In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response

Journal name:
Nature Medicine
Volume:
16,
Pages:
718–722
Year published:
DOI:
doi:10.1038/nm.2155
Received
Accepted
Published online

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.

At a glance

Figures

  1. In vivo imaging of color-coded T cells.
    Figure 1: In vivo imaging of color-coded T cells.

    (a) FACS sorting of DsRed+CD4+GFP red Teff cells from DsRed–knock-in mice and CD4+GFP+ green nTreg cells from the original knock-in mice. (b) Graft survival curves of mice treated with CD154-specific mAb plus rapamycin and untreated rejecting controls. The difference in the survival curves is significant, as calculated by either log-rank (Mantel-Cox) (P = 0.0004) or Gehan-Breslow-Wilcoxon (P = 0.0012) tests. (c) Representative image of allograft-infiltrating nTreg (green), Teff (red) and iTreg cells (yellow) acquired by intravital microscopy. Scale bar, 50 μm.

  2. Analysis of infiltrating T cells within islet allografts.
    Figure 2: Analysis of infiltrating T cells within islet allografts.

    (a) Representative intravital microscopy images showing T cell infiltration within islet allografts in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin on week 1 and week 2 after transplantation. Scale bar, 100 μm. (bd) Summary of cell density of islet allograft–infiltrating nTreg (b), iTreg (c) and Teff (d) cells, as detected by intravital imaging. (e,f) Summary of the ratios of islet allograft–infiltrating nTreg to Teff (e) and iTreg to Teff (f) cells, as detected by intravital imaging. Error bars represent means ± s.d.

  3. Serial endomicroscopy of infiltrating T cells within islet allografts.
    Figure 3: Serial endomicroscopy of infiltrating T cells within islet allografts.

    Representative endomicroscopy images within islet allografts on days 3, 5, 7, 10, 12 and 14 after transplantation in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin. Each row of images is from the same mouse at the given time points. Infiltrating nTreg (green), Teff (red) and iTreg cells (green + red) accumulate in the allograft over time. Scale bar, 50 μm.

  4. Detection of nTreg, Teff and iTreg cells by in vivo flow cytometry in the peripheral blood.
    Figure 4: Detection of nTreg, Teff and iTreg cells by in vivo flow cytometry in the peripheral blood.

    (a) A representative in vivo flow cytometry trace showing the identification of single positive nTreg (green box), Teff (red boxes) and double-positive iTreg (yellow box) cells. The second peak in the DsRed channel occurred about 45 ms before the second peak in the GFP channel. As this time difference was greater than the uncertainty of the measurements, these two peaks were distinguished as separate cells and not a double-positive iTreg cell. (b) In vivo flow cytometry showing Teff (red), nTreg (green) and iTreg cells (yellow) in the peripheral blood. There is a ten-fold difference in scale between mice treated with CD154-specific mAb plus rapamycin and untreated mice. Each curve represents serial analysis of the same blood vessel of the same animal. (c) Summary of the ratio of circulating Treg to Teff cells, as detected by in vivo flow cytometry. Error bars represent means ± s.d.

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Author information

  1. These authors contributed equally to this work.

    • Zhigang Fan &
    • Joel A Spencer

Affiliations

  1. Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

    • Zhigang Fan,
    • Yan Lu,
    • Gurbakhshish Singh,
    • Vasilis Toxavidis,
    • Terry B Strom &
    • Maria Koulmanda
  2. Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Joel A Spencer,
    • Costas M Pitsillides &
    • Charles P Lin
  3. Department of Biomedical Engineering, Science and Technology Center, Tufts University, Medford, Massachusetts, USA.

    • Joel A Spencer
  4. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.

    • Costas M Pitsillides
  5. Advanced Microscopy Program, Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Pilhan Kim &
    • Seok H Yun

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (3M)

    Supplementary Figures 1–5 and Supplementary Table 1

Movies

  1. Supplementary Video 1 (10M)

    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 μm.

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