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Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity

Nature Immunology volume 22, pages 880–892 (2021)Cite this article

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Abstract

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or ‘mixed’ priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell–induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.

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Fig. 1: Provenance mapping of CNS T cells to distinct peripheral priming sites.
Fig. 2: Clonal expansion of i-T cells versus m-T cells.
Fig. 3: Imprinting of helper T cell signatures in i-T cells versus m-T cells.
Fig. 4: Core signatures of i-T cells versus m-T cells.
Fig. 5: Transcriptional modules of i-T cells versus m-T cells.
Fig. 6: The i- and m-signatures of antigen-specific T cells are robust under various priming conditions.
Fig. 7: Distinct functional phenotypes of i-T cells versus m-T cells in the CNS.

Data availability

Next-generation sequencing raw data and processed gene expression data have been deposited into the GEO repository under the accession numbers GSE156718 (scRNA-seq mouse), GSE172003 (scRNA-seq human), GSE172513 (ATAC-seq mouse) and GSE171122 (bulk RNA-seq mouse). Clinical data for human samples can be found in Supplementary Table 2. All other data generated or analyzed during this study are included in the published article or are available from the corresponding author upon reasonable request.

Code availability

No custom code or algorithms were used in this study.

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Acknowledgements

We thank V. Husterer and M. Pfaller for skillful technical assistance as well as A. Domadenik, V. Kavaka and M. Hammel for help with library preparation. This study was supported by funds from the European Research Council (ERC; CoG 647215 to T. Korn); the Deutsche Forschungsgemeinschaft (SFB1054-B06 (T. Korn), SFB1054-B05 (D.H.B., V.R.B. and S.G.), SFB1371-P04 (D.H.B., K.S. and S.J.), GA 2913/1-1 (C.G.), DO 420/7-1 (K.D.), EXC 2145 (SyNergy) ID 390857198 (T. Korn and K.D.), TRR128-A07 and TRR128-A12 (T. Korn), TRR274-A01 (T. Korn), TRR274-B03 (T.M.), TRR274-C02 (T.M.) and TRR274-Z02 (T. Korn and T.M.)); the Gemeinnützige Hertie-Stiftung (Hertie Network of Excellence in Clinical Neuroscience to C.G. and T. Korn); and the Langmatz-Foundation (to C.G.).

Author information

Authors and Affiliations

  1. Institute for Experimental Neuroimmunology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Michael Hiltensperger, Ravi Kant, Sofia Tyystjärvi, Gildas Lepennetier, Helena Domínguez Moreno, Andreas Muschaweckh, Christopher Sie, Lilian Aly, Garima Garg, Ali M. Afzali & Thomas Korn

  2. Institute of Clinical Neuroimmunology, University Hospital and Biomedical Center, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany

    Eduardo Beltrán, Isabel J. Bauer, Lisa Ann Gerdes, Tania Kümpfel, Naoto Kawakami & Klaus Dornmair

  3. Department of Neurology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany

    Gildas Lepennetier, Christiane Gasperi, Lilian Aly, Benjamin Knier, Ali M. Afzali, Bernhard Hemmer & Thomas Korn

  4. Institute for Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany

    Simon Grassmann, Sebastian Jarosch, Kilian Schober, Veit R. Buchholz & Dirk H. Busch

  5. Institute of Neuronal Cell Biology, Technical University of Munich, Munich, Germany

    Selin Kenet & Thomas Misgeld

  6. Institute of Molecular Oncology and Functional Genomics, TranslaTUM Cancer Center, Technical University of Munich, Munich, Germany

    Rupert Öllinger & Roland Rad

  7. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    Lisa Ann Gerdes, Bernhard Hemmer, Thomas Misgeld, Klaus Dornmair & Thomas Korn

  8. Institute of Clinical Molecular Biology, Kiel University, Kiel, Germany

    Sören Franzenburg

  9. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany

    Thomas Misgeld

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  1. Michael Hiltensperger
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Contributions

M.H. designed experiments, performed most of the experiments, analyzed data and drafted the manuscript. E.B. performed the human scRNA-seq experiments and analyzed the mouse and human scRNA-seq data. R.K. performed histology and analyzed data. G.L. analyzed the mouse scRNA-seq and bulk RNA-seq experiments. H.D.M. performed and analyzed the ATAC-seq experiments. S.T., S.K., A.M., C.S., L.A., B.K., G.G., A.M.A., L.A.G. and S.F. performed experiments and analyzed data. I.J.B. performed and analyzed the Ca2+ imaging experiments. R.Ö. performed the mouse bulk RNA-seq experiments and analyzed data. S.G., S.J. and K.S. performed the mouse scRNA-seq experiments and analyzed data. C.G. analyzed the human scRNA experiments. V.R.B., R.R., T. Kümpfel, N.K., B.H., D.H.B., T.M. and K.D. designed experiments and analyzed data. T. Korn conceptualized and directed the study, supervised the experiments, analyzed data and wrote the manuscript.

Corresponding author

Correspondence to Thomas Korn.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Britta Engelhardt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Site-specific labeling of T cells by photoconversion in inguinal and mesenteric lymph nodes in vivo.

a, Schematic of photoconversion of T cells in the iLN and mLN of PhAMT mice. b, Flow cytometric gating strategy for mD2GREEN or mD2RED CD4+ T cells. The DUMP channel comprised LIVE/DEADTM-Near-IR and CD8α-APC-Cy7. c, Flow cytometric assessment in the indicated LN of iLN-labeled (top) and mLN-labeled (bottom) PhAMT mice immediately after photoconversion (photo) or without photoconversion (dark). Representative plots of n = 3 mice per group. d, e, Flow cytometric proliferation readout of in vitro-activated PhAMT CD4+ T cells 4 days after photoconversion (d) and mD2RED signal over time (e). Representative plots from two independent experiments. f, Experimental design of iLN (top) or mLN (bottom) irradiation in MOG(35-55)/CFA immunized PhamT mice. g, Flow cytometric assessment of mD2REDCD44high frequencies (top) and absolute numbers (bottom) in different tissues in iLN-labeled PhAMT EAE mice 2 days after photoconversion at indicated time points. Representative plots from two mice per time point. h, i, Flow cytometric assessment in the spleen of iLN-labeled (left) and mLN-labeled (right) PhAMT EAE mice 2 days after photoconversion at disease onset (h) and of the CNS in non-photoconverted (dark) PhAMT EAE mice (i). Representative plots from spleen n = 15 mice per group, CNS n = 7 mice. j, Frequencies of regulatory and activated conventional mD2GREENCD4+ T cells in PhAMT EAE mice for different tissues. n = 2 mice per group, representative plot from three independent experiments for Foxp3 and CD44 and two for CD69. k, Frequency and duration of Ca2+ signaling in iLN (top) or mLN (bottom) of mice immunized on days 3 or 4 post transfer, that is days 13 or 14 post immunization. Left panels: individual Ca2+ signaling durations of TCRMOG Twitch-2B T cells. The dotted line indicates the cut-off (2 minutes) to distinguish between short- and long-term Ca2+ signaling. Cumulative results from iLN(PBS) and mLN(PBS) n = 2, iLN(MOG) and mLN(MOG) n = 3 mice. Right panels: fractions of T cells presenting short- and long-term Ca2+ signaling or no Ca2+ spikes. Data shown as mean. l, Representative images of TCRMOG Twitch-2B T cells from intravital time-lapse two-photon microscopy for Ca2+ imaging depicted by a fluorescence overlay of T cells (left) and a pseudocolor Ca2+ ratio image (right) from iLN(PBS) and mLN(PBS) n = 2, iLN(MOG) and mLN(MOG) n = 3 mice (see Supplementary Videos 1–4). Scale bars 25 μm.

Extended Data Fig. 2 TCR repertoire analysis of i-T cells and m-T cells.

a, Schematic of single cell sequencing of sorted iLN- or mLN-derived photoconverted T cells from MOG(35-55)/CFA immunized PhAMT mice in combination with TotalSeq hashtag barcoding antibodies. b, Unsupervised clustering t-distributed stochastic neighbour embedding (t-SNE) plot of all single mD2RED CD4+CD44high T cells analyzed. In the upper plot only single T cells from i-stream are colored (purple). In the lower plot only single T cells from m-stream are colored (orange). n = 5 PhAMT EAE mice per group, n = 14621 cells, i-T cells n = 7228 cells, m-T cells n = 7393 cells. c, Repertoire overlap analysis using hierarchical clustering. Dendrogram shows weighted clonal overlaps for TRB-CDR3 sequences among mice, analyzed using F pairwise similarity metric in VDJtools. Branch length shows the distance between repertoires. n = 3 PhAMT EAE mice per group. d, Average gene expression of all single mD2RED CD4+CD44high T cells analyzed for the TOP10 differentially expressed genes of cluster 0 to 7.

Extended Data Fig. 3 Single cell transcriptome analysis in i-T cells and m-T cells.

a, Average gene expression of all single mD2RED CD4+CD44high T cells analyzed from i- and m-stream in spleen and CNS grouped into T cell subsets based on key signature genes (see Fig. 3). b, Flow cytometric assessment in iLN-labeled (left) and mLN-labeled (right) PhAMIL17 EAE mice 2 days after photoconversion at disease onset. LN (top row), spleen (middle row), and CNS (bottom row). Representative plots of n = 3 mice per group. c, Unsupervised clustering t-SNE plot, colored according to i- and m-stream cell group and key gene marker (Tbx21, Rorc, Ccr6, Csf2, Ifng, Il17a, Il10) expression. n = 2 PhAMT EAE mice per group, n = 4169 cells.

Extended Data Fig. 4 Core signatures and transcription factor regulons of i-T cells and m-T cells.

a, b, Gene expression (violin plots) of i-stream signature genes Cxcr6 and Itgb1 and m-stream signature genes P2rx7 and Itga4 in the spleen (a) and CNS (b). c-e, Transcriptional module analysis of scRNAseq data from Fig. 4 based on SCENIC algorithm in i- and m-T cells. All genes controlled by a given transcription factor build a regulon. The regulon specificity score (RSS) was calculated using Jensen-Shannon divergence. c, Genome-wide regulons with RSS > 0.2 in at least one condition are displayed for i-T cells and m-T cells (i-stream and m-stream across all organs). d, e, Regulons that control Cxcr6 (d) and Itga4 (e) with no RSS threshold requirement.

Extended Data Fig. 5 ATACseq of i-T cells and m-T cells isolated from the spleen.

a, Genome browser view of key gene markers (Tbx21, Rorc, Ifng, Csf2, Il21, and Ccr6). Displayed tracks correspond to ATACseq for i-T cells (purple) and m-T cells (orange). In all regions, differential peaks (if existing) are highlighted with arrows. b, c, Ranked list of the top transcription factor motifs predicted by HOMER based on cumulative hypergeometric distribution testing for differential ATAC peaks associated to signature genes for splenic i-T cells (b) and m-T cells (c). For i-T cells 48 transcription factor sequences out of 154 are shown and for m-T cells, 16 sequences out of 30 are shown.

Extended Data Fig. 6 Robust i-T cell and m-T cell signatures in various immunization protocols.

a, Flow cytometric assessment of mD2REDCD4+CD44high T cells in the spleen of iLN-labeled (purple) and mLN-labeled (orange) PhamT mice, for Cxcr6 (top), P2rx7 (middle), and CD49d (Itga4) vs CD29 (Itgb1) (bottom). PhamT mice were subjected to different s.c. immunization regimens at the base of tail plus pertussis toxin i.v. on day 0 and day 2 after immunization (as indicated) and analyzed 2 days after photoconversion on day 11 after immunization. Representative plots from n = 3 PhamT mice per group (iLN- and mLN-labeled) and immunization condition. Numbers indicate mean fluorescent intensities. PGN, peptidoglycan. b, i-T cells and m-T cells do not cross-traffic after i.v. immunization. 2D2 PhamT mice were injected with 40 μg MOG(35-55) i.v., photoconverted at iLN (left) or mLN (right) on day 2, and analyzed for the fraction of CD44high mD2RED T cells in iLN and mLN on day 4 after injection. Representative plots from n = 4 2D2 PhamT mice per group.

Extended Data Fig. 7 Distinct functional phenotypes of i-T cells and m-T cells in the CNS compartment both in mice and humans.

a, EAE progression in iLN-labeled and mLN-labeled PhAMT EAE mice. Photoconversion at disease onset and analysis 2 days later. iLN-labeled n = 5 mice, mLN-labeled n = 3 mice, representative plot from five independent experiments. Data shown as mean ± s.d. b, Flow cytometric assessment of CD49d (Itga4) vs CD44 in transferred TCRMOG mD2REDCD4+CD44high i- and m-T cells isolated from the spleen of secondary recipient Rag1–/– mice (approximately 20 days after transfer). n = 3 mice per group. c, EAE progression in wild-type (WT) and Cxcr6–/– mice. WT n = 5 mice, Cxcr6–/– n = 6 mice, representative plot from three independent experiments. Data shown as mean ± s.d. d, Unsupervised clustering t-distributed stochastic neighbour embedding (t-SNE) plot of cerebrospinal fluid (CSF) single CD4+ T cells isolated from untreated MS patients, colored according to T helper cell and Treg cell signature gene expression. Th1 (CSF1, CXCR6, IL12B2, IL18R1, KLRC1, HOPX), Th2 (IL4, TNFSF13B, BATF, NFIL3, ATF5), Th17 (IL17A, IL21, IL2, TNFRSF13B, PTGFRN, AHR, IRF4, RORA, PLAGL2), and Treg (FOXP3, IKZF2, NRP1, FOSB, TNFSF11, IRF8). e, Gene marker expression of a selection of i-stream core signature genes (IL2RB, IL18RAP, SYTL2) (top row), and m-stream core signature genes (NT5E, DST, AIG1) (bottom row). d, e, n = 6 CSF samples from untreated MS patients, n = 14339 cells.

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Supplementary Tables 1–3.

Supplementary Video 1

Intravital imaging of Ca2+ signaling in iLN of a PBS/CFA-immunized mouse after transfer of TCRMOG Twitch-2B T cells. A fluorescence overlay (left) and a pseudocolor ratio image (right) are depicted. Blue/green, TCRMOG Twitch-2B T cells; red, phagocytes visualized by detecting autofluorescence and i.v. infusion of fluorescent dextran; yellow, high Ca2+; purple, low Ca2+.

Supplementary Video 2

Intravital imaging of Ca2+ signaling in iLN of a MOG35–55/CFA-immunized mouse after transfer of TCRMOG Twitch-2B T cells. A fluorescence overlay (left) and a pseudocolor ratio image (right) are depicted. Blue/green, TCRMOG Twitch-2B T cells; red, phagocytes visualized by detecting autofluorescence and i.v. infusion of fluorescent dextran; yellow, high Ca2+; purple, low Ca2+.

Supplementary Video 3

Intravital imaging of Ca2+ signaling in mLN of a PBS/CFA-immunized mouse after transfer of TCRMOG Twitch-2B T cells. A fluorescence overlay (left) and a pseudocolor ratio image (right) are depicted. Blue/green, TCRMOG Twitch-2B T cells; red, phagocytes visualized by detecting autofluorescence and i.v. infusion of fluorescent dextran; yellow, high Ca2+; purple, low Ca2+.

Supplementary Video 4

Intravital imaging of Ca2+ signaling in mLN of a MOG35–55/CFA-immunized mouse after transfer of TCRMOG Twitch-2B T cells. A fluorescence overlay (left) and a pseudocolor ratio image (right) are depicted. Blue/green, TCRMOG Twitch-2B T cells; red, phagocytes visualized by detecting autofluorescence and i.v. infusion of fluorescent dextran; yellow, high Ca2+; purple, low Ca2+.

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Hiltensperger, M., Beltrán, E., Kant, R. et al. Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity. Nat Immunol 22, 880–892 (2021). https://doi.org/10.1038/s41590-021-00948-8

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