Developmental regulation of alpha beta T cell antigen receptor expression results from differential stability of nascent TCR alpha proteins within the endoplasmic reticulum of immature and mature T cells.

The alpha beta T cell antigen receptor (TCR) that is expressed on most T lymphocytes is a multisubunit transmembrane complex composed of at least six different proteins (alpha, beta, gamma, delta, epsilon and zeta) that are assembled in the endoplasmic reticulum (ER) and then transported to the plasma membrane. Expression of the TCR complex is quantitatively regulated during T cell development, with immature CD4+CD8+ thymocytes expressing only 10% of the number of surface alpha beta TCR complexes that are expressed on mature T cells. However, the molecular basis for low TCR expression in developing alpha beta T cells is unknown. In the present study we report the unexpected finding that assembly of nascent component chains into complete TCR alpha beta complexes is severely impaired in immature CD4+CD8+ thymocytes relative to their mature T cell progeny. In particular, the initial association of TCR alpha with TCR beta proteins, which occurs relatively efficiently in mature T cells, is markedly inefficient in immature CD4+CD8+ thymocytes, even for a matched pair of transgenic TCR alpha and TCR beta proteins. Inefficient formation of TCR alpha beta heterodimers in immature CD4+CD8+ thymocytes was found to result from the unique instability of nascent TCR alpha proteins within the ER of immature CD4+CD8+ thymocytes, with nascent TCR alpha proteins having a median survival time of only 15 min in CD4+CD8+ thymocytes, but > 75 min in mature T cells. Thus, these data demonstrate that stability of TCR alpha proteins within the ER is developmentally regulated and provide a molecular basis for quantitative differences in alpha beta TCR expression on immature and mature T cells. In addition, these results provide the first example of a receptor complex whose expression is quantitatively regulated during development by post-translational limitations on receptor assembly.     

Susceptibility to cell death is a dominant phenotype: triggering of activation-driven T-cell death independent of the T-cell antigen receptor complex.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.     

Activation of immature cortical thymocytes through the T11 sheep erythrocyte binding protein

Two major pathways, the T cell receptor and the T11 alternate pathway, allow for T cell activation. In the human thymus, the T cell antigen receptor complex is reduced or absent on immature thymocytes, whereas the T11 glycoprotein is present at high cell surface density on all thymocytes. To determine whether activation through the T11 pathway induces similar or different changes in mature and immature thymocytes, we fractionated thymocytes according to their surface expression of the T3-T cell receptor (T3/Ti) complex. We report that two populations, one with high and one with low T3/Ti expression, can be activated through the T11 pathway to undergo nuclear activation and express IL 2 receptors. Moreover, in the absence of accessory cells, only the most mature population, expressing high T3 density, could be induced to proliferate, whereas the subset representing immature cortical thymocytes required accessory cells for proliferation. These findings suggest that the cellular microenvironment may have a critical role in regulating the activation of immature cortical thymocytes and that this cell population may not represent "nonfunctional" dead end cells, but rather a valid intermediate in human thymic differentiation.     

Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.     

Thymocyte sensitivity and supramolecular activation cluster formation are developmentally regulated: a partial role for sialylation

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca(2+) flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.     

Differential responsiveness of immature- and mature-stage murine B cells to anti-IgM reflects both FcR-dependent and -independent mechanisms.

Mature and immature B cells differ in their responses to antigen receptor crosslinking. Whereas mature B cells enter cell cycle in response to such stimulation, immature B cells exhibit proliferative unresponsiveness and undergo induced tolerance following surface immunoglobulin (sIg) engagement. Previous studies evaluating antigen receptor-mediated negative signaling have utilized intact goat anti-immunoglobulin (anti-Ig) antibodies as polyclonal ligands based upon observations that the Fc portion of these reagents does not interact with and mediate negative signaling through the FcR on mature B cells. Thus, the negative effects of goat anti-Ig on immature B cells have been attributed solely to signals mediated via their antigen receptors. In the studies reported here we show that the activation unresponsiveness inherent to immature B cells is FcR independent. However, we also show that immature B cells are sensitive to FcR-mediated inhibition and that these effects can be mediated by intact goat antibodies at concentrations that promote positive activation signals in mature B cells. Our results demonstrate that inhibition of immature B cell LPS responses by anti-Ig antibodies, used in previous studies as an in vitro model for B cell tolerance induction, is an FcR-mediated phenomenon. We show that developmentally associated anti-Ig-mediated inhibition of LPS requires the use of intact antibodies, and that this inhibition can be blocked by the anti-FcR monoclonal antibody 2.4G2. Flow cytometric analysis of FcR-positive B cells indicates that both mature and immature B cells express equivalent levels of FcR gamma. Therefore, the sensitivity of immature, but not mature, cells to intact goat anti-mu antibodies suggests that either FcRs or their associated inhibitory pathways change during B cell development.     

MicroRNA与变应性鼻炎

在变应性鼻炎(allergic rhinitis,AR)发病的免疫细胞分化及免疫应答过程中,micro RNA(miRNA)发挥着非常重要的调控作用。本文旨在对AR中miRNA研究进行回顾,以期为AR的有效治疗提供新的视角。首先,变应性鼻炎患者鼻粘膜中miR-7和miRPlus-E1194等差异表达。其次,在T辅助细胞发育、分化与活化过程中,miR-181a、miR-155、miR-21、miR-1、miR-31、miR-223、miR-139-3p、miR-126、Let-7家族成员会发生显著上调或下降,而且某些miRNA的相关作用靶点也得到验证。再次,嗜酸粒细胞定向分化祖细胞向成熟嗜酸粒细胞的分化过程中,miR-21和miR-223都有所上调。最后,在肥大细胞激活和脱颗粒过程中,miR-221和miR-222以及其他miRNA显著上调。以上miRNA参与了T辅助细胞分化与活化、嗜酸粒细胞发育、肥大细胞脱颗粒等AR病理进程的各个环节,而且,miRNA还会参与前期的抗原提呈等环节。可见miRNA对AR调控呈现出复杂性和多重性。     

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.     

miRNA profiling of naïve, effector and memory CD8 T cells

microRNAs have recently emerged as master regulators of gene expression during development and cell differentiation. Although profound changes in gene expression also occur during antigen-induced T cell differentiation, the role of miRNAs in the process is not known. We compared the miRNA expression profiles between antigen-specific na?ve, effector and memory CD8+ T cells using 3 different methods--small RNA cloning, miRNA microarray analysis and real-time PCR. Although many miRNAs were expressed in all the T cell subsets, the frequency of 7 miRNAs (miR-16, miR-21, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) alone accounted for approximately 60% of all miRNAs, and their expression was several fold higher than the other expressed miRNAs. Global downregulation of miRNAs (including 6/7 dominantly expressed miRNAs) was observed in effector T cells compared to na?ve cells and the miRNA expression levels tended to come back up in memory T cells. However, a few miRNAs, notably miR-21 were higher in effector and memory T cells compared to na?ve T cells. These results suggest that concomitant with profound changes in gene expression, miRNA profile also changes dynamically during T cell differentiation. Sequence analysis of the cloned mature miRNAs revealed an extensive degree of end polymorphism. While 3'end polymorphisms dominated, heterogeneity at both ends, resembling drosha/dicer processing shift was also seen in miR-142, suggesting a possible novel mechanism to generate new miRNA and/or to diversify miRNA target selection. Overall, our results suggest that dynamic changes in the expression of miRNAs may be important for the regulation of gene expression during antigen-induced T cell differentiation. Our study also suggests possible novel mechanisms for miRNA biogenesis and function.     

miR-362靶向ZNF644基因调控猪未成熟支持细胞的增殖和凋亡

睾丸组织中未成熟支持细胞的增殖能力决定成熟支持细胞的数量,进而制约成年雄性动物的精子生成能力。研究表明microRNA (miRNA)参与调控猪未成熟支持细胞的增殖和凋亡,但大部分鉴定出的miRNA功能仍不明确。本文基于前期RNA-seq数据筛选结果,研究了miR-362对猪未成熟支持细胞增殖和凋亡的调控作用。首先利用生物信息学方法预测miR-362的靶基因,通过qRT-PCR技术检测miR-362和ZNF644基因在不同发育阶段的猪睾丸组织中的表达水平以及在猪未成熟支持细胞中过表达或抑制表达miR-362后ZNF644基因的表达水平,采用双荧光素酶报告基因系统验证miR-362与ZNF644基因之间的靶向关系。结果显示,miR-362与ZNF644基因3′UTR具有一个潜在的结合位点,miR-362和ZNF644基因在猪睾丸组织中的mRNA表达水平显著负相关(r=-0.723, P<0.01),miR-362和psiCHECK2-ZNF644-WT 3′UTR共转染组的双荧光活性显著降低,且miR-362显著调节ZNF644基因的表达水平,表明miR-362靶向ZNF644基因并抑制其表达水平。为进一步检测过表达miR-362或抑制表达ZNF644基因对猪未成熟支持细胞增殖和凋亡的影响,通过流式细胞术检测细胞周期,CCK8和EdU试剂盒检测细胞增殖情况,Annexin V-FITC/PI方法和qRT-PCR技术检测细胞凋亡情况及凋亡相关基因的表达水平。结果表明,过表达miR-362后,猪未成熟支持细胞周期被阻滞在G₁期,抑制表达ZNF644基因后,猪未成熟支持细胞被阻滞在G₂期,细胞增殖能力显著减弱,细胞凋亡率显著提高,细胞凋亡相关基因呈促进凋亡的差异表达。本研究结果证实miR-362靶向ZNF644基因抑制猪未成熟支持细胞的增殖而促进其凋亡,为深入研究miR-362在猪精子生成过程中的生物学功能提供了理论基础。     

BEN, a novel surface molecule of the immunoglobulin superfamily on avian hemopoietic progenitor cells shared with neural cells.

BEN is a novel molecule of the immunoglobulin superfamily that we previously identified by means of a monoclonal antibody on neural cell populations during avian development and epithelial cells of the bursa of Fabricius. In this paper, we describe the expression of BEN by hemopoietic cells during ontogeny. In the thymus, BEN is expressed as early as E9, and from E12 until just after hatching 30-60% of thymocytes are BEN positive. Thus the cells expressing BEN are immature thymocytes and not yet differentiated T cells. In the spleen, BEN expression parallels the myelopoietic activity. It is present on 75% of splenocytes during embryonic development and falls rapidly to 20% of cells during the first week after hatching when the spleen is becoming a secondary lymphoid organ. BEN is also found on a large proportion (about 80% positive cells) of bone marrow cells during ontogeny. Post hatching, BEN is present on 40-50% of bone marrow cells. The population of BEN-positive cells in the bone marrow includes myeloid and erythroid progenitor cells, identified by their ability to form colonies in vitro. BEN expression is lost as progenitor cells proliferate and differentiate to develop mature colonies in the clonal assay. Mature myeloid cells, such as macrophages, granulocytes, thrombocytes, and erythrocytes do not express the BEN antigen. Taken together, these data demonstrated that BEN is a stage-specific rather than a lineage-specific differentiation antigen expressed by immature hemopoietic cells.     

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a "mature erythrocyte" antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of "mature erythrocyte" antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long periods of time express intermediate levels of MHC antigens, suggesting a "leaky" expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.     

Differential post-translational modification of CD63 molecules during maturation of human dendritic cells.

The capacity of dendritic cells to initiate T cell responses is related to their ability to redistribute MHC class II molecules from the intracellular MHC class II compartments to the cell surface. This redistribution occurs during dendritic cell development as they are converted from an antigen capturing, immature dendritic cell into an MHC class II-peptide presenting mature dendritic cell. During this maturation, antigen uptake and processing are down-regulated and peptide-loaded class II complexes become expressed in a stable manner on the cell surface. Here we report that the tetraspanin CD63, that associates with intracellularly localized MHC class II molecules in immature dendritic cells, was modified post-translationally by poly N-acetyl lactosamine addition during maturation. This modification of CD63 was accompanied by a change in morphology of MHC class II compartments from typical multivesicular organelles to structures containing densely packed lipid moieties. Post-translational modification of CD63 may be involved in the functional and morphological changes of MHC class II compartments that occur during dendritic cell maturation.     

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.