Abstract
Here we present methods to longitudinally track islet allograft–infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (Teff cells) and discrimination between natural and induced regulatory T cells (nTreg and iTreg cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance.
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Acknowledgements
This work was supported by grants from the US National Institutes of Health (R21 AI081010 to S.H.Y. and R01 EY14106 to C.P.L. and P01-AI041521 and P01-AI041521-S1 to T.B.S.) and the Juvenile Diabetes Research Foundation (JDRF 7-2005-1329 to T.B.S. and M.K.).
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Z.F. and J.A.S. designed the experiments, conducted research, collected and analyzed data and wrote the manuscript; Y.L., C.M.P., G.S. and V.T. helped conduct research and collected and analyzed data; P.K. and S.H.Y. developed and performed endoscopic microscopy; T.B.S., C.P.L. and M.K. designed the experiments, sponsored the project and wrote the manuscript.
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Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Table 1 (PDF 3048 kb)
Supplementary Video 1
Z-stack reconstructed movie showing yellow iTreg (green + red), green nTreg and red Teff cells (red) in a tolerized islet allograft at week 2. Z axis step size is 2 ÎĽm. (AVI 10288 kb)
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Fan, Z., Spencer, J., Lu, Y. et al. In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response. Nat Med 16, 718–722 (2010). https://doi.org/10.1038/nm.2155
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DOI: https://doi.org/10.1038/nm.2155
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