Abstract
Tissue-selective chemoattractants direct lymphocytes to epithelial surfaces to establish local immune environments, regulate immune responses to food antigens and commensal organisms, and protect from pathogens. Homeostatic chemoattractants for small intestines, colon and skin are known1,2, but chemotropic mechanisms selective for respiratory tract and other non-intestinal mucosal tissues remain poorly understood. Here we leveraged diverse omics datasets to identify GPR25 as a lymphocyte receptor for CXCL17, a chemoattractant cytokine whose expression by epithelial cells of airways, upper gastrointestinal and squamous mucosae unifies the non-intestinal mucosal tissues and distinguishes them from intestinal mucosae. Single-cell transcriptomic analyses show that GPR25 is induced on innate lymphocytes before emigration to the periphery, and is imprinted in secondary lymphoid tissues on activated B and T cells responding to immune challenge. GPR25 characterizes B and T tissue resident memory cells and regulatory T lymphocytes in non-intestinal mucosal tissues and lungs in humans and mediates lymphocyte homing to barrier epithelia of the airways, oral cavity, stomach, and biliary and genitourinary tracts in mouse models. GPR25 is also expressed by T cells in cerebrospinal fluid and CXCL17 by neurons, suggesting a role in central nervous system (CNS) immune regulation. We reveal widespread imprinting of GPR25 on regulatory T cells, suggesting a mechanistic link to population genetics evidence that GPR25 is protective in autoimmunity3,4. Our results define a GPR25–CXCL17 chemoaffinity axis with the potential to integrate immunity and tolerance at non-intestinal mucosae and the CNS.
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Data availability
Raw and processed scRNA-seq data generated in this study are available from the NCBI Gene Expression Omnibus repository under the accession number GSE273397. Supplementary Table 1 lists all published external datasets used in this study. Integrated scRNA-seq datasets used for the analyses can be accessed at http://med.stanford.edu/butcherlab/data/GPR25.html. Source data are provided with this paper.
Code availability
Code for computational analyses is available upon request.
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Acknowledgements
This study was funded by NIH grants no. R01 AI178113 and no. R01 AI047822, MERIT award no. I01 BX-002919 from the United States Department of Veterans Affairs Biomedical Laboratory R&D Service (VA BLR&D), grant no. 1903-03787 from The Leona M. & Harry B. Helmsley Charitable Trust and the Regents of the University of California Tobacco Related Disease Research Program (TRDRP) grants no. T31IP1880 and no. T33IR6609 to E.C.B.; grants no. R21 AI149369 and no. R21 AI156662 to I.K.; grants no. R01 AI161880 and no. R01 GM136202 to I.K. and T.H.; and grant no. R01 MH125244 to S.M. B.A.Z. was supported by Merit Review Award Number I01 BX004115 from the VA BLR&D and by TRDRP grants no. T32IP5349 and no. T33IP6514. A.A. was supported by the California Institute for Regenerative Medicine (CIRM), award no. EDUC2-12677. F.M. was supported by the Department of Excellence 2023–2028 DNBM, the Cariverona Foundation–Research and Development Grant 2022 and the #NEXTGENERATIONEU and the Italian Ministry of University and Research, National Recovery and Resilience Plan (PNRR), project MNESYS (grant no. PE0000006). M.X. was supported by the TRDRP grant no. T31FT1867. Y.B. and B.O. were Research Fellow Awardees of the Crohn’s and Colitis Foundation of America (grants no. 835171 and no. 574148), and B.O. was a postdoctoral fellow of the Ramon Areces Foundation (Madrid, Spain). We thank Y. Yao for statistics, B. Xu for technical advice, G. Ramos for mouse colony maintenance and L. Magalhaes for administrative support. The schematic of GPR25 in Fig. 3a created with Biorender (https://BioRender.com).
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B.O., Y.B., A.A., M.K., M.H., J.P., M.X., K.B., M.L., N.L. and F.M. performed experiments. M.X. and K.B. performed scRNA-seq analysis. M.X., C.Z., J.R.D.D., I.K. and J.P. performed in silico analysis and protein modelling. S.T. performed RNAscope. S.M. and T.S. performed population genetics analyses. J.E.H. collected autopsy samples. M.H. and T.H. provided reagents. E.C.B., M.X., B.O. and Y.B. wrote, and T.H., B.A.Z. and K.B. edited, the manuscript. E.C.B. and J.P. conceived and E.C.B. supervised the study.
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Extended data figures and tables
Extended Data Fig. 1 Tissue- and subset-selective expression of GPR25 by lymphocytes.
a. Comparison of GPR25, GPR15 and CCR9 in the airway, colon, and SI, respectively, in total CD4 or CD8 T cells. Data from healthy adult and pediatric donors (n = 12–16). Boxplots of GPR25, GPR15 and CCR9 mean imputed expression per patient sample in total CD4 or CD8 T cells are shown, with each dot representing the mean value per sample. Hinges of box correspond to the first and third quartiles. Whisker extends from the corresponding hinge to the max/min value no further than 1.5x interquartile range from the hinge. Samples with fewer than 10 cells are not plotted. *: p-value < 0.05; ****: p-value < 0.0001, two-tailed T-test. b. Normalized transcript per million (TPM) of GPR25 from scRNAseq of all human cell types from the Human Protein Atlas. C. Mean TPM of GPR25 from bulk RNAseq of immune cell types sorted from PBMC of 4 healthy donors. Sample source information is provided in Supplementary Table 1.
Extended Data Fig. 2 GPR25 expression and association with neurodegeneration.
scRNAseq violin plots of imputed GPR25 expression in T cell subsets and myeloid cells in CSF samples from healthy donors and patients with mild cognitive impairment/Alzheimer’s Disease (MCI/AD). Data from all patients with more than 1000 cells are presented with means of individual donors (open circles) and mean values of the donor means (solid circles) ± SEM (n = 28). *: p-value < 0.05; **: p-value < 0.01; ****: p-value < 0.0001; n.s.: non-significant, multivariate regression. Trending differences between healthy and diseased samples are not statistically significant. Sample source information is provided in Supplementary Table 1.
Extended Data Fig. 3 Expression of GPR25 in T cells in the MLN, TLN and small intestines.
a. GPR25+ cells are enriched in mature Tregs during CD4 T cell differentiation in MLN and TLN. CD4 T cells aligned along a developmental path from CD4 naive cells illustrating sequential expression of CCR9 and GPR25 by T cells along a developmental (pseudotime) trajectory seeded from naive CD4 cells. Mature FOXP3-high IL2RA-hi Tregs (Treg hi) emerge late and are enriched in GPR25 + CCR9+ cells in MLN. Cells are pooled from 14 MLN and 9 TLN samples from healthy donors. b. GPR25 is expressed by subsets of Treg and TEM in the small intestines. Violin plots illustrating CCR9 and GPR15 expression by GPR25 + (GPR25 > 0.2) vs GPR25- (GPR25 < 0.2) T cells in SI, pooled from 12 healthy donors and presented with means of individual donors (open circles) and mean values of donor means (solid circles) ± SEM. All gene expression imputed. Sample source information is provided in Supplementary Table 1.
Extended Data Fig. 4 Predicted structure of the GPR25 complex with CXCL17.
a. The overall view of the complex. Receptor and the CXCL17 C-terminal helix are shown in white and black ribbons, respectively, and viewed along the plane of the membrane. b. The acidic C-terminus of CXCL17 is predicted to insert into the predominantly positively charged orthosteric binding pocket of GPR25. The receptor is viewed along the plane of the membrane as in (A) and is shown as a cut-away space-filling mesh colored by electrostatic potential (blue: positive, red: negative). The C-terminal part of CXCL17 is shown as black ribbon (backbone) and sticks (for the carboxyl group and residue side-chains only). c. The amino-acid residue environment in the receptor binding pocket is complementary to the molecular composition of the distal C-terminus of CXCL17, which ensures favorable hydrophobic packing against W952.60 and prominent hydrogen bonding interactions with the network of S1163.29, R1784.64, E19345.52, and R2646.55. Receptor is viewed across the plane of the membrane from the extracellular side and shown in white ribbon and sticks; the two C-terminal residues of CXCL17 are shown in black. Cyan dotted lines denote hydrogen bonds. The model was built using AlphaFold 2.3.2 Multimer89,90,91. Structure was refined and visualized in ICM 3.9-3b92.
Extended Data Fig. 5 CXCL17 is a chemoattractant ligand for GPR25 but not GPR15 or CMKLR1.
a. Human GPR15 transfectants migration to GPR15LG (250 nM) and CXCL17 (10–300 nM). b. Human CMKLR1 transfectants migration to chemerin and CXCL17 (10–300 nM). c. Checkerboard assay with human 4CysCXCL17 250 nM and human GPR25 transfectants. d. Pertussis toxin (100 ng/ml, 2 h pre-treatment before migration assay) inhibits CXCL17-induced chemotaxis on human GPR25 L1-2 transfectants. e. Intact mouse 6CysCXCL17 (3 nM - 1μM) is an active chemoattractant on human GPR25. f. Intact human 4CysCXCL17 is an active chemoattractant on mouse GPR25. g. mGPR25 transduced cells, but not the empty vector transduced counterparts robustly chemotax to mouse and human CXCL17 in in vitro transwell-based migration assays. Results with 3–9 replicates pooled from at least two independent experiments are shown as mean ± SEM. ****; P < 0.0001 vs no chemokine control in a two-tailed T-test.
Extended Data Fig. 6 Subset selective T cell chemotaxis to CXCL17.
a. Table showing % of migration to no chemokine in Fig. 4a and c. b-d. Tonsil cells were migrated in transwells to human 4CysCXCL17 or human GPR15LG for 3 hrs. Migrated and input cells were counted and phenotyped by flow cytometry. b-d. Naive (CD45RO− CD45RA +) or indicated effector/memory (CD45RO + CD45RA−) TCRαβ + CD4+ subsets were defined with MAbs to intracellular Foxp3 and CD25 (Tregs), and CD161, a marker of mucosal tissue homing T cells. Mucosal-associated invariant T cells are Vα7.2 +. NK cells were defined as CD14−, HLA/DR−, CD3−, CD19−, CD56 +, CD16−. NKT shared the same immunophenotyping but were gated as CD3 +. Conventional dendritic cells (DC) were defined as CD3−, CD19−, CD14−, HLA/DR + , CD11c +. Plasmacytoid dendritic cells (pDC) were defined as CD3−, CD19−, CD14−, HLA/DR +, CD123 +. Data are % of input cells migrated above mean “no chemokine/NC control” migration (which defines 0). e. Table showing % of migration to no chemokine in panels b-d. Results pooled from three independent experiments and shown as mean ± SEM of % of specific migration, except for hGPR15LG (two experiments). N ≥ 5. *; P < 0.05, **; p < 0.01, ***; p < 0.001, ****; p < 0.0001. One way ANOVA analysis with Dunnet post hoc test was performed to each cell subset comparing the indicated condition vs no chemokine control (NC).
Extended Data Fig. 7 CXCL17 expression in the human and mouse CNS.
a. UMAP of scRNAseq data of the human brain from Human Protein Atlas. Cells with CXCL17 expression are denoted in black. b. Violin plots illustrating CXCL17 expression by subsets in the hippocampus from healthy donors (n = 2). c. Violin plots of Cxcl17 expression by CNS cells from whole brains of mice at 4-week (n = 2) or 90-week (n = 2). d. Violin plots of Cxcl17 in mouse spinal cord subsets in injury models (n = 3). In B-D mean imputed expression values from individual donors (open circles) and mean values of the donor means (solid circles) are shown with SEM. Sample source information is provided in Supplementary Table 1.
Extended Data Fig. 8 CXCL17 expression in the airway and the gut.
a. Violin plots showing CXCL17 expression in airway epithelial populations of healthy donors and COVID-19 patients. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001, multivariate regression between healthy and severe COVID-19 samples. b. Violin plots illustrating low expression level of CXCL17 in the gut of healthy donors. Selective expression of GPR15LG in colon and CCL25 in SI are shown for comparison. Mean imputed expression values from individual donors (open circles) and mean values of the donor means (solid circles) are shown with SEM. Sample source information is provided in Supplementary Table 1.
Extended Data Fig. 9 CXCL17 immunohistology of the human cerebellum.
a. CXCL17 immunoreactivity highlights granule neurons (g). b. Reactivity of Purkinje (P) neurons and white matter (wm) surrounding a vessel (v). Methods: Sections of formalin fixed paraffin embedded normal human cerebellum were processed for antigen retrieval and staining with monoclonal mouse IgG anti-human CXCL17 (clone 422204, R&D) using the polymerized goat anti mouse IgG ImmPRESS (peroxidase) kit. DAB shown without counterstain. Isotype control (clone G3A1 mouse IgG1, Cell Signaling) is shown as inset in A. Results representative of 3 or more sections from 2 independent donors.
Supplementary information
Supplementary Fig. 1
Flow cytometry gating strategies for immune cell subsets analysed in this study.
Supplementary Table 1
Detailed sample information of all sequencing data used in this study.
Supplementary Table 2
Molecular weight and pI of human secreted proteins in Fig. 3c.
Supplementary Table 3
Mean BLAST bit-scores from pairwise alignments of the C-terminal six amino acids of each human protein in Fig. 3d and its orthologs in mouse, rat, rabbit, dog and cow.
Supplementary Table 4
Pearson correlation between GPR25 expression and genes in Fig. 3e across 49 non-lymphoid tissues.
Supplementary Table 5
Sequences of CXCL17 variants used in Fig. 3g–i.
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Ocón, B., Xiang, M., Bi, Y. et al. A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and CNS. Nature 635, 736–745 (2024). https://doi.org/10.1038/s41586-024-08043-2
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DOI: https://doi.org/10.1038/s41586-024-08043-2
