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Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF

Nature Immunology volume 24, pages 474–486 (2023)Cite this article

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Abstract

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

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Fig. 1: TSP2- and HPC-annotated CD34+ human thymocytes express low levels of IRF8 and are bi-phenotypic and potent for T- and DC-lineages.
Fig. 2: IRF8 expression is induced during human T-lineage specification but is silenced in the subsequent commitment stage.
Fig. 3: Low dose of active IRF8 promotes generation of tmTNF-expressing CD7+CD123+ progenitors and T cell precursors.
Fig. 4: IRF8-dependent DC-biased CD123+CD127+ progenitors express tmTNF and augment generation of T cell precursors via cellular cross-talk.
Fig. 5: TNFR2 expression precedes induction of CD7 during early human T cell development.
Fig. 6: Selective targeting of TNFR2 enhances in vitro generation of T cell precursors.
Fig. 7: TNF-activated lymphoid progenitors downregulate expression of IFN-related genes and are competent in T-lineage development.

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Data availability

The datasets generated and/or analyzed in this study are available in the Gene Expression Omnibus with the following accession numbers: ex vivo human CD34+ thymocytes for scRNA-seq (GSE144870), 4-OHT-treated OP9-DLL4 cocultures for ATAC-seq (GSE179534) and RNA-seq (GSE179381) and TNF-activated ATO cultures for scRNA-seq (GSE211400). GRCh38 reference genome is publicly accessible at the NCBI Datasets Genomes. Source data are provided with this paper.

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Acknowledgements

We thank C. de Bock (KU Leuven) for the ATAC-seq protocol, J.C. Zúñiga-Pflücker (University of Toronto) for OP9-DLL4-7FS stromal cell line, K. Francois and G. Van Nooten (Department of Human Structure and Repair, Ghent University Hospital) for thymus tissue, the Red Cross Flanders and the Ghent University Hospital Hematopoietic Biobank for cord blood and buffy coat, M. Guilliams (VIB, Ghent University) for an aliquot of IRF8 antibody, K. Weening and A. Kuchmiy (Ghent University) for assistance with molecular cloning, S. Vermaut and K. Reynvoet (Ghent University) for assistance with flow cytometry and cell sorting, F. Branco Madeira (Ghent University) for C57/BL6 mice, M. De Smedt and Jean Plum (Ghent University) for assistance in processing and collection of human tissue, E. De Meester (NXTGNT, Ghent University) for assistance in the preparation of samples for scRNA-seq and R. Colman (Ghent University) for assistance with statistical analyses. This work was supported by the Fund for Scientific Research Flanders (FWO, grants G053816N and G053916N to T.T.), The Concerted Research Action from the Ghent University Research Fund (GOA, BOF18-GOA-024 to T.T.), The Foundation against Cancer (Stichting Tegen Kanker, 2016-094 and 2020-114 to T.T.) and the Chan Zuckerberg Initiative (CZF2019-002445 to T.T.). The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation—Flanders (FWO) and the Flemish Government—department EWI. Research reported in this publication was performed at the CORE Flow Cytometry and NXTGNT sequencing facilities of Ghent University, Belgium.

Author information

Author notes

  1. Juliette Roels

    Present address: Genentech Inc., South San Francisco, CA, USA

  2. Marieke Lavaert

    Present address: Laboratory of Genome Integrity, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

  3. Inge Van de Walle

    Present address: argenx BVBA, Zwijnaarde, Belgium

  4. Roman Fischer

    Present address: BioNtech, Mainz, Germany

  5. These authors contributed equally: Juliette Roels, Marieke Lavaert.

Authors and Affiliations

  1. Department of Diagnostic Sciences, Ghent University, Ghent, Belgium

    Kai Ling Liang, Juliette Roels, Marieke Lavaert, Tom Putteman, Lena Boehme, Imke Velghe, Inge Van de Walle, Bart Vandekerckhove, Georges Leclercq & Tom Taghon

  2. Cancer Research Institute Ghent, Ghent, Belgium

    Kai Ling Liang, Bart Vandekerckhove, Georges Leclercq, Pieter Van Vlierberghe, Filip Van Nieuwerburgh & Tom Taghon

  3. Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium

    Laurentijn Tilleman & Filip Van Nieuwerburgh

  4. Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany

    Valentina Pegoretti, Roman Fischer, Roland E. Kontermann & Klaus Pfizenmaier

  5. Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University Medical School, Aachen, Germany

    Stephanie Sontag & Martin Zenke

  6. Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany

    Stephanie Sontag & Martin Zenke

  7. VIB Center for Inflammation Research (IRC), Technologiepark, Ghent, Belgium

    Jolien Vandewalle & Claude Libert

  8. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Jolien Vandewalle & Claude Libert

  9. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    Pieter Van Vlierberghe

  10. Leeds Institute of Medical Research, University of Leeds, Leeds, UK

    Gina Doody

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K.L.L. conceived the study, designed and performed experiments, analyzed data and wrote the manuscript. J.R. analyzed bulk ATAC-seq data. M.L. and T.P. analyzed the previously published scRNA-seq data. T.P. and L.B. analyzed the scRNA-seq data generated in this study. L.T. analyzed bulk RNA-seq data. I.V. assisted to set up experiments. I.V.W. performed an experiment related to regulation of IRF8 expression. J.V., B.V., G.L., P.V.V. and C.L. provided reagents. F.V.N provided expertise in ATAC- and RNA-seq. V.P., R.F., R.E.K. and K.P. provided TNFR2-selective TNF mutein (EHD2-scTNFR2). G.D. provided IRF8-related constructs. S.S. and M.Z. provided IRF8+/+ and IRF8−/− iPSCs. T.T. supervised the study, designed experiments and wrote the manuscript. All authors have seen, reviewed and approved the final version of the manuscript.

Corresponding author

Correspondence to Tom Taghon.

Ethics declarations

Competing interests

K.L.L. and T.T. filed a PCT application (PCT/EP2022/063712: Generating T cell precursors via agonizing tumor necrosis factor receptor 2) with the European Patent Office on 20th May 2022. K.P., R.F. and R.E.K. are named inventors on a patent covering the TNFR2-specific agonist (EZH2-scTNFR2). R.F. and R.E.K. received funding from Resano, a company that has licensed the technology to generate TNFR2-specific agonist. R.E.K. is a consultant for Immatics, Oncomatryx, Roche and SunRock. K.P. is a consultant for Oncomatryx, Resano and SunRock. The remaining authors declare no competing interests.

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Peer review information

Nature Immunology thanks Matthew Collin, Ivan Maillard and Christopher Seet for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Immunophenotyping of ex vivo murine and human immature thymocytes.

a, Scheme summarizes the developmental trajectories of the previously annotated CD34+ postnatal thymocytes for T- and DC-lineages. b,c, Scheme illustrates the development of murine thymocytes during T lineage specification and commitment (top), and their identification by flow cytometric gating (bottom) (b). Expression of IRF8 and GATA3 (n = 2) for the thymocyte subsets (top) shown in (b) and for the lin− and lin+ fractions of thymocytes (bottom) (c). d, Four cellular subsets of human immature thymocytes (n = 2) that express different levels of IRF8 and CD1a were overlaid in order to show their expression for PU.1 and CD123. e-g, Flow cytometry gating of ex vivo lin−CD4−CD34+ thymocytes (n = 9) based on their expression levels of CD123 and CD1a (e). Frequencies of the cellular subsets (n = 9) identified in (e) (f). These subsets were sorted (top) and their expression for CD123, CD34 and IRF8 were subsequently examined by flow cytometry (bottom) (n = 2) (g). Data are representatives of one (b-d), nine (e) and two (g) independent experiments. Data are presented as mean±s.d. of nine (f) independent experiments. DN, double negative; n, biological replicates (f).

Source data

Extended Data Fig. 2 Impact of low dose IRF8 activity on early human T cell development.

a, Scheme illustrates the downstream independent analyses of empty vector (control)- or IRF8-ERT2-transduced HSPCs that were cocultured with OP9-DLL4 stromal cells. b, Flow cytometric staining of IRF8 expression in empty vector (control)- and IRF8-ERT2-transduced cord blood HSPCs at day 2 post transduction. The transduced cells were not treated with 4-OHT. c,d, Volcano plots depict genes that were differentially expressed in 0 nM (c) and 50 nM (d) of 4-OHT treated IRF8-ERT2-expressing cells compared to the untreated control. Genes with a FDR of <0.05 and a log2 fold change of differential expression of >1 or < -1 were considered significant. e, RNA-seq revealed changes in expression of Notch target genes in empty vector (control)- and IRF8-ERT2 (treated with 50 nM 4-OHT)-expressing CD7+ cells from matched donors (n = 3). The normalized counts are CPM. f, The enrichment scores of IFN-related gene signature for the previously annotated populations of human CD34+ thymocytes. g,h, Transcription factor motif analyses for the chromatin regions that are significantly and uniquely opened (g) or closed (h) in IRF8-ERT2-expressing CD7+ cells that were treated with 50 nM of 4-OHT. i,j, Flow cytometric analysis to examine the expression of tmTNF and TNFR1 during in vitro human early T cell development (i) and their expression patterns (n = 3) were quantified proportionally (j). Data are representatives of two (b) and one (i) independent experiments. Data are presented as mean±s.d. of one experiment (j). Motif enrichment was determined by HOMER algorithm93 that uses ZOOPS scoring coupled with the hypergeometric or binomial enrichment calculations (g,h). Data are analyzed by two-way ANOVA with Šídák’s multiple comparisons test (j). FDR, false discovery rate; CPM, counts per million; TF, transcription factor; n, biological replicates (e,j); exact P values are provided in the Source data.

Source data

Extended Data Fig. 3 Impact of IRF8 deficiency on early human hematopoiesis and T cell development.

a,b, Flow cytometric identification of CD326−CD56+ EMPs that were generated from IRF8+/+ and IRF8-/- induced pluripotent stem cells (a), and their frequency (n = 6 per genotype) (b). c, Flow cytometric analysis to determine the impact of IRF8 loss on the generation of TNFR2+CD123+CD127+ hematopoietic progenitors. d-h, Scheme illustrates the collection of conditioned medium from EMO cultures of different conditions at the indicated time points (d). sTNF was measured using flow cytometric bead-based immunoassay in which a standard curve was generated (e). Concentration of sTNF detected in the conditioned medium (n = 6 per condition) (f). The fraction above the bars denotes the actual n from each condition that had detectable sTNF. Flow cytometric analysis of EMOs (n = 4 per condition), cultured with fresh or conditioned medium from day 7 onwards, to identify TNFR2+CD123+CD127+ hematopoietic progenitors (g) and their frequency (h). i,j, Frequency (i) and cell count (j) of CD7+CD5+ T cell precursors (n = 3) that were generated from tmTNF-expressing CD123+CD127+HLA-A2+ progenitors in the ATO-spiking experiment. k,l, Frequency (k) and cell count (l) of CD7+CD5+ T cell precursors (n = 3) that were generated from HLA-A2− HSPCs in the ATO-spiking experiment. Data are representatives of six (a), three (c) and two (g) independent experiments. Data are presented as mean±s.d. of six (b), two (h) and one (i-l) independent experiments. Data are presented as mean of two independent experiments (f: WT). Data are analyzed by two-tailed paired t test (b,k) and one-way ANOVA with Dunnett’s multiple comparisons test (h). WT, wild type; KO, knockout; MFI, median fluorescent intensity; n, EMP induction (b), pool of 2 EMOs (f,h), pool of 2 ATOs (i,j) and biological replicates (k-l); exact P values are provided in the Source data.

Source data

Extended Data Fig. 4 Immunophenotypic analysis of TNF-signaling components in hematopoietic precursors and during early T cell development.

a, Flow cytometric dot plots showing expression of TNFR2 and tmTNF (n = 4) on the 5 cellular subsets of ex vivo human immature thymocytes as shown in (Fig. 5a, b). b, ATOs were assembled using human HSPCs derived from fetal liver (CD34hiCD45+; n = 2), cord blood (lin−CD34+CD38−; n = 2) or adult buffy coats (lin−CD34+; n = 4), and harvested for flow cytometry analyses at day 2, 4, 7 and 10 post culture. For each ontogenetic stage, expression of TNFR1 across different time points is compared to the FMO control and is shown in representative offsetting histograms. c,d, Flow cytometric analysis illustrates the TNFR2 expression profiles of human HSPCs from fetal liver, cord blood and adult buffy coat (n = 2 donors for each) (c). Median fluorescent intensity of TNFR2 for HSPCs from different sources (d). ATOs from each ontogenetic stage were assembled and analyzed independently (b). Data are representatives (a,c) and presented as mean (d) of one experiment; n, biological replicates (d).

Source data

Extended Data Fig. 5 Generation of a MS5 cell line that co-expresses human DLL4 and tmTNF (MS5-DLL4/tmTNF).

a, 6 weeks after sorting, based on co-expression of GFP (reporter for DLL4-encoding construct) and BFP (reporter for TNF-encoding construct), expression of human tmTNF and DLL4 on the transduced MS5 cells was determined by flow cytometry. The MS5 parental cell line served as a negative control. Previously generated MS5-DLL4 cell line served as a positive control for DLL4 expression. b, Expression of reporters, DLL4 and tmTNF on MS5-DLL4/tmTNF cells over time after sorting. Data are representative of one experiment.

Source data

Extended Data Fig. 6 Impact of specific TNFR2 activation on the generation of early T cell precursors.

a,b, Frequency (a) and cell count (b) of CD7+CD5+ T cell precursors that were generated from adult buffy coat lin−CD34+ HSPCs (n = 7) at day 10, without (control) and with TNFR2 activation (EHD2-scTNFR2 and tmTNF at 1% density), in the ATO system. Data are analyzed by two-tailed paired t test (a). n, biological replicates (a,b); exact P values are provided in the Source data.

Source data

Extended Data Fig. 7 sTNF-mediated TNFR1-specific signaling is dose-dependent and accelerates differentiation of human HSPCs into T cell precursors at the expense of their maintenance.

a, Scheme of assembly for the ATOs that were treated without (control) or with sTNF for 10 days. b, Absolute counts of CD45+ cells harvested from an ATO that was aggregated with a normalized amount of 7,500 cord blood-derived lin−CD34+CD38− HSPCs (n = 10 donors for all conditions except n = 3 for treatment with 0.25 ng/mL of sTNF). c-e, Flow cytometric analysis to identify CD7+CD5+ T cell precursors that were generated from the ATOs (c), of which some remained undifferentiated and expressed CD34 (d). Quantification of the impact of sTNF treatment on the cell counts of T cell precursors and undifferentiated CD34+ HSPCs compared to the control (n = 10 for all conditions except n = 3 for treatment with 0.25 ng/mL of sTNF) (e). f,g, Flow cytometric analysis to determine HLA-DR expression on CD1a-expressing T-lineage committed precursors (f) and quantification of the cellular fractions (n = 10 for all conditions except n = 3 for treatment with 0.25 ng/mL of sTNF) that were positive (g). Data are presented as mean±s.d. (b,e,g) and representatives (c,d,f) of three independent experiments. Data are analyzed by two-tailed mixed-effects analysis with Dunnett’s post-hoc test (b,g) and two-tailed mixed-effects analysis with linear trend test (e). n, biological replicates (b,e,g); exact P values are provided in the Source data.

Source data

Extended Data Fig. 8 Differential impacts of sTNF and tmTNF on early T cell precursor generation.

a, Overlays of the UMAP for cells derived from specific (red: control, tmTNF or sTNF) and all conditions (gray). b, UMAP visualization of the cell cycle status for all 26 cellular clusters shown in Fig. 7a. c, The enrichment scores of IFN-related gene signature for all the annotated populations of hematopoietic cells shown in Fig. 7b. d, The relative distribution of cells, derived from ATOs without and with TNF stimulus, in cluster 4 and 15 that are annotated as lymphoid progenitors. e, Heatmap shows the average expression of IFN-related genes by the previously annotated populations of human CD34+ thymocytes. f-l, Scheme illustrates the experimental design where CD7+ T-specified progenitors, derived from control or TNF activated (sTNF at 0.25 ng/mL or tmTNF at 1% density)-ATOs assembled with cord blood lin−CD34+ HSPCs, were examined for their maturation potential towards later stages of T-lineage development (f). Frequency (g) and cell count (h) of CD4+CD8b+ thymocytes (n = 5) that were generated from the secondary ATO cultures. Frequency (i) and cell count (j) of CD3+TCRαβ+ T cells (n = 5) that were generated from the secondary ATO cultures. Frequency (k) and cell count (l) of CD3+TCRγδ+ T cells (n = 5) that were generated from the secondary ATO cultures. n, biological replicates (g-l).

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Supplementary Tables 1–3.

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Liang, K.L., Roels, J., Lavaert, M. et al. Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF. Nat Immunol 24, 474–486 (2023). https://doi.org/10.1038/s41590-022-01417-6

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