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Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR–induced stop signal

Nature Immunology volume 10, pages 1185–1192 (2009)Cite this article

Abstract

Programmed death 1 (PD-1) is an inhibitory molecule expressed on activated T cells; however, the biological context in which PD-1 controls T cell tolerance remains unclear. Using two-photon laser-scanning microscopy, we show here that unlike naive or activated islet antigen–specific T cells, tolerized islet antigen–specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DCs) in pancreatic lymph nodes. Inhibition of T cell antigen receptor (TCR)-driven stop signals depended on continued interactions between PD-1 and its ligand, PD-L1, as antibody blockade of PD-1 or PD-L1 resulted in lower T cell motility, enhanced T cell–DC contacts and caused autoimmune diabetes. Blockade of the immunomodulatory receptor CTLA-4 did not alter T cell motility or abrogate tolerance. Thus, PD-1–PD-L1 interactions maintain peripheral tolerance by mechanisms fundamentally distinct from those of CTLA-4.

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Figure 1: Antigen-specific tolerance blocks diabetes, TCR signaling and Ca2+ flux in a PD-L1-dependent manner.
Figure 2: PD-1–PD-L1 but not CTLA-4 prevents the T cell stop signal.
Figure 3: PD-L1 inhibition requires antigen.
Figure 4: PD-L1 inhibits T cell movement in the islets.
Figure 5: PD-L1 blockade promotes prolonged T cell–DC interactions and T cell activation.

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Acknowledgements

We thank M. Hara and G. Bell (University of Chicago) for C57BL/6 MIP.eGFP mice; M. Nussenzweig (Rockefeller University) for C57BL/6 CD11c-YFP mice; A. Sharpe (Harvard Medical School) for C57BL/6 PD-L1-deficient mice; C. Benoist and D. Mathis (Harvard Medical School) for NOD BDC2.5 TCR–transgenic mice; R. Locksley (University of California, San Francisco) for Yeti (IFN-γ reporter) mice; N. Martenier for animal care; C. Allen, E. Finger, E. Peterson, R. Freidman, K. Hogquist, C. Penaranda and X. Zhou for scientific discussions; G. Szot and P. Koudria for islet transplantation; C. McArthur for cell sorting; and A. Bullen and M. Jenkins for multiphoton imaging support. Supported by LifeScan, the US National Institutes of Health (AI35297 to J.A.B., and P30 DK63720 for core support), the Juvenile Diabetes Research Foundation (10-2006-799 to B.T.F.), the American Diabetes Association (7-09-JF-21 to B.T.F.) and the University of Minnesota Medical School (B.T.F.).

Author information

Authors and Affiliations

  1. Department of Medicine, UCSF Diabetes Center, University of California, San Francisco, California, USA

    Brian T Fife, Todd N Eagar, Jenny Wu, Qizhi Tang & Jeffrey A Bluestone

  2. Department of Medicine, Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA

    Brian T Fife, Kristen E Pauken & Takashi Obu

  3. Department of Neurology, University of Texas Southwestern, Dallas, Texas, USA

    Todd N Eagar

  4. Department of Surgery, University of California, San Francisco, California, USA

    Qizhi Tang

  5. Department of Molecular Immunology, Tokyo Medical and Dental University, Tokyo, Japan

    Miyuki Azuma

  6. Department of Pathology, University of California, San Francisco, California, USA

    Matthew F Krummel

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Contributions

B.T.F. and J.A.B. designed and conceptualized the research project; B.T.F., K.E.P, T.O. and J.W. did the experiments; B.T.F., T.N.E., Q.T. and M.F.K. analyzed the data; B.T.F prepared the figures; B.T.F. and J.A.B. interpreted the data and wrote the manuscript; and M.A. provided anti-PD-L1 hybridoma.

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Correspondence to Brian T Fife.

Supplementary information

Supplementary Movie 1

Activated antigen specific T cells stop in antigen-draining PLN. A time-lapse sequence of 50 μm z-projection images shows the dynamics of in vitro activated BDC2.5 CD4+ T cells (labeled red using CMTMR) in a PLN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient mouse. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 15 min in real-time, which is compressed to 3 sec in this video. (MOV 1549 kb)

Supplementary Movie 2

Dynamic movement of tolerized T cells in islet antigen-draining PLN. A time-lapse sequence of 40 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and isotype antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 15 min in real-time, which is compressed to 3 sec in this video. (MOV 2206 kb)

Supplementary Movie 3

PD-L1 blockade causes tolerized T cells to stop in islet antigen-draining PLN. A time-lapse sequence of 36 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and anti-PD-L1 antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 15 min in real-time, which is compressed to 3 sec in this video. (MOV 2119 kb)

Supplementary Movie 4

CTLA-4 blockade does not alter tolerized T cell movement in islet antigen-draining PLN. A time-lapse sequence of 44 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and anti-CTLA-4 antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 15 min in real-time, which is compressed to 3 sec in this video. (MOV 1796 kb)

Supplementary Movie 5

Dynamic movement of activated antigen specific T cells in islet antigen-deficient ILN. A time-lapse sequence of 42 μm z-projection images shows the dynamics of in vitro activated BDC2.5 CD4+ T cells (labeled red using CMTMR) in a inguinal LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1371 kb)

Supplementary Movie 6

Tolerized T cells move freely in islet antigen-deficient ILN. A time-lapse sequence of 44 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a inguinal LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and isotype control antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1246 kb)

Supplementary Movie 7

PD-L1 blockade does not alter tolerized T cell movement in islet antigen-deficient ILN. A time-lapse sequence of 46 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a inguinal LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and anti-PD-L1 injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1143 kb)

Supplementary Movie 8

CTLA-4 blockade does not alter tolerized T cell movement in islet antigen-deficient ILN. A time-lapse sequence of 40 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a inguinal LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and anti-CTLA-4 injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 3116 kb)

Supplementary Movie 9

PD-L1 blockade causes tolerized T cells, but not naïve polyclonal T cells to stop in islet antigen-draining PLN. A time-lapse sequence of 28 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a pancreatic LN explant 18h after transfer to a NOD.CD4-eGFP (green naïve T cells) recipient and anti-PD-L1 antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1588 kb)

Supplementary Movie 10

Dynamic movement of tolerized T cells in pancreatic islet tissue. A time-lapse sequence of 46 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in pancreatic islet grafts of NOD.SCID transplant recipients receiving NOD.MIP-GFP islets (green β-cells) and isotype control antibody injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 2122 kb)

Supplementary Movie 11

PD-L1 blockade causes tolerized T cells to stop in pancreatic islet tissue. A time-lapse sequence of 42 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in pancreatic islet grafts of NOD.SCID transplant recipients receiving NOD.MIP-GFP islets (green β-cells) and anti-PD-L1 injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 3370 kb)

Supplementary Movie 12

CTLA-4 blockade does not alter tolerized T cell movement in pancreatic islet tissue. A time-lapse sequence of 44 μm z-projection images shows the dynamics of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in pancreatic islet grafts of NOD.SCID transplant recipients receiving NOD.MIP-GFP islets (green β-cells) and anti-CTLA-4 injection. Elapsed time is shown as hh:mm:ss:sss. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1808 kb)

Supplementary Movie 13

Investigation of T cell-DC conjugates in pancreatic LN. A time-lapse sequence of 34 μm 3D projection shows the dynamic movement of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP (yellow and green DC) recipient and isotype control antibody injection. Elapsed time is shown as hh:mm:ss:sss. Randomly selected cells were tracked and the trailing line following the cells represents the cell path over time. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1276 kb)

Supplementary Movie 14

PD-L1 blockade induces stable T cell-DC conjugates in pancreatic LN. A time-lapse sequence of 36 μm 3D projection shows the stable interactions of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) with DC (yellow and green) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP recipient and anti-PD-L1 injection. Elapsed time is shown as hh:mm:ss:sss. Randomly selected cells were tracked and the trailing line following the cells represents the cell path over time. The duration of the imaging is 10 min in real-time, which is compressed to 2 sec in this video. (MOV 1690 kb)

Supplementary Movie 15

Dynamic T cell-DC interactions following T cell tolerance. A magnified time-lapse sequence illustrating the dynamic and short interactions of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) with DC (yellow and green) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP recipient and isotype antibody injection. Elapsed time is shown as hh:mm:ss:sss. Brief conjugates can be seen with co localization of the cells in yellow. The duration of the imaging is 30 min in real-time, which is compressed to 6 sec in this video. (MOV 2493 kb)

Supplementary Movie 16

PD-L1 blockade induces stable T cell-DC conjugates and breaks tolerance. A magnified time-lapse sequence illustrating stable and long lasting interactions of in vivo tolerized BDC2.5 CD4+ T cells (labeled red using CMTMR) with DC (yellow and green) in a pancreatic LN explant 18h after transfer to a NOD.CD11c-eYFP recipient and anti-PD-L1 injection. Elapsed time is shown as hh:mm:ss:sss. Stable conjugates can be seen with co localization of the cells in yellow. The duration of the imaging is 30 min in real-time, which is compressed to 6 sec in this video. (MOV 2725 kb)

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Fife, B., Pauken, K., Eagar, T. et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR–induced stop signal. Nat Immunol 10, 1185–1192 (2009). https://doi.org/10.1038/ni.1790

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