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A molecular threshold for effector CD8+ T cell differentiation controlled by transcription factors Blimp-1 and T-bet

Abstract

T cell responses are guided by cytokines that induce transcriptional regulators, which ultimately control differentiation of effector and memory T cells. However, it is unknown how the activities of these molecular regulators are coordinated and integrated during the differentiation process. Using genetic approaches and transcriptional profiling of antigen-specific CD8+ T cells, we reveal a common program of effector differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. The loss of both T-bet and Blimp-1 leads to abrogated cytotoxic function and ectopic IL-17 production in CD8+ T cells. Overall, our data reveal two major overlapping pathways of effector differentiation governed by the availability of Blimp-1 and T-bet and suggest a model for cytokine-induced transcriptional changes that combine, quantitatively and qualitatively, to promote robust effector CD8+ T cell differentiation.

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Figure 1: Inflammation can compensate for IL-2 during Blimp-1 induction.
Figure 2: IL-2R signaling regulates SLEC differentiation in a Blimp-1–dependent manner.
Figure 3: Blimp-1–dependent and Blimp-1–independent transcriptional signatures induced by IL-2 signaling.
Figure 4: IL-2Rα and Blimp-1 collaborate in regulating the differentiation of effector CD8+ T cells.
Figure 5: T-bet and Blimp-1 regulate overlapping and distinct transcriptional signatures during effector CD8+ T cell differentiation.
Figure 6: T-bet and Blimp-1 are required for protective antiviral CD8+ T cell responses.
Figure 7: T-bet and Blimp-1 cooperate during CD8+ T cell effector differentiation and prevent aberrant differentiation of IL-17–producing CD8+ T cells in response to viral infection.
Figure 8: T-bet can partially compensate for the loss of Blimp-1 in SLEC differentiation.

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Acknowledgements

We thank M. Camilleri and L. Mackiewicz for technical support, and M. Ghisi (Peter MacCallum Cancer Centre) for assistance with MetaCore bioinformatics analysis. Supported by the National Health and Medical Research Council of Australia (program grant 1054618 to T.P.S. and G.K.S.; fellowship 1058892 to G.K.S.; project grant 1023454 to G.K.S. and W.S.; project grant 637345 to A.K. and G.T.B.; project grant 1042582 to G.T.B. and M.P.; project grant 603122 to M.E.), the Sylvia and Charles Viertel Foundation (A.K. and G.T.B.), the Australian Research Council (S.L.N., G.T.B. and A.K.), the US National Institutes of Health (NIH) (R01AI066232 and R0O1AI074699 to S.M.K.), the Howard Hughes Medical Institute (S.M.K.), the Howard Hughes Medical Institute International Student Fellowship (T.G.), the Division of Intramural Research and the DNA Sequencing Core, National Heart, Lung, and Blood Institute, NIH (W.J.L.), the Adam J. Berry Memorial Fund, jointly provided by the Australian Academy of Science and NIH (A.X.) and the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support scheme.

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Authors

Contributions

A.X. and F.M. planned and performed most experiments; S.P., R.G. and T.G. performed additional experiments; Y.L., W.S. and G.K.S. analyzed most RNA and ChIP sequencing experiments; M.O., J.-X.L., P.L. and T.P.S. contributed to the RNA sequencing analysis; M.E., W.J.L., S.M.K. and M.P. contributed to the scientific planning and organization of experiments; S.L.N., G.T.B. and A.K. conceived the idea for the study and designed experiments; A.K. oversaw and designed the study; A.X., F.M. and A.K. wrote the manuscript.

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Correspondence to Axel Kallies.

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Integrated supplementary information

Supplementary Figure 1 IL-2Rα is required for optimal CD8+ T cell response in the spleen during influenza infection.

(a-c) F1 (Ly5.2+Ly5.1+) mice were lethally irradiated and reconstituted with bone marrow from Blimp1GFP (Ly5.1+) and Il2ra−/−Blimp1GFP (Ly5.2+) mice. (b) Representative flow cytometry plot of NP366 tetramer staining in CD8+ T cells from influenza virus (HKx31) infected mice without prior priming with PR8 influenza virus. (c) Blimp1-GFP expression in splenic KLRG1+ or KLRG1- NP366-specific CD8+ T cells of the indicated genotypes of chimeric mice 9-10 after primary infection with HKx31 influenza virus. Data are from two experiments with at least each with 2-3 mice per group. (d) Analysis of STAT5 phosphorylation by flow cytometry in a mixed culture of splenic wild-type (Ly5.1+) and Il2ra−/− (Ly5.2+) CD8+ T cells. The CD8+ T cells were pre-activated with anti-CD3 and anti-CD28 antibodies for two days and rested in medium for 24 hrs. IL-2, IL-15 or PBS was added to the culture and STAT5 phosphorylation was examined 15 min later. Flow cytometry plots are representative of 3 independent experiments with 3-4 mice per group. (e) Proliferation of wild-type and IL-2-deficient CD8+ T cells. Naïve (CD62LhiCD44low) CD8+ T cells were sorted by flow cytometry and labeled with Cell Trace Violet (CTV). They were activated with anti-CD3 and anti-CD28 antibodies and cultured with cytokines as indicated. Representative of three experiments. (f) Blimp-1-GFP expression (geometric mean fluorescence intensity, GMFI) three days after activation of Blimp1GFP CD8+ T cells with anti-CD3/CD28 and cross-titrated concentrations of IL-15 and IL-12 in the presence of an antibody neutralizing murine IL-2. Data are from experimental duplicates and representative of two independent experiments. ***P<0.001; NS – not significant (U-Mann-Whitney test).

Supplementary Figure 2 IL-2 regulates CD8+ effector T cell differentiation through Blimp-1–dependent and independent pathways.

(a-b) Analysis of splenic CD8+ T cells of the genotypes as indicated in mixed bone marrow chimeric mice infected with influenza virus without prior priming. Data are from three independent experiments each with 2-3 mice per group. (a) Mean ± S.E.M. of the frequency of PA224-specific CD8+ T cells as determined by flow cytometric analysis 9-10 days after primary HKx31 infection. (b) Frequency of granzyme B expressing cells in NP366-specific CD8+ T cells of the genotypes as indicated (mean ± S.E.M.). (c) Enrichment analysis of indicated gene set in Masson_Id2-regulated genes34. The indicated gene set was derived from the RNA-sequencing analysis as in Fig. 4a. Graph shows enrichment score for upregulated (red) and downregulated (blue) genes. *P<0.05; **P<0.01; ***P<0.001; NS – not significant (U-Mann-Whitney test).

Supplementary Figure 3 Genes regulated by Blimp-1 and/or T-bet.

NP366-specific CD8+ T cells of the indicated genotypes were sorted from mixed bone marrow chimeric mice that were primed and infected with influenza virus. Expression of selected genes as determined by RNA-sequencing. (a) Metacore analysis of biological processes relevant to immune function for genes specifically differentially expressed in Tbx21−/− NP366-specific CD8+ T cells, or commonly deregulated in Tbx21−/− and Il2ra−/−Blimp1fl/flLckCre NP366-specific CD8+ T cells. (b) Expression of selected differentially expressed genes as identified by RNA-sequencing analysis. (c) Heatmap showing relative expression levels (Z-score) for DE genes in antigen-specific CD8+ T cells of the indicated genotypes that belong to the ImmGen_10 clusters dynamically regulated during a CD8+ T cell response10. Data are from RNA-sequencing of two independent biological samples each pooled from 5-6 mice (a-c).

Supplementary Figure 4 Combined loss of Blimp-1 and T-bet results in defective function of antigen-specific CD8+ T cells during influenza infection.

Wild-type, Tbx21−/−, Blimp1fl/flLckCre and Tbx21−/−Blimp1fl/flLckCre mice were infected with influenza virus. (a) 9 days after infection splenic CD8+ T cells of the indicated genotypes analyzed by NP366 tetramer staining (a, upper panel) or stimulated with NP366-peptide and after intracellular staining analyzed by flow cytometry for the expression of IFN-γ and granzyme B (a, lower panel). (b) Analysis of splenic NP366-specific CD8+ T cells from influenza virus infected mice of the indicated genotypes for expression of KLRG1 and IL-7R. Data are representative of two independent experiments, with each 3-4 mice per genotype.

Supplementary Figure 5 T-bet and Blimp-1 are required for protective antiviral CD8+ T cell response and to prevent the development of IL-17–producing antigen-specific CD8+ T cells..

(a-e) Analysis of wild-type (WT), Tbx21−/−, Blimp1fl/flLckCre or Tbx21−/−Blimp1fl/flLckCre mice as indicated 8 days post infection with 3000 PFU LCMV. (a) Frequency of KLRG1+ cells amongst gp33-specific CD8+ T cells (mean ± S.E.M.). (b) LCMV virus titres in spleen (left panel) and kidney (right panel). Each symbol represents an individual mouse; horizontal lines are the mean ± S.E.M. (c) Spleen weight (mean ± S.E.M.). (d) Frequency of CD25+ cells (mean ± S.E.M.) among gp33-specific CD8+ T cells. (e) PD1 expression (geometric mean fluorescence intensity, GMFI; mean ± S.E.M.) of gp33-specific CD8+ T cells. Data in (a-e) are cumulative of two independent experiments with each 2-3 mice per group. (f). Body weight of mice of the indicated genotypes following infection with 300 PFU LCMV (n=7-8 mice per group). Data are cumulative of two independent experiments with each 3-4 mice. * depicts mice succumbed to disease. (g) Survival of wild-type mice following CD8+ or CD4+ T cell depletion and infection (n=6 per group pooled from two experiments). (h). Flow cytometric analysis of IFN-γ and IL-22 production after restimulation with gp33 peptide and intracellular staining (left panel) and frequencies of IL22+ cells within the IFNγ+ population of splenic CD8+ T cells 9-10 days after 300 PFU LCMV infection. Data are from one experiment representative of two experiments each with n=2-4 mice per group (h, left panel) or cumulative of two experiments with each 2-4 mice per group (h, right panel, mean ± S.E.M). (i-j) Tbx21−/−Blimp1fl/flLckCre (Ly5.2) / WT (Ly5.1) mixed bone marrow chimeric mice were infected with 300 PFU LCMV and analyzed 10 days later. Flow cytometry plot showing IFN-γ and IL-17A production from CD8+ T cells (i) and frequencies of IL-17A+ cells within total CD8+ T cells (j) after restimulation with PMA and ionomycin and intracellular staining. Data are the mean ± S.E.M. from four mixed bone marrow chimeric mice. *P<0.05; **P<0.01; NS – not significant (a-e, U-Mann-Whitney test); ***P<0.001 (j, paired Student’s t-test).

Supplementary Figure 6 Neutrophil accumulation and immunopathology in T-bet–deficient Blimp1fl/flLck-Cre mice after LCMV infection.

Analysis of wild-type (WT), Tbx21−/−, Blimp1fl/flLckCre or Tbx21−/−Blimp1fl/flLckCre mice as indicated 9-10 days post infection with 300 PFU LCMV. (a) Cytokine concentration in the serum as identified by bead array. Each symbol represents an individual mouse; horizontal lines are the mean ± S.E.M. (b-c) Frequency (b) and enumeration (c) of neutrophils (CD11b+ Ly6g+ Ly6clow) within the spleen of LCMV infected mice from the indicated genotypes. (d) H&E staining of liver sections from LCMV infected mice of the indicated genotypes. Scale bar 100 µm. **P<0.01; NS – not significant (U-Mann-Whitney test). Data are from one experiment representative (d) or cumulative of two experiments each with n=2-4 mice per group (b-c, mean ± S.E.M).

Supplementary Figure 7 Transcripts regulated by Blimp-1 and/or T-bet.

Heatmap showing relative expression levels (Z-score) for DE genes in antigen-experienced CD8+ T cells of the indicated genotypes that belong to the ImmGen_10 clusters dynamically regulated during a CD8+ T cell response10. Data are from RNA-sequencing of two independent biological samples each pooled from two mice.

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Xin, A., Masson, F., Liao, Y. et al. A molecular threshold for effector CD8+ T cell differentiation controlled by transcription factors Blimp-1 and T-bet. Nat Immunol 17, 422–432 (2016). https://doi.org/10.1038/ni.3410

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