Development of reagents to study the turkey's immune response: Identification and molecular cloning of turkey CD4, CD8α and CD28

The cDNAs of three turkey CD markers, CD4, CD8α and CD28, were identified by screening a turkey cDNA library. The coding regions of the chicken and turkey genes are highly conserved, with 91.3–96.1% nucleotide (nt) and 84.2–95.5% amino acid (aa) identity. Identity was less conserved between avian CD markers and their mammalian homologues, ranging from 44.7 to 59.8% and 22.4 to 50.4% at the nt and aa levels, respectively. Anti-chicken CD8α and CD28 monoclonal antibodies were demonstrated to specifically cross-react with turkey CD8α and CD28, respectively.     

The Role of CD40–CD154 Interactions in Autoimmunity and the Benefit of Disrupting this Pathway

Many tissue injuries and immune mediated pathologies such as graft allo-rejections were found to involve CD40–CD40 ligand (CD40L, CD154) signaling pathway. The disruption of this pathway in many animal models led to the improvement of graft survival in these models. CD40–CD154 interactions were also shown to play a significant role in the maintenance of autoimmunity, and the production of auto-antibodies in systemic lupus erythematosus (SLE). High-level expression of CD154 has been detected on T cells from patients with active SLE, rheumatoid arthritis (RA) and other autoimmune diseases, indicating that such cells could account for the high-level expression of immune accessory molecules on B cells of patients with active disease. An increased serum level of soluble CD154 was also reported in SLE, RA, and Sjogren's disease in correlation with the relevant auto-antibodies and with the clinical disease activity.

Anti-CD154 antibody therapy prevents auto-antibody production and renal immune complex deposition in lupus nephritis, indicating that disruption of this pathway could be a beneficial treatment in SLE. However, the etiology of the higher than expected number of thromboembolic events in anti-CD154 treated SLE patients should be investigated and preventive measures should be considered.     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.     

CD27: marker and mediator of T-cell activation?

CD27 is a lymphocyte-specific member of the tumour necrosis factor receptor (TNF-R) family, expression of which is tightly regulated during T-cell ontogeny. Recently, the ligand for CD27 was identified and was shown to be identical to CD70, a novel member of the TNF family. Functional experiments show that the interaction between CD27 and its ligand generates a co-stimulatory signal for T-cell activation. Here, Rogier Hintzen and colleagues integrate the phenotypic and functional data available on CD27 and its ligand, and propose a role for CD27 in the amplification of T-cell responses.     

Generation and Regulation of CD8＋ Regulatory T Cells

Research into the suppressive activity of CD4＋FoxP3＋ T regulatory cells （Treg） has defined a sublineage of CD4＋ cells that contribute to self-tolerance and resistance to autoimmune disease. Much less attention has been given to the potential contribution of regulatory sublineages of CD8＋ cells. Analysis of a small fraction of CD8＋ cells that target autoreactive CD4＋ cells through recognition of the MHC class Ib molecule Qa-1 in mouse and HLA-E in human has revitalized interest in CD8＋ Treg. Here we summarize recent progress and future directions of research into the role of this CD8＋ sublineage in resistance to autoimmune disease. Cellular ＆ Molecular Immunology. 2008; 5（6）：401-406.     

A CD40–CD95L fusion protein interferes with CD40L-induced prosurvival signaling and allows membrane CD40L-restricted activation of CD95

We analyzed a novel bifunctional fusion protein, CD40ed–CD95Led, consisting amino-terminally of the extracellular domain of CD40 and carboxy-terminally of the extracellular domain of CD95L. On cells lacking CD40L, this fusion protein is poorly active with respect to CD95 activation [median effective dose (ED50)>1 μg/ml], but it stimulates CD95 signaling with high efficiency upon binding to membrane-expressed CD40L (ED50<1 ng/ml). Thus, cell surface immobilization mediated by the CD40 part of the molecule unmasks the high-latent, CD95-stimulating capacity of the otherwise poorly active CD95L fusion protein. Moreover, interaction of the CD40 part of CD40ed–CD95Led with CD40L prevents the activation of cellular CD40. The CD40ed–CD95Led fusion protein therefore simultaneously blocks antiapoptotic CD40 activation and induces CD95-mediated apoptosis. Indeed, T47D cells displaying an antiapoptotic autocrine CD40–CD40L signaling loop were significantly more sensitive toward CD40ed–CD95Led than toward soluble CD95L artificially activated by crosslinking. Fusion proteins of RANK and CD95L (RANKed–CD95Led) and CD40 and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (CD40ed–TRAILed), with domain architectures similar to CD40ed–Cd95Led, displayed RANKL-dependent CD95 and CD40L-dependent TRAILR2 activation, respectively, indicating the principle feasibility of this fusion protein design.Klaus Pfizenmaier and Harald Wajant contributed equally to this work.     

Effects of PGE2 upon differentiation and programmed cell death of suspension cultured CD4− CD8− thymocytes

Recently, several works have focused on the modulation of the immune response by arachidonic acid metabolites. Some of these metabolites, such as prostaglandins, have been shown to influence thymocyte “education” in vitro. However, the effect of one of them, prostaglandin E₂ (PGE₂), in the education of CD4⁻ CD8⁻ double negative immature thymocytes (DN cells) remained unclear. Using a flow cytometry analysis of DN cells cultured for 24 h in the presence of PGE₂, we observed, compared with DN thymocytes cultured without PGE₂, an increase in the CD4⁺ CD8⁻ CD3⁻ immature thymocytes and in the CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature single positive thymocytes and a decrease in the DN and CD4^high CD8^high double positive thymocytes. Other differentiation thymocyte surface markers, such as CD3, CD5, TCRαß, TCRδγ and HSAg, revealed an increasing number of thymocytes bearing these first four markers and a lower expression of the HSAg. Furthermore, we observed an accumulation of CD4^low CD8^low thymocytes and an increasing proportion of hypodiploid nuclei. These two findings have been shown to be markers of the programmed cell death process. These findings suggest that PGE₂ probably acts on thymocyte differentiation through at least two distinct pathways. On the one hand, PGE₂ seems to promote differentiation of DN cells into CD4⁺ CD8⁻ CD3⁻ immature cells and drive CD4⁺ CD8⁺ CD3⁺ thymocyte to a CD4⁺ CD8⁻ and CD8⁺ CD4⁻ mature phenotype. On the other hand, PGE₂ is probably implicated directly or indirectly in the increase or the acceleration of the programmed cell death process of immature CD4⁺ CD8⁺ CD3⁺ thymocytes, which is linked to the positive and/or negative selection.     

Post-Natal Ontogenesis of the T-Cell Receptor CD4 and CD8 Vβ Repertoire and Immune Function in Children with DiGeorge Syndrome

DiGeorge syndrome (DGS) is a congenital disorder characterized by typical facial features, hypoparatyroidism, conotruncal cardiac defects and thymic hypoplasia. Although there are some reports addressing lymphocytes counts and function in DGS children over time, few data have been reported on the T-cell receptor Vβ (TCRBV) repertoire in relation to disease progression. The aim of this study was to evaluate the degree and nature of immunodeficiency and to investigate a possible correlation to clinical findings.We used third complementary region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire diversity in 7 DGS’s children. The rate of thymic output, the phenotype and function of peripheral T-cells and the humoral immunity were also investigated. At baseline a profound alteration of the TCR repertoire was noted, mainly in the CD8+ T-cells, in DGS patients when compared to a control group. Furthermore, analysis of thymic output showed a significant decrease in TCR rearrangement excision circles (TRECs) levels in the patient group. Immunoglobulin abnormalities were also detected. The observed TCR repertoire alterations, although not statistically significant, may suggest an increased susceptibility to infections. A parallel increase in the TCR repertoire diversity and clinical improvement occurred during the follow-up. Our results confirm that the extent of immunodeficiency is highly variable and could improve through childhood, and indicate that TCR repertoire may be a useful marker to clinically monitor thymic function in this primary immunodeficiency.C. Cancrini and M.L. Romiti contributed equally to this work.     

Regulation of Con A – dependent cytokine production from CD4+ and CD8+ T lymphocytes by autosecretion of histamine

Objectives: Previously we have shown that both CD4+ T cells and CD8+ T cells produce histamine when activated with Con A. The aim of this study was to examine whether cytokine production by these cells is regulated by autosecretion of histamine.Materials: CD4+ and CD8+ T cells were separated from spleen cells of C57BL/6 mice and mice lacking the H1 receptor (H1R) or H2R, using anti – CD4+ – and anti – CD8+ – coupled magnetic beads, respectively.Results: Depletion of the H1R resulted in decreases in the release of IL-2 and IL-10 from both CD4+ and CD8+ cells and increases in the release of IL-4 from CD4+ T cells and IFN- from CD8+ cells. Mice lacking the H2R showed up – regulation of IFN- secretion from CD8+ cells and of IL-4 from CD4+ and CD8+ T cells. Release of IL-2 and IL-10 from CD4+ as well as CD8+ cells was down – regulated in these mice. Both CD4+ and CD8+ T cell fractions synthesized histamine, which was enhanced in the H1R - deficient CD8+ T cells. Treatment of the cells with -fluoromethyl-histidine, a specific inhibitor of HDC, or histaminase increased IFN- from CD8+ cells, whereas it had no appreciable effect on IL-4 secretion from CD4+ cells.Conclusions: These results suggest that cytokine production by CD4+ and CD8+ T lymphocytes is regulated by autosecretion of histamine.Received 4 July 2003; returned for revision 23 September 2003; returned for final revision 13 October 2003, accepted by M. Parnham 17 October 2003     

Origin of CD8^＋ Effector and Memory T Cell Subsets

It is well accepted that CD8＋ T cells play a pivotal role in providing protection against infection with intracellular pathogens and some tumors. In many cases protective immunity is maintained for long periods of time （immunological memory）. Over the past years, it has become evident that in order to fulfill these multiple tasks, distinct subsets of effector and memory T cells have to be generated. Until today, however, little is known about the underlying mechanisms of subset differentiation and the timing of lineage fate decisions. In this context, it is of special importance to determine at which level of clonal expansion functional and phenotypical heterogeneity is achieved. Different models for T cell subset diversification have been proposed; these differ mainly in the time point during priming and clonal expansion （prior, during, or beyond the first cell division） when differentiation programs are induced. Recently developed single-cell adoptive transfer technology has allowed us to demonstrate that individual precursor cell still bears the full plasticity to develop into a plethora different T cell subsets. This observation targets the shaping of T cell subset differentiation towards factors that are still operative beyond the first cell division. These findings have important implications for vaccine development, as the modulation of differentiation patterns towards distinct subsets could become a powerful strategy to enhance the efficacy and quality of vaccines. Cellular ＆ Molecular Immunology.     

Role of CD3ε-mediated signaling in T-cell development and function

Receptor-mediated signal transduction plays an important role in T-cell differentiation and function. The pre-T-cell receptor (pre-TCR) and TCR complexes are the most critical receptors for T-cell biology. Signals induced by pre-TCR and TCR in a ligand-independent or a ligand-dependent manner, respectively, are essential for thymocyte maturation. CD3 proteins, which are γ, δ, ε, and ζ polypeptides, play pivotal role in intracellular assembly, surface expression, and signal transduction via the pre-TCR and TCR complexes. Recent studies have suggested central and multiple roles for CD3ε in T-cell development and function. We review the role of the CD3ε chain in T-cell biology.     

Differential expression of regulator of G-protein signalling transcripts and in vivo migration of CD4+ naïve and regulatory T cells

The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.     

Stem and Progenitor Cell Expansionin Co-culture of Mobilized CD34~ Cells and Osteopetrotic Mouse Stroma

1 IntroductionCulture systems capable of expanding and/or maintaining hematopoietic stem cells will not only facilitate our understanding of stem cell biology, but also broaden clinical applications. Among various in vitro hematopoietic culture systems, co-cultures of marrow or CD34~ cells with an adherent stromal layer that can produce cytokines and extracellular matrix components most effectively supports long-term hematopoiesis (LTC), mimicking the bone marrow micro-environment.The OP-9…     

Expression and clinical significance of CD151 and integrin α3β1 in colorectal tumors

目的:探讨CD151和整合素α3β1在直肠腺瘤及结直肠腺癌组织中的表达及其与临床各个病理因素之间的关系,并且研究两者的相关性.方法:应用免疫组织化学双染方法对正常结直肠黏膜、结直肠腺瘤及结直肠腺癌组织各120例进行CD151和整合素α3β1检测,并进行Kaplan-Meier生存分析.采用Spearman等级相关分析CD151和整合素αβ1之间的相关性.结果:CD151结直肠正常黏膜、腺瘤、腺癌组织的阳性率分别为21.7％、52.5％、72％,腺瘤和腺癌组织分别与正常黏膜比较均具有统计学意义.整合素α3β1在结直肠正常黏膜、腺瘤、腺癌组织的阳性率分别为34.2％、55％、70％,腺瘤和腺癌组织分别与正常黏膜比较均具有统计学意义.在结直肠腺癌中,CD151和整合素α3β1的表达与患者年龄、性别和肿瘤的部位、大小无相关性,与肿瘤的分化程度、浸润深度、淋巴结转移及Duke's分期有关.CD151和整合素α3β1在大肠正常黏膜、腺瘤及腺癌组织中的表达经双变量相关分析,表达呈正相关.从图的Kaplan-Meier生存曲线及Log-Rank检验可知,CD151+、α3β1+、CD151+α3β1+与大肠癌患者5年生存期密切相关,是影响大肠癌预后的因素.结论:CD151和整合素α3β1在大肠癌的表达密切相关,提示CD151与整合素α3β1复合物存在于大肠癌,其表达对预后产生明显的影响.CD151与整合素α3β1联合表达是临床预后判断的可靠指标.     

Coexpression of Fcγ receptor IIIA and interleukin-2 receptor β chain by a subset of human CD3+/CD8+/CD11b+ lymphocytes

In this study we identify and characterize a subset of human peripheral blood T cells, present in all individuals, that has features previously described for T cells either separately or in special circumstances. These cells are found in purified suspensions of resting peripheral blood lymphocytes within the CD8⁺ T lymphocytes, express T cell receptor (TCR), and can be identified and isolated because of high-density expression of surface CD11b (TCR⁺/CD3⁺/CD8⁺/CD11b⁺ cells). They coexpress constitutively the IL-2 receptor chain, FcRIIIA, and CD56. Although they do not mediate spontaneous cytotoxicity, CD3⁺/CD8⁺/CD11b⁺ cells have cytotoxic potential, demonstrated in redirected cytotoxicity assays with P815 target cells in the presence of anti-FcRIII (CD16) or anti-CD3 monoclonal antibodies. Stimulation of CD3⁺/CD8⁺/CD11b⁺ cells with rIL-2 induces proliferation, cytotoxicity against NK-sensitive and NK-resistant target cells, and expression of surface activation antigens, including IL-2 receptor chain (CD25). CD3⁺/CD8⁺/CD16⁺/CD56⁺ cell clones with cytotoxic functions including those mediated by engagement of surface CD16 were obtained by limiting-dilution cloning of purified CD3⁺/CD8⁺/CD11b⁺ cells in the presence of rIL-2 and autologous feeder cells. Our data support the hypothesis that the CD3⁺/CD8⁺/CD11b⁺/CD16⁺ cells represent a discrete peripheral blood lymphocyte subset that could be the physiological counterpart of that expanded in several pathological conditions and in large granular lymphocyte lymphocytosis.     

What's the difference between CD80 and CD86?

CD28 and CD152 have crucial yet opposing functions in T-cell stimulation, in which CD28 promotes but CD152 inhibits T-cell responses. Intriguingly, they share two ligands, CD80 and CD86, but at present there is no clear model for understanding whether a ligand will promote or inhibit responses. Current perceptions are based around the concept that CD86 is the initial co-stimulatory ligand based on its more abundant and earlier expression pattern; CD80 has a role following antigen-presenting-cell activation. We describe an alternative view in which CD80 is the initial ligand, responsible for maintaining aspects of immune tolerance through interactions with CD152. These inhibitory functions can then be over-ridden by the upregulation of CD86 on dendritic cells as a result of inflammatory stimuli, leading to immune activation.     

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of na?ve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Na?ve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for na?ve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when na?ve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.