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The transcription factor BCL-6 controls early development of innate-like T cells

Abstract

Innate T cells, including invariant natural killer T (iNKT) and mucosal-associated innate T (MAIT) cells, are a heterogeneous T lymphocyte population with effector properties preprogrammed during their thymic differentiation. How this program is initiated is currently unclear. Here, we show that the transcription factor BCL-6 was transiently expressed in iNKT cells upon exit from positive selection and was required for their proper development beyond stage 0. Notably, development of MAIT cells was also impaired in the absence of Bcl6. BCL-6-deficient iNKT cells had reduced expression of genes that were associated with the innate T cell lineage, including Zbtb16, which encodes PLZF, and PLZF-targeted genes. BCL-6 contributed to a chromatin accessibility landscape that was permissive for the expression of development-related genes and inhibitory for genes associated with naive T cell programs. Our results revealed new functions for BCL-6 and illuminated how this transcription factor controls early iNKT cell development.

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Fig. 1: The gene expression profile of ST0 and ST1 iNKT cells.
Fig. 2: BCL-6 is expressed specifically in ST0 iNKT cells.
Fig. 3: BCL-6-deficient mice lack iNKT and MAIT cells.
Fig. 4: Accumulation of immature iNKT cells in the absence of BCL-6.
Fig. 5: BCL-6-deficient mice lack mature iNKT cells.
Fig. 6: Impaired progression from ST0 to ST1 in the absence of BCL-6.
Fig. 7: BCL-6 is required for repression of a subset of PLZF target genes.
Fig. 8: Chromatin accessibility around developmentally regulated genes depends on BCL-6.

Data availability

RNA-seq and ATAC-seq data have been deposited to the Gene Expression Omnibus with the accession code GSE134212. Source data for Figs. 25, 7 and 8 and Extended Data Figs. 16 are provided with the paper. The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

This work was supported by a European Commission H2020-MSCA-IF grant (no. 655271) and a Hellenic Foundation for Research and Innovation grant (no. 486) to M.V. and grants from the National Institute of Allergy and Infectious Diseases (R56 AI104303 and R01 AI123396) to B.L.K. M.V. and P.M. were supported by a Stavros Niarchos Foundation start-up grant to BSRC Alexander Fleming (GRA-14451), as part of the Foundation’s initiative to support the Greek research ecosystem. M.S. was supported by Knut and Alice Wallenbergs Foundation and Cancerfonden. A.L.D. was supported by NIH 5R01 AI32771. This work benefited from the project “Strategic Development of the Biomedical Research Institute Alexander Fleming” (MIS 5002562) to P.H., which was implemented under “Action for the Strategic Development on the Research and Technological Sector”, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the European Union. We thank A. Melnick (Weill Cornell Medicine) for advice and C. Dagla, G. van der Voort, A. Rao and L. Lenner for technical support. We also thank the InfrafrontierGR Infrastructure for providing mouse and flow cytometry facilities, BSRC Fleming Flow Facility, the University of Chicago Genomics Core Facility and the Cytometry and Antibodies Core Facility, the University of Chicago Comprehensive Cancer Center (P30 CA014599) and the NIH Tetramer Facility.

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Authors

Contributions

M.G. designed, performed and analyzed experiments. A.G. performed bioinformatic analysis of RNA-seq and ATAC-seq data and analyzed experiments under the supervision of P.M. and P.H. S.G. assisted with flow cytometry cell sorting and flow cytometry analysis. A.L.D. provided the Bcl6F/F mouse strain. M.S. performed the RNA-seq experiments. B.L.K. obtained funding, performed ATAC-seq experiments, interpreted data and reviewed and edited the manuscript. M.V. conceptualized the project, obtained funding, supervised research, interpreted data, performed and analyzed experiments and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Barbara L. Kee or Mihalis Verykokakis.

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The authors declare no competing interests.

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Editor recognition statement L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Bcl6 mRNA expression in ST0 and ST1 cells.

a, Flow cytometry plots showing the sorting strategy and purity of ST0 and ST1 cells after sorting. Shown one representative experiment out of three. b, Flow cytometry plots showing expression of PLZF, T-BET and RORγt in ST1 wild-type cells. Graphs show mean percentage and number ± SEM of the indicated populations. n = 7 (PLZFhi, PLZFhiT-BET+, and PLZFloT-BET+), or n = 3 (PLZFintRORγt+) independent experiments c, Graphs showing the normalized counts of Bcl6 mRNA in ST0 and ST1 cells based on RNA-seq data. n = 3 independent experiments (DESeq, **P < 0.01). d, Flow cytometry plots showing the expression of BCL-6 and CD69 (left) or BCL-6 and EGR-2 (right) in Tetr+TCRβ+CD24+CD44 iNKT cells. Shown one representative experiment out of four (CD69) or two (EGR-2). Source data

Extended Data Fig. 2 The iNKT phenotype is cell-intrinsic.

a, The indicated populations were FACS-sorted from Cd4Cre-expressing heterozygous Bcl6 floxed mice and DNA was submitted to PCR genotyping. In ST3 iNKT, CD4 and DP thymocytes only the germline Bcl6 allele was amplified, whereas in DN and tail samples, both the floxed and the germline alleles were amplified. One experiment shown out of one. b, Flow cytometry plots showing the percentage of iNKT cells in CD45.1+ (wild-type competitor) and CD45.2+ controls (Bcl6F/F) in the thymus from competitive BM chimeric mice. Graphs show the mean percentage ± SEM of iNKT cells in the indicated chimeric mice. n = 5 independent experiments. c, Flow cytometry plots showing the percentage of iNKT cells in CD45.1+ (wild-type competitor) and CD45.2+ (Bcl6Δ/Δ) in the spleen from competitive BM chimeric mice. Graphs showing the mean percentage ± SEM of iNKT cells in the indicated chimeric mice. n = 3 independent experiments. d, Flow cytometry plots showing the percent of Tetr+TCRβ+ iNKT cells after MACS enrichment in the indicated mice. These experiments were repeated twice with similar results. e, f, Flow cytometry plots showing the percent of ST0 (e) and ST1–3 (f) cells in CD45.1+ (competitor) and CD45.2+ control (Bcl6F/F) iNKT cells in competitive bone marrow chimeras. Graphs indicate the mean percentage ± SEM. n = 3 (ST0) or n = 4 (ST1–3) independent experiments. Statistical analysis was performed with two-tailed unpaired t-test. ** P < 0.01. Source data

Extended Data Fig. 3 Conventional T cell development is normal in the absence of BCL-6.

a, FACS plots showing the expression of CD4 and CD8 (upper panel), and TCRβ (lower panel) in total thymocytes from the indicated mouse strains. b, Histograms showing the total number of the indicated thymocyte populations in control and Bcl6Δ/Δ mice. Graphs show the mean ± SEM. n = 5 independent experiments. c, Histograms showing the abundance of Tcra Vα14-Jα18 and Sh2d1a transcripts in control and Bcl6Δ/Δ sorted DP thymocytes. Graphs show the mean ± SEM. n = 3 independent experiments. d, Histograms showing the expression of CD1D, CD150, and LY108 in DP thymocytes from control and Bcl6Δ/Δ mice. These experiments were repeated three times with identical results. Statistical analysis was performed with two-tailed unpaired t-test. Source data

Extended Data Fig. 4 BCL-6—deficient spleens lack mature iNKT cells.

a, FACS plots of Tetr+TCRβ+ splenocytes showing the percentage of NKT1 (T-BET+PLZFlo), NKT2 (PLZFhiT-BET) and NKT17 (RORγt+TBET) cells in the indicated mouse strains. b, Cell number and percentage of NKT1, NKT2 and NKT17 cells in control and Bcl6Δ/Δ spleens. Graphs show mean ± SEM. n = 5 (NKT1 and NKT2), or n = 4 (NKT17) independent experiments. Statistical analysis was performed with two-tailed unpaired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001. c, Histograms showing the fold reduction of Rorc mRNA in Bcl6Δ/Δ ST1 cells compared to wild-type, based on the RNA-seq data. n = 3 independent experiments (DESeq, **P < 0.01). Source data

Extended Data Fig. 5 Proliferation and apoptosis of immature iNKT cells is independent of BCL-6.

a, Graphs show the percentage of EDU+ and Ki67+ cells in ST0 and ST1 cells in control littermates and Bcl6Δ/Δ mice. Data represent mean ± SEM. n = 3 independent experiments. b, FACS plots showing expression of IL-7 receptor and LEF1, in control littermates and Bcl6Δ/Δ mice. Experiments were repeated three times with identical results. c, Percentage of FLICA+ cells in the indicated iNKT cell stages, in control littermates and Bcl6Δ/Δ mice. Graphs show mean ± SEM. n = 3 independent experiments with 3 (control) and 4 (Bcl6Δ/Δ) mice. d, Flow cytometry plots showing the percent of total iNKT thymocytes, as Tetramer+TCRβ+ cells, in 10-days old control littermates and Bcl6Δ/Δ mice. e, Numbers and percentage of iNKT cells in the indicated mouse strains. Graphs represent mean ± SEM. n = 2 independent experiments with 5 control and 9 Bcl6Δ/Δ mice. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical analysis was performed with two-tailed unpaired t-test (a, e), or one-tailed unpaired t-test (c). Source data

Extended Data Fig. 6 PLZF expression is reduced in the absence of BCL-6.

a, qPCR analysis showing the transcript levels of Zbtb16 in sorted ST0 and ST1 cells from control and Bcl6Δ/Δ mice. Graphs represent the mean ± SEM. n = 3 independent experiments with 3–5 pooled thymi for each genotype. b, FACS plots showing the expression of PLZF protein in ST0 (upper panel) and ST1 (lower panel) cells from the indicated mouse strains. Graphs show the mean percentage ± SEM of PLZF+ cells in the indicated populations. n = 5 (ST0) and n = 8 (ST1) independent experiments. c, Graph showing the geometric MFI of PLZF expression in PLZF-expressing ST0 cells. Graphs represent the mean ± SEM. n = 4 independent experiments. d, Histograms showing the expression of PLZF in ST2 and ST3 cells in the indicated mouse strains. Graphs show the mean percentage ± SEM of PLZF cells. n = 5 independent experiments. e, Histograms showing the expression of CD69 and CD5 in ST0 iNKT cells from the indicated mouse strains. The corresponding graphs show the mean geometric MFI ± SEM. n = 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical analysis was performed with one-sample t-test (a), two-tailed unpaired t-test (b, d, and e) or two-tailed paired t-test (c). Source data

Extended Data Fig. 7 Impaired progression from ST0 to ST1 in the absence of BCL-6.

a, GSEA analysis showing enrichment of NKT0-associated genes in ST0 cells, compared to ST1 cells. b, GSEA analysis showing enrichment of NKT17-associated genes in wild-type ST1 cells, compared to Bcl6Δ/Δ ST1 cells. c, GSEA analysis showing enrichment of NKT2-associated genes in Bcl6Δ/Δ ST1 cells, compared to wild-type ST1 cells. n = 3 independent experiments. Normalized enrichment scores (NES) and FDR as implemented by GSEA, based on 1,000 permutations.

Extended Data Fig. 8 Chromatin accessibility profiles near genes that are developmentally regulated.

a, Genome track views of several loci of developmentally-regulated genes in the indicated populations from wild-type mice. b, Heatmaps showing aligned ATAC-seq reads around the center of peaks that are more accessible in ST1 cells (left), or in ST0 cells (right), in the indicated populations. c, Genome track views of Bach2 and Sell genes in wild-type and Bcl6Δ/Δ ST1 cells. d, Genome track views of several loci of developmentally-regulated genes in wild-type and Bcl6Δ/Δ ST0 cells.

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Gioulbasani, M., Galaras, A., Grammenoudi, S. et al. The transcription factor BCL-6 controls early development of innate-like T cells. Nat Immunol 21, 1058–1069 (2020). https://doi.org/10.1038/s41590-020-0737-y

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