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Abstract

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1–P3). P1-pDCs (PD-L1+CD80) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.

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Acknowledgements

We thank the Cytometry Core facility of IC for cell sorting. We thank INSERM U932, particularly P. Michea, for frequent discussions. We thank S. Amigorena, N. Manel and L. Pattarini for critical reading of the manuscript. This work was supported by funding from INSERM (BIO2014-08), FRM, ANR-13-BSV1-0024-02, ANR-10-IDEX-0001-02 PSL* and ANR-11-LABX-0043, ERC (IT-DC 281987, HEALTH 2011-261366 and 2013/COG/616180 DARK) and CIC IGR-Curie 1428. S.G.A. was supported by an IC fellowship and LabEx DCbiol. V.S-A. is supported as a Fondation pour la Recherche Médicale fellow (ARF20150934193). High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie, supported by grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the Agence Nationale de la Recherche (‘Investissements d’Avenir’ program), by the Canceropole Ile-de-France and by the SiRIC-Curie program, SiRIC Grant INCa-DGOS-4654.

Author information

Affiliations

  1. Institut Curie, Centre de Recherche, PSL Research University, Paris, France

    • Solana G. Alculumbre
    • , Violaine Saint-André
    • , Pablo Vargas
    • , Philemon Sirven
    • , Pierre Bost
    • , Mathieu Maurin
    • , Paolo Maiuri
    • , Maxime Wery
    • , Mabel San Roman
    • , Antonin Morillon
    •  & Vassili Soumelis
  2. INSERM U932, Immunity and Cancer, Paris, France

    • Solana G. Alculumbre
    • , Philemon Sirven
    • , Pierre Bost
    • , Mathieu Maurin
    • , Mabel San Roman
    •  & Vassili Soumelis
  3. CNRS UMR 3244, ncRNA, Epigenetic, and Genome Fluidity, Université Pierre et Marie Curie, Paris, France

    • Violaine Saint-André
    • , Maxime Wery
    •  & Antonin Morillon
  4. Department of Dermatology, University Hospital CHUV, Lausanne, Switzerland

    • Jeremy Di Domizio
    • , Curdin Conrad
    •  & Michel Gilliet
  5. CNRS UMR144, Paris, France

    • Pablo Vargas
  6. Department of Biology, Ecole Normale Supérieure, PSL Research University, Paris, France

    • Pierre Bost
  7. IFOM Foundation, Institute FIRC of Molecular Oncology, Milan, Italy

    • Paolo Maiuri
  8. UMR7211 and Inflammation–Immunopathology–Biotherapy Departement (DHU i2B), Sorbonne Universités, UPMC Université de Paris, Paris, France

    • Léa Savey
    •  & David Saadoun
  9. Assistance Publique-Hôpitaux de Paris (AP-HP), Groupe Hospitalier Pitié Salpétrière, Department of Internal Medicine and Clinical Immunology, National Reference Center for Autoimmune and Systemic Diseases, Paris, France

    • Léa Savey
    •  & David Saadoun
  10. AURA Paris Plaisance, Paris, France

    • Maxime Touzot
  11. Department of Internal Medicine, National Referral Center for Rare Autoimmune and Systemic Diseases, Cochin Hospital, AP-HP, Université Paris Descartes, Paris, France

    • Benjamin Terrier
  12. CIC IGR-Curie 1428, Paris, France

    • Vassili Soumelis

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Contributions

S.G.A. designed and performed experiments, analyzed results and wrote the manuscript. V.S.-A. analyzed results and wrote the manuscript. P.B., M.M. and P.M. analyzed results. P.V. performed experiments and analyzed results. P.M. analyzed results. M.W. performed experiments. J.D.D., M.S.R. and P.S. performed experiments. L.S., D.S., M.T., B.T., C.C. and M.G. performed clinical management, selected the patients and provided clinical samples. A.M. designed experiments and supervised the research. V.S. designed experiments, supervised the research and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Vassili Soumelis.

Integrated Supplementary Information

  1. Supplementary Figure 1 Sorting strategy for the isolation of CD2CD5AXL pDCs.

    a PBMC from human healthy donors were enriched for panDC and pDC were isolated as Linage (Lin)(CD14, CD16, CD19, CD20, CD3, CD56), CD4+, CD11c, CD2, CD5 and AXL. b The population (LinCD4+CD11c) CD2+and/or CD5+and/or AXL+ was isolated and cultured with Flu for 24h. PD-L1 and CD80 expression (left panel) and quantification of populations (right panel). Results shown as median of 3 independent donors.

  2. Supplementary Figure 2 Activated pDC populations show similar viability.

    PDCs were activated with Flu for 24h and the apoptotic marker annexin V was analyzed among each pDC subpopulation (a) and quantified the annexin+ cells (b) Median. n=3 independent donors. ns=non-significant (Friedman test + Dunn’s post-hoc).

  3. Supplementary Figure 3 P3 pDCs showed increased polarization and decreased endoplasmic reticulum.

    After 24h Flu activation, pDCs were sorted as P1-, P2- and P3-pDCs. a and b Immunofluorescence, phalloidin: red and DAPI: blue. Similar results were obtained for 3 independent donors b cells Aspect ratio. Median. P1 n= 230; P2 n=530; P3 n=338 cells. Unpaired t test. *p < 0.05; **p < 0.001; ***p < 0.0001. c EM images of the three pDC populations. Arrows denote the endoplasmic reticulum. Scale bars 2μm. Similar results were obtained for 2 independent donors.

  4. Supplementary Figure 4 IFN production by CD2CD5AXL pDC–derived subpopulations.

    PDC were sorted as CD2CD5AXL and culture 24h with Flu. PDC subpopulations were sorted and kept in culture for extra 24h in medium. IFN-α was measured in the culture supernatants. Results shown as the median of 6 independent donors. (Friedman test + Dunn's post-hoc). *p < 0.01.

  5. Supplementary Figure 5 T helper polarization by P1-, P2- and P3-pDCs.

    a ICOS, CD127, L-Selectin and CCR7 surface expression by CD4 T cells after coculture with pDC activated populations b PDC were sorted as CD2 CD5 AXL and culture 24h with Flu. PDC subpopulations were sorted and coculture with heterologous CD4 naive T cells. T cell expansion was measured after 6 days with P1-, P2- or P3-pDC (P1-T, P2-T and P3-T respectively). Results shown as the median of 3 independent donors. (ANOVA + Tukey’s post-hoc) c T helper cytokines induced by the activated pDC sub populations. Cytokines were measured after 24h polyclonal restimulation of the pDC polarized TH cells. Results include the median for 6 independent donors. (Friedman test + Dunn's post-hoc) d PDC-polarized TH cells were restimulated with PMA and Ionomicine. Intracellular TNF (left) and quantification of TNF producing cells (right). Results shown as the median for 5 donors. ns=non-significant; (ANOVA+ Tukey’s post-hoc). *p < 0.05; **p < 0.01.

  6. Supplementary Figure 6 PDC-derived type I IFN does not affect pDC diversification.

    PDCs were cultured with flu during 24h in the presence of antibodies blocking IFNAR2, IFN-α, and IFN-β or their corresponding isotype controls. a PD-L1 and CD80 expression and b quantification of pDC populations. Results shown as median of 3 independent donors. (paired t test). ns= non-significant.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Tables 1 and 2.

  2. Life Sciences Reporting Summary

Videos

  1. Supplementary Video 1: P1-pDC migration

    P1-pDC subpopulation migration in collagen gels towards a CCL21 gradient.

  2. Supplementary Video 2: P2-pDC migration

    P2-pDC subpopulation migration in collagen gels towards a CCL21 gradient.

  3. Supplementary Video 3: P3-pDC migration

    P3-pDC subpopulation migration in collagen gels towards a CCL21 gradient.

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https://doi.org/10.1038/s41590-017-0012-z