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A central role for Notch in effector CD8+ T cell differentiation

Nature Immunology volume 15, pages 1143–1151 (2014)Cite this article

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Abstract

Activated CD8+ T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8+ T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

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Figure 1: TEC-promoting signals induce Notch expression on CD8+ T cells.
Figure 2: Notch ligands are expressed on APCs during infection with influenza virus.
Figure 3: Notch is required for effector function.
Figure 4: Notch is required for TEC differentiation.
Figure 5: Notch regulates the TEC transcriptome.
Figure 6: Notch regulates pathways that control TEC differentiation.

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Acknowledgements

We thank J.C. Zuniga Pflucker (Sunnybrook Research Institute) and D. Vignali (University of Pittsburgh) for the pMIY and pMIY-myr-Akt retroviral expression constructs; T.N.M. Schumacher (Netherlands Cancer Institute) for OVA-influenza; J. den Haan (Free University of Amsterdam) for MyD88-deficient mice; M. Suresh and E.H. Kim for advice on the staining of phosphorylated proteins for flow cytometry; M. Wolkers, M. Nolte, R. van Lier and M. van Ham for suggestions and proofreading of the manuscript; and the tetramer facility of the US National Institutes of Health for tetramers of major histocompatibility complex. Supported by NWO-ALW, the Landsteiner Foundation for Blood Research, the Academic Medical Center (D.A.), the US National Institutes of Health (AI095245 and DK072201 to J.M.B.), the Burroughs Wellcome Trust Fund (J.M.B.), the Leukemia and Lymphoma Society (J.M.B.), the Irma-Hirschl Charitable Trust and Monique Weill-Caulier Charitable Trust (J.M.B.).

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Author notes

  1. Ronald A Backer

    Present address: Present address: TRON Translationale Onkologie an der Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany.,

  2. Ronald A Backer and Christina Helbig: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, the Netherlands

    Ronald A Backer, Christina Helbig, Rebecca Gentek, Yevan S de Souza & Derk Amsen

  2. Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands

    Ronald A Backer, Christina Helbig, Klaas van Gisbergen & Derk Amsen

  3. Department of Medicine, The Icahn School of Medicine at Mount Sinai, Immunology Institute and Tisch Cancer Institute, New York, New York, USA

    Andrew Kent & J Magarian Blander

  4. Department of Immunobiology and Howard Hughes Medical Institute, Yale University, School of Medicine, New Haven, Connecticut, USA

    Brian J Laidlaw, Claudia X Dominguez & Susan M Kaech

  5. Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands,

    Stella E van Trierum, Ruud van Beek & Guus F Rimmelzwaan

  6. Viroclinics Biosciences BV, Rotterdam, the Netherlands.,

    Stella E van Trierum, Ruud van Beek & Guus F Rimmelzwaan

  7. Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands

    Anja ten Brinke

  8. Bioinformatics Laboratory, Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, Amsterdam, the Netherlands

    A Marcel Willemsen & Antoine H C van Kampen

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Contributions

R.A.B. designed, performed and analyzed experiments and wrote the manuscript; C.H., R.G., A.K., B.J.L., C.X.D., Y.S.d.S., S.E.v.T., R.v.B. and A.t.B. performed and analyzed experiments; A.M.W. and A.H.C.v.K. analyzed data from high-throughput RNA sequencing; G.F.R., S.M.K., J.M.B. and K.v.G. designed and analyzed experiments; and D.A. supervised the study, designed experiments and wrote the manuscript.

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Correspondence to Derk Amsen.

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Integrated supplementary information

Supplementary Figure 1 TLR ligands induce a soluble factor that promotes the expression of Notch in CD8+ T cells.

(a) Naïve CD8+ T cells were stimulated with 10 µg/ml plate bound anti-CD3 alone (black bars) or in the presence of R-848 (white bars) or LPS (grey bars) without DC. Cell surface expression of Notch1 (left) and Notch2 (right) was determined after 16 h of culture by FACS and displayed as geo Mean Fluorescence Intensities (MFI) + s.e.m. (b) Naïve CD8+ T cells were stimulated o/n with plate-bound anti-CD3 mAb with supernatants from unstimulated (black bars), R-848 stimulated (white bars) or LPS stimulated (grey bars) WT BM-DC (left) or MyD88ko BM-DC cultures (right) and average Notch1 surface expression was determined by FACS. (c) Naïve CD8+ T cells, isolated from WT (black bars), MyD88ko (white bars) or Notch1-2-KO mice (grey bars), were stimulated o/n with plate bound anti-CD3 mAb and supernatants isolated from non-stimulated (DC sup) or R-848 stimulated BM-DC cultures (R-848 DC sup). Expression of Notch1 was determined as in (a). Triplicates, representative of 2 experiments. *** P < 0.001, One-way ANOVA with Bonferroni corrections.

Supplementary Figure 2 Notch surface levels are elevated on early TECs in vivo.

Mice were infected with HKx31 influenza, and after 3 days, different CD8+ T cell subpopulations (grey bars-CD44+KLRG1+; white bars-CD44+KLRG1–; black bars-CD44–) were stained for N1 and N2 expression. Results represent 4 pooled mice. Shown are mean + s.e.m. * P < 0.05; ** P < 0.01; unpaired, two-tailed t-test.

Supplementary Figure 3 Expression of Notch ligands on mediastinal lymph nodes and lung APCs during infection with influenza virus.

C57BL/6 mice were infected intranasally with A/HKx31 influenza. (a) Five days post infection (p.i.) expression of DLL1, DLL4, Jagged1 and Jagged2 expression on mediastinal LN (mLN) APC subsets was measured by flow cytometry. Two main APC subsets in the mLN were defined as mDCs (migratory DC, MHCIIhiCD11c+, red gate) or macrophages (MΦ, MHCIImedCD11chi, green gate). (b) Bar graph shows average cell surface expression of DLL1, DLL4, Jagged1 and Jagged2 on mLN macrophages. Shown is Δ-MFI (corrected for background staining with isotype control mAb) for uninfected (UI-white bars) and infected mice (black bars). (c) Gating strategy for SSChiMHCIImedCD11c- cells (gate I), alveolar MΦ (SSChiMHCIImedCD11chi, gate I), and mDCs (migratory DC, MHCIIhiCD11chi, gate III) in lung following (Helft, J. et al. Cross-presenting CD103+ dendritic cells are protected from influenza virus infection. J Clin Invest 122, 4037-4047 (2012).), 5 days post infection (p.i.). Expression of DLL1, DLL4, Jagged1 and Jagged2 on these cell subsets was measured by flow cytometry. Bar graphs represent average Δ-MFI (corrected for background staining with isotype control mAb) for non-infected (white bars) and infected mice (black bars). Results represent 2 (uninfected) or 4 (infected) separately processed mice from a representative of 5 experiments. Shown are the mean + s.e.m. * P < 0.05; two-tailed t-test.

Supplementary Figure 4 TEC differentiation is dependent on Notch gene dose.

Notch1flox/+Notchflox/+ wild type (grey bars) or Notch1flox/+Notchflox/+ CD4-Cre+ double heterozygous littermate mice (open bars) were infected with A/HkX31 influenza and after 10 days the expression of KLRG1 and CD127 was determined on H-2Db–NP366-374 binding CD8+ T cells. Results are cumulative from 2 experiments with 10 mice per group total. Shown are the mean + s.e.m. (* P < 0.05; two-tailed t-test).

Supplementary Figure 5 Efficient CD8+ T cell effector functions during viral infection depend on RBPJ.

RBPJflox/floxCd4-Cre+ (RBPJ-KO) and WT control littermate mice were infected intranasally with A/HKx31 influenza. Ten days post-infection, CD8+ T cell responses were analyzed. (a) Average frequencies of H-2Db–NP+ CD8+ T cells in blood, 10 days after infection. Black bars represent WT and white bars RBPJ-KO mice. (b) Single cell suspensions from lungs of WT and RBPJ-KO mice, were stimulated with NP366-374 peptide for 4 hours in the presence of brefeldin A. Percentages of CD8+ T cells producing IFNγ (left) and TNF (right) are shown. (3 mice/group, representative experiment of 2). * P < 0.05; two-tailed t-test.

Supplementary Figure 6 The role of Notch in TEC differentiation is independent of the strain of influenza virus.

Mice were infected intranasally with A/PR/8/34 (200xTCID50) and analyzed after 10 days. (a) Representative FACS profiles and (b) average frequencies of H-2 Db–NP-binding CD8+ T cells in blood from WT (grey bars) and Notch1-2-KO (white bars) mice. (c) Average frequencies (left) and total cell numbers (right) of H-2 Db–NP+CD8+ T cells in spleens and lungs. (d) Representative FACS profiles for KLRG1 and CD127 and (e) percentages of H-2 Db–NP+CD8+ TECs and MPCs in blood, spleens and lungs from wild type (grey bars) and Notch1-2-KO (white bars) mice. (f) FACS profile for Granzyme-B production by WT (black line) and Notch1-2-KO (grey line) lung CD8+ T cells after NP366-374 peptide stimulation. Filled grey histogram represents isotype control staining. (g) Average percentages of CD8+ T cells from lungs producing (from left to right) GZMB, IFNγ and TNF after stimulation with NP366-374 peptide. Shown are mean + s.e.m. of 4 mice per group. * P < 0.05; ** P < 0.01; *** P<0.001; unpaired, two-tailed t-test.

Supplementary Figure 7 The requirement for Notch in TEC differentiation is independent of TCR specificity.

Seven hundred fifty wild type (grey bars) or Notch1-2-KO (white bars) OT-I CD45.2+ CD8+ T cells were adoptively transferred into CD45.1 congenic wild type mice infected with WSN-Ova. After 10 days, the percentage of CD45+ OT-I cells among CD8+ T cells (left) or KLRG1+ cells among the OT-I T cells (right) was determined by FACS. Result is representative of 3 experiments. *** P<0.001.

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Supplementary Figure 8 Notch controls pathways that promote TEC differentiation.

(a) Phosphorylation of FoxO1/3 is reduced in Notch1-2-KO CD8 T cells. FACS profile (left) and average Δ-MFI (right - corrected for background staining) for Phosphorylation of FoxO1/3 (Thr24/Thr32) in transferred WT (black line, grey bars) and Notch1-2-KO (grey line, white bars) OT-I T cells, 5 days post-infection with OVA-influenza. Black bars indicate pFoxO1/3 staining on CD44–CD8+ T cells. Results are cumulative from two different experiments with a total of 8 individually tested mice per group. * P < 0.05, unpaired, two-tailed t-test. (b) Model showing signal integration and positive feedback of the Notch hub.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 635 kb)

Supplementary Dataset 1

Gene sets GSEA (XLSX 298 kb)

Supplementary Dataset 2

TEC and MPC genes (XLSX 143 kb)

Supplementary Dataset 3

Go categories (XLS 17 kb)

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Backer, R., Helbig, C., Gentek, R. et al. A central role for Notch in effector CD8+ T cell differentiation. Nat Immunol 15, 1143–1151 (2014). https://doi.org/10.1038/ni.3027

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