Identification of CD3ε, CD4, CD8β splice variants of Atlantic salmon

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.     

Characterization and expression analysis of CD3ɛ and CD3γ/δ in fugu, Takifugu rubripes

CD3 is an essential component of the CD3-TCR complex. In this report, we describe the cloning, characterization, and expression analysis of the CD3 and CD3/ chain genes from fugu, Takifugu rubripes. Two distinct CD3 homologue cDNAs, designated as CD3-1 and CD3-2, and a CD3/ homologue cDNA were isolated from the fugu thymus. The deduced amino acid sequences of these cDNAs exhibit conserved essential CD3 chain motifs and overall structures. RT-PCR analysis demonstrated that the CD3 and CD3/ genes were expressed in lymphoid organs (e.g. thymus, head kidney, trunk kidney and spleen), mucosal tissues (gill, skin, and intestine), and peripheral blood leucocytes (PBL). The CD3 and TCR genes were expressed only in the surface IgM^– population, which were separated from PBL using an anti-fugu IgM monoclonal antibody. In addition, in situ hybridization confirmed that CD3-expressing cells were distributed randomly in the head kidney, trunk kidney, and spleen, but in the thymus were restricted to the lymphoid outer zone and epithelioid inner zone only. Collectively, these results suggest that CD3 molecules are useful markers for the identification of T cells in teleost fish. The present study thus provides a critical step in identifying T cells in this model organism.Nucleotide sequence data reported in this paper are available in the DBJ/EMBL/GenBank databases and have been assigned the accession numbers AB166798 (CD3-1), AB166799 (CD3-2), and AB166800 (CD3/).     

CD3AK细胞抗肿瘤作用的研究进展

抗ＣＤ３单克隆抗体激活的杀伤细胞ＣＤ３ＡＫ是一种新型抗肿瘤免疫活性细胞,具有体外扩增能力强,细胞毒活性高,体内抗肿瘤效果好,低依赖ＩＬ－２等特点。本文综述了ＣＤ３ＡＫ细胞的诱导和扩增、形态学和免疫学特性、体内外抗肿瘤作用和ＣＤ３ＡＫ细胞的活性增强物质,并讨论了ＣＤ３ＡＫ细胞的作用机制     

抗CD3mAb诱导TN细胞向过渡型CD8单阳性细胞分化

本实验观察了在IL-7维持TN细胞生长的体外细胞培养系统中,抗CD 3 mAb对TN细胞分化的作用。发现抗CD 3 mAb在体外能诱导TN细胞分化产生CD 4~-CD 8~ 及部分CD 4~(lo)CD 8~ 细胞。由于多数CD 8~ 细胞为TCRβ~-,故抗CD 3 mAb诱导产生的CD 4~-CD 8~ 细胞是DN向DP分化的不成熟过渡状态细胞。抗TCR βmAb也能诱导CD 4~-CD 8~ 细胞产生,但其诱导分化能力弱于抗CD3 mAb。在体外细胞培养条件下,抗CD 3mAb诱导的DN→DP转化伴有IL-2 Rα表达下调,并且IL-7诱导的TCRβ分子表达也受到抑制。当TN细胞在IL-7条件下培养3天后,抗CD 3 mAb便失去诱导TN细胞分化的能力,表明抗CD 3 mAb诱导的TN细胞分化可能是通过细胞表面pre-TCR复合体进行的。     

CD8 T cell activation after intravenous administration of CD3×CD19 bispecific antibody in patients with non-Hodgkin lymphoma

A bispecific antibody directed to T and B cells (CD3×CD19 bsAb) was daily infused intravenously in escalating doses from 10 g up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed at _1/2 of 10.5 h with peak levels of 200–300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon , IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1ß in serum). No gross changess in histology or number of IL-2R⁺, CD4⁺ or CD8⁺ cells were found in the lymph nodes after therapy, but one patient showed activated CD8⁺ T cells within the tumour nodules. In conclusion, after intravenously administered CD3×CD19 bsAb only moderate toxicity was found, probably due to CD8⁺ T cell activation and cytokine release, without CD4⁺ T cell activation.     

CD3 × CD28 cross-interacting bispecific antibodies improve tumor cell dependent T-cell activation

Bispecific antibodies (Bs-Abs) containing an anti-CD3 and an anti-TAA specificity can recruit T cells to the tumor for cancer immunotherapy. To be effective, efficient activation at the tumor site is a prerequisite. This can be achieved by triggering both the T-cell receptor and the co-stimulatory molecule CD28. We engineered two recombinant cross-interacting Bs-Abs (CriBs-Abs) by incorporating a peptide tag and its cognate single-chain variable fragment (scFv), respectively, into a pair of (tumor × CD3) and (tumor × CD28) binding Bs-Abs. A 30-fold lower concentration of the activating CriBs-Ab as compared to non interacting Bs-Ab was sufficient for strong T-cell activation in the presence of tumor cells. One thousand-fold higher concentrations of both CriBs-Abs were required for marginal T-cell activation (70-fold below maximal response) in the absence of tumor cells. An optimized stoichiometry (1 : 1000) of activating versus co-stimulating CriBs-Ab thus allowed low doses of activating CriBs-Ab to induce tumor-cell dependent T-cell activation when used in combination with high concentrations of the pre-targeted co-stimulating CriBs-Ab in vitro. This indicates a large window of operation in which only tumor cell dependent T-cell activation is induced and systemic tumor cell independent T-cell activation is avoided, while ensuring optimal activation with a low concentration of the activating CriBs-Ab, which has the highest potential to induce toxic effects in vivo.     

Unprimed CD4+ and CD8+ T cells can be rapidly activated by a CD3×CD19 bispecific antibody to proliferate and become cytotoxic

We previously reported that a CD3×CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3×CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3×CD19 bsAb. Within the same time spaancytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3×CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.This work was supported by grant IKMN 90-10 from the Dutch Cancer Society. M.C. was supported by a grant from the UK Medical Research Couneil     

CD4+CD25-Foxp3+ T cells: a marker for lupus nephritis?

Introduction

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4⁺CD25^-Foxp3⁺ T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations.

Methods

Phenotypic analyses, proportions and absolute cell numbers of CD4⁺CD25^-Foxp3⁺ T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4⁺CD25^-Foxp3⁺ T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4⁺CD25^-Foxp3⁺ T cells were analyzed in urine sediment samples. Time course analyses of CD4⁺CD25^-Foxp3⁺ T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone.

Results

CD4⁺CD25^-Foxp3⁺ T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients with renal involvement. CD4⁺CD25^-Foxp3⁺ T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria.

Conclusion

CD4⁺CD25^-Foxp3⁺ T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4⁺CD25^-Foxp3⁺ T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.     

抗CD3/CD28单克隆抗体联合PHA对T淋巴细胞活化和增殖的影响

目的探讨抗人CD3/CD28单克隆抗体联合植物血凝素(PHA)对人外周血T淋巴细胞活化增殖的影响。方法通过Ficoll密度梯度离心法分离健康成人外周血单核细胞后,用CD3免疫磁珠分选T淋巴细胞,抗CD3/CD28单抗、PHA及重组人白介素-2(recombinant human IL-2, rhIL-2)刺激T淋巴细胞。用CCK-8试剂盒(CCK-8 cell counting kit)检测细胞增殖情况, ELISA测定细胞上清液干扰素γ(interferon-γ, IFN-γ)的含量。结果 CCK-8结果显示在增殖3 d时,CD3/CD28+PHA组A_(450 nm)值与PHA组、IL-2组与空白组比较,差异有统计学意义(P<0.05);比较各组在6、9、12 d的增殖情况,CD3/CD28+PHA组的A_(450 nm)值与其他各组差异均有统计学意义(P<0.05)。刺激3 d各组分泌的IFN-γ质量浓度是最高的。刺激9 d时,CD3/CD28+PHA组、CD3/CD28组、PHA组上清液中IFN-γ浓度分别为(235.27±27.80)、(189.23±24.79)和(124.66±3.39)pg/mL,CD3/CD28+PHA组上清液中IFN-γ的质量浓度高于其余2组(P<0.05)。结论抗CD3/CD28单抗联合PHA比单用抗CD3/CD28单抗、PHA或rhIL-2能更好地激活T细胞及持久地促使T细胞增殖。     

巨型引物PCR法对抗CD3改型单链抗体

亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.     

CD3单抗诱导幼龄小鼠胸腺细胞凋亡研究

用抗小鼠ＣＤ３单抗刺激幼龄小鼠胸腺细胞,培养不同时间后,检测小鼠胸腺细胞的凋亡情况。结果表明,胸腺细胞呈现了凋亡的典型形态学改变。流式细胞仪检测可见凋亡细胞特有的ＡＰ峰,ＣＤ３单抗刺激未成熟胸腺细胞可以通过内源性的凋亡途径引起细胞死亡。     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

抗CD3/抗CD20双特异性单链抗体的表达、鉴定及活性分析

设计并合成多个60bp左右的DNA小片段 ,经重叠延伸PCR扩增获得1640bp的抗CD3 抗CD2 0双特异性单链抗体完整基因片段 ,将其克隆入真核定点表达载体pcDNA5 FRT中 ,脂质体法转染Flp-In^TM CHO细胞 ,获得稳定表达细胞株 ,目的蛋白在上清中的表达量约为 300μg/L。采用Ni_NTA柱对其进行了纯化 ,经SDS-PAGE蛋白电泳及Western-blot分析结果表明 ,含组氨酸标签的目的蛋白的分子量约为 70kD ,与预期结果一致。活细胞间接免疫荧光实验和玫瑰花环实验证明抗CD3抗CD20双特异性单链抗体具有与Ramous(CD20+)及Jurkat(CD3+)细胞特异性结合的活性。光学显微镜下可以观察到抗CD3 抗CD2 0双特异性单链抗体可以有效介导人外周血淋巴细胞裂解B淋巴瘤细胞Ramous。以上工作为进一步了解抗CD3 抗CD2 0双特异性单链抗体的体内体外生物学活性奠定了基础。     

哮喘患者诱导痰和外周血CD3+CD56+NKT 细胞的检测及意义

目的:研究CD3+CD56+NKT细胞在哮喘患者急性发作期诱导痰和外周血中的比例改变,并探讨其临床意义.方法:以28例哮喘急性发作期患者为研究组,22名正常人作为对照组,采用二色直接荧光素标记法和多参数流式细胞仪检测诱导痰和外周血CD3+CD56+NKT细胞的比例,同时检测外周血IL-4、Ig-E及INF-γ等水平.结果:哮喘患者急性发作期诱导痰和外用血CD3+CD56+NKT细胞明显高于健康对照组(P<0.01).哮喘患者急性发作期外周血IL-4、Ig-E及INF-γ等水平明显高于健康对照组(P<0.05).哮喘患者外周血中CD3+CD56+NKT细胞比例与IL-4、Ig-E及INF-γ升高成正相关.结论:哮喘患者急性发作期诱导痰和外周血中的CD3+CD56+NKT细胞明显增高,CD3+CD56+NKT细胞可能通过调节IL-4、Ig-E及INF-γ等细胞因子从而在哮喘的发病机制发挥重要作用.