摘要
目的：
ZCH-2B8a单克隆抗体由本研究室采用经典的杂交瘤技术制备而成，经HLDA8协作组鉴定后认为属国际上尚未认识的造血细胞膜新的分化抗原或新的CD分子，前期研究表明该抗原(Ag)可能为一活化分子。因此有必要对该Ag进一步研究以明确该其蛋白序列及其表达谱、是否为一新的活化分子及它的功能，开发其潜在的应用价值。
方法：
本研究主要包括以下四部分：(1)制备2B8a FITC直标抗体：采用SPASepharose亲和层析法从腹水中纯化2B8a抗体；聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定抗体纯度及分子量；采用改良Marshall法制备2B8a FITC直标抗体，并通过流式细胞术鉴定。(2)检测2B8a Ag的分布情况：采用流式细胞术检测2B8a Ag在外周组织、正常外周血、白血病细胞、细胞株上的分布；采用激光共聚焦显微镜观察2B8a Ag在细胞上的定位。(3)2B8a Ag的功能研究：通过流式细胞术检测PHA刺激后T细胞活化过程中2B8a的表达规律；检测再生障碍性贫血等病人外周血标本2B8a Ag的表达情况及与其他活化标志的相关性；利用流式细胞微球技术(CBA)检测2B8a Ab对活化T细胞分泌细胞因子的影响；通过2B8a抗体封闭Raji细胞的Ag表位检测它与骨髓基质细胞粘附能力的变化；通过木瓜酶酶切法结合流式细胞术检测2B8a Ag介导抗体内化的能力；通过补体依赖的细胞毒实验(CDC)了解2B8a Ab与Ag特异性结合及激活补体的能力。(4)2B8a Ag蛋白的鉴定：将Raji细胞蛋白裂解液经免疫共沉淀法纯化后行SDS-PAGE检测，胶上的Ag蛋白条带送肽质量指纹图及串联质谱分析；Western blot法鉴定2B8a Ag蛋白的分子量。
结果：
经SDS-PAGE鉴定，2B8a抗体的重链和轻链分子量分别为52 kDa和9 kDa。2B8a直标法(2B8a FITC)和间标法(2B8a+GAM-FITC)与Raji细胞阳性反应率分别为98．48%和98．35%，与Molt-3细胞阳性反应率分别为9．35%和10．80%。2B8a Ag在正常外周血的T细胞表面不表达(5．4±3．8%)，B细胞表面弱表达(29．9±14．8%)，而中性粒细胞(92．2±8．7%)和单核细胞(81．9±19．3%)表面表达较强。在B系急性淋巴细胞白血病细胞表面，该Ag的表达较对照组显著增强(中位值99．0%vs．43．9%)。该Ag在所检测B系、T系、髓系的外周血细胞和肿瘤细胞株的胞浆内呈高表达。2B8a Ag在活化T细胞上的表达规律与CD69、CD25和HLA-DR不同，在T细胞受PHA刺激后12h内即可表达于胞膜上，72～96h达高峰。再生障碍性贫血(AA)等免疫相关疾病的T细胞表面，该Ag的表达明显上调(AA：CD4~+T细胞阳性率90．9%，CD8~+T细胞阳性率72．7%)。采用2B8a抗体封闭抗原表位后并不能减少Raji细胞与骨髓基质细胞的粘附(2h、4h、6h和12h时2B8a组与对照组的粘附率比分别为0．87、1．02、1．02和0．96，均为P＞0．05)，也并不影响T细胞活化后细胞因子的分泌水平和活化状态(PHA组与PHA+2B8a组IL-2中位值：425pg/ml vs．442pg/ml，P=0．465)。与3A4抗体在37℃时才能逐渐内化不同，2B8a抗体在4℃和37℃均可以快速“内化”(2B8a FITC与Raji细胞4℃和37℃孵育5min后再行木瓜酶酶切，流式检测阳性反应率分别达到76．7±2．5%和89．2±1．5%)。2B8a抗体能激活补体，介导CDC作用杀伤Raji细胞(按0．1μg抗体：10μl补体：5×10~4细胞的比例，Raji细胞死亡率88．6±7．9%)，而对Molt-3细胞基本无杀伤作用(死亡率3．6±0．6%)。将Raji细胞裂解液经免疫共沉淀纯化2B8a Ag后，Western blot鉴定证实该Ag的分子量为50 kDa左右。质谱分析未能明确得到唯一、可靠的蛋白序列，通过蛋白数据库比对及功能研究，冠蛋白的可能性较大，但它在表达谱上与2B8a Ag仍有较大不同。
结论：
(1)本研究自行研制的2B8a FITC荧光抗体具有良好的敏感性和特异性；
(2)2B8a Ag在淋巴结、扁桃体、肝脏及胰腺组织中可检测到；在外周血，它主要分布在活化T细胞、B细胞、单核细胞、嗜中性粒细胞膜和细胞浆，而在静息T细胞膜上不表达，但在其胞浆中表达也很强；
(3)2B8a Ag是一个早期稳定的T细胞活化标志；在再生障碍性贫血等免疫相关疾病中表达上调；
(4)2B8a Ag可以介导抗体迅速内化；
(5)2B8a Ab能激活补体，发挥明显的CDC作用；
(6)2B8a Ag不能介导细胞间的粘附；对活化T细胞的细胞因子分泌无明显影响；
(7)质谱分析未能在现有的蛋白质数据库中检索到与2B8a Ag相匹配的蛋白，该抗原可能为一新的蛋白分子。
OBJECTIVES
Monoclonal antibody (mAb) ZCH-2B8a was generated in our laboratory by classichybridoma technique.The antigen (Ag) bound by this mAb was recognized as a noveldifferentiation antigen or CD molecule at the surface of hematopoietic cells by the 8~(th)International Workshop and Conference on Human Leukocyte Differentiation Antigens(HLDA8) and as an activation Ag of T cells by previous studies.So it is of greatimportance to illustrate the protein or DNA consequences,the expression pattern ontissues and hematopoietic cells and the biological functions of the Ag,to prove whetherthis is a novel CD molecule or activation Ag and to explore the potential applicationvalue.
METHODS
This study included four parts:1.Preparation and identification of 2B8a FITC:Theascites containing 2B8a antibody (Ab) derived from Balb/c mice was purified byaffinity chromatography using SPA Sepharose column.The purity and molecular weight(MW) of the heavy and light chains were identified by SDS-PAGE.2B8a FITC wasprepared according to Marshall's method and evaluated by flow cytometry (FCM).2.The reactivity of 2B8a Ab with tissue,normal hematopoietic cells,leukemic blasts andcell lines,and the subcellular localization were identified by FCM and laser scanningconfocal microscope.3.Function analysis:we assessed the expression kinetics of 2B8aAg on in vitro activated T cells after stimulation with PHA by FCM;evaluated the 2B8aexpression level on the surface of T cells in patients with aplastic anemia (AA),hemophagocytic lymphohistiocytosis (HLH) and so on;observed the variation ofTh1/Th2 cytokine levels those secreted by activated T cells after co-incubated with2B8a Ab during PHA stimulation;detected the variation of adhesion capacity of Raji tobone marrow stromal cells after blockage of 2B8a epitopes by specific antibody;identified the internalization of 2B8a FITC using a novel method comprising enzymatic hydrolysis of 2B8a FITC by papain and FCM detection;determined the capacity of2B8a Ab specifically binding with 2B8a Ag and activating complement to executecomplement dependent cytotoxicity (CDC);4.Consequence of 2B8a Ag protein:2B8aAg was separated and purified from Raji cell lysate by co-immunoprecipitation andassessed using SDS-PAGE.The Ag band on the gel was harvested and delivered forsequencing by mass spectrometry (MS) including peptide mass fingerprinting (PMF)and tandem MS.The MW of the Ag was evaluated using SDS-PAGE followed bywestern blot.
RESULTS
The 2B8a Ab turned out to be two bands of approximately 52 kDa and 29 kDawhen identified by SDS-PAGE.The reactivity of 2B8a FITC and 2B8a±GAM-FITC toRaji and Molt-3 cells were comparable (positive rates of Raji:98.48% vs.98.35%;ofMolt-3:9.35% vs.10.80%).FCM analysis carried out with 2B8a FITC and otherfluorescent Abs specific to leukocytic antigens revealed that 2B8a Ag was not expressedon the surface of resting T cells (5.4±3.8%),whereas it was low expressed on B cells'(29.9±14.8%),highly expressed on granulocytes' (92.2±8.7%) and monocytes'(81.9±19.3%) surfaces.However,the Ag density within the cytoplasm was extremelyhigh in all above hematopoietic cells regardless of surface expression level.In the blastsof B lineage acute lymphoblastic leukemia,2B8a Ag was highly expressed on themembrane when compared with normal B cells (median:99.0% vs.43.9%).2B8ashowed a different activation pattern when compared with CD69,CD25 and HLA-DR,which could up-regulate within 12h after stimulation of PHA and reach peak after72～96h.The 2B8a level on the surface of T cells in patients with AA,HLH and so onwas significantly higher than those of control population (AA:positive rate in CD4~+Tcells 90.9%,positive rate in CD8+T cells 72.7%).The previously binding of 2B8aantigen with specific antibody showed no effect on adhesion capacity of Raji to bonemarrow stromal cells (the adhesion ratios of 2B8a groups to controls in 2h,4h,6h and 12h were 0.87,1.02,1.02 and 0.96,respectively;all P＞0.05) or on cytokine productionof activated T cells (median levels of IL-2 in PHA and PHA+2B8a groups:425pg/ml vs.442pg/ml,P = 0.465).2B8a Ag could mediate the internalization of 2B8a FITC rapidlyafter their binding both at 4℃and 37℃.2B8a Ab could specifically bind to 2B8a Agand activate complement to kill Raji cells through CDC (0.1μg 2B8a Ab:10μlcomplement:5×10~4 cells:The mortality of Raji cells and Molt-3 cells were 88.6±7.9% and 3.6±0.6%,respectively,P＜0.05).The Ag was identified as a protein with aMW of 50 kDa or so.However,PMF and tandem MS failed to provide the exact anddefinite sequence of the Ag protein,however,coronin was a possible candidate butshowed some differences on expression pattern from 2B8a Ag.
CONCLUSIONS
1.The 2B8a FITC fluorescent antibody is an excellent reagent with a very highsensitivity and specificity;
2.2B8a Ag is detectable in many tissues such as lymph node,tonsil,liver andpancreas;It expresses both on the surface and in the cytoplasm of B cells,neutrophils,monocytes and activated T cells,but only in the cytoplasm of the resting T cells;
3.2B8a is an early and steady activation marker of T cells and can up-regulate indiseases related to immune disorder,such as AA and HLH;
4.2B8a Ag canmediate quick internalization of the binding antibody;
5.2B8a Ab can activate complement and selectively kill the cells expressing 2B8aAg by CDC;
6.2B8a Ag shows no effect on cell-cell adhesion and cytokine production ofactivated T cell;
7.2B8a Ag may be a novel protein as no matched protein was found in the proteindatabase by MS analysis.
引文
1 Dougan M,Dranoff G.Immune therapy for cancer.Annu Rev Immunol,2009,27:83-117.
2 Cheever MA.Twelve immunotherapy drugs that could cure cancers.Immunol Rev,2008,222:357-368.
3 Waldmann TA.Effective cancer therapy through immunomodulation.Annu Rev Med,2006,57:65-81.
4 Zafir-Lavie I,Michaeli Y,Reiter Y.Novel antibodies as anticancer agents. Oncogene,2007,26:3714-3733.
5 Khanna C,Helman LJ.Molecular approaches in pediatric oncology.Annu Rev Med,2006,57:83-97.
6 Maggon K.Monoclonal antibody “gold rush”.Curr Med Chem,2007,14:1978-1987.
7 Reichert JM,Valge-Archer VE.Development trends for monoclonal antibody cancer therapeutics.Nat Rev Drug Discov,2007,6:349-356.
8 Tang YM,Ning BT,Cao J,et al.Construction and expression of single-chain antibody derived from a new clone of monoclonal antibody against human CD14 in CHO cells.Immunopharmacol Immunotoxicol,2007,29:375-386.
9 宁铂涛．鼠抗人CD14新克隆ZCH-7-2F9单链抗体的研究[博士学位论文]．杭州：浙江大学，2005．
10 徐卫群．人鼠嵌合型基因工程抗体Hm2E8的研究[博士学位论文]．杭州：浙江大学，2008．
11 郭莉．一个新的白细胞膜抗原ZCH-2B8a编码基因及其生物学特性的研究[博士学位论文]．杭州：浙江大学，2006．
1.Fox DA,Hussey RE,Fitzgerald KA,et al.Tal,a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.J Immunol.1984 133:1250-1256.
2.Song J,So T,Croft M.Activation of NF-kappaBl by OX40 contributes to antigen-driven T cell expansion and survival.J Immunol.2008 180:7240-7248.
3.Croft M.Co-stimulatory members of the TNFR family:keys to effective T-cell immunity.Nat Rev Immunol.2003 3:609-620.
4.Castro AG,Hauser TM,Cocks BG,et al.Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM):differential expression and responsiveness in Thl and Th2 cells.J Immunol.1999 163:5860-5870.
5.So T,Song J,Sugie K,Altaian A,Croft M.Signals from OX40 regulate nuclear factor of activated T cells cl and T cell helper 2 lineage commitment.Proc Natl AcadSci USA.2006 103:3740-3745.
6.Howie D,Okamoto S,Rietdijk S,et al.The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production.Blood.2002 100:2899-2907.
7.Marelli-Berg FM,Okkenhaug K,Mirenda V.A two-signal model for T cell trafficking.Trends Immunol.2007 28:267-273.
8.Mody PD,Cannon JL,Bandukwala HS,et al.Signaling through CD43 regulates CD4 T-cell trafficking.Blood.2007 110:2974-2982.
9.Chen L.Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity.Nat Rev Immunol.2004 4:336-347.
10.Greenwald RJ,Freeman GJ,Sharpe AH.The B7 family revisited.Annu Rev Immunol.2005 23:515-548.
11.Pistillo MP,Tazzari PL,Palmisano GL,et al.CTLA-4 is not restricted to the lymphoid cell lineage and can function as a target molecule for apoptosis induction of leukemic cells.Blood.2003 101:202-209.
12.Fiorentino S,Chopin M,Dastot H,et al.Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.Eur Cytokine Netw.2005 16:300-308.
13.Yanagita S,Hori T,Matsubara Y,Ishikawa T,Uchiyama T.Retroviral transduction of acute myeloid leukaemia-derived dendritic cells with OX40 ligand augments their antigen presenting activity.Br J Haematol.2004 124:454-462.
14.Valzasina B,Guiducci C,Dislich H,Killeen N,Weinberg AD,Colombo MP.Triggering of OX40(CD134)on CD4(+)CD25+ T cells blocks their inhibitory activity:a novel regulatory role for OX40 and its comparison with GITR.Blood.2005 105:2845-2851.
15.Messmer D,Kipps TJ.CD154 gene therapy for human B-cell malignancies.Ann N YAcad Sci.2005 1062:51-60.
16.Zeiser R,Marks R,Bertz H,Finke J.Immunopathogenesis of acute graft-versus-host disease:implications for novel preventive and therapeutic strategies.Ann Hematol.2004 83:551-565.
17．金伯泉主编．细胞和分子免疫学实验技术．西安：第四军医大学出版社，2002：26-28．
18．J．萨姆布克鲁，E．F．弗里奇，T．曼尼阿蒂斯著．金冬燕，黎孟枫译．分子克隆实验指南(第二版)．北京：科学出版社，1992：888-897．
19．陈志南，刘名培．抗体分子与肿瘤．北京：人民军医出版社，2002：241-249．
20．汤永民，郭莉，杨世隆，等．白细胞新单克隆抗体ZCH-2B8a的表达谱分析及其意义．中国实验血液学杂志，2006 14(5)：990-994．
21．马雄华，韩颖超，李世普．生物标记用发光材料研究现状．化工中间体，2006 23(8)：4-7．
22.van SAP,Machielsen PA,Gribnau TC.Effects of temperature,flow rate and composition of binding buffer on adsorption of mouse monoclonal IgG1 antibodies to protein A Sepharose 4 Fast Flow.Prep Biochem.1992 22:135-149.
23.Bastien Y,Toledano BJ,Mehio N,et al.Detection of functional platelet-activating factor receptors on human tonsillar B lymphocytes.J Immunol.1999 162:5498-5505.
24.Kong Y,Yoshida S,Saito Y,et al.CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL.Leukemia.2008 22:1207-1213.
25.Spiridon CI,Ghetie MA,Uhr J,et al.Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity of a human breast cancer cell line in vitro and in vivo.Clin Cancer Res.2002 8:1720-1730.
26．李丽娥．人骨髓基质细胞的生物学特性及其分化的研究[硕士学位论文]．苏州：苏州大学．2001：3-8．
27．庄俊玲．骨髓基质细胞和黏附分子对骨髓瘤细胞生长的影响作用初探[博士学位论文]．北京：中国协和医科大学．2004：32-33．
28.Tauro S,Hepburn MD,Bowen DT,Pippard MJ.Assessment of stromal function,and its potential contribution to deregulation of hematopoiesis in the myelodysplastic syndromes.Haematologica.2001 86:1038-1045.
29．张晶，马月霞，韩忠朝．血小板第四因子对CD34～+白血病细胞系KG1a粘附功能的影响．中国医学科学院学报，2002 24(2)：160-164．
30．王江芳，张毅，茅子均，等．MTT法定量检测白血病细胞株与基质细胞粘附率的方法．上海免疫学杂志，1998 18(3)：182．
31．R．I．弗雷谢尼著．章静波，徐存拴译．动物细胞培养-基本技术指南(第四版)．北京：科学出版社，2004：398-401．
32.Rea IM,McNedan SE,Alexander HD.CD69,CD25,and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha,IFN-gamma,and sIL-2R levels in aging.Exp Gerontol.1999 34:79-93.
33.Caruso A,Licenziati S,Corulli M,et al.Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.Cytometry.1997 27:71-76.
34.Chatenoud L,Primo J,Bach JF.CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice.J Immunol.1997 158:2947-2954.
35.Bisikirska B,Colgan J,Luban J,Bluestone JA,Herold KC.TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs.J Clin Invest.2005 115:2904-2913.
36.Shevach EM,McHugh RS,Piccirillo CA,Thornton AM.Control of T-cell activation by CD4+ CD25+ suppressor T cells.Immunol Rev.2001 182:58-67.
37.Piccirillo CA,Shevach EM.Cutting edge:control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells.J Immunol.2001 167:1137-1140.
38.Eichler W,Hamann J,Aust G.Expression characteristics of the human CD97 antigen.Tissue Antigens.1997 50:429-438.
39.Valk E,Rudd CE,Schneider H.CTLA-4 trafficking and surface expression.Trends Immunol.2008 29:272-279.
40.Veninga H,Becker S,Hoek RM,et al.Analysis of CD97 expression and manipulation:antibody treatment but not gene targeting curtails granulocyte migration.J Immunol.2008 181:6574-6583.
41.Miyamoto YJ,Mitchell JS,McIntyre BW.Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes.Mol Immunol.2003 39:739-751.
42.Krummel MF,Allison JP.CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation.J Exp Med.1995 182:459-465.
43.Grewal IS,Flavell RA.CD40 and CD154 in cell-mediated immunity.Annu Rev Immunol.1998 16:111-135.
44.Zhou D.OX40 signaling directly triggers the antitumor effects of NKT cells.J Clin Invest.2007 117:3169-3172.
45.Monfardini E,Paape MJ,Wang Y,et al.Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition.Vet Res.2002 33:271-281.
46．何维．医学免疫学．北京：人民卫生出版社，2005：116-123．
47.Funaro A,Reinis M,Trubiani O,Santi S,Di PR,Malavasi F.CD38 functions are regulated through an internalization step.J Immunol.1998 160:2238-2247.
48.Willmarth NE,Baillo A,Dziubinski ML,Wilson K,2nd RDJ,Ethier SP.Altered EGFR localization and degradation in human breast cancer cells with an amphiregulin/EGFR autocrine loop.Cell Signal.2009 21:212-219.
49.Harata M,Soda Y,Tani K,et al.CD19-targeting liposomes containing imatinib efficiently kill Philadelphia chromosome-positive acute lymphoblastic leukemia cells.Blood.2004 104:1442-1449.
50.Poussin C,Foti M,Carpentier JL,Pugin J.CD14-dependent endotoxin internalization via a macropinocytic pathway.J Biol Chem.1998 273:20285-20291.
51.Nal B,Carroll P,Mohr E,et al.Coronin-1 expression in T lymphocytes:insights into protein function during T cell development and activation.Int Immunol.2004 16:231-240.
52.Yan M,Di CC,Grinstein S,Trimble WS.Coronin function is required for chemotaxis and phagocytosis in human neutrophils.J Immunol.2007 178:5769-5778.
53.Olszewski AJ,Grossbard ML.Empowering targeted therapy:lessons from rituximab.Sci STKE.2004 2004:pe30.
54.Sacchi S,Federico M,Dastoli G,et al.Treatment of B-cell non-Hodgkin's lymphoma with anti CD 20 monoclonal antibody Rituximab.Crit Rev Oncol Hematol.2001 37:13-25.
55．E．哈洛，D．莱恩 著．沈关心，龚非力 译．抗体技术实验指南．北京：科学出版社，2002：131-157．
56．刘清萍，刘中华，唐新科，等．串联质谱在多肽测序中的应用．生命科学研究，2004，(02)：112-116．
57．阮松林，马华升，王世恒，等．植物蛋白质组学研究进展——Ⅰ．蛋白质组关键技术．遗传，2006，(11)：1472-1486．
58．周新文，张玲，谢锦云，等．蛋白质的肽质谱指纹图分析方法的优化．中国生物化学与分子生物学报，2005，(06)：114-122．
59.Bustelo XR.Regulatory and signaling properties of the Vav family.Mol Cell Biol.2000 20:1461-1477.
60.Hall AB,Gakidis MA,Glogauer M,et al.Requirements for Vav guanine nucleotide exchange factors and Rho GTPases in FcgammaR-and complement-mediated phagocytosis.Immunity.2006 24:305-316.
61.Tybulewicz VL.Vav-family proteins in T-cell signalling.Curr Opin Immunol.2005 17:267-274.
62.Trenkle T,McClelland M,Adlkofer K,Welsh J.Major transcript variants of VAV3,a new member of the VAV family of guanine nucleotide exchange factors.Gene.2000 245:139-149.
63.Movilla N,Bustelo XR.Biological and regulatory properties of Vav-3,a new member of the Vav family of oncoproteins.Mol Cell Biol.1999 19:7870-7885.
64.Charvet C,Canonigo A J,Billadeau DD,Altman A.Membrane localization and function of Vav3 in T cells depend on its association with the adapter SLP-76.J Biol Chem.2005 280:15289-15299.
65.Trenkle T,McClelland M,Adlkofer K,Welsh J.Major transcript variants of VAV3,a new member of the VAV family of guanine nucleotide exchange factors.Gene.2000 245:139-149.
66．盛泉虎．串联质谱蛋白质鉴定方法的研究[硕士学位论文]．上海：中国科学院研究生院(上海生命科学研究院)．2004：33-41．
67.Leung MF,Lin TS,Sartorelli AC.Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.Cancer Res.1992 52:3063-3066.
68.Rybakin V,Clemen CS.Coronin proteins as multifunctional regulators of the cytoskeleton and membrane trafficking.Bioessays.2005 27:625-632.
69.de Hostos EL.The coronin family of actin-associated proteins.Trends Cell Biol.1999 9:345-350.
70.Nal B,Carroll P,Mohr E,et al.Coronin-1 expression in T lymphocytes:insights into protein function during T cell development and activation.Int Immunol.2004 16:231-240.
71.Yan M,Collins RF,Grinstein S,Trimble WS.Coronin-1 function is required for phagosome formation.Mol Biol Cell.2005 16:3077-3087.
72.Yah M,Di CC,Grinstein S,Trimble WS.Coronin function is required for chemotaxis and phagocytosis in human neutrophils.J Immunol.2007 178:5769-5778.
73.Maniak M,Rauchenberger R,Albrecht R,Murphy J,Gerisch G.Coronin involved in phagocytosis:dynamics of particle-induced relocalization visualized by a green fluorescent protein Tag.Cell.1995 83:915-924.
74.Foger N,Rangell L,Danilenko DM,Chan AC.Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis.Science.2006 313:839-842.
75.Combaluzier B,Pieters J.Chemotaxis and phagocytosis in neutrophils is independent of coronin 1.J Immunol.2009 182:2745-2752.
1.Fox DA,Hussey RE,Fitzgerald KA,et al.Ta1,a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.J Immunol 1984;133:1250-1256.
2.Abbas AK,Lichtman AH,Pillai S.Cellular and Molecular Immunology.6th ed.Philadelphia:Saunders Elsevier,2007:194-213.
3.Rea IM,McNerlan SE,Alexander HD.CD69,CD25,and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha,IFN-gamma,and sIL-2R levels in aging.Exp Gerontol 1999;34:79-93.
4.Jones AT,Federsppiel B,Ellies LG,et al.Characterization of the activation-associated isoform of CD43 on murine T lymphocytes./ Immunol.1994;153:3426-3439.
5.Maino VC,Suni MA,Ruitenberg JJ.Rapid flow cytometric method for measuring lymphocyte subset activation.Cytometry.1995;20:127-133.
6.Eichler W,Hamann J,Aust G Expression characteristics of the human CD97 antigen.Tissue Antigens.1997;50:429-438.
7.Diaz LA Jr,Friedman AW,He X,Kuick RD,Hanash SM,Fox DA.Monocyte-dependent regulation of T lymphocyte activation through CD98.Int Immunol 1997;9:1221-1231.
8.Gramaglia I,Weinberg AD,Lemon M,Croft M.Ox-40 ligand:a potent costimulatory molecule for sustaining primary CD4 T cell responses.J Immunol 1998;161:6510-6517.
9.Wang N,Morra M,Wu C,et al.CD 150 is a member of a family of genes that encode glycoproteins on the surface of hematopoietic cells.Immunogenetics.2001; 53:382-394.
10.Krummel MF,Allison JP.CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation.J Exp Med.1995;182:459-465.
11.Roy M,Waldschmidt T,Aruffo A,Ledbetter JA,Noelle RJ.The regulation of the expression of gp39,the CD40 ligand,on normal and cloned CD4+ T cells.J Immunol.1993;151:2497-2510.
12.Egen JG,Allison JP.Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength.Immunity.2002;16:23-35.
13.Greenwald RJ,Oosterwegel MA,der Woude D v,et al.CTL.A-4 regulates cell cycle progression during a primary immune response.Eur J Immunol.2002;32:366-373.
14.Fraser JH,Rincon M,McCoy KD,Le GG.CTLA4 ligation attenuates AP-1,NFAT and NF-kappaB activity in activated T cells.Eur J Immunol.1999;29:838-844.
15.Grohmann U,Orabona C,Fallarino F,et al.CTLA-4-Ig regulates tryptophan catabolism in vivo.Nat Immunol.2002;3:1097-1101.
16.Ruby CE,Montler R,Zheng R,Shu S,Weinberg AD.IL-12 is required for anti-OX40-mediated CD4 T cell survival.J Immunol.2008;180:2140-2148.
17.Song J,So T,Croft M.Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival.J Immunol.2008;180:7240-7248.
18.Redmond WL,Gough MJ,Charbonneau B,Ratliff TL,Weinberg AD.Defects in the acquisition of CD8 T cell effector function after priming with tumor or soluble antigen can be overcome by the addition of an OX40 agonist.J Immunol.2007;179:7244-7253.
19.Sugamura K,Ishii N,Weinberg AD.Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40.Nat Rev Immunol.2004;4:420-431.
20.Croft M.Co-stimulatory members of the TNFR family:keys to effective T-cell immunity.Nat Rev Immunol.2003;3:609-620.
21.Hendriks J,Xiao Y,Rossen JW,et al.During viral infection of the respiratory tract,CD27,4-1BB,and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion.J Immunol.2005;175:1665-1676.
22.Gorrell MD,Gysbers V,McCaughan GW.CD26:a multifunctional integral membrane and secreted protein of activated lymphocytes.Scand J Immunol.2001;54:249-264.
23.Femandez-Cabezudo MJ,Vijayasarathy C,Pflugh DL,Bothwell AL,al-Ramadi BK.Evidence for a dual pathway of activation in CD43-stimulated Th2 cells:differential requirement for the Lck tyrosine kinase.Int Immunol.2004;16:1215-1223.
24.Abbott RJ,Spendlove I,Roversi P,et al.Structural and functional characterization of a novel T cell receptor co-regulatory protein complex,CD97-CD55.J Biol Chem.2007;282:22023-22032.
25.Castro AG,Hauser TM,Cocks BG,et al.Molecular and functional characterization of mouse signaling lymphocytic activation molecule (SLAM):differential expression and responsiveness in Thl and Th2 cells./ Immunol.1999;163:5860-5870.
26.Williams CA,Murray SE,Weinberg AD,Parker DC.OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28,CD40,IL-12Rbeta2,or T-bet.J Immunol.2007;178:7694-7702.
27.So T,Song J,Sugie K,Altman A,Croft M.Signals from OX40 regulate nuclear factor of activated T cells cl and T cell helper 2 lineage commitment.Proc Natl Acad Sci USA.2006;103:3740-3745.
28.Wang N,Satoskar A,Faubion W,et al.The cell surface receptor SLAM controls T cell and macrophage functions.J Exp Med.2004;199:1255-1264.
29.Howie D,Okamoto S,Rietdijk S,et al.The role of SAP in murine CD150(SLAM)-mediated T-cell proliferation and interferon gamma production.Blood.2002;100:2899-2907.
30.Rethi B,Gogolak P,Szatmari I,et al.SLAM/SLAM interactions inhibit CD40-induced production of inflammatory cytokines in monocyte-derived dendritic cells.Blood.2006;107:2821-2829.
31.Hancock WW,Sayegh MH,Zheng XG,Peach R,Linsley PS,Turka LA.Costimulatory function and expression of CD40 ligand,CD80,and CD86 in vascularized murine cardiac allograft rejection.Proc Natl Acad Sci U S A.1996;93:13967-13972.
32.Stuber E,Strober W,Neurath M.Blocking the CD40L-CD40 interaction in vivo specifically prevents the priming of T helper 1 cells through the inhibition of interleukin 12 secretion.J Exp Med.1996;183:693-698.
33.Grewal IS,Flavell RA.CD40 and CD154 in cell-mediated immunity.Annu Rev Immunol.1998;16:111-135.
34.Ohnuma K,Dang Nil,Morimoto C.Revisiting an old acquaintance:CD26 and its molecular mechanisms in T cell function.Trends Immunol.2008;29:295-301.
35.Cannon JL,Collins A,Mody PD,et al.CD43 regulates Th2 differentiation and inflammation.J Immunol.2008;180:7385-7393.
36.Marelli-Berg FM,Okkenhaug K,Mirenda V.A two-signal model for T cell trafficking.Trends Immunol.2007;28:267-273.
37.Alcaide P,King SL,Dimitroff CJ,Lim YC,Fuhlbrigge RC,Luscinskas FW.The 130-kDa glycoform of CD43 functions as an E-selectin ligand for activated Th1 cells in vitro and in delayed-type hypersensitivity reactions in vivo.J Invest Dermatol.2007;127:1964-1972.
38.Mody PD,Cannon JL,Bandukwala HS,et al.Signaling through CD43 regulates CD4 T-cell trafficking.Blood.2007;110:2974-2982.
39.Carlow DA,Ziltener HJ.CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.J Immunol.2006;177:6450-6459.
40.Veninga H,Becker S,Hoek RM,et al.Analysis of CD97 expression and manipulation:antibody treatment but not gene targeting curtails granulocyte migration.J Immunol 2008;181:6574-6583.
41.Capasso M,Durrant LG,Stacey M,Gordon S,Ramage J,Spendlove I.Costimulation via CD55 on human CD4+ T cells mediated by CD97.J Immunol.2006;177:1070-1077.
42.Miyamoto Y J,Mitchell JS,McIntyre BW.Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes.Mol Immunol.2003;39:739-751.
43.Warren AP,Patel K,Miyamoto Y,Wygant JN,Woodside DG,McIntyre BW.Convergence between CD98 and integrin-mediated T-lymphocyte co-stimulation.Immunology.2000;99:62-68.
44.Sancho D,Gomez M,Sanchez-Madrid F.CD69 is an immunoregulatory molecule induced following activation.Trends Immunol.2005;26:136-140.
45.Sancho D,Gomez M,Viedma F,et al.CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis.J Clin Invest.2003;112:872-882.
46.Esplugues E,Sancho D,Vega-Ramos J,et al.Enhanced antitumor immunity in mice deficient in CD69.J Exp Med.2003;197:1093-1106.
47.Han Y,Guo Q,Zhang M,Chen Z,Cao X.CD69+ CD4+ CD25-T cells,a new subset of regulatory T cells,suppress T cell proliferation through membrane-bound TGF-betal./7/wmmo/.2009;182:111-120.
48.Wing K,Onishi Y,Prieto-Martin P,et al.CTLA-4 control over Foxp3+ regulatory Tcell function.Science.2008;322:271-275.
49.Chen L.Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity.Nat Rev Immunol.2004;4:336-347.
50.Zheng SG,Wang JH,Stohl W,Kim KS,Gray JD,Horwitz DA.TGF-beta requires CTLA-4 early after T cell activation to induce FoxP3 and generate adaptive CD4+CD25+ regulatory cells.J Immunol.2006;176:3321-3329.
51.Li R,Perez N,Karumuthil-Melethil S,Prabhakar BS,Holterman MJ,Vasu C.Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25-TGF-beta 1+ adaptive regulatory T cells./ Immunol.2007;179:5191-5203.
52.So T,Croft M.Cutting edge:OX40 inhibits TGF-beta-and antigen-driven conversion of naive CD4 T cells into CD25+Foxp3+ T cells./ Immunol.2007;179:1427-1430.
53.Xiao X,Kroemer A,Gao W,Ishii N,Demirci G,Li XC.OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo.J Immunol.2008;181:3193-3201.
54.Valzasina B,Guiducci C,Dislich H,Killeen N,Weinberg AD,Colombo MP.Triggering of OX40 (CD134) on CD4(+)CD25+ T cells blocks their inhibitory activity:a novel regulatory role for OX40 and its comparison with GITR.Blood. 2005;105:2845-2851.
55.Scalapmo KJ,Daikh DI.CTLA-4:a key regulatory point in the control of autoimmune disease.Immunol Rev.2008;223:143-155.
56.Blaha P,Bigenzahn S,Koporc Z,et al.The influence of immunosuppressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade.Blood.2003;101:2886-2893.
57.Nohara C,Akiba H,Nakajima A,et al.Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody:a critical role for OX40 ligand in migration,but not development,of pathogenic T cells.J Immunol.2001;166:2108-2115.
58.Weinberg AD,Bourdette DN,Sullivan TJ,et al.Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis.Nat Med.1996;2:183-189.
59.Wang N,Campo M,Ting L,Fleming C,Terhorst C,Finn PW.The costimulatory molecule SLAM is critical for pulmonary allergic responses.Am J Respir Cell Mol Biol 2006;35:206-210.
60.Abbas AK,Lichtman AH,Pillai S.Cellular and Molecular Immunology.6th ed.Philadelphia:Saunders Elsevier,2007:410-416.
61.Zhou D.OX40 signaling directly triggers the antitumor effects of NKT cells.J Clin Invest.2007;117:3169-3172.
62.Yanagita S,Hori T,Matsubara Y,Ishikawa T,Uchiyama T.Retroviral transduction of acute myeloid leukaemia-derived dendritic cells with OX40 ligand augments their antigen presenting activity.Br J Haematol.2004;124:454-462.
63.Leach DR,Krummel MF,Allison JP.Enhancement of antitumor immunity by CTLA-4 blockade.Science.1996;271:1734-1736.
64.Egen JG,Kuhns MS,Allison JP.CTLA-4:new insights into its biological function and use in tumor immunotherapy.Nat Immunol.2002;3:611-618.
65.van EA,Hurwitz AA,Allison JP.Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4)and granulocyte/macrophage colony-stimulating factor(GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.J Exp Med.1999;190:355-366.
66.Sutmuller RP,van DLM,van EA,et al.Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25(+)regulatory T cells in antitumor therapy reveals alternative pathways for suppression of autoreactive cytotoxic T lymphocyte responses.JExp Med.2001;194:823-832.
67.Fiorentino S,Chopin M,Dastot H,et al.Disruption of T cell regulatory pathways is necessary for immunotherapeutic cure of T cell acute lymphoblastic leukemia in mice.Eur Cytokine Netw.2005;16:300-308.
68.Perez-Garcia A,Brunet S,Berlanga JJ,et al.CTLA-4 genotype and relapse incidence in patients with acute myeloid leukemia in first complete remission after induction chemotherapy.Leukemia.2009;23:486-491.
69.Messmer D,Kipps TJ.CD154 gene therapy for human B-cell malignancies.Ann N YAcad Sci.2005;1062:51-60.
70.D'Amico G,Marin V,Biondi A,Bonamino MH.Potential use of CD40 ligand for immunotherapy of childhood B-cell precursor acute lymphoblastic leukaemia.Best Pract Res Clin Haematol.2004;17:465-477.
71.Chen HR,Ji SQ,Wang HX,et al.Humanized anti-CD25 monoclonal antibody for prophylaxis of graft-vs-host disease(GVHD)in haploidentical bone marrow transplantation without ex vivo T-cell depletion.Exp Hematol.2003; 31:1019-1025.
72.Jacobsohn DA.Novel therapeutics for the treatment of graft-versus-host disease.Expert Opin Investig Drugs.2002;11:1271-1280.
73.Zeiser R,Marks R,Bertz H,Finke J.Immunopathogenesis of acute graft-versus-host disease:implications for novel preventive and therapeutic strategies.Ann Hematol.2004;83:551-565.
74.Edinger M,Hoffmann P,Ermann J,et al.CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation.Nat Med.2003;9:1144-1150.
75.Cheever MA.Twelve immunotherapy drugs that could cure cancers,Immunol Rev.2008;222:357-368.