Molecular Identification And Functional Characterization Of A Novel T Cell Activation Marker Recognized By A Monoclonal Antibody ZCH-2B8a

OBJECTIVESMonoclonal 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^thInternational 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.METHODSThis 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.RESULTSThe 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:5×10⁴ 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.CONCLUSIONS1.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.