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Antigen presentation by CD40<sup>+</sup>MHC-II<sup>+</sup> astrocytes promotes CNS autoimmunity

Nature作者：Joon-Hyuk Lee2026年8月5日正文已收录本站

Data availability

The analysis results and bioinformatics data have been deposited into the Zenodo61 repository 19803100 (https://zenodo.org/records/19803100). scRNA-seq or single-nucleus RNA-seq datasets of astrocytes were obtained from the Gene Expression Omnibus with accession numbers GSE150858 (ref. 2), GSE130119 (ref. 8), GSE180759 (ref. 29) and the BioProject repository PRJNA544731 (ref. 30). All other data are available from the corresponding author on reasonable request. Correspondence and request for materials should be addressed to F.J.Q. Source data are provided with this paper.

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Acknowledgements

We thank all members of the Quintana laboratory for helpful advice and discussions; L. Ding for technical assistance with confocal microscopy; the Research Computing Group at Harvard Medical School for access to the O2 High Performance Compute Cluster; R. Krishnan for technical assistance with flow cytometry and FACS; and R. Tomaino for technical assistance with mass spectrometry.

Funding

This work was supported by grants NS142904, NS129778, ES02530 and ES029136 from the US National Institutes of Health (NIH), Mayer Foundation and PA-1604-08459 from the International Progressive MS Alliance. J.-H.L. was supported by the Basic Science Research Program funded by the National Research Foundation of Korea (NRF)/Ministry of Education (2022R1A6A3A03071157) and a long-term postdoctoral fellowship funded by the Human Frontier Science Program (LT0015/2023-L). A.-R.K. was supported by a postdoctoral fellowship program (Nurturing Next-generation Researchers) through the NRF funded by the Ministry of Education (2021R1A6A3A14039622). N.P. is an investigator of the Howard Hughes Medical Institute. J.E.K. was supported by the DoD Multiple Sclerosis Research Program EIRA no. MS230278 and the NIH T32 Cancer Neuroscience Training grant no. T32CA272386. J.J.Y. was supported by the UE5 NIH NINDS Research Education Program. H.-G.L. was supported by a Basic Science Research Program through the NRF funded by the Ministry of Education (2021R1A6A3A14039088). M.A.W. was supported by R01MH130458, R01MH132632, R01DA061199 and R00NS114111 from the NIH. C.F.A. was supported by a scholarship from the German Academic Exchange Service (DAAD). T.I. was supported by the EMBO Postdoctoral Fellowship (ALTF: 1009-2021) and the Jerome-Lejeune Postdoctoral Fellowship. C.M.P. was supported by the National Multiple Sclerosis Society (FG-2307-42209) and the Mayer Foundation. M.K. was supported by a Leopoldina Research Fellow Grant of the German Academy of Sciences. B.M.A. was supported by the Training Program in Nervous System Tumors (K12CA090354) from the National Cancer Institute (NCI)/NIH, the Career Enhancement Program for the NCI/NIH SPORE at the Harvard Cancer Center (P50CA165962) from the NCI/NIH, the Post-Doctoral Fellowship in Translational Medicine from the PhRMA Foundation, Cancer Neuroscience grant T32CA272386 from the NCI/NIH, and 1IK2BX006568-01A1 from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. P.D.-A. was supported by the EMBO Postdoctoral Fellowship (ALTF: 953-2025). B.S.K. and J.S.S. were supported by NS111583. J.L. was supported by the Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2025-02653596).

Author information

Authors and Affiliations

  1. Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Joon-Hyuk Lee, Zhaorong Li, Tomer Illouz, Carolina M. Polonio, Michael Kilian, Jessica E. Kenison, Anton M. Schüle, Camilo Faust Akl, Hong-Gyun Lee, Brian M. Andersen, Jessica J. Ye, Joseph M. Rone, Gavin Piester, Lena Srun, Jazmin Martinez, Austin Danko, Jinsu Lee, Tae Hyun Heo, Elizabeth N. Chung, Landon K. Oetjen, Pere Duart-Abadia, Nakyung Koo, Michael A. Wheeler & Francisco J. Quintana

  2. The Gene Lay Institute of Immunology and Inflammation, Brigham and Women’s Hospital, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Joon-Hyuk Lee, Zhaorong Li, Tomer Illouz, Carolina M. Polonio, Michael Kilian, Jessica E. Kenison, Anton M. Schüle, Camilo Faust Akl, Hong-Gyun Lee, Brian M. Andersen, Jessica J. Ye, Joseph M. Rone, Gavin Piester, Lena Srun, Jazmin Martinez, Jinsu Lee, Tae Hyun Heo, Elizabeth N. Chung, Landon K. Oetjen, Pere Duart-Abadia, Nakyung Koo, Michael A. Wheeler & Francisco J. Quintana

  3. Department of Physiology, University of California, Los Angeles, CA, USA

    Joselyn S. Soto & Baljit S. Khakh

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Ah-Ram Kim & Norbert Perrimon

  5. Department of Neurology, Veterans Affairs Medical Center, Harvard Medical School, Boston, MA, USA

    Brian M. Andersen

  6. Howard Hughes Medical Institute, Boston, MA, USA

    Norbert Perrimon

  7. Department of Neurobiology, University of California, Los Angeles, CA, USA

    Baljit S. Khakh

  8. Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada

    Stephanie E. J. Zandee

  9. Neuroimmunology Research Lab, CRCHUM and Department of Neuroscience, Faculty of Medicine, Universite de Montreal, Montreal, Quebec, Canada

    Stephanie E. J. Zandee & Alexandre Prat

  10. The Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Francisco J. Quintana

Authors

  1. Joon-Hyuk Lee
  2. Zhaorong Li
  3. Joselyn S. Soto
  4. Ah-Ram Kim
  5. Tomer Illouz
  6. Carolina M. Polonio
  7. Michael Kilian
  8. Jessica E. Kenison
  9. Anton M. Schüle
  10. Camilo Faust Akl
  11. Hong-Gyun Lee
  12. Brian M. Andersen
  13. Jessica J. Ye
  14. Joseph M. Rone
  15. Gavin Piester
  16. Lena Srun
  17. Jazmin Martinez
  18. Austin Danko
  19. Jinsu Lee
  20. Tae Hyun Heo
  21. Elizabeth N. Chung
  22. Landon K. Oetjen
  23. Pere Duart-Abadia
  24. Nakyung Koo
  25. Norbert Perrimon
  26. Michael A. Wheeler
  27. Baljit S. Khakh
  28. Stephanie E. J. Zandee
  29. Alexandre Prat
  30. Francisco J. Quintana

Contributions

J.-H.L. and F.J.Q. designed the research. J.-H.L., H.-G.L., M.K., A.M.S., C.F.A., B.M.A., J.E.K., T.I., C.M.P., J.J.Y., J.M.R., G.P., L.S., J.M., A.D., J.L., E.N.C., T.H.H., L.K.O., P.D-.A. and N.K. performed the experiments. J.-H.L., Z.L., C.F.A., M.A.W. and F.J.Q. performed the bioinformatic analyses. S.E.J.Z. and A.P. provided unique resources and discussed and/or interpreted the findings. J.-H.L., J.S.S., B.S.K. and F.J.Q. performed the proteomics experiments. A.-R.K. and N.P. performed the AlphaFold-Multimer prediction for protein–protein interaction analysis. J.-H.L. and F.J.Q. wrote the paper with input from co-authors. F.J.Q. directed and supervised the study.

Corresponding author

Correspondence to Francisco J. Quintana.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Manuel Friese, Naomi Habib, Kendrick Li, E. Moseman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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

Extended data figures and tables

Extended Data Fig. 1 CD40+MHC-II+ astrocytes are located adjacent to blood vessels.

a, Representative images showing CD40+MHC-II+ astrocytes along with CD31+ blood vessels. Scale bar = 100 μm. b, Sholl mask showing the distribution of CD40+MHC-II+ (left) or total astrocytes (right). c, Quantified cross-section of total astrocytes (top), CD40+MHC-II+ astrocytes (middle) and relative CD40+MHC-II+ astrocytes cross-section per total astrocytes (bottom). n = 7 mice. Differences between the data point and its counterpart at 475 nm were evaluated by One-way ANOVA, followed by multiple comparisons. Any non-significant P-values are listed in the Source Data. Data shown as mean ± s.e.m.

Source data

Extended Data Fig. 2 CD40+MHC-II+CNS APCs.

a, Gating strategy. b, Fraction of CNS APCs out of total cells (left) and CD40+MHC-II+ cells (right). c, Representative histogram (left) and quantification (right) of MHC-II expression in CD40+MHC-II+ CNS APCs. d, Representative histogram (left) and quantification (right) of CD40 expression in CD40+MHC-II+ CNS APCs. (c-d) n = 5 mice per group. (c-d) The differences between astrocytes and other APCS were assessed by One-way ANOVA, followed by multiple comparisons. e-g, EAE development. GfaABC1D promoter (e), Itgam promoter (f) and Itgax promoter (g) driven Cd40 or H2-ab1 inactivation. n = 5 mice per group. Analyzed by Two-way ANOVA, followed by multiple comparisons at dpi20. h, Quantification of CD40+ population in sgCd40 LV injected group. i, Quantification of MHC-II+ population in sgH2-ab1 LV injected group. (h-i) n = 5 mice per group. Analyzed by Mann-Whitney test. (c-i) Data shown as mean ± s.e.m.

Source data

Extended Data Fig. 3 CD40+MHC-II+ astrocytes uptake myelin.

a, Experimental approach. b-c, Representative histogram (b) and MFI of engulfed myelin in astrocytes. n = 4 for CD40-MHC-II-, CD40+, MHC-II+ astrocytes and n = 8 mice for CD40+MHC-II+ astrocytes. Analyzed by One-way ANOVA, followed by multiple comparisons. d-e, Representative flow cytometry plots (d) and percentage (e) of myelin+ astrocytes in vitro. n = 6 biological replicates per group. Analyzed by One-way ANOVA, followed by multiple comparisons. f, Astrocyte H2-ab1 (left) and Cd40 (right) expression. n = 7 biological replicates per group. Analyzed by Two-way ANOVA, followed by multiple comparisons. g, Ex vivo myelin phagocytosis assay (h-i, m-n). h-i, Representative flow cytometry plots (h) and quantification (i) of myelin+ astrocytes ex vivo. Naïve vs EAE. n = 4 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. j, In vivo myelin phagocytosis assay (k-l, o-p). k-l, Representative flow cytometry plots (k) and quantification (l) of myelin+ astrocytes in vivo. Naïve vs EAE. n = 5 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. m-n, Representative histograms (m) and quantification (n) of myelin+ astrocytes ex vivo. CD40-MHC-II- vs CD40+MHC-II- vs CD40-MHC-II+ vs CD40+MHC-II+. n = 4 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. o-p, Representative histograms (o) and quantification (p) of myelin+ astrocytes in vivo. CD40-MHC-II- vs CD40+MHC-II- vs CD40-MHC-II+ vs CD40+MHC-II+. n = 5 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. For all graphs, Data shown as mean ± s.e.m. Schematics in panels a,g,j created in BioRender; Lee, J.-H. https://biorender.com/1ml2yme (2026).

Source data

Extended Data Fig. 4 Antigen presentation by astrocytes.

a, Tgfb1, Il23a and Il6 expression measured by qPCR in CD40-MHC-II-, CD40+, MHC-II+ and CD40+MHC-II+ astrocytes. n = 5 mice per group (Tgfb1, Il6). n = 3 per group (Il23a). Analyzed by One-way ANOVA, followed by multiple comparisons. b, Representative flow cytometry plots (left) and quantification (right) of CD4+ T cells before and after sorting. n = 4 biological replicates per group. Analyzed by Mann-Whitney test. c, Representative flow cytometry plots (left) and quantification (right) of MHC-II+ cells before and after sorting. n = 4 biological replicates per group. Analyzed by Mann-Whitney test. d, Gating strategy for CD45, CD11b-negative astrocytes from astrocyte-enriched mixed glia. e, Representative images of CD45negCD11bnegO4neg primary astrocytes and CD45posCD11bposO4pos microglia and oligodendrocytes at div 7. Scale bars = 200 μm. f, Quantification of GFAP+ astrocytes and IBA1+ microglia. n = 4 biological replicates per group. Analyzed by One-way ANOVA, followed by multiple comparisons. g, Gating strategy of CD4+ T cells from astrocyte-T cell co-culture. Schematic in panel g created in BioRender; Lee, J.-H. https://biorender.com/xg1vkv9 (2026). h, Experimental design for i-l. i, Representative flow cytometry plot (left) and quantification of proliferating total CD4+ T cells after co-culture with pan dendritic cells (Pan DC). n = 5 biological replicates. Analyzed by Mann-Whitney test. j-k Representative flow cytometry plot (j) and quantification (k) of CD4+ T cell proliferation. Blue dotted line indicates the proliferation of total CD4+ T cell by dendritic cells (Extended Data Fig. 9i). n = 6 (0ug/ml), 9 (CD4+ T only), 8 (astrocyte, astrocyte+TNF + IL-1β in 40ug/ml groups) biological replicates. Analyzed by Two-way ANOVA, followed by multiple comparisons. l, Fraction of IL-17A/IFNγ/GM-CSF/Foxp3+CD4+ T Cells out of total CD4+ T cells after astrocyte-T cell co-culture. n = 6 biological replicates per group. Analyzed by One-way ANOVA followed by multiple comparisons. m-n, Effect of MHC-II-blocking antibodies. Representative flow cytometry plot (m) and quantification (n) of CD4+ T cell proliferation. n = 4 biological replicates per group. Analyzed by Mann-Whitney test. o-p, Effect of CD40-agonizing antibodies. Representative flow cytometry plot (o) and quantification (p) of CD4+ T cell proliferation. n = 6 (TNF+IL-1b+isotype, control+anti-CD40), 7 (control+isotype, TNF+IL-1b+anti-CD40) biological replicates. Analyzed by Mann-Whitney test. q-r, Astrocyte presentation of mouse recombinant MOG (rmMOG) epitopes. Representative flow cytometry plot (q) and quantification (r) of CD4+ T cell proliferation. n = 4 biological replicates per group. Analyzed by One-way ANOVA followed by multiple comparisons. s, IL-17A, IFNγ, GM-CSF or Foxp3+CD4+ T cells after co-culture of total CD4+ T cells with rmMOG-pulsed astrocytes. n = 4 biological replicates for group. Analyzed by One-way ANOVA followed by multiple comparison. For all graphs, Data shown as mean ± s.e.m.

Source data

Extended Data Fig. 5 uLIPSTIC-based investigation of CNS cell interactions in EAE.

a, Gating strategy. b, Quantification of SrtA+ cells at peak EAE in uLIPSTICAldh1l1-CreERT2 mice. n = 4 per group. The differences between groups were assessed by Mann-Whitney test. Data shown as mean ± s.e.m. c, Fraction of each cell type out of total SrtA+ Cells. n = 4 per group. Data shown as mean ± s.e.m. d, Experimental desing for CD40L or MHC-II blockade in CNS. e, Representative flow cytometry plots of biotinylated CD4+ T cells. f, Quantification of biotin+ CD4+ T cells. n = 4 per group. Analyzed by One-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. g-h, Representative flow cytometry plot (g) and quantification (h) showing biotinylation of CNS-resident or infiltrating cells at peak EAE. n = 3 and 7 mice for uLIPSTIC and uLIPSTICAldh1l1-CreERT2 group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. i, Fraction of each cell type out of total biotin+ cells. Bar graph showing individual value per biological replicate (left). Pie chart of quantification (right) n = 7 mice. Data shown as mean ± s.e.m. j, Quantification of cytokine-producing CD4+ T cells from brefeldin A injected uLIPSTICAldh1l1-CreERT2. n = 5 mice. Analyzed by Paired t test. Data shown as mean ± s.e.m. k, RosauLIPSTIC (uLIPSTICAldh1l1-CreERT2) mice were injected with AAV5::GFAP-Cre or PBS and induced with EAE. At disease peak, biotin-LPETG was administered. l-m, Validation of uLIPSTICAldh1l1-CreERT2. (l) Representative flow cytometry plot showing SrtA expression in each cell type. (m) Quantification of SrtA+ population in each cell type (right) at EAE peak. n = 3 for PBS and n = 6 for AAV5::GFAP-Cre injected mice. Analyzed by Two-way ANOVA. Data shown as mean ± s.e.m. n-o, Representative flow cytometry plot showing fraction of biotin+ CD4+ T cells at EAE peak (n) and quantification (o) n = 3 for PBS and n = 6 for AAV5::GFAP-Cre injected mice. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. p, Quantification of biotin+ CD4+ T cells in the mouse CNS. n = 3 for PBS and n = 6 for AAV5::GFAP-Cre injected mice. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. q, Representative flow cytometry plot showing Foxp3+ and IL-17A+ fraction in total CD4+ and biotin+ CD4+ population. r, Quantified Th17/Treg ratio. s, Quantification of cytokine-producing CD4+ T cells. t, Percentage of Th17 subtypes. (r-t) n = 6 mice. Analyzed by Paired t test. u, Experimental design. v, Representative flow cytometry plot and quantification of in vitro T cell differentiation. w, Gating strategy of transferred in vitro differentiated helper T cells. x-y, Representative plots (x) and quantification (y) of biotinylation of transferred helper T cells. n = 5 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. z, Quantification showing fraction of CD4+ T cell subtypes in biotin+ and- ICV-transferred helper T cells. n = 5 mice per group. Analyzed by Paired t test. Schematics created in BioRender: k, Lee, J.-H. https://www.biorender.com/93e5olq (2026); u, Lee, J.-H. https://biorender.com/kl947j9 (2026).

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Extended Data Fig. 6 BioID2 analysis of CD40 interactors in EAE astrocytes.

a, Kinetics of astrocyte H2-ab1 expression upon anti-CD40 treatment or co-culture with activated CD4+ T cells. n = 6 for isotype control, anti-CD40, TNF+IL-1b and anti-CD40+TNFa+IL-1b, and n = 4 biological replicates for activated CD4+ T cells and activated CD4+ T cells+ TNF+IL-1b. Analyzed by Two-way ANOVA, followed by multiple comparisons. For exact p-values, please see Source Data. Data shown as mean ± s.e.m. b-c, Upstream regulators in anti-CD40 treated astrocytes (b) and DC (c) in vitro. n = 4 mice per group. (b-c) p-value was calculated by Qiagen IPA. d, Western blot showing biotinylated protein enrichment in CD40-BioID2 injected mouse compared to CD40-GFP injected mouse. n = 4 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. e, Representative images and quantification of GfaABC1D promoter-driven CD40-BioID2 expression in astrocytes (S100b) and neuron (NeuN). Scale bars = 20 μm. n = 5 mice per groups. Mann-Whitney test. f, Representative images and quantification of biotinylated proteins (left) and biotinylated area (right). Scale bars = 100 μm. n = 5 mice per groups. Mann-Whitney test. Data shown as mean ± s.e.m. g-n, Venn diagrams comparing protein interactors detected with CD40-BioID2 to those identified (Soto et al.4) using (g) whole astrocyte BioID2, (h) astrocyte LCK-BioID2, (i) astrocyte SAPAP3-BioID2, (j) astrocyte AQP4-BioID2, (k) astrocyte EZR-BioID2, (l) astrocyte GLT1-BioID2. (m) astrocyte Kir4.1-BioID2, (n) astrocyte CX43-BioID2.

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Extended Data Fig. 7 Astrocyte CD40 activation promotes LD accumulation.

a, Western blot verifying immunoprecipitation (IP) of PLIN4 (IP: PLIN4). b, Western blot analysis of serine phosphorylation of IP: PLIN4 (pPLIN4) and total PLIN4 in astrocytes after treatment with isotype control and anti-CD40 for 18 h. c, pPLIN4/PLIN4 ratio. n = 8 biological replicates. Analyzed by Mann-Whitney test. d, Expression of Plin4 in vehicle, isotype control and anti-CD40 treated astrocytes. n = 8 biological replicates per group. Analyzed by One-way ANOVA, followed by multiple comparisons. e, Experimental design for (f-g). f-g, Representative flow cytometry plot (l, 5uM oleic acid group) and quantification (f) of LD+ astrocytes after treatment with anti-CD40 and 0-500uM oleic acid. n = 4 biological replicates per group. Analyzed by One-way ANOVA, followed by multiple comparisons. h-i, Representative images (h) and quantification (i) of LD (BODIPY 493/503, green) contents in astrocytes (GFAP, magenta) after stimulated with isotype IgG2a or anti-CD40. Scale bars = 10 μm. n = 5 mice per groups. Analyzed by Mann-Whitney test. j-k, Representative images (j) and quantification (k) of PLIN4 (green) expression in astrocytes (GFAP, magenta) in naïve and EAE spinal cord. Scale bars = 10 μm. n = 4 mice per groups. Analyzed by Mann-Whitney test. l, Representative images showing isolation of LD signals of BODIPY493/503 staining. m-n, Representative images (m) and quantification (n) of LD (BODIPY493/503, green)-positive astrocytes (GFAP, magenta) in sgScramble and sgCd40 EAE group. Scale bar = 10 μm. n = 4 mice per group. Analyzed by Mann-Whitney test. For all graphs, Data shown as mean ± s.e.m.

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Extended Data Fig. 8 T cell CD40L-triggered CD40 signaling drives LD accumulation in astrocytes.

a, Gating strategy of ACSA2+ astrocytes. b, Representative flow cytometry plots showing biotin+ astrocytes, OPCs, microglia, DCs, Macrophage/monocytes and B cells. c, Quantification of biotin+ cells out of parental cells. n = 3 for No SrtA control and n = 7 mice for oLIPSTIC. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. d, Quantification of biotin+ cells out of total biotin+ CNS APCs (left). Fraction out of total biotin+ CNS APCs (right) n = 7 mice. Data shown as mean ± s.e.m. e-g, Representative histogram (e) and quantification of CD40 (f) and MHC-II (g) expression in biotin+ or biotin- astrocytes. n = 7 mice per group. Analyzed by Paired t test. h-i, Representative flow cytometry plots (h) and quantification (i) of CD40+MHC-II+ astrocytes in biotin+ astrocytes. n = 4 mice per group. Analyzed by Paired t test. j, Experimental design for CD40L or MHC-II blockade in CNS of oLIPSTIC. k, Representative flow cytometry plots of biotinylated astrocytes. l, Quantification of biotin+ astrocytes. n = 6 mice for vehicle group and n = 4 mice for anti-MHC-II and anti-CD40L groups. Analyzed by One-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. m, Representative images (left) and quantification (right) of biotinylated CNS parenchyma of biotin injected (biotin+) and non-biotin injected (biotin−) oLIPSTIC at the EAE peak. n = 4 mice per group. Scale bar=10 μm. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. n, Representative images of biotin+ astrocytes interacting with RORyT+ CD4+ T cells. Scale bar=10 μm. o, Quantification of RORyT+ CD4+ T cells or RORyT- CD4+ T cells locate nearby biotin+ astrocytes, biotin- astrocytes. n = 4 mice per group. Analyzed by Two-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. p-q, Representative flow cytometry plot (m) and quantification of BODIPY493/503 MFI (n) of ACSA2+ astrocytes in EAE and naïve CNS. n = 5 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m.

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Extended Data Fig. 9 PLIN4-dependent astrocyte LDs promote CNS inflammation.

a-b, Representative flow cytometry plots (a) and quantification of CD40+MHC-II+ astrocytes (b) in LD Low and LD High astrocyte fraction. n = 4 mice per group. Analyzed by Paired T test. c-d, Representative histogram (c) and quantification (d) of BODIPY immunoreactivity in each astrocyte population. n = 4 mice per group. Analyzed by One-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. e, EAE development in sgil6, sgil23a and sgScramble groups. n = 10 mice per group. Analyzed by Two-way ANOVA, followed by multiple comparisons. Data shown as mean ± s.e.m. f-g, Il6 (f) and Il23a (g) expression in astrocytes. n = 7 for sgScramble, n = 10 for sgCd40 and n = 6 mice for sgH2-ab1. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. h, Representative images and quantification of PLIN4 expression (green) in astrocytes (GFAP, magenta). Scale bars = 10 μm. n = 4 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. i, Plin4, Il6 and Il23a expression measured by qPCR. n = 6 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. j, Representative images and quantification of demyelination. Scale bars = 100 μm. n = 12 biological replicates from 4 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. k, Representative images and quantification of spinal cord CD4+ T cell infiltration (CD4, red, Fluormyelin, cyan). Scale bars = 100 μm. n = 12 biological replicates from 4 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. l, Plin4 expression in astrocytes after transfection with LV::GfaABC1D-Cas9-sgScramble or sgPlin4. n = 4 (sgScramble) and 8 (sgPlin4) biological replicates per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. m, Representative image of Spyro Ruby-stained LD proteome. n, Representative western blot showing PLIN4 in lysates and purified LDs of isotype control, anti-CD40 antibody or oleic acid (OA) treated astrocytes. o, PCA of astrocyte LD proteome. p, Pearson correlation. (o-p) n = 3 mice per group. q, IPA pathway analysis of anti-CD40 treated astrocyte LD proteome. P-value was calculated by Qiagen IPA. r, Western blot analysis of ACLY expression in lysates and purified LDs of anti-CD40 treated astrocytes. s, ACLY-acetyl-CoA-NF-κB axis. Schematic in panel s created in BioRender; Lee, J.-H. https://biorender.com/e0ldkjq (2026). t-u, Representative histogram (t) and quantification (u) of p65 (K310)ac+ astrocyte population. n = 4 mice per group. Analyzed by Paired T test. v, Representative flow cytometry plots showing CD40+MHC-II+ astrocytes in sgScramble and sgPlin4 injected EAE CNS. w-x, Quantification of CD40+MHC-II+ astrocytes (w) and p65 (K310)ac+ astrocytes (x). n = 4 mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. y, Representative images and quantified number of SOX9+ astrocytes. Scale bars=100 μm. n = 4 mice. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m.

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Extended Data Fig. 10 CD40-LD-acetyl NF-κB axis in CNS APCs.

a, c, e, Representative histogram showing BODIPY 493/503 (a) or p65 (K310)ac (c, e) signal in biotin+/− APCs (a, c), or LD high and LD low APCs (e). b, d, f, Quantification of BODIPY 493/503 (b) or p65 (K310)ac (d, f) signal in biotin+/− APCs (b, d), or LD high and LD low APCs (f). n = 7 mice per group. Analyzed by Paired t test. g, Representative histogram showing PLIN4 signal in biotin+/− APCs. h-i, PLIN4 expression in biotin+/− CNS-resident APCs (h) or CNS-recruited myeloid APCs. j, BODIPY493/503 signal in PLIN4 high (top 20%) and PLIN4low (bottom 20%) CNS-resident APCs. (h-j) n = 6 mice per group. Analyzed by Paired t test. k-l, Fraction of BODIPY 493/503 (b) or p65 (K310)ac-positive APCs in isotype control or anti-CD40 treated primary APCs. n = 6 (DCs, OPCs and microglia) or 7 (macrophages) mice per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m. m, p65 (K310)ac-positive APCs in LD+ or LD- APCs. n = 6 (DCs, OPCs and microglia) or 7 (macrophages) mice per group. Analyzed by Paired t test. n, Plin4 expression in isotype control or anti-CD40 treated primary APCs. n = 10 (DCs), 7 (macrophages and microglia) and 6 (OPCs) biological replicates per group. Analyzed by Mann-Whitney test. Data shown as mean ± s.e.m.

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Lee, JH., Li, Z., Soto, J.S. et al. Antigen presentation by CD40+MHC-II+ astrocytes promotes CNS autoimmunity. Nature (2026). https://doi.org/10.1038/s41586-026-10860-6

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