Early Activiation And Cross Cytotoxicity Effect Of Gammadelta T Cell Against Influenza Virus

BackgroundSince the first worldwide large outbreak of influenza in1918, a total of4times worldwide pandemic has caused human infection and definetly caused deaths ofpeople’s lives and serious impact to human health and social economy in its over100years history. Tough the vaccine has been the classic method for human preventionand treatment against influenza virus infection, the rapid antigen variation ofinfluenza virus make it difficult to develop a high cross-protection vaccine. Influenzavaccine research usually mainly concentrated in the the humoral immune, but alwayslack of cross protection to various subtypes and new variant strains. In cell-mediatedimmunity, virus-specific memory T cells have the proficiency to providecross-reactivity to each subtype virus and thus play a protective role, but it isdemonstrated that vaccinesproduction based on memory β T cells are difficult.Vγ9V2T cells are main cells group which can specifically identify individualantigens (including peptide antigen and non-peptide antigen) in human peripheralblood and characterized as non MHC strictive in antigen recognition stage. It has beenstated that Vγ9V2T cells took part in opening adaptive immunity and killinginfected cell in infection. The phenomenon that Vγ9V2T cells can be activiated byinfluenza A and play an important role in killing target cell in early time has beenreported recently. All its characterics and related research indicate the cross immunity protective effect of γ T cells and provide a new direction for the development ofcross immunity effect vaccine.However, what’s the potential cross-protective immunity mechanism?,Whether γ T cell’s cross-protective immunity mechanism is the same in other highvariated influenza virus? To study the broad spectrum activiation and cross-protectionabilities of γ T cells against influenza virus A and B which based on free MHCrecognition are important for vaccine research and delvelopment.PurposeTo explore the possibility of γ T cells as a broad-spectrum anti-influenzavirus immune cells. To provide the scientific basis for universal vaccine developmentand broad-spectrum anti-influenza virus treatment strategies. To establish thefoundation for depth understanding the recogniton and immune response mechanismof γ T cells against influenza virus.Methods1. The activation of γ T cells by various types and subtypes of influenza virusvia flow cytometry: Albuginea which collected from10healthy volunteers peripheralblood was separated into human peripheral blood munonuclear cells (PBMC) bydensity gradient centrifugation method. PBMC stimulated by H1N1、H3N2、BV(MOI=2) for24hours were re-harvest for the flow cytometry to test the activationmarkes (CD25and CD69) and differentiation marks (CD4and CD8) on the surface ofT cells and γ T cells.2. The cytotoxicity of γ T cells against influenza virus via lactatedehydrogenase release test (LDH): PBMC separated from6health volunteers weredifferentiated into MDM by9days stimulated with recombinant human granulocytemacrophage colony stimulating factor (rhGM-CSF). Co-cultured the target cells(H1N1、H3N2、BV infected MDM) and effect cells (influenza virus stimulated γ Tcells) at ratio of10:1, and LDH was used to detect the direct and cross cytotoxicity ofγ T cells against various influenza virus infection. 3. The influence of mevalonate pathway to the activiation and cytotoxicity of γT cells stimulated by influenza virus: PBMC were separated from10healthyvolunteers. Before the virus stimulation, PBMC were pre-cultured with fluvastatin(10μM/mL) for4hours. After24hours virus stimulation, activiation anddifferentiation marks were test as forward. Before the virus stimulation,6volunteers’PBMC were pre-cultured with fluvastatin (10μM/mL) for4hours. Target cells(H1N1、H3N2、BV infected MDM) and effect cells (influenza virus stimulated γ Tcells) were co-cultured at ratio of10:1. LDH was used to detect the direct and crosscytotoxicity of γ T cells against various influenza virus infection so as to explore theimpact of mevalonate pathway for γ T cells’ cytotoxicity.Results1. For normal population, T cells and γ T cells are usually in the unactivatedstate with the low expression of CD25、CD69and CD25+CD69+marks (T cells:9.76%,14.09%and2.79%; γ T cells:5.88%、8.46%and2.77%). Both of these twotypes of cells are mainly in initial state with high level of CD4-CD8-(T cells:31.48%;γ T cells:61.02%) and low level of CD4+CD8+(T cells:1.00%; γ T cells:2.64%).2. γ T cells can be early activiated after various influenza virus stimulating in24hours. The expression of CD25、CD69and CD25+CD69+marks for H1N1、H3N2and BV stimulation are respectively24.60%、40.11%、22.23%；17.79%、29.81%、15.24%；16.87%、29.84%、15.63%. There are a certain degree of CD4+CD8+functional cells in H1N1、H3N2and BV stimulated γ T cells (6.22%、4.14%and3.48%).3. There are high direct cytotoxicity of activiated γ T cells to H1N1、H3N2andBV infect two different target cells (MDCK:43.59%、36.76%、21.59%; MDM:42.94%、38.15%、23.13%). The H1N1activiated γ T cells showed the higest crosscytotoxicity with the36.99%and18.16%cytotoxicity in H3N2and BV infectedMDM groups. The H3N2activiated γ T cells also showed a certain degree crosscytotoxicity to H1N1infected MDM (29.99%) and BV infected MDM (10.63%). 4. The activiation of γ T cells to H1N1、H3N2and BV are declined afterfluvastatin pretreatment which showed decrease of CD25、CD69and CD25+CD69+(H1N1decreased by56.14%,57.71%and62.12%; H3N2decreased by40.92%、57.63%and57.55%; BV decreased by30.88%、39.24%and42.87%). The directcytotoxicity to H1N1、H3N2and BV infected MDM are decreased by42.77%、75.66%and60.63%. The cross cytotoxicity of H1N1activiated γ T cells to H3N2and BV infected MDM respectively decreased by67.96%and51.28%; the crosscytotoxicity of H3N2activiated γ T cells to H1N1and BV infected MDMrespectively decreased by80.17%and56.52%; the cross cytotoxicity of BV activiatedγ T cells to H1N1and H3N2infected MDM respectively decreased by10.06%and14.48%.Conclusion1. The T cells and γ T cells in peripheral blood are in an inactive state. Thoughthere is certain part of CD8+population cells, they are mainly characterized asCD4CD8double negative and non CD4+cells.2. Various influenza viruses can early and rapidly activate γ T cells without thetype difference. The activated γ T cells are trend to differentiate into effector T cellsmajored in CD4+and CD4+CD8+cells. The differentiations degrees are influenced byvirus type which is most obvious in H1N1stimulation group with CD4CD8doublepositive cells.3. The activated γ T cells open its direct and cross cytotoxicity progress andthese cytotoxicities are obvious in influenza A goups (H1N1and H3N2).4. Mevalonate pathway may be the potential way to influence the activiationand cytotoxicity of γ T cells.