| γδT cells which are one kind of human T lymphocytes in 1-5 percentages express theγδTCRs and recognize antigens without MHC restriction.γδT cells have potent antiviral activities against different viruses, but few data are available concerning their responses against influenza viruses and their hemagglutinins. Therefore we design different methods to study whetherγδT cells could recognize influenza A viruses hemagglutins and reveal the responses against hemgglutinins.The present study focused on four scientific questions as follows:First, to demonstrate whether y8T cells in PBMCs can response to influenza A virus hemagglutinins(HAs).Second, if y8T cells response to the stimulation of influenza A virus HAs,,whether HA proteins could be recognized byγδT cells directly Whether HA proteins could be recoganized via TCRγδor NKG2D?Thirdly, could HA proteins stimulateγδT cells dependent on other mechanisms,if there is no direct recognization between HA proteins andγδT cells?Finaly, which is the possible pathway mediate the cytotoxicity of activatedγδT cells to virus infected host cells in the infection tissues than the peripheral blood?To solve the scientific questions as mentioned above, we performed the investigations and got the results as follows.(1) We successfully expressed and purified recombinant extracellular segments of HA proteins in baculovirus expression system and packaged successfulfy influenza A pseudotypes coating with HA proteins. The proteins and pseudotypes make foundation for further researches.(2) HA proteins rapidly upregulate the expression of early activation markers onγδT cells like CD69 and the production of activation cytokines like IFN-y. The same as the pseudotypes.(3)The data of ELISA,FCM and Confocal assays display that HA proteins can bind withγδT cells. Rresults also indicate that the receptors onγδT cells binding with HAs are not TCRγδ. Blocking assay show that the receptors binding with HA proteins are not NKG2D, TLRs or other potential HA receptors have reported on other cells. CoIP of membrane proteins could not find the specific receptor of HA proteins..At last, we found that non-specific receptors binding with HA proteins are a-2,3 and a-2,6 SA.(4) We got the conclusion that the activation ofγδT cells triggered by HA proteins needs help from the other cells of PBMCs.. The help effects are are probablely mediated by the soluble molecules like cytokines.(5) ActivatedγδT cells efficiently killed pseudotypes-infeceted macrophages mediated by NKG2D-stressed moleculars pathway.Taken these data together, influenza A virus HA proteins can quickly trigger the activation of humanγδT cells in peripheral blood through indirect ways, probablely depend on soluble cytokines from other immune cells. ActivatedγδT cells show cytotoxicity activity to pseudotype-infected macrophages mediated by NKG2D pathway.We verified the influenza A virus HAs involved in the immune responses when the virus invading human bodies and interpreted the possible mechanism during the immune responses,. The study improve people's knowledgement about biological function especially the anti-virus ability ofγδT cells, which may help people develop new alternative therapy and prevention ways against the influenza virus especially pandemic or high pathogenic influenza virus.
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