OVA_257-264 and capable of selecting transgenic T cells in thymic organ culture. Therefore, both peripheral T cell activation and thymic deletion have an overall similar pattern of peptide specificity which differs from that required for positive selection. This suggests that a subset of major histocompatibility complex-presented peptides could promote positive selection without causing either thymic deletion or peripheral activation of those selected T cells.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides

The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an αβ TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2D^b MHC molecule. Both peptides were shown to antagonize specifically the T cell cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands.     

T cell subset distribution and B cell hyperreactivity in mice expressing interleukin-4 under the control of major histocompatibility complex class I regulatory sequences

Transgenic mice in which interleukin-4 (IL-4) is expressed under the control of the major histocompatibility complex (MHC) class I regulatory sequences show low level expression of IL-4 in all organs investigated. Several weeks after birth the animals develop thymus hypoplasia with a loss of CD4⁺CD8⁺ double-positive cells and a relative increase in the mature population, especially, and in contrast to previously published lines, the CD4⁺ single-positive cells. In the periphery, T lymphocytes eventually decline, CD8⁺ cells being more strongly affected. Many of the residual T cells exhibit the CD44^highMel-14^low phenotype of antigenically experienced T cells. B cells also show an activated phenotype with respect to size, MHC class II and CD23 expression, are more readily stimulated by anti-μ F(ab′)₂ antibodies than are B cells from control littermates, and show a higher spontaneous and antigen-induced production of IgG1 and IgE.     

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1^−/−, TCRβ^−/−, and p56^lck−/− mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRβ and p56^lck gene mutations but partially decreased in RAG-1^−/− mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRβ chain. Genetic blocks in CD8 lineage commitment (CD8^−/− and IFN regulatory factor-1^−/− mice) and positive and negative T cell selection (CD45^−/−, TCRα^−/−, and CD30^−/− mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4^−/− mice, but not MHC class II^−/− mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4^−/− block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.     

尼古丁对卵蛋白致敏大鼠CD4+T细胞Th1/Th2细胞因子和T-bet/GATA-3表达的影响

目的 研究尼古丁对卵白蛋白致敏大鼠CD4+T淋巴细胞Th1/Th2平衡的影响.方法 卵清白蛋白(OVA)腹腔注射致敏Wistar大鼠,CD4+T淋巴细胞纯化柱分离大鼠脾脏CD4+T细胞,体外培养,将细胞随机分为4组:对照组、1 μg/ml尼古丁组、10 μg/ml尼古丁组、100μg/ml尼古丁组(各组加等浓度的OVA),不同浓度尼古丁刺激24 h,酶联免疫吸附试验(ELISA)检测细胞培养上清液IFN-γ和IL-4含量,实时荧光定量PCR检测培养细胞T-bet和GATA-3 mRNA的表达.结果 (1)不同尼古丁干预组IFN-γ的表达分别为(113.78±6.06) ng/L、(70.31±7.26) ng/L、( 20.00±2.14)ng/L,均较对照组[(142.30±5.89) ng/L]明显减少,差异有统计学意义(F=265.52,P＜0.01);不同尼古丁干预组IL-4的表达分别为(50.97±3.07) ng/L、(69.49±3.91) ng/L、( 93.63±4.56)ng/L,均较对照组[ (36.91±3.24) ng/L]明显增加,差异有统计学意义(F=128.67,P＜0.01).(2)不同尼古丁干预组T-bet mRNA分别为0.73±0.03、0.57±0.04、0.31 ±0.00,均较对照组(0.98±0.09)明显减少,差异有统计学意义(F=75.76,P＜0.01);不同尼古丁干预组GATA-3 mRNA的表达分别为4.31±0.26、5.16±0.23、1.56±0.14,均较对照组(1.00±0.07)明显增加,差异有统计学意义(F=348.41,P＜0.01).结论 尼古丁可能通过促进转录因子GATA-3 mRNA的表达同时抑制T-bet mRNA的表达,在哮喘Th2过敏性气道炎症中具有一定的作用.     

Cdk2 activation acts upstream of the mitochondrion during glucocorticoid induced thymocyte apoptosis

Thymocytes undergo apoptosis during negative selection in vivo and following treatment with glucocorticoids or DNA-damaging drugs in vitro. The post-mitochondrial biochemical steps leading to apoptosis induced by these stimuli are well characterized, however, much less is known about the pathways connecting receptor triggering, apical caspase activation and induction of mitochondrial dysfunction. These stimuli specifically activate the kinase Cdk2 and this step is obligatory for these forms of thymocyte apoptosis. We report here that Cdk2 activation is a very early step during thymocyte apoptosis preceding apical caspase activation and phosphatidylserine exposure. Furthermore, Cdk2 activation is required for mitochondrial permeability disruption, cytochrome c release and, as a consequence, activation of the downstream caspases 9 and 3. Our data allow an integrated linear pathway regulating DNA damage and glucocorticoid-induced thymocyte apoptosis to be proposed.     

Effect of reduced EPHB4 expression in thymic epithelial cells on thymocyte development and peripheral T cell function

The Eph kinase (EPH) and ephrin (EFN) families are involved in a broad range of developmental processes. Increasing evidence is demonstrating the important roles of EPHBs and EphrinBs in the immune system. In this study on epithelial cell-specific Ephb4 knockout (KO) mice, we investigated T-cell development and function after EPHB4 deletion. KO mice presented normal thymic weight and cellularity. Their thymocyte subpopulation percentages were in the normal range. KO mice had normal T-cell numbers and percentages in the spleen, and T cells were activated and proliferated normally upon TCR ligation. Furthermore, naïve spleen CD4 cells from KO and wild type mice were capable of differentiating, in a comparable manner, into Th1, Th17 and Treg cells. In vivo, KO mice mounted effective delayed type hypersensitivity responses, indicating that thymocytes develop normally in the absence of TEC EPHB4, and T cells derived from EPHB4-deleted thymic epithelian cells (TEC) have normal function. Our data suggest that heavy redundancy and promiscuous interaction between EPHs and EFNs compensate for the missing EPHB4 in TECs, and TEC EPHB4's role in T cell development might only be revealed if multiple EPHs are ablated simultaneously. We cannot exclude the possibility that (1) some immunological parameters not examined in this study are affected by the deletion; (2) the deletion is not complete due to the leaky Cre-LoxP system, and the remaining EPHB4 in TEC is sufficient for thymocyte development; or (3) EPHB4 expression in TEC is not required for T cell development and function.     

Transgenic expression of a CD83-immunoglobulin fusion protein impairs the development of immune-competent CD4-positive T cells

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell response. CD83 deficiency leads to a block in the thymic maturation of CD4-positive T cells, and interference with peripheral CD83/CD83 ligand interaction by addition of soluble CD83 suppresses immune responses in vivo and in vitro. Here we report the generation of a mouse transgenic for a fusion protein consisting of the extracellular domain of murine CD83 fused to the constant part of human IgG1 heavy chain. Thymic selection of CD4-positive T cells was unchanged in CD83Ig transgenic and in CD83Ig/OT-2 double-transgenic mice. However, thymic and peripheral CD4-positive T cells derived from CD83Ig/OT-2 transgenic mice displayed a reduced cytokine response to antigenic stimulation in vitro, whereas CD83Ig/OT-1-derived CD8-positive T cells showed normal cytokine secretion. The T cell defect was relevant in vivo, since a sub-lethal infection with Trypanosoma cruzi led to an increased parasitemia and reduced survival rate of CD83Ig transgenic mice compared to wild-type C57BL/6 mice. In contrast, in vivo application of recombinant CD83Ig did not result in an increase in parasitemia. Taken together our data suggest that thymic selection in the presence of CD83Ig leads to an intrinsic T cell defect of CD4-positive T cells resembling the phenotype described for CD4-positive T cells derived from CD83-deficient mouse strains.     

Interleukin-4 induces expression of the CD45RA antigen on human thymocyte subpopulations

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)