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Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a

Nature Immunology volume 24, pages 110–122 (2023)Cite this article

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Abstract

Expressed on epidermal Langerhans cells, CD1a presents a range of self-lipid antigens found within the skin; however, the extent to which CD1a presents microbial ligands from bacteria colonizing the skin is unclear. Here we identified CD1a-dependent T cell responses to phosphatidylglycerol (PG), a ubiquitous bacterial membrane phospholipid, as well as to lysylPG, a modified PG, present in several Gram-positive bacteria and highly abundant in Staphylococcus aureus. The crystal structure of the CD1a–PG complex showed that the acyl chains were buried within the A′- and F′-pockets of CD1a, while the phosphoglycerol headgroup remained solvent exposed in the F′-portal and was available for T cell receptor contact. Using lysylPG and PG-loaded CD1a tetramers, we identified T cells in peripheral blood and in skin that respond to these lipids in a dose-dependent manner. Tetramer+CD4+ T cell lines secreted type 2 helper T cell cytokines in response to phosphatidylglycerols as well as to co-cultures of CD1a+ dendritic cells and Staphylococcus bacteria. The expansion in patients with atopic dermatitis of CD4+ CD1a–(lysyl)PG tetramer+ T cells suggests a response to lipids made by bacteria associated with atopic dermatitis and provides a link supporting involvement of PG-based lipid-activated T cells in atopic dermatitis pathogenesis.

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Fig. 1: CD1a tetramers identify populations of human T cells.
Fig. 2: CD1a-lysylPG tetramer+ T cells functionally respond to lysylPG antigen.
Fig. 3: Phospholipid reactivity of T cells binding lysylPG-treated CD1a tetramers.
Fig. 4: Cytokine gene expression of CD4+CD1a-(lysyl)PG tetramer+ T cells.
Fig. 5: Single-cell RNA-sequencing of CD1a–(lysyl)PG tetramer+CD4+ T cells.
Fig. 6: Response to staphylococcal bacteria and CD1a–(lysyl)PG tetramer+ T cells in atopic dermatitis.

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Data availability

The CD1a-PG final model and structure factor files were deposited in the PDB under ID 7SH4. Single-cell and bulk RNA-seq files are available in the Gene Expression Omnibus under accession no. GSE186459.Source data are provided with this paper.

Code availability

The Seurat object (RDS file format), intermediate output files and differential expression files (csv format) along with the code used in this manuscript for single-cell and bulk RNA-seq analysis is available in GitHub project repository (https://github.com/IshaMonga/CD1a-single-cell).

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Acknowledgements

We thank the NIH Tetramer Core Facility for CD1 proteins, P. Sims and M. Finlayson for their advice regarding the analysis of single-cell RNA-seq data, A. Peschel for providing the S. aureus strain, Q. Cremers for assistance with ChemDraw and I. van Rhijn for critical reading of the manuscript. The work is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01 AR074037, K01 AR068475 and P30 AR069632 to A.d.J. and R01 AR048632 to D.B.M.) and the Wellcome Trust Collaborative Award (to D.B.M., G.O. and J.R.) as well as an Irving Scholarship (to A.d.J.). J.R. is supported by a National Health and Medical Research Council Investigator award. G.O. receives funding from the Medical Research Council UK and National Institute for Health and Care Research Oxford Biomedical Research Centre. This work was supported by the NCI Cancer Center Support Grant (P30 CA013696), used the Genomics and High Throughput Screening Shared Resource and was also supported by the National Center for Advancing Translational Sciences (UL1 TR001873), as well as funding to Columbia University Irving Medical Center flow core facilities through S10RR027050 and S10OD020056. We thank the staff of the MX1 beamline of the Australian Synchrotron, part of Australian Nuclear Science and Technology.

Author information

Author notes

  1. These authors contributed equally: D. Branch Moody, Jamie Rossjohn, Annemieke de Jong.

Authors and Affiliations

  1. Department of Dermatology, Columbia University Irving Medical Center, New York, NY, USA

    Gwennaëlle C. Monnot, Brigitte N. Sallee, Reka Chakravarthy, Ioanna Maria Karantza, Alexandra E. Khaleel, Isha Monga, Laura N. Uwakwe, Soundos Youssef, Lindsey A. Bordone & Annemieke de Jong

  2. Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Marcin Wegrecki, Shin Yi Tin, Adam Shahine & Jamie Rossjohn

  3. Division of Rheumatology, Inflammation and Immunity, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Tan-Yun Cheng & D. Branch Moody

  4. Medical Research Council Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Yi-Ling Chen, Soo Weei Ng & Graham Ogg

  5. Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA

    Alice Tillman, Javier A. Garcia-Vilas, Anne-Catrin Uhlemann & Arnold Han

  6. Microbiome and Pathogen Genomics Core, Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA

    Alice Tillman & Anne-Catrin Uhlemann

  7. Department of Biostatistics, Columbia University Irving Medical Center, New York, NY, USA

    Bin Cheng

  8. Department of Surgery, Columbia University Irving Medical Center, New York, NY, USA

    Christine H. Rohde

  9. Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK

    Jamie Rossjohn

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  1. Gwennaëlle C. Monnot
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Contributions

G.C.M. and A.D.J. conceived the project. G.C.M., R.C., I.M.K. and A.E.K. performed T cell and tetramer assays. G.C.M. and I.M. performed analysis of RNA-seq data and data submission. M.W., S.Y.T. and A.S. performed structural analysis and SPR studies. T.-Y.C. performed lipid elutions and quantification by HPLC–MS and A.H. and J.A.G.V. performed single-cell sequencing. B.N.S., L.N.U., S.Y., C.H.R. and L.A.B. recruited and enrolled study participants and collected clinical specimens. A.T. and A.C.U. provided cultured S. aureus and S. epidermidis for lipid analysis. Y.L.C., S.W.N. and G.O. completed tetramer analyses of the second patient cohort. B.C. performed statistical analyses. A.d.J., J.R. and D.B.M. provided oversight for experiments and input for the study. A.d.J. and G.C.M. prepared the manuscript with input from all authors.

Corresponding author

Correspondence to Annemieke de Jong.

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Competing interests

A.d.J. and D.B.M. provide consulting to Pfizer. G.O. and Y.L.C. have relevant research collaborations with UCB and Janssen and a patent related to CD1a. The other authors declare no competing interests.

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Nature Immunology thanks Paolo Dellabona, Mikael Karlsson, Steven Porcelli and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1

(a) Flow cytometric analysis of CD1a-autoreactive DermT cell line stained with the indicated CD1a tetramers: unloaded (endo), PC, PG and lysylPG, co-stained for CD4 and gated on live cells. Indicated in the dotplots is the percentage of tetramer+ T cells in the tetramer gate. (b) Electron density maps of phosphatidylglycerol C18:1, C18:0 in the cleft of CD1a. Left: Unbiased (green, Fo-Fc at 2.5 σ. Right: Refined (blue, 2Fo-Fc at 0.9 σ) (c) Co-receptor expression determined on CD1a tetramer+ T cells measured in Fig. 1f. Indicated are the percentages of tetramer+ T cells expressing CD4 (red), CD8 (blue), neither (white) of both (grey) of the co-receptors. Right graph: Percentage of CD4 + T cells among tetramer+ cells. *p < 0.05 as determined by Wilcoxon matched-pairs signed-rank test (2-sided).

Source data

Extended Data Fig. 2

(a) Three T cells lines containing CD1a-lysylPG tetramer+ T cells were staining with lysylPG-treated CD1b, CD1c and CD1d tetramers co-stained with anti-CD4 and analyzed by flow cytometry. Plots are gated on live T cells. (b) Human CD1/β2m isoforms were expressed in Expi293F GnTI- cells and purified by nickel-affinity and size exclusion chromatography. CD1 proteins carrying endogenous lipids (CD1-endo) were incubated with lysylPG 16 hrs at 25 °C in 0.1 M MES pH 5.5, 150 mM NaCl, 0.05% CHAPS. 1 µg of CD1-endo and CD1-lysylPG samples were loaded on a 3–9 gel (Cytiva), and displacement of endogenous lipid and/or shifts towards more electropositive values indicate efficient loading of lysylPG. (c) GM-CSF concentration in supernatant of CD1a-lysylPG tetramer+ T cell line (921a) after 24 h of incubation with plate-bound CD1a and indicated concentrations of PC, PG and lysylPG. Indicated are mean ± SD of triplicate values. P values were based on paired two-tailed t-test. * p < 0.05 ** p < 0.01.

Source data

Extended Data Fig. 3

(a) LysylPG was treated at the indicated pH at 25 °C overnight (16 hr) or 37 °C for 1 hr, followed by lipid extraction and HPLC–MS analysis. The PG and lysylPG were quantified as percentage of the sum of both lipids, based on the curve fitting of external standards. (b) Isoelectric focusing gel showing the migration pattern of human CD1a carrying heterogenous mixture of lipids derived from the mammalian expression system (CD1a-endo) upon incubation with lysylPG or PG at low (citrate pH 5.5) or high (tris pH 8) pH. Presence of negatively charged lipids (for example PG) in the cleft of CD1a shifts the PI of the protein towards electronegative values (CD1a−1). Degradation of lysylPG to PG at high pH results in migration pattern identical to CD1a-PG. (c) CD1a proteins treated with lysylPG (pH 5.5 or pH 8) described in (b) were extracted and the eluted lipids, lysylPG and PG were analyzed by HPLC–MS. (d) Isoelectric focusing gel showing distinct migration pattern of CD1a incubated with CHAPS, PG or lysylPG based on the pI of the protein. LysylPG-loaded CD1a was further incubated overnight in MMT buffer at different pH to assess the stability of the lysylated headgroup. The lack of band corresponding to more negatively charged species of CD1a (CD1a-1) suggests that lysylPG is stable once bound to CD1a.

Source data

Extended Data Fig. 4

FACS sorting of CD4 + CD1a-PG tetramer+ T cells from donor 211 PBMC. Sorted T cells were expanded in vitro for 14 days after which the cells were tested for specificity using dual tetramer staining with CD1a-PG (APC-labeled) and CD1a-lysylPG (PE-labeled).

Extended Data Fig. 5

(a) S. aureus PGs were saponified and methylated to form FAMEs. The bacterial C15 fatty acid was determined as branched by co-elution of its FAME with iso-C15 FAME and anteiso-C15 FAME external standards, but not with straight chain n-C15 FAME. Data are presented as an overlay of mass chromatograms of m/z. 257.2476 [C15 FAME + H]+ by the positive mode reversed-phase HPLC-QTOF-MS analysis. (b) CID_MS spectra for PG and lysylPG supports assignments in Fig. 6b.

Extended Data Fig. 6

(a, b) Mass chromatogram of eight most abundant PG and lysylPG species from S. aureus and S. epidermidis are depicted. (c) The detected PGs and lysylPGs by the QToF mass spectrometer are summarized. The quantity (ng) of each lipid species in injected total lipid extracts (5 µg) was determined by curve fitting of the chromatogram area to the external standard curve. The measurements were performed in triplicate (mean ± SD).

Source data

Extended Data Fig. 7 Supernatants from the IL-13 Elispot assays of DC – S. aureus/ S. epidermidis co-cultures with CD1a-(lysyl)PG tetramer+ CD4 + T cells (Fig. 6c) were analyzed by LEGENDplex in duplicate.

This panel measured 12 different human T cell cytokines, 11 of which are depicted (IL-13 was measured using Elispot and could therefore not be reliably measured in the supernatant). Concentrations in the supernatant for indicated cytokines are depicted for each culture condition (mean ± SD).

Extended Data Fig. 8 Three T cell lines (834, 325, 921a) and one T cell clone (2114.1) were stained with mock-treated, S.aureus PG-treated and C18:1 PG-treated CD1a tetramers.

The lines were co-stained with CD4 antibody and DAPI, and live cells were analyzed for tetramer staining by flow cytometry.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Table 1 and Notes.

Reporting Summary

Peer Review File

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Source Data Extended Data Fig. 3

Unprocessed gels.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

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Monnot, G.C., Wegrecki, M., Cheng, TY. et al. Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a. Nat Immunol 24, 110–122 (2023). https://doi.org/10.1038/s41590-022-01375-z

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