| ObjectiveAccording to the research of actuality of collaboration between macrophages anddendritic cells(DCs) involved in adaptive immune response in vivo, interaction andmechanism of splenic macrophages and DCs is the key scientific problem, and the moderncell/molecular immunologic methods are used as the core technology. Combinedapplication of kinds of knockout mice such as Baft3-/-and CX3CR1GFP/GFPmice, thisexperiment studied the role of macrophages subsets and DCs subsets in theantigen-induced CD4+T immune response, and further investigate the mechanism ofsplenic macrophages transferring Ag to DCs. This study helps to provide the theoreticalbasis for state the mechanism of antigen presenting cells(APCs) to induce and regulateadaptive immune response.Methods1. To prepare for cell-associated antigen, thymocytes were collected from C57BL/6mice, then were exposed to2000cGy of X-radiation and cultured for4hours at37°C inRPMI1640media plus1%BSA and10mg/ml OVA VI protein.2. To analyze localization of cell-associated antigen in the spleen, C57BL/6micewere sacrificed and splenic single cell suspension was collected in1hour after injectinge450-labeled CD45.1+thymocytes intravenously. Moreover, a part of spleen was keepedfor frozen section. The phagocytosis of cell-associated antigen were analyzed by FACS andimmunofluorescence.3. C57BL/6mice were injected with200ul CL or PBS,on0d、4d、11d. Then transfer106-CD4+Vβ5+Vα2+T cell s labeled with e450derived from OT II.CD45.1+miceTwenty-four hours later, mice were injected with cell-associated antigen.3d later,single-cell suspension of spleen were prepared, stained and examined the proliferation of CD45.1+CD4+T cells with dilusion of e450and absolute numbers. by FACS.4. Batf3-/-or C57BL/6mice were transferred with106CD4+Vβ5+Vα2+T cells derivedfrom OT II.CD45.1+mice intravenously, twenty-four hours later, the proliferation ofCD45.1+CD4+T cells with dilusion of e450and absolute numbers were analyzed byFACS.5. C57BL/6mice were injected cell-associated antigen intravenously,1hours later,splenic macrophages and DC were sorted, and cocultured with e450-labeledCD4+Vβ5+Vα2+T cells derived from OT II mice.96hours later, the proliferation ofCD45.1+CD4+T cells with dilusion of e450and absolute numbers were analyzed byFACS.6. C57BL/6mice were injected cell-associated antigen intravenously,1hours later,splenic macrophages were sorted, then incubated with Naive DC derived from C57BL/6mice for1h、3h、4h、5h, the percentage of antigen from macrophage to DC were measuredby FACS.7. C57BL/6mice were transferred106e450-labeled CD4+Vβ5+Vα2+T cells derivedfrom OT II.CD45.1+mice intrabavenously, the next day, mice were injected with500ng/mouse pertussis toxin(PTx) intraperitoneally which inhibits Gαi protein-coupledreceptors including chemokine receptor signaling.8hours later, mice were injected withcell-associated antigen and were injected with500ng/mouse PTx i.p.again. Three dayslater, the proliferation of CD45.1+CD4+T cells with dilusion of e450and absolutenumbers were analyzed by FACS.8. C57BL/6mice were injected cell-associated antigen intravenously,1hours later,splenic macrophages and na ve DC were sorted separately, then cocultured withe450-labeled CD4+Vβ5+Vα2+T cells separately or together.96hours later, the proliferationof CD45.1+CD4+T cells with dilusion of e450and absolute numbers were analyzed byFACS.9. CX3CR1GFP/GFPwere transferred with106e450-labeled CD4+Vβ5+Vα2+T cellsderived from OT II.CD45.1+mice intrabavenously.. The next day mice were injected withcell-associated antigen, and three days later, the proliferation of CD45.1+CD4+T cellswith dilusion of e450and absolute numbers were analyzed by FACS.10. For two days, C57BL/6mice were intraperitoneally injected with GW4869(1.25mg/kg/day), an inhibitor of exosome secretion，and mice with DMSO (solvent ofGW4869) as a control, for3days then were transferred with106e450-labeledCD4+Vβ5+Vα2+T cells from OT II.CD45.1+mice intrabavenously.Twenty-four hours later, mice were injected with cell-associated antigen and the same time intraperiton-ealinjected with GW4869or DMSO for3days. the fourth day, the proliferation of CD45.1+CD4+T cells with dilusion of e450and absolute numbers were analyzed by FACS.Results1. Splenic macrophage, mDC, LDC,pDC and neutrophils have the abilities tophagotose cell-associated antigen, the ratio of phagocytosis is16%,1.5%，19%，3.2%and18.6%respectively.2. Compared to control, clearance ratio of mDC, LDC and F4/80+Фis50%,99%and100%after injection of CL for1d; clearance ratio of F4/80+MΦis100%after injection ofCL for5d, however the ratio of cDC subsets has been recovered; clearance ratio of F4/80+MΦis33%after injection of CL for12d.3. The ratio of antigen-specific CD4+T cells proliferation reduced80%、60%、20%respectively after injection of CL for1d,5d, and12d.4. The percentage of antigen-specific CD4+T cells proliferation reduced70%inBatf3-/-mice compared to that of control mice.5. Macrophage, DC or both were cocultured with OTII T cells for3d, the ratio of Tcells proliferation is3.39%,22.6%and38.1%respectively analyzed by dilution of e-450.6. The ratio of DC which contained cell-associated Ag derive from macrophage is0.12%,0.7%,1.07%and1.55%respectively after incubated na ve DC and macrophagephagocytosing Ag.7. na ve DC, macrophage phagocytosing Ag or both were cocultured with OTII Tcells for3d, the ratio of T cells proliferation analyzed by dilution of e-450is2.97%,33.5%and55.6%respectively.8. The ratio of specific CD4+T cells proliferation reduced70%when mice wereinjected PTx which inhibit migration of cells. The proliferation of CX3CR1GFP/GFPmicewhose splenic DC and F4/80macrophage both defecte CX3CR1specific CD4+T cellsreduced20%。9. The proliferation of specific CD4+T cells reduced20%when mice was ip. injectedwi-th GW4869compared with DMSO.Conclusion:Splenic macrophages can transfer antigen to dendritic cells, and play an import role inenhancing CD4+T cell immune response. The methods of transferring antigen includemigration of immune cells, releasing of exosome which encompass antigen. |
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