We have been revealing immune system based of spatiotemporal regulation of immune cells in the entire body by using photoconvertible fluorescent protein (FP), Kaede and KikGR mice. Flow cytometric analysis of green and red signals of non-photoconverted and photoconverted Kaede or KikGR expressing cells with multiple fluorochrome-conjugated mAbs have been difficult and unable to increase color panels, because of the fluorescence signal overlap between FPs and fluorochromes. We have newly developed a spectral flow cytometer (FCM) based on a novel measurement principle. Unlike a polychromatic FCM, the spectral FCM with 32 channel linear array PMT detects the fluorescence derived from every fluorescent probe. The data of acquired spectra is analyzed with a unique algorithm. We detected spectral changes of KikGR color from green to red during photoconversion. We separated EGFP and Venus. One of the feature advantages of this instrument is that it can recognize spectral shape of each fluorescent probes and we could separate EGFP and FITC, their peaks of wavelength are almost same and only spectral shape of GFP is slightly broader than FITC. Finally, we successfully separated 11-colors including kikGR-green and -red simultaneously. Taken together, spectral FCM allow us to detection of FPs with multicolor fluorochromes, and thus, the system which uses KikGR mice with spectral FCM is a powerful tool to investigate spatiotemporal regulation of immune cells in the entire body.
Detailes of spatiotemporal regulation of antigen-carring DCs migrated from immunized site and antigen-specific T cell prolifeaion in the dLN is unknown. Kaede-Tg mice can monitor cellular movement by labeling cells by changing color from green to red by violet light and Fucci-Tg mice can visualize cell-cycle progression. Footpads of Kaede-Tg mice were immunized with CFA/OVA and labeled cells in the footpad and followed their migration to the dLN. After immunization, 100 times of MHC class IIHighDC migrated from immunized footpad compared with that in the steady state and lasted more than 14d. Whereas, MHC class IIInt DCs and pDCs increasead in dLN, which was sustained by increase of precursor immigration from blood circulation, migration from immunized site and proliferation. However, almost all migrated DCs from immunized site in the dLN replaced within less than 24h. Thus rapid replacement of DCs enable to control fine-tune of immune responses and spatiotemporal regulation of DCs under immune response is like “Large supply and mass consumption”. Although antigen-specific T cell proliferation already terminated 7d after immunization, skin-derived DCs continued to carry antigen from immunized site and dLN had capability to induce proliferation of re-transferred naive T cells. Thus termination of antigen-specific T cell proliferation is controlled by T cell intrinsic manner, not for the result of the absence of antigen-presenting DCs in dLN.
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