Mechanism And Biological Effect Of Human γδT Cell Activation Via Lipid A

TCRγδ-expressing cells are believed to play an important role in immune defense and immunoregulation.However,we have little knowledge about theγδT cell-recogized antigens so far,especially for lipid ones.Clarification of the mechanism of antigen recognition byγδT cells is essential for better understanding biological function ofγδT cells.Moreover,it will deepen the understanding of the essence of immune response and also set up a novel strategy for immune therapy.The present study focused on the antigen recognition mechanism of humanγδT cells to LA,elementarily explored the length and sequence features of TCRδ-CDR3,and further demonstrated the immunological effects of LA-reactiveγδT cells.1.Study on the antigen recognition mechanism of humanγδT cells to LA. LA is the most conservative membrane anchor of LPS and is regarded to be responsible for LPS-induced biological effects.It has strong antigenicity.In this study,we examined the following questions:What indeed is the mechanism of LA recognition of humanγδT cells? Can a lipid antigen induce humanγδT cells proliferate in CD1-restricted manner? Which CD1 family member is responsible for LA-induced activation of theγδT cells? How about the features of TCRγδspecific to LA? How LA-reactiveγδT cells play an immunological function during bacterial invasion? Therefore,we firstly detected the proliferation of humanγδT cells from peripheral blood induced by LA in the presence or absence of monocyte-derived dendritic cells(moDC) by flow cytometry,~3H-TdR incorporation assay and Carboxyfluorescein diacetate succinimidyl ester(CFSE) staining technique.Secondly,we blocked the proliferation ofγδT cells induced by LA with antibodies against CD1a,CD1b,CD1c,CD1d,TCRγδ,LA,MHCⅠor MHCⅡ, respectively.Thirdly,we analyzed the TCRδCDR3 sequences of LA-reactiveγδT cells by sequencing and gene scanning;Finally,we measured IFN-γ,TNF-α,IL-2,IL-4,IL-5 or IL-10 secreted by LA-reactiveγδT cells before or after the blockade with anti-CD1a,CD1b,CD1c,CD1d,CRγδor LA antibodies.Our data indicate that LA loaded on moDC can induce significantly proliferation ofγδT cells,however,there is basically no proliferation ofγδT cells observed whenγδT cells is stimulated with LA alone;anti-CD1b, -CD1c,-TCRγδor-LA antibodies completely blocked the response ofγδT cells to LA-loaded moDC,but other antibodies have no such effect;furthermore,we find there is no cross-blocking effect between antibodies against CD1b and CD1c;the results obtained from gene scan and sequencing show that there is no specific amino sequence in TCRδCDR3 of LA-reactiveγδT cells,but there are preponderant sequences which are about 75bp in length and TDRV2D-J1 in amino constitution;LA-loaded moDCs induceγδT cells to produce Th1 cytokine,especially IFN-γand TNF-α,and in agreement to antibody blocking assay,anti-CD1b,-CD1c,-TCRγδor -LA antibodies completely block the secretion of cytokines fromγδT cells.Taken together,our data demonstrate that humanγδT cells recognize LA presented by CD1b or CD1c on DCs in a TCRγδ-dependent manner.The findings provide the evidence for the pattern of antigen recognition ofγδT cells for lipid antigen.The results about TCRδCDR3 provide clues and foundations for seeking TCRγδsequences with high specificity and appetency.2.Study on the function of LA-reactiveγδT cellsIn this part,we mainly studied on the cytotoxity of LA -reactiveγδT cell to E.coli,and the immunoregulatory effects of LA-reactiveγδT cells on phagocytosis of macrophages and the anti-LA antibody production of B lymphocytes.Firstly,we used redox p-iodonitrotetrazolium(INT) and 5-cyano-2,3-ditolyl tetrazolium chlorid(CTC) to detect the cytotoxity of LA-reactiveγδT cells to E.coli,results showed that respiratory activity of E.coli significantly reduced after incubated with LA-reactiveγδT cells according to the formazan production,indicating that LA-reactiveγδT cells truly can kill E.coli directly and effectively,but the mechanism for this inhibitory effect is needed to study further.Secondly,through constructing the expressing vector for green fluorescent protein(GFP) in E.coli and inducing its expression in E.coli BL21 by IPTG;we obtained GFP expressing E.coli(E.coli-GFP) which was used to detect the phagocytosis of macrophages. Autologous macrophages were given E.coli-GFP in the presence or absence of LA-activatedγδT cells before flow cytometrical analysis.Results showed that purified LA-activatedγδT cells induced a significant increase of E.coli-GFP internalization by macrophages when compared to those without LA-activatedγδT cells,suggesting LA-activatedγδT cells enhance phagocytosis of autologous macrophages.Finally,we checked the regulatory effects of LA-reactiveγδT cells on anti-LA antibody production of B lymphocytes in hu-PBMC-SCID mice.We examined the anti-human Ig G in SCID peripheral blood after 2 wk since given human PBMC.Then,the successfully reconstructed ones were immunized with LA plus LA-reactiveγδT cells or not plus,and measured the anti-LA antibodies after 2,3,5,8 or 10 wk,respectively,using ELISA.Results showed that LA-reactiveγδT cells enhanced the antibody producing function of B lymphocytes.All the above results demonstrate that LA-reactiveγδT cells can clear E.coli directly,as well as by regulating other effective immune cells,such as macrophages and B cells,which provide evidences for the notion thatγδT cells bridge the gap between innate and acquired immunity,andγδT cells in inflammatory setting are useful targets for manipulation in vaccine research and for the development of LA /LA-reactiveγδT cell-based immunotherapies.