Stability and function of regulatory T cells is maintained by a neuropilin-1–semaphorin-4a axis

Journal name:
Nature
Volume:
501,
Pages:
252–256
Date published:
DOI:
doi:10.1038/nature12428
Received
Accepted
Published online

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis1. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections1. The transcription factor Foxp3 has a major role in the development and programming of Treg cells2, 3. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject4, 5, 6. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a–Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

At a glance

Figures

  1. Sema4a binds Nrp1 to potentiate Treg-cell function and survival in vitro.
    Figure 1: Sema4a binds Nrp1 to potentiate Treg-cell function and survival in vitro.

    a, Transwell suppression assay in which Treg cells were co-cultured in the top chamber of a transwell plate with anti-CD3- and anti-CD28-coated beads in the presence or absence of CD4+ or CD8+ Tconv cells that had been transfected previously with scrambled or siRNA to Sema4a. Proliferation of Tconv cells stimulated with anti-CD3- and anti-CD28-coated beads in the bottom chambers was measured by [3H]-thymidine uptake. b, Transwell suppression assay with Treg cells co-cultured in top chamber with CD4+, CD8+ or CD11c+ cells including anti-Sema4a or its isotype control. c, Transwell suppression assay in which Treg cells were co-cultured in the absence of Tconv cells but in the presence of beads coated with Sema4a-IgG1 or its isotype control. d, ELISA-based binding assay in which plates coated with Nrp1 were incubated with Sema4a-IgG1 or its isotype control in the presence of various blocking antibodies. Sema4a-IgG1 was detected using an isotype specific antibody. e, Transwell suppression assay in which Treg cells were purified by flow cytometry from Foxp3Cre or Nrp1f/fFoxp3Cre mice. f, Annexin V-7-AAD staining of Treg cells stimulated for 48h in vitro in the presence of Sema4a-IgG1 or its isotype control. Results represent the mean of five independent experiments. *P<0.05, **P<0.01, ***P<0.001 by unpaired t-test. Error bars indicate s.e.m.

  2. Nrp1-deficient Treg cells fail to suppress anti-tumour immune responses.
    Figure 2: Nrp1-deficient Treg cells fail to suppress anti-tumour immune responses.

    a, Tumour growth curve (top) and survival plot (bottom) of Foxp3Cre, Nrp1f/fFoxp3Cre, or Foxp3DTR-GFP mice receiving 1.25×105 MC38 melanoma cells subcutaneously and (for Foxp3DTR-GFP) 100µg diphtheria toxin (DT) intraperitoneally, twice weekly. b, As in a, but mice received 1.25×105 EL4 thymoma intradermally. c, As in a, but mice received 1.25×105 B16 melanoma intradermally. d, Tumour growth curve of C57/BL6 mice receiving 1.25×105 B16 melanoma intradermally concomitant with injections of isotype control, anti-Sema4a, or anti-Nrp1 (100µg) twice weekly. e, Tumour growth curve as in d except mice received Sema4a-IgG1 twice weekly. f, Tumour growth curve of C57/BL6 mice receiving 1.25×105 B16 melanoma intradermally. When tumours were palpable (day 5, indicated by arrow), mice began receiving injections of anti-Nrp1 or its isotype control (400µg initially, 200µg every 3days). g, Lung metastasis counts from Foxp3Cre or Nrp1f/fFoxp3Cre mice injected with 2.5–10×105 B16 cells intravenously, 17–20days earlier. h, Tabulation of flow cytometric analysis of tumour-infiltrating lymphocytes from Foxp3Cre or Nrp1f/fFoxp3Cre mice injected intradermally with B16 18days earlier. i, Sema4a expression of various immune cells in nondraining lymph node (ndLN), draining lymph node (dLN) or tumour-infiltrating lymphocytes (TIL). Results represent the mean of five (a–c, n = 10–25 mice), three (d–h, n = 8–20 mice), or three (i) experiments. *P<0.05, **P<0.01, ***P<0.001, by (a–f) one-way analysis of variance (ANOVA) or (g–i) unpaired t-test. CR, complete response. Error bars indicate s.e.m.

  3. Ligation of Nrp1 by Sema4a promotes Treg-cell stability through modulation of Akt-mTOR signalling.
    Figure 3: Ligation of Nrp1 by Sema4a promotes Treg-cell stability through modulation of Akt–mTOR signalling.

    a, Total internal reflection fluorescence (TIRF) microscopic analysis of immunologic synapse Akt phosphorylation in Treg cells stimulated 20min on a lipid bilayer coated with anti-TCR antibodies in the presence or absence of Sema4a-IgG1. b, Transwell suppression assay using Treg cells retrovirally expressing wild-type (WT) or dominant-negative (DN)-Akt. Transductants were selected and expanded using puromycin and IL-2. NS, not significant. c, Immunoprecipitation (IP) analysis of Nrp1 using in vitro expanded Treg cells that were serum starved for 3h, then stimulated as indicated for 6h before IP. d, Transwell suppression assay using Foxp3Cre or Ptenf/fFoxp3Cre Treg cells. e, TIRF microscopic analysis of immunologic synapse Akt phosphorylation as in a, of Nrp1f/fFoxp3Cre Treg cells retrovirally reconstituted with wild-type (WT) or SEA-deficient (ΔSEA) Nrp1. f, Foxo3a cytoplasmic (top) and nuclear (bottom) localization signals, as defined by masking using actin and DNA staining. Arbitrary Units represent fluorescence intensity calculated volumetrically through 20 to 30 slices of Treg cells. n = 70–93. US, unstimulated (control). g, Heat map (right) of genes regulated by Nrp1. Foxp3Cre and Nrp1f/fFoxp3Cre CD45RBlo Foxp3 (YFP)+ CD4+ T cells were stimulated for 48h with anti-CD3, anti-CD28, 100 units per ml rhIL-2, and immobilized IgG1 or Sema4a-IgG1. RNA was subjected to Affymetrix gene profiling. Treg-cell signature genes are in bold. All genes shown met false discovery rate (FDR) <0.10 and compared using two-way ANOVA. Results represent at least three independent experiments (a, c, e, f) or represent means of three (b, d) or seven (g) experiments. *P<0.05, ** P<0.01 by unpaired t-test. Error bars indicate s.e.m.

  4. Tumour-infiltrating Treg cells bear a signature similar to Sema4a-Nrp1 ligation.
    Figure 4: Tumour-infiltrating Treg cells bear a signature similar to Sema4a–Nrp1 ligation.

    a, Akt phosphorylation in Treg cells. TIL (blue and maroon) and ndLN (green and gold) were collected from tumour-bearing Foxp3Cre or Nrp1f/fFoxp3Cre mice. Cells were immediately fixed and stained for phosphorylated Akt (pAkt). bh, Shaded histogram indicates isotype control. Results are tabulated normalized to isotype control staining. IRF4 and RORγT (b), Ki67 and BrdU (c), cleaved caspase-3 (d), Bcl2 (e), ICOS (f), IL-10 (g) and CD73 (h) staining from ndLN, dLN, or TIL from tumour-bearing Foxp3Cre or Nrp1f/fFoxp3Cre mice. Ki67 and BrdU analysis included injection with BrdU 14h before harvest. IL-10 staining included restimulation with phorbol myristate acetate (PMA) and ionomycin for 16h in the presence of brefeldin A. Results represent the mean of 3–5 independent experiments. *P<0.05, **P<0.01, ***P<0.001 by paired t-test (a, n = 10–11) or unpaired t-test (b–h n = 7–17). MFI, mean fluorescence intensity. Error bars indicate s.e.m.

Accession codes

Referenced accessions

Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Greg M. Delgoffe &
    • Seng-Ryong Woo

Affiliations

  1. Department of Immunology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

    • Greg M. Delgoffe,
    • Seng-Ryong Woo,
    • Meghan E. Turnis,
    • David M. Gravano,
    • Cliff Guy,
    • Abigail E. Overacre,
    • Matthew L. Bettini,
    • Creg J. Workman &
    • Dario A. A. Vignali
  2. Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA

    • Abigail E. Overacre
  3. Department of Pathology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

    • Peter Vogel
  4. Computational Biology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA

    • David Finkelstein
  5. R&D Systems Inc., Minneapolis, Minnesota 55413, USA

    • Jody Bonnevier

Contributions

G.M.D. designed and performed most of the experiments and wrote the manuscript. S.-R.W. performed critical initial experiments and identified Sema4a and Nrp1 as the ligand–receptor pair. M.E.T. conducted many of the tumour experiments. D.M.G. performed a substantial portion of the colitis experiments. C.G. performed TIRF microscopy. M.L.B. assisted with the Foxp3-deficiency rescue experiments. A.E.O. assisted with several experiments. P.V. performed histological analysis. D.F. performed computational analysis of the microarray data. J.B. provided the blocking monoclonal antibodies to Sema4a and Nrp1. C.J.W. conducted and curated the initial microarray analysis. D.A.A.V. conceived the project, directed the research and wrote the manuscript. All authors edited and approved the manuscript.

Competing financial interests

J. Bonnevier is an employee of R&D Systems.

Corresponding author

Correspondence to:

The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE41185.

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Supplementary information

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  1. Supplementary Information (1.3 MB)

    This file contains Supplementary Figures 1-15.

Excel files

  1. Supplementary Table 1 (44 KB)

    This file contains geneset enrichment analysis for Nrp1-upregulated genesets.

  2. Supplementary Table 2 (167 KB)

    This file contains geneset enrichment analysis for Nrp1-downregulated genesets.

Additional data