Lymphocyte trafficking receptor and ligand

Abstract

Compositions and methods are provided for modulating, targeting and tracking lymphocyte localization associated with the chemokine CXCL17 and its receptor, identified as GPR25. It is shown herein that GPR25 is a chemoattractant receptor that binds to its ligand chemokine CXCL17, which mediates lymphocyte localization to the airways and non-intestinal mucosal tissues.

Description

LYMPHOCYTE TRAFFICKING RECEPTOR AND LIGAND CROSS REFERENCE TO OTHER APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 524,152 filed June 29, 2023, and U.S. Provisional Application No. 63 / 565,806 filed March 15, 2024, the contents of which are hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract AI47822 awarded by the National Institutes of Health. The Government has certain rights in the invention. SEQUENCE LISTING

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “S22-499_STAN- 2065WO_Seqlist” created June 28, 2024 and having a size of 3,239 Bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. BACKGROUND

[0004] Lymphocyte trafficking is essential to the establishment, organization and function of the immune system. Innate lymphocytes, including γδT cells, are primed in the thymus to migrate to frontline barriers in mucosal tissues and skin. Naive conventional T and B cells are pre-programmed to home to secondary lymphoid organs where they are activated in response to antigens arriving through afferent lymphatics. During activation they are 'imprinted' by environmental factors (presented by stromal cells and migratory dendritic cells) to migrate to target tissues where they carry out effector, regulatory and surveillance functions.

[0005] Adhesion receptors for vascular addressins provide regional (e.g. GI tract vs skin) specificity, but constitutively expressed "homeostatic" chemoattractant cytokines ("chemokines") expressed by epithelial cells are pivotal in regulating lymphocyte homing and establishing distinct lymphocyte compartments in various barrier tissues. Secreted chemokines bind glycosaminoglycans and are presented by stromal elements and endothelial cells, providing “chemoaffinity” cues for tissue-specific endothelial recognition by blood-borne lymphocytes, and for haptotactic migration within tissues. Chemokine / receptor pairs have been identified that recruit lymphocytes to the small intestine (CCL25 / CCR9) and colon (GPR15LG / GPR15), where they establish specialized immunologic environments and mediate immune tolerance and inflammation. Lymphocyte trafficking also disseminates immune responses to distant sites: Intranasal vaccination provides protective immunity in the vagina and cervix, a process that involves migration of antigen-specific T cells via the blood to the genitourinary mucosa where subsets establish long term resident memory. Intestinal exposureto food antigens and intestinal flora induces regulatory T cells (Tregs) that provide systemic immune tolerance, preventing allergic reactions to pulmonary exposure and suppressing inflammatory responses to cross-reactive autoantigens in the CNS.

[0006] The mammalian lung features terminal alveoli, creating a vast surface area for air exchange but also for exposure to pathogens and allergens. The lung and respiratory tract are uniquely susceptible to airborne agents, but are also exposed to food antigens and oral microbiota inhaled during mouth breathing, as well as intestinal microbes delivered by hand- to-mouth or -nose transmission or contamination. The respiratory immune system must maintain a strict balance between tolerance and inflammation in the face of this broad challenge with antigens and organisms.

[0007] The mechanisms for dissemination of immune responses and cross-talk between organs remain unclear. Moreover, mechanisms of lymphocyte localization to some of the most clinically important organs in the body including the respiratory tract and genitourinary system have remained poorly understood. Compositions and methods to direct homing are of great interest. SUMMARY

[0008] Compositions and methods are provided for modulating, targeting and tracking lymphocyte trafficking and positioning, i.e. localization within the body, associated with expression of the chemokine CXCL17 and its receptor, identified herein as GPR25. It is shown that GPCR25 is a chemoattractant that binds to its ligand chemokine CXCL17. The interaction between CXCL17 and GPR25 mediates lymphocyte chemotaxis to tissues that express the ligand which include mucosal tissues, including the airways, upper GI tract, conjunctiva, exocrine glands, stomach, esophagus, pancreas, urinary tract, serous, mucous and sebaceous glands, prostate, oral and vaginal squamous mucosae and inflamed intestines, and th CNS. The chemokine CXCL17 / GPR25 axis provides an important target for therapeutic intervention in pathophysiological or inflammatory processes of the CNS, respiratory and non- intestinal mucosal tissues (NIMT) or intestines in inflammation

[0009] In some embodiments, methods are provided for manipulation of CXCL17 / GPR25 activity by targeting cells to these tissues expressing CXCL17 for suppression or moderation of pathologic inflammation in these tissues, without altering immunity systemically. Methods of targeting include, for example, modifying cells to express GPR25, providing a GPR25 targeting moiety on a cell surface protein of interest, etc. Cells of interest for targeting include, without limitation, lymphocytic immune cells, such as T cells, NK cells, Treg cells, CAR T cells, and the like. Cells may be targeted to CXC17 expressing tumors. Cells may be targeted to CNS, respiratory and non-intestinal mucosal tissues (NIMT).

[0010] In some embodiments, methods are provided for reducing trafficking of an immune cell of interest to CXCL17-expressing tissues, by administering an effective dose of a GPR25 / CXCL17 blocking agent. Blocking agents include, for example and without limitation, antibodies or derivatives thereof specific for CXCL17, antibodies or derivatives thereof specific for GPR25, soluble non-activating fragments of CXCL17, activating fragments of CXCL17 without gradient, soluble fragments of GPR25 (decoys), organic molecules that interact with the receptor and the like. Inhibitors can reduce immune responses by inhibiting trafficking of, for example, T helper cells, cytotoxic T cells, NK cells, and the like. Inhibitors can enhance immune responses by inhibiting trafficking of, for example, regulatory T cells (Treg). Targeted tissues include any tissue in which CXCL17 is expressed, for example CNS, airways, upper GI tract, conjunctiva, exocrine glands, stomach, esophagus, pancreas and urinary tract, etc. Conditions of interest include autoimmune disease, inflammatory bowel disease, allergic responses, fibrosis, hypertension, diabetes, MS, primary biliary cholangitis, celiac disease, arthritis, cancer, CNS disorders etc. For example, autoimmune diseases including MS, Crohn’s disease, ulcerative colitis, arthritis, type 1 diabetes, celiac disease, narcolepsy etc. have been linked GPR25 expression and / or SNPs. Administration can be topical, local, or systemic; inhaled as an aerosol or mist; in combination with another therapeutic, etc.

[0011] In some embodiments, methods are provided for modulating GPR25 signaling, by inhibiting or enhancing the interaction between CXCL17 and GPR25, by contacting: GPR25 with a CXCL17 antagonist; contacting CXCL17 with a blocking agent; and / or contacting a cell expressing GPR25 with a CXCL17 antagonist. The CXCL17 antagonist can be selected from: a) an antibody (or fragment thereof) which binds to GPR25; a CXCL17 variant that binds, but does not signal; a CXCL17 variant that binds and generates an inhibitory signal; or a small molecule compound. The blocking agent can be selected from: an antibody (or fragment thereof, including for example nanobodies) that binds to CXCL17; a fragment of GPR25, which can be a soluble portion of the receptor; and / or a small molecule compound, e.g. aptamers, etc.

[0012] GPR25 receptor is selectively expressed in immune cells, particularly lymphocytes e.g. T and B cells, and lymphocytes expressing the receptor can be associated with antigenic challenge in a target tissues, e.g. expressing CXCL17. Measurement of cells expressing GPR25 can be used to isolate and characterize lymphocytes engaged in immune responses in these sites, for diagnostic purposes, monitoring of responses, characterization of responding cells, and the like.

[0013] Methods are provided for screening candidate agents for activity in modulating lymphocyte trafficking to and positioning in tissues expressing CXCL17, the methods comprising determining the activity of a candidate agent in altering the interaction between CXCL17 and GPR25. Candidate agents include, for example, antibodies, polypeptides,aptamers, small molecules, etc., for example antibodies binding GPR25, including species variants or counterparts; polypeptide sequence variants of CXCL17, including species variants; non-peptide conjugation variants of CXCL17, e.g., glycosylation or other modifications; small molecule antagonist candidates; aptamer libraries, etc.

[0014] In some embodiments an immunogenic composition is provided, e.g. a vaccine composition, comprising a CXCL17 targeting moiety, e.g. a binding moiety of GPR25. In other embodiments an immunogenic composition may comprise an agent that inhibits GPR25 signaling. The composition may comprise protective antigens such as those in vaccines for SARS-CoV2, hepatitis viruses, human papilloma virus, DPT, influenza, etc. In other embodiments, a target antigen is a tumor associated antigen, e.g. a tumor antigen associated with cancers of the airways, and NIMT. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0016] FIG. 1. GPR25 is closely related in the ligand binding pocket to the colon homing receptor GPR15, and shares similarity to other GPCRs activated by other C-terminal peptides of attractants including chemerin receptors CMKLR1 and GPR1. COINpocket algorithm.

[0017] FIG.2. AlphaFold modeling predicts insertion of the conserved C-terminal peptide of CXCL17 into the signaling pocket of GPR25. (4CysCXCL17 modeled; full length CXCL17 model also shows FALPL insertion into the receptor pocket).

[0018] FIG. 3. CXCL17 selectively attracts Gpr25+ lung homing Vγ4+ γδ T cells. A. Gene expression showing selective Gpr25 expression by Vγ4+ γδ T cells, and Gpr15 by Vγ5+ skin / colon homing cells. Data from Immgen.org. B. Flow cytometry. Left: 2X2 plot of input thymocytes for chemotaxis assays. Right: GPR15 expression by Vγ5+ cells but not Vγ4+ cells. C. Flow cytometry of cells migrated to the indicated chemokine or control. Representative data from 2-3 transwells in each of 2 biologic replicates. Percent of Vγ4+ and Vγ5+ cells among gated γδ T cells is shown. D. Results of chemotaxis assay illustrating selective CXCL17 recruitment of Vγ4+ γδ T cells. Data presented as mean percent migration of input cells ± SEM of 2-3 replicate wells from one (Vγ5 cells) or two (Vγ4 cells) replicate experiments. Recombinant CXCL17 from R&D systems was used.

[0019] FIG. 4. CXCL17 triggers calcium signaling through GPR25. CHO cells were transfected with human GPR25 and Gα15, or only Gα15 (control). For experiments, the cellswere loaded with Fura Red in the presence of Pluronic F-127 and prepared for flow cytometry acquisition. After running each sample for 30 seconds, human CXCL17 (full length from R&D) was added at the indicated final concentration. The Fura Red BV650 / PE-Cy5.5 emission ratio was calculated over time during the acquisition. CXCL17 induced a sharp and concentration-dependent increase in signal reflecting GPR25 activation.

[0020] FIG. 5. Left: CXCL17 and GPR25 are detected in a common set of non-intestinal mucosal tissues and exocrine glands. Bulk RNAseq data from GTEx Portal (https: / / gtexportal.org) comparing GPR25 (top) and CXCL17 expression. Right: Gastric scRNAseq data shows that tissue GPR25 is limited to lymphocytes; while CXCL17 is expressed by epithelial cells. Clusters and scRNAseq data from the Human Protein Atlas. Note lack of expression by macrophages.

[0021] FIG.6. Airway lymphocytes. Violin plots showing T cells in airways express GPR25 but not genes for epidermotropic (CCR10), colon (GPR15) or SI (CCR9) chemoattractant receptors. Profiles in nasal cavity and lower airways (bronchi and trachea) are similar, suggesting that nasal cavity lymphocytes are representative of airway cells in general. scRNAseq data from Yoshida. Subsets modified and simplified from author annotation. Raw data processed through our computational pipeline. Results are pooled from 9 healthy children and adults with generally similar profiles.

[0022] FIG.7. Epithelial cells. LEFT panel: Constitutive and selective CXCL17 expression by airway epithelial cells. Violin plots of gene expression from the study in Fig 6. Expression was similar in trachea, bronchial and nasal cavity samples, and not altered in COVID patients. RIGHT panel: In contrast, colonic epithelium expresses GPR15L but not CXCL17. Data from the gut cell atlas. Data processed through our pipeline.

[0023] FIG.8. PBMC: GPR25 is expressed by a small fraction of circulating CD4 and CD8 Teff and Tregs, but is enhanced in dividing CD4 and CD8 T cell blasts (although GPR25 on blasts was variable, and blasts were not present in all patients). Cells with expression >0.4 (arbitrary units), a level comparable to that of GPR25 expression by T cells in airways (Fig 6) are highlighted in black. Monocytes and dendritic cells are negative. Pooled scRNAseq data from the same cohort as in Fig 6.

[0024] FIG.9. GPR25 regulation during T cell activation and differentiation in the tonsil: We sorted activated and differentiating CD4 T cells from a tonsil, mixed them with ~10% total CD4 cells, and submitted them for scRNA sequencing (10x, 5 prime). ~3000 single cells are displayed in UMAP dimensionality reduction of a tSpace trajectory matrix. LEFT panel: The graph illustrates 'pseudotime' trajectories from activated naive T cells (light grey) to Treg (FOXP3, IL2RA hi; IL7R low) and Th17 (IL17A hi, IL7R hi) effectors, and to T follicular helper cells (CXCR5 hi). RIGHT panel: GPR25 increases along Treg and Th17 branches, suggesting upregulation during Treg and Th17 differentiation. Cells with intermediate (blue) or high (red)GPR25 are colored. Methods: 50K cells were sorted as CD45RO / CD45RA double positive (transitional) CD4 cells; 20K HLA-DR+, CD38+ / low (activated) CD4 cells; and 10K total CD4 cells were combined for 10x analysis.

[0025] FIG. 10. Blood CD4 T cell migration to CXCL17 (transwell assays). Migration to GPR15L is shown for comparison. A. CD4 T effector cells (CD45RO+, CD127hi, CD4+) and Tregs (CD45RO+, CD127low, IL2RA+, CD4+) show dose-dependent migration to CXCL17, comparable to migration to GPR15L. B. TCR γδ T cells migrate more efficiently to CXCL17, but in this donor sample they failed to migrate to GPR15L. Data is presented as mean of 4-6 technical replicates from a single pilot study of cells from a healthy donor. P values by Student's T test, compared to no chemokine control.

[0026] FIG.11. Robust migration of blood borne antibody secreting cells to CXCL17. PBMC of a healthy donor were allowed to migrate to 300nM 4Cys or full length CXCL17, or CCL28 for comparison, in the bottom wells of transwell chambers. Input and migrated IgA (blue) or IgG (red) antibody secreting cells (ASC) were quantified by ELISPOT assay. Results are presented as % of input ASC migrated with SEM (N=3 technical replicates from one preliminary experiment). **P<0.01. *P<0.02 vs no chemokine control.

[0027] FIG.12. CXCL17 binding to human T cells.4CysCXCL17 was conjugated to AF488 and incubated with PBMC for one hour. Cells were counterstained with a panel of antibodies to define T cell subsets. Shown are plots of gated CD45RO+ memory CD4 T cells. LEFT: Distinct GPR15+ and CXCL17-binding subsets. MIDDLE: gated CXCL17+ T cells comprise memory / effector and (CD127lo CD25hi) Tregs. RIGHT: Total non-CXCL17 binding cells for comparison.

[0028] FIG.13. IgG1 Fc-4CysCXCL17 chimera binds GPR25. The Fc-CXCL17 was incubated for 30 min with GPR25+ CHO cells and bound chimera detected with a goat anti-human IgG Fc antibody. Controls include WT CHO cells, no Fc-chimera (second stage only), and blockade of Fc-CXCL17 binding by preincubation of CHO-GPR25 cells with a 10x molar excess of cold 4CysCXCL17.

[0029] FIGS.14A-14D. GPR25 is expressed by lymphocytes in lungs, non-intestinal mucosae and CNS. A. Expression of GPR25, GPR15 and CCR9 (black, labeled) and other class A GPCRs (gray) in total CD4 / CD8 T cells in airway vs colon (left), and in airway vs small intestine (SI) (right). B. Violin plots of GPR25, GPR15, and CCR9 in CD4 T cell subsets in indicated target tissues. Subsets with fewer than 10 cells were not plotted. C. Violin plots of GPR25 in B cell subsets in indicated target tissues. D. Violin plots of GPR25 in T innate subsets in indicated target tissues.

[0030] FIGS.15A-15G. Single-cell transcriptomes reveal GPR25 induction on innate T cells in the thymus and on regulatory and effector T and B cells during peripheral immune activation. A. GPR25 expression by maturing innate T cells in the thymus. UMAPs of postnatalthymocytes based on 5' scRNAseq colored by subset and gene expression. Violin plots show prominent expression by NKT cells and CD8aa(II) mucosal intraepithelial lymphocyte precursors. Cells with GPR25 expression less than 0.1 are colored light gray. B. tUMAPs of activated (CD38+ and / or HLA-DR+) tonsil CD4 T cells showing subsets and GPR25 expression on differentiating CD4 Teff and Treg cells. Cells with GPR25 expression less than 0.1 are colored light gray. C. tUMAPs of tonsil B cells showing subsets and GPR25 expression by plasma cells and FCRL4+ tissue homing B memory cells. B cells were enriched for activated (CD38+) cells by flow cytometry. Cells with GPR25 expression less than 0.05 are colored light gray. D-G. Expression of GPR25 and CCR9 by CD4 and CD8 T cells from lung- draining thoracic LN (TLN) and small intestine-draining mesenteric LN (MLN). D. UMAP illustrating GPR25 single positive (SP; GPR25+,CCR9-), CCR9 SP (GPR25-, CCR9+) and double positive (GPR25+, CCR9+) T cells. GPR25+ and CCR9+ are defined as GPR25 or CCR9 expression level greater than 0.1, respectively. E. Mean GPR25 expression and percent GPR25+ cells within CD4 and CD8 T cell subsets from 14 independent MLN donors and and 9 TLN donors. Values are mean ± SEM. F. Representation of subsets among MLN and TLN T cells expressing different levels of GPR25. G. Dot plots of GPR25 vs CCR9 expression by T subsets from independent MLN and TLN donors, illustrating coexpression of CCR9 and GPR25 by Tregs and Teff in the MLN. Ellipses represent 1 standard deviation around mean.

[0031] FIGS. 16A-16H. Bioinformatic discovery of a GPR25-CXCL17 lymphocyte chemoattractant axis. A. Schematic of GPR25 protein structure, highlighting a negatively charged N terminal extracellular peptide and a DRYLAVV motif. B. Similarity of ligand binding pocket of GPR25 to that of other Class A GPCRs. Receptors with closely related binding pockets activated by C-terminal peptides of ligands are labeled. C. Isoelectric point (pI) and molecular weight (MW) of human secreted proteins. Chemokines 21 and targets of interest (colored) are highlighted. Square indicates a gate for pI > 8 and MW < 25kD. D. C-terminal conservation of proteins gated in C. Upper panel: mean BLAST bit-scores calculated from pairwise alignment of C-terminal 6 amino acids of the human protein and orthologs in mouse, rat, rabbit, dog, and cow. Square indicates a gate with BLAST score > 4. Lower panel: C- terminal amino acid sequence of CXCL17 in the indicated species. Red indicates mismatch with the human protein sequence. E. Correlation of GPR25 and CXCL17 gene expression across 49 non-lymphoid tissues. Upper panel: Pearson 7 correlation of cross-tissue gene expression profiles between secreted proteins gated in D and GPR25. Lower panel: scaled log-transformed gene expression profiles of GPR25 and CXCL17 across tissues. F. AlphaFold model of 4Cys CXCL1722 interaction with GPR25 (blue), illustrating insertion of C-terminal peptide of CXCL17. G. CXCL17 activation of RhoA signaling pathway through GPR25. Serum response factor response element (SRFRE)- driven luciferase reporter activity of GPR25 transfected L1.2 cells incubated with the indicated amounts of human 4-Cys CXCL17, mouseCxcl1726aa C-terminus, human 4-Cys CXCL17 C-terminal-truncated and human GPR15LG. Mean ± SEM, n = 3 for each condition and representative of three independent experiments. H. Transwell chemotaxis of GPR25 transfected L1.2 cells to indicated chemoattractants or ligands illustrating selective migration to CXCL17 variants with intact C terminus. NC: no chemokine control.

[0032] FIGS. 17A-17C. Subset- and tissue-selective epithelial and neuronal expression of CXCL17. A. Pseudo-bulk expression of CXCL17 by indicated epithelial cell subsets and tissues at log scale (upper) and linear scale (lower). Data represent normalized gene expression by distinct subsets (clusters) of the indicated cell types and are shown with means. B. Violin plots illustrating expression of CXCL17, GPR15LG and CCL25 by airway epithelial cells in healthy donors. Subsets with fewer than 10 cells were not plotted. N, nasal cavity; T, trachea; B, bronchus. C. Histologic localization of CXCL17 gene expression: RNAscope was performed using probes for CXCL17 (blue) and GPR15LG (red) in sections of the indicated tissues. Asterisk adjacent to CXCL17+ alveolar epithelial cells. Arrows highlight CXCL17+ conjunctival epithelium: epithelium overlying the cornea is negative. Neurons are cerebellar granule neurons.

[0033] FIGS. 18A-18H. CXCL17 and GPR25 in T cell localization to airways, upper GI, biliary and GU tracts. A. Ratio of GPR25-transduced (GPR25) and control vector-transduced (control) donor cells within tissues 1 week or 7 weeks after i.v. injection into Rag1- / - mice. GPR25 vs control cells were distinguished by CD45.1 vs CD45.2 allotype, switched in different experiments and analyzed by flow cytometry. Normalized to input ratio. Results pooled from 3 independent experiments with 2-3 mice per experiment and shown as mean ± SEM. *; P<0.05, **; p<0.01, ***; p<0.001. Two tailed t-test. B. Ratio of GPR25 to control donor cells, distinguished by fluorescent labels, 10-12h after i.v. transfer. Cells within vessels (intravascular) or extravasated within mucosal sites determined by confocal microscopy of whole mount tissues or frozen sections. Ratios in control spleen determined by flow cytometry. Fisher's exact test comparing cell counts in indicated target tissues vs spleen in WT recipients (+); or comparing counts in target tissues in CXCL17- / - vs WT recipients (*). (+) P values < 0.001 vs spleen for in-vessel and extravasated cell counts in trachea, uterus, stomach and tongue of wild type recipients; <0.001 for extravasated counts in gallbladder. Not significant for SI, colon and cells in HEV of LN; or for in vessel cells in gallbladder (only 11 cells total). (*) P<0.05 vs WT for in- vessel and extravasated cell counts in trachea, tongue, uterus, and stomach; P<0.05 for extravasated cells in gallbladder. Cell counts pooled from 1 (gallbladder) or 2-4 independent experiments with one mouse per condition and experiment. Mean ratios shown. C-E. Representative microscopic images of PLN (C), trachea (D), a cross-section of the lung (E) taken 10 hours after injection of an ~1:1 ratio of GPR25 (green) and control (red) CD4 T cells. Arrowheads in E point to dispersed donor GPR25 cells localized to bronchus (Br). Asterisksindicate cells near veins (V). F. Ratio of GPR25 to control donor cells within indicated pulmonary microenvironments: Bronchi: within 25-30 µm of bronchial basement membrane. Vein: within 25-30 µm of or in contact with venous endothelium. Alveoli: within alveolar spaces not adjacent to veins or bronchi. Each dot is the ratio within 2-4 independent 10x fields for WT and Cxcl17- / - representing ~ 4 mm2. Spleen ratios duplicated from B for comparison. Results show 3 independent experiments with 1-2 mice per experiment and shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001 vs spleen; WT vs Cxcl17- / - . Two tailed t-test. G-H. Immunofluorescence visualization and quantification of intraepithelial CD8 and CD4 T cells in lung bronchioles and stomach of WT vs Cxcl17- / - mice. Representative high power images of lung bronchiole and stomach cross-sections showing intraepithelial CD8αβ T cells in WT vs Cxcl17- / -, are show on the right side of the figure. Asterisks indicate CD103+ CD8αβ T cells and arrows indicate CD103- CD8αβ T cells in stomach epithelium. Data from 4-5 sections from each of 2 WT and 2 Cxcl17- / - mice, mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, P < 0.0001. One tailed t test.

[0034] FIGS. 19A-19G. GPR25 mediates T cell homing to airways, upper GI tract and GU tract. Analysis and visualization of GPR25-transduced and control-transduced donor cells within tissues 7 days after i.v. injection into Rag1- / - mice (A), or 10-12 hours after transfer into WT C57BL6 / N or Cxcl17- / - mice A. Ratios of donor GPR25 vs control cells distinguished by CD45.1 vs CD45.2 allotype, switched in different experiments and analyzed by flow cytometry. Normalized to input ratio. Results pooled from 3 independent experiments with 2-3 mice per experiment and shown as mean ± SEM. *; P<0.05, **; p<0.01, ***; p<0.001. Two tailed t-test. B. Ratio of GPR25 to control donor cells, distinguished by fluorescent labels, 10-12h after i.v. transfer. Cells within vessels (intravascular) or extravasated within mucosal sites determined by confocal microscopy of whole mount tissues or frozen sections. Ratios in control spleen determined by flow cytometry. P values < 0.001 vs spleen for in-vessel and extravasated cell counts in trachea, uterus, stomach and tongue of wild type recipients; <0.001 for extravasated counts in gallbladder. Not significant for SI, colon and cells in HEV of LN; or for in vessel cells in gallbladder (only 11 cells total). Results are based on Fisher exact test comparing cell counts vs spleen for each site pooled from 2-4 independent experiments with one mouse per condition and experiment. Comparing cell counts between WT and Cxcl17- / - for each site, P<0.05 for invessel and extravasated cell counts in trachea, uterus, and stomach, Fisher exact test on counts pooled from 2-3 independent experiments with one mouse per condition and experiment. Mean ratios shown. C-F. Representative microscopic images of trachea (C), stomach (D), PLN (E) and a cross-section of the lung (F) taken 10 hours after injection of an ~1:1 ratio of GPR25-transduced (green) and control vector-transduced (red) CD4 T cells. Arrowheads in F point to dispersed donor GPR25 transduced cells localized to bronchus (Br).Asterisks indicate cells near veins (V). (G) Ratio of GPR25-transduced to control-transduced donor cells within indicated pulmonary microenvironments: Bronchi: within 25-30 μm of bronchial basement membrane. Vein: within 25-30 μm of or in contact with venous endothelium. Alveoli: within alveolar spaces not adjacent to veins or bronchi. Each dot is the ratio within 2-3 independent 10x fields for WT and 1 representative 10x field for Cxcl17- / - representing ~ 4 square mm. Spleen ratios duplicated from B for comparison. N=3 *, P < 0.05; **, P < 0.01; ***, P <0.001; ****, p < 0.0001. Two tailed t-Test.

[0035] FIG. 20 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. Boxplots of GPR25, GPR15 and CCR9 mean expression per patient sample in total CD4 or CD8 T cells are shown, with each dot representing the mean value per sample. Samples with fewer than 10 cells are not plotted. *: p-value < 0.05; ****: p-value < 0.0001, 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.

[0036] FIG.21. GPR25 expression and association with neurodegeneration. scRNAseq violin plots of 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. *: 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.

[0037] FIG. 22. 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 22 healthy donors and presented with means of individual donors (open circles) and mean values of donor means (solid circles) ± SEM.

[0038] FIG.23. 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-terminusof 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 W952.60and 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.

[0039] FIG.24. CXCL17 is a chemoattractant ligand for GPR25 but not GPR15 or CMKLR1. A. Human GPR15 transfectants migration to GPR15LG (250nM) and CXCL17 (250nM). B. Human CMKLR1 transfectants migration to chemerin and CXCL17 (250nM). C. Checkerboard assay with human 4CysCXCL17250nM and human GPR25 transfectants. D. Pertussis toxin (100 ng / ml, 2 hours pre-treatment before migration assay) inhibits CXCL17-induced chemotaxis on human GPR25 L1-2 transfectants. E. Mouse 6CysCXCL17 (300nM) 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 from at least two independent experiments are shown as mean + / - SEM. ****; P<0.0001 vs no chemokine control in a two-tailed T-test.

[0040] FIG. 25. Subset selective T cell chemotaxis to CXCL17. A. Table showing % of migration to no chemokine in figure 3J and L. B-D. Tonsil cells were migrated in transwells to human 4CysCXCL17 or human GPR15L 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-) TCR⍺β+ 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 are pooled from three independent experiments and shown as mean + / - SEM of % of specific migration. *; P<0.05, **; p<0.01, ***; p<0.001, ****; p<0.0001. One way ANOVA analysis with Dunnetpost hoc test was performed to each cell subset comparing the indicated condition vs no chemokine control (NC).

[0041] FIG.26. 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. C. Violin plots of Cxcl17 expression by CNS cells from whole brains of mice at 4-week or 90-week. D. Violin plots of Cxcl17 in mouse spinal cord subsets in injury models. In B-D mean expression values from individual donors (open circles) and mean values of the donor means (solid circles) are shown with SEM.

[0042] FIG.27. 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 expression values from individual donors (open circles) and mean values of the donor means (solid circles) are shown with SEM.

[0043] FIG. 28. 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. DETAILED DESCRIPTION

[0044] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is alsoencompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0047] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0048] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0049] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0050] Compounds can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail thesynthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif.1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0051] "Comparable cell" shall mean a cell whose type is identical to that of another cell to which it is compared. Examples of comparable cells are cells from the same cell line.

[0052] "Inhibiting" the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.

[0053] "Treating" a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent.

[0054] "Inhibiting" the expression of a gene in a cell shall mean either lessening the degree to which the gene is expressed, or preventing such expression entirely. "Specifically inhibit" the expression of a protein shall mean to inhibit that protein's expression (a) more than the expression of any other protein, or (b) more than the expression of all but 10 or fewer other proteins.

[0055] "Antibody" shall include, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, this term includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, this term includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.

[0056] "Anti-sense nucleic acid" shall mean any nucleic acid which, when introduced into a cell, specifically hybridizes to at least a portion of an mRNA in the cell encoding a protein ("target protein") whose expression is to be inhibited, and thereby inhibits the target protein's expression.

[0057] "Specifically hybridize" to a nucleic acid shall mean, with respect to a first nucleic acid, that the first nucleic acid hybridizes to a second nucleic acid with greater affinity than to any other nucleic acid.

[0058] "Subject" or "patient" shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.

[0059] "Suitable conditions" shall have a meaning dependent on the context in which this term is used. That is, when used in connection with an antibody, the term shall mean conditions that permit an antibody to bind to its corresponding antigen. When this term is used in connection with nucleic acid hybridization, the term shall mean conditions that permit a nucleic acid of at least 15 nucleotides in length to hybridize to a nucleic acid having a sequence complementary thereto. When used in connection with contacting an agent to a cell, this term shall mean conditions that permit an agent capable of doing so to enter a cell and perform its intended function. In one embodiment, the term "suitable conditions" as used herein means physiological conditions.

[0060] Determining a therapeutically or prophylactically effective amount of an agent, e.g. an antibody, can be done based on animal data using routine computational methods. In one embodiment, the therapeutically or prophylactically effective amount contains between about 0.01 mg and about 1 g of inhibitor / kg, e.g. nucleic acid, protein or peptide, etc., as applicable. In another embodiment, the effective amount contains between about 1 mg / kg and about 100 mg / kg of agent, as applicable. In a further embodiment, the effective amount contains between about 10 mg / kg and about 50 mg / kg of the agent, as applicable.

[0061] Administering the instant compositions can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, intramuscularly, intrathecally, and subcutaneously. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.

[0062] Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone. Nucleic acids of the invention can also be administered attached to particles using a gene gun.

[0063] Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate andcellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).

[0064] Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).

[0065] Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

[0066] Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, xanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

[0067] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In an embodiment, the mammal is a human. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc.

[0068] The definition of an appropriate patient sample encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s sample cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s sample cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / orpolypeptides); and a sample comprising sample cells from a patient. A biological sample comprising a sample cell from a patient can also include normal, non-diseased cells.

[0069] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease.

[0070] Treating may refer to any indicia of success in the treatment or amelioration or prevention of disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with the disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0071] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic (i.e., first therapeutic agent) and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0072] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of the therapeutic agent and inhibitor agent at such time that both the known therapeutic agent and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0073] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increasesas well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0074] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0075] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0076] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0077] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0078] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)

[0079] Lymphocyte homing, or trafficking, refers to the process whereby phagocytic cells and lymphocytes adhere to and migrate across vascular endothelium into an organ or site of inflammation. The exquisite specificity of such lymphocyte trafficking is determined by tissue- specific guidance signals expressed by the vascular endothelial cells, combined with counter receptors expressed by circulating lymphocytes. For example, high endothelial venules can selectively recruit naive lymphocytes into the lymph nodes and Peyer's patches by expressing a unique combination of vascular addressins and chemoattractants. The inflamed postcapillary venules in extralymphoid tissues also use a distinct array of endothelial adhesion molecules and tissue selective chemokines to support the recruitment of effector and memory lymphocytes that express appropriate trafficking receptors. Exit of lymphocytes from lymphoid and extralymphoid tissues into circulation is actively regulated by signals through specific receptors for sphingosine-1-phosphate and a certain chemokine(s), respectively.

[0080] Chemokines are defined by their primary amino acid sequence and the arrangement of specific structurally important cysteine residues within the mature protein. These form disulfide bonds that maintain the structure of the chemokine monomer, which consists of a central three stranded β‐sheet, an overlying C‐terminal α‐helix, and a short unstructured N terminus that plays a critical role in receptor activation. Variation in the precise configuration of the two cysteines closest to the N terminus allows chemokines to be split into four subfamilies: CC, CXC, CX3C, and XC. In CC chemokines, these cysteines are directly juxtaposed, while CXC chemokines have a single variable amino acid between them. The sole CX3C chemokine has three amino acids between these two cysteines, while XC chemokines, of which there are two forms in humans and one in mice, lack the first and the third cysteines of the motif.

[0081] While chemokines are active as monomers, they also form homodimers, heterodimers, and higher order aggregates that can contain one or more chemokine species, and this can be influenced by interactions with GAGs. The full chemokine ‘interactome’ reveals complex and extensive interactions. Chemokines are also profoundly affected by post‐translational modifications such as citrullination, nitration / nitrosylation, and cleavage by matrix metalloproteinases (MMPs), cathepsins, thrombin, plasmin, elastase, the dipeptidyl peptidase CD26, and other proteases. These changes can substantially modify chemokine activity.

[0082] Proteases are key chemokine regulators. CD26‐mediated trimming of two amino acids off a chemokine's N terminus can, depending on the chemokine, change receptor specificity, substantially alter receptor affinity or convert agonists into antagonists. Many MMPs can also modify this part of certain chemokines, and the impact of these modifications depends on the identity of the chemokine being studied. Proteases are also thought to help create and modifyinterstitial chemokine gradients, and have the capacity to degrade chemokines, or the ECM to which they are bound, so are important in regulating chemokine half‐life and distribution.

[0083] The chemokine CXCL17 is found in human (NP_940879.1), provided herein as SEQ ID NO:2, Mouse (NCBI gene ID: 284340) (NP_705804.2), Chimpanzee (XP_001154726.1), etc. Natural sequences may in many cases be substituted by variants thereof, including in certain embodiments at least about 80% identity, about 85%, or about 90% identity or more, including at least about 95% or 100% identity. For example a segment of comparison may be about 95% of the amino acids in length, or about 90%, 85%, or 80% of the amino acids of the length for comparison. The length of comparison may be at least about 20, 30, 40, 50, 55, 60, 65, 70, or 75 amino acids. The variants may conserve particular physicochemical or functional features as the prevailing natural sequence, while other variants may have modified combinations of structural and functional features. In some embodiments, the variants do not include sequences identical to naturally occurring human sequences, or naturally occurring sequences of other animals. Truncated versions, or fusions with other segments are provided, which exhibit a function as described. Embodiments of the present invention allow for evaluating function corresponding to structural changes. Monoclonal antibodies specific for CXCL17 are known and commercially available, for example MA5-24157 (Thermo Fisher); MAB4207 and MAB4270 (R&D Systems); ABIN7434647 (Antibodies online); 18108-1-AP (Proteintech); LS-C125470 (LS Bio); etc.

[0084] GPR25 is a previously orphan receptor, the human sequence may be accessed at NP_005289.2, provided herein as SEQ ID NO:1. Natural sequences may in many cases be substituted by variants thereof, including in certain embodiments at least about 80% identity, about 85%, or about 90% identity or more, including at least about 95% or 100% identity. For example a segment of comparison may be about 95% of the amino acids in length, or about 90%, 85%, or 80% of the amino acids of the length for comparison. The length of comparison may be at least about 20, 30, 40, 50, 55, 60, 65, 70, or 75 amino acids. The variants may conserve particular physicochemical or functional features as the prevailing natural sequence, while other variants may have modified combinations of structural and functional features. In some embodiments, the variants do not include sequences identical to naturally occurring human sequences, or naturally occurring sequences of other animals. Truncated versions, or fusions with other segments are provided, which exhibit a function as described. Embodiments of the present invention allow for evaluating function corresponding to structural changes. In some embodiments a soluble variant of the receptor is provided, in which the transmembrane and instracellular domains are deleted, for use as a decoy, etc.

[0085] Embodiments of the invention describe the identification of GPR25 as the receptor for CXCL17 chemokine. CXCL17 is expressed in the CNS, airways and NIMT sites where itrecruits cells of the immune system expressing GPR25, including B and T lymphocytes to maintain homeostasis and to regulate inflammatory responses in these tissues, among other functions. There are a number of inflammatory conditions in these tissues that cause human disease, and regulating the CXCL17 / GPR25 axis is therefore important for therapeutic purposes. Gene Expression

[0086] A GPR25 or CXCL17 protein may be produced recombinantly directly, or as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression the native signal sequence may be used, or other mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.

[0087] Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to a protein coding sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.

[0088] Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.

[0089] Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linkedto DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0090] Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence but is preferably located at a site 5' from the promoter.

[0091] To achieve expression of the recombinant protein, a nucleic acid encoding the protein (and / or CAR) is inserted into a replicable vector for expression. Many such vectors are available. Expression vectors for expression of the protein and optionally CAR in the T-cell may be viral vectors or non-viral vectors. Plasmids are examples of non-viral vectors. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non- viral vector may under conditions that facilitate uptake of the non-viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo- Fisher Scientific), high salt, and magnetic fields (electroporation). As an alternative to introducing an expression vector, the genome of the targeted cell may be modified, e.g. by CRISPR / Cas9 techniques, to upregulate expression.

[0092] In one embodiment, a non-viral vector may be provided in a non-viral delivery system. Non-viral delivery systems are typically complexes to facilitate transduction of the target cell with a nucleic acid cargo wherein the nucleic acid is complexed with agents such as cationic lipids (DOTAP, DOTMA), surfactants, biologicals (gelatin, chitosan), metals (gold, magnetic iron) and synthetic polymers (PLG, PEI, PAMAM). Numerous embodiments of non-viral delivery systems are well known in the art including lipidic vector systems (Lee et al. (1997) Crit Rev Ther Drug Carrier Syst.14:173-206); polymer coated liposomes (Marin et al., U.S. Pat. No.5,213,804, issued May 25, 1993; Woodle, et al., U.S. Pat. No.5,013,556, issued May7, 1991); cationic liposomes (Epand et al., U.S. Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A., U.S. Pat. No. 5,578,475, issued Nov. 26, 1996; Rose et al, U.S. Pat. No. 5,279,833, issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No. 5,334,761, issued Aug. 2, 1994).

[0093] In another embodiment, the expression vector may be a viral vector. When a viral vector system is to be employed for CAR and expression of the receptor , retroviral or lentiviral expression vectors are preferred. In particular, the viral vector is a gamma retrovirus (. (Pule, et al. (2008) Nature Medicine 14(11):1264-1270), self-inactivating lentiviral vectors ( June, et al. (2009) Nat Rev Immunol 9(10):704-716) and retroviral vectors as described in Naldini, et al. (1996) Science 272: 263-267; Naldini, et al. (1996) Proc. Natl. Acad. Sci. USA Vol.93, pp. 11382-11388; Dull, et al. (1998) J. Virology 72(11):8463–8471; Milone, et al. (2009) 17(8):1453-1464; Kingsman, et al. United States patent No 6,096,538 issued August 1, 2000 and Kingsman, et al. United States patent No. 6,924,123 issued August 2, 2005. In one embodiment of the invention, the CAR expression vector is a Lentivector® lentiviral vector available from Oxford Biomedica.

[0094] Transduction of immune cells with an expression vector may be accomplished using techniques well known in the art including but not limited co-incubation with host cells with viral vectors, electroporation, and / or chemically enhanced delivery. Immune Cells for Engineering

[0095] Engineered T cells include, for example, T-cells which may have been recombinantly modified by transduction with an expression vector encoding a CAR in substantial accordance with the teaching above. Engineered immune cells also include cells in which a genetic construct encoding GPR25 has been introduced. Engineered cells include Treg cells, which may be modified by introduction of a genetic construct encoding GPR25, alone or in combination with a chimeric antigen receptor.

[0096] T-cells contemplated herein include naïve T-cells, central memory T-cells, effector memory T-cells, effector T cells, Treg cells, or combination thereof. T cells for engineering as described above are collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads,affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0097] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS). The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0098] In some embodiments, the engineered cells comprise a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol.42(4):626-39 “Adoptive Cell Therapy- Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01174121, “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641-645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0099] In some embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor and a cellular product suitable for all patients independent of HLA type provides an alternative. A universal ‘off the shelf’ CAR T cell product provides advantages in uniformity of harvest and manufacture. Antigen-Binding Proteins

[0100] Antigen binding region (ABR). As used herein, the term ABR refers to a combination of variable heavy (VH and variable light (VL) polypeptides to associate to form a variable region domain. An ABR of interest may be specific for CXCL17, or for GPR25. Antibodies of interest may be agonist antibodies; antagonist antibodies; or antibodies that bind to CXCL17 or GPR25 at a site that does not affect binding of the two proteins to each other, or their physiologic activity.

[0101] An ABR is the minimum antibody fragment that contains a complete antigen- recognition and binding site. This region consists of heavy- and one light-chain variable domain in tight, non-covalent association, as a single polypeptide or as a dimer. It is in this configuration that the three CDRS of each variable domain interact to define an antigen- binding site on the surface of the domain. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0102] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0103] An antibody or ABR “which binds” an antigen of interest, is one that binds the antigen with sufficient affinity such that the antibody or binding molecule is useful as a diagnostic and / or therapeutic agent in targeting the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or other binding molecule to a non-targeted antigen will usually be no more than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0104] An ABR may also be formulated as a single chain antibody. An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061. The generation of ScFvs based on monoclonal antibody sequences is well known in the art. See, e.g. The Protein Protocols Handbook, John M. Walker, Ed. (2002) Humana Press Section 150 “Bacterial Expression, Purification and Characterization of Single-Chain Antibodies” Kipriyanov, S. Antibodies used in the preparation of scFvs may be optimized to select for those molecules which possess particular desirable characteristics (e.g. enhanced affinity) through techniques well known in the art such as phage display and directed evolution

[0105] In another embodiment, the ABR is a single domain antibody obtained through immunization of a camel or llama with a target cell derived antigen, in particular a tumor antigen. See, e.g. Muyldermans, S. (2001) Reviews in Molecular Biotechnology 74: 277-302. Alternatively, the ABD may be generated wholly synthetically through the generation of peptide libraries and isolating compounds having the desired target cell antigen binding properties in substantial accordance with the teachings or Wigler, et al. United States Patent No.6303313 B1 issued November 12, 1999; Knappik, et al., United States Patent No 6,696,248 B1 issued February 24, 2004, Binz, et al. (2005) Nature Biotechnology 23:1257-1268, and Bradbury, et al. (2011) Nature Biotechnology 29:245-254. Multi-Target ABDs

[0106] The ABD may have affinity for more than one target antigen. For example, an ABD of the present invention may comprise chimeric bispecific binding members, i.e. have capable of providing for specific binding to a first target cell expressed antigen and a second target cell expressed antigen. Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann (2012) MAbs.4(2): 182–197; Stamova et al. (2012) Antibodies, 1(2), 172-198; Farhadfar et al. (2016) Leuk Res.49:13-21; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1):178-92; Fan et al. (2015) J Hematol Oncol.8:130; May et al. (2016) Am J Health Syst Pharm.73(1):e6-e13. In some embodiments, the chimeric bispecific binding member is a bivalent single chain polypeptides. See, e.g. Thirion, et al. (1996) European J. of Cancer Prevention 5(6):507-511; DeKruif and Logenberg (1996) J. Biol. Chem 271(13)7630-7634; and Kay, et al. United States Patent Application Publication Number2015 / 0315566 published November 5, 2015. In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3. In some instances, a chimeric bispecific binding member may be a CAR T cell adapter. As used herein, by “CAR T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR T cell adapter will have to binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR T cell adapters include but are not limited to e.g., those described in Kim et al. (2015) J Am Chem Soc.137(8):2832-5; Ma et al. (2016) Proc Natl Acad Sci U S A.113(4):E450-8 and Cao et al. (2016) Angew Chem Int Ed Engl.55(26):7520-4.

[0107] Antibodies, also referred to as immunoglobulins, conventionally comprise at least one heavy chain and one light, where the amino terminal domain of the heavy and light chains is variable in sequence, hence is commonly referred to as a variable region domain, or a variable heavy (VH) or variable light (VH) domain. The two domains conventionally associate to form a specific binding region, although as well be discussed here, a variety of non-natural configurations of antibodies are known and used in the art.

[0108] A “functional” or “biologically active” antibody or antigen-binding molecule is one capable of exerting one or more of its natural activities in structural, regulatory, biochemical or biophysical events. For example, a functional antibody or other binding molecule may have the ability to specifically bind an antigen and the binding may in turn elicit or alter a cellular or molecular event such as signaling transduction or enzymatic activity. A functional antibody or other binding molecule may also block ligand activation of a receptor or act as an agonist or antagonist. The capability of an antibody or other binding molecule to exert one or more of its natural activities depends on several factors, including proper folding and assembly of the polypeptide chains.

[0109] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, three chain antibodies, single chain Fv, nanobodies, etc., and also include antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species.

[0110] The term antibody may reference a full-length heavy chain, a full length light chain, an intact immunoglobulin molecule; or an immunologically active portion of any of thesepolypeptides, i.e., a polypeptide that comprises an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule, including engineered subclasses with altered Fc portions that provide for reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species. In one aspect, the immunoglobulin is of largely human origin.

[0111] The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0112] The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region may comprise amino acid residues from a “complementarity determining region” or “CDR”, and / or those residues from a “hypervariable loop”. “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0113] Variable regions of interest include 3 CDR sequences, which may be obtained from available antibodies with the desired specificity, or may be obtained from antibodies developed for this purpose. One of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see “Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol.2010;47:694–700), which is based on sequence variability and is the most commonly used. The Chothia definition is based on the location of the structural loop regions (Chothia et al. “Conformations of immunoglobulin hypervariable regions.” Nature.1989;342:877–883). Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol.2001;309:657–670; Ofran et al. “Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol. 2008;181:6230–6235; Almagro “Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires.” J Mol Recognit. 2004;17:132–143; and Padlanet al. “Identification of specificity-determining residues in antibodies.” Faseb J. 1995;9:133–139., each of which is herein specifically incorporated by reference.

[0114] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0115] The antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.

[0116] An “intact antibody chain” as used herein is one comprising a full length variable region and a full length constant region. An intact “conventional” antibody comprises an intact light chain and an intact heavy chain, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, hinge, CH2 and CH3 for secreted IgG. Other isotypes, such as IgM or IgA may have different CH domains. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequencevariants thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc constant region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); and down regulation of cell surface receptors. Constant region variants include those that alter the effector profile, binding to Fc receptors, and the like.

[0117] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy- chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Ig forms include hinge-modifications or hingeless forms (Roux et al (1998) J. Immunol. 161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US 2005 / 0048572; US 2004 / 0229310). The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequences of their constant domains. ScFv ABDs and CARs

[0118] In one embodiment, the ABD is a single chain Fv (ScFv), which optionally finds use in a chimeric antigen receptor (CAR) for T cell therapies. CAR T cells can be generated with a CAR specificity for an antigen of interest, while the T cell also expresses GPR25 for localization / targeting purposes.

[0119] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to facilitate antigen binding. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. In one embodiment, the linker is the hinge region from an immunoglobulin, e.g. the hinge from any one of IgG1, IgG2a, IgG2b, IgG3, IgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In those instances where the ABD is an scFv, an IgG hinge is effective. In some embodiments the linker comprises the amino acid sequence (G4S)n where n is 1, 2, 3, 4, 5, etc., and in some embodiments n is 3.

[0120] CARs useful in the practice of the present invention further comprise a transmembrane domain joining the ABD (or linker, if employed) to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membranespanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e.g Pace, et al. (1998) Biophysical Journal 75: 422-427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3ζ, CD4, CD8, CD28, etc.

[0121] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor ζ-chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), p110α, β, or δ catalytic subunit of PI3 kinase, the human CD3 ζ- chain, cytoplasmic domain of CD134 (also referred to as OX40 and TNFRSF4), FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptides (δ, Δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28.

[0122] In some embodiments, the CAR may also provide a co-stimulatory domain. The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of co- stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM- 1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.

[0123] CARs are often referred to as first, second, third or fourth generation. The term first- generation CAR refers to a CAR wherein the cytoplasmic domain transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FcεRIγ, or the CD3ζ chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen- dependent T-cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding. Second- generation CARs include a co-stimulatory signal in addition to the CD3ζ signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain is usually be membrane proximal relative to the CD3ζ domain. Third-generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3ζ, OX40 or 4-1BB signaling region. In fourth generation, or “armored car” CAR T-cells are further gene modified to express or block molecules and / or receptors to enhance immune activity.

[0124] Examples of intracellular signaling domains comprising may be incorporated into the CAR of the present invention include (amino to carboxy): CD3ζ; CD28 – 41BB - CD3ζ; CD28 – OX40 – CD3ζ; CD28 – 41BB – CD3ζ; 41BB –CD-28 -- CD3ζ and 41BB – CD3ζ. Compositions and Dosing

[0125] The effective dose of an agent to modulates the CXCL17 / GPR25 axis may range up to about 30 mg / kg, up to about 20 mg / kg, up to about 10 mg / kg, up to about 5 mg / kg; up to about 1 mg / kg, up to about 0.5 mg / kg; up to about 0.1 mg / kg; up to about 0.05 mg / kg; where the dose may vary with the specific antibody and recipient.

[0126] The agents may be administered one or a plurality of days, and in some embodiments is administered daily, every two days, semi-weekly, weekly, etc. for a period of from about 1, about 2, about 3, about 4, about 5, about 6, about 7 or more weeks, up to a chronic maintenance level of dosing. Therapeutic entities of the present invention are usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the therapeutic entity in the patient. Alternatively, therapeutic entities of the present invention can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.

[0127] In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0128] In still yet some other embodiments, for prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.

[0129] In still yet some other embodiments, for therapeutic applications, therapeutic entities of the present invention are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and / or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved.

[0130] According to the present invention, compositions can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal, aerosol, or intramuscular means. The most typical route of administration is intravenous although other routes can be equally effective.

[0131] For parenteral administration, compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies and / or polypeptides can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises polypeptide at 1 mg / mL, formulated in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or NaOH.

[0132] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated inliposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0133] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0134] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0135] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Preferably, a therapeutically effective dose will provide therapeutic benefit without causing substantial toxicity.

[0136] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

[0137] In some embodiments, methods are provided for reducing trafficking of an immune cell of interest to CXCL17-expressing tissues, by administering an effective dose of a GPR25 / CXCL17 blocking agent. Blocking agents include, for example and without limitation, antibodies or derivatives thereof specific for CXCL17, antibodies or derivatives thereof specific for GPR25, soluble non-activating fragments of CXCL17, activating fragments of CXCL17 without gradient, soluble fragments of GPR25 (decoys), and the like. Inhibitors can reduce immune responses by inhibiting trafficking of, for example, T helper cells, cytotoxic T cells, NKcells, and the like. Inhibitors can enhance immune responses by inhibiting trafficking of, for example, regulatory T cells (Treg). Targeted tissues include any tissue in which CXCL17 is expressed, for example airways, upper GI tract, conjunctiva, exocrine glands, stomach, esophagus, pancreas and urinary tract, etc.

[0138] Conditions of interest include autoimmune disease, inflammatory bowel disease, allergic responses, fibrosis, hypertension, diabetes, MS, arthritis, cancer, CNS disorders etc. For example, autoimmune diseases including MS, Crohn’s ulcerative colitis, arthritis, type 1 diabetes, celiac disease, etc. have been linked to GPR25 expression and / or SNPs, for example see Ricaño-Ponce et al. J Autoimmun. 2016 Apr;68:62-74; Surya Koturan. Pathogenic mechanism of chronic inflammatory diseases : linking genotype, immune cell function and pathology in Spondyloarthritis. Molecular biology. Université Paris Cité, 2020. Administration can be topical, local, or systemic; inhaled as an aerosol or mist; in combination with another therapeutic, etc.

[0139] Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. The kit can further contain a least one additional reagent. Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0140] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0141] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0142] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.EXPERIMENTAL

[0143] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXAMPLE 1

[0144] We have identified a novel lymphocyte chemoattractant receptor and its chemoattractant ligand, which is selectively expressed in lung, airways, upper GI tract, genitourinary mucosae and mucosa-associated glands including the pancreas and salivary glands. Lymphocytes migrate from the blood into the various tissues of the body where they provide immunity to infection and control cancer, but can also cause autoimmune and other inflammation associated diseases. The newly discovered molecules disclosed herein direct lymphocytes to some of the most clinically important organs in the body, including the lungs and airways, stomach, genitourinary tract and the pancreas. Use of these lymphocyte trafficking molecules provides vaccine strategies and preventative, therapeutic or curative treatment strategies for autoimmune, inflammatory and neoplastic diseases of these organs.

[0145] The nature and indeed the existence of chemoattractant mechanisms for tissue specific homing of lymphocytes to non-intestinal mucosal tissues and lungs has been obscure and lacking a mechanistic understanding of homeostatic chemoattractants for the airways, the stomach and upper GI tract, for mucosae-associated exocrine glands (salivary, pancreas) and the genitourinary tract. The disclosure provided herein provides a mechanistic understanding of an integrated non-intestinal mucosal immune system.

[0146] The orphan chemokine CXCL17 is identified as a ligand for GPR25. GPR25 is a poorly characterized GPCR with homology to GPR15 and CMKLR1. CXCL17 expression (by epithelial cells) and GPR25 expression (by tissue T and B cells) are selectively and coordinately enriched in the airways, upper GI tract and the GU tract, and in the pancreas and salivary glands (collectively, the CXCL17+ non-lymphoid tissues or NLT). Taking advantage of the organotypic homing properties of developing γδ T cell subsets in the mouse thymus, we show that CXCL17 selectively attracts lung / genitourinary tract homing (Vδ4) T cells whichexpress Gpr25. CXCL17-GPR25 axis directs lymphocyte homing to the non-intestinal mucosae, lungs, salivary glands and pancreas.

[0147] Identification of GPR25 as a lymphocyte homing receptor. GPR25 is an evolutionarily conserved orphan GPCR with orthologues in all vertebrates. Using similarity metrics based on canonical ligand binding sequences of class A GPCRs (FIG.1), we found that its closest GPCR relatives include GPR15, a lymphocyte receptor which we previously identified as a skin and colon homing receptor and deorphaned. Other close relatives include CMKLR1 (which we identified as a dendritic cell / NK cell homing receptor for the chemoattractant chemerin), GPR1 (a second chemerin receptor) and the Apelin receptor. Like chemokine receptors in general, GPR25 has a negatively charged N terminus with the potential to bind positively charged ligands.

[0148] Identification of CXCL17 as a candidate GPR25 ligand. Based on the close relationship of GPR25 with GPR15 and CMKLR1, we hypothesized that a functional ligand for GPR25 would be chemokine-like (e.g. MW of 15-25 kD, pI>8.5 to support interaction with the negatively charged N terminus of the receptors and for binding to glycosaminoglycans). In addition, we predicted that, like the GPR15 ligand GPR15L and CMKLR1 ligand chemerin, a GPR25 ligand would have a conserved C-terminal tail for receptor activation. Analyses of global gene and predicted protein databases identified CXCL17 as a candidate. CXCL17 has a MW of ~13 kD, a pI of ~11, similar to most chemokines, and a highly conserved C terminus. AlphaFold molecular modeling of interaction between CXCL17 and GPR25 predicts insertion of the C-terminal peptide FALPL into the signaling pocket of the receptor (FIG.2).

[0149] As mentioned above, CXCL17 is highly and selectively expressed by epithelium in the airways (including in lung alveolar type 2 cells), in the upper GI tract (esophagus, stomach), and the genitourinary (GU) tract (vagina, cervix, fallopian tubes, bladder) and mucosae- associated glands (salivary, pancreas). We refer to these as CXCL17+ non-lymphoid tissues (NLT). In general CXCL17 expression is largely mutually exclusive with expression of the organotypic small intestinal (CCL25) and colon (GPR15L) chemokines.

[0150] Lung / GU tract homing Vγ4+ γδ T cells express Gpr25 and migrate to CXCL17. In the mouse, waves of γδ T cells, identified by Vγ TCR usage develop in the thymus before and after birth. Each wave populates specific peripheral organs. By analyzing microarray data from sorted γδ T cell subsets, we noted selective expression of the gene encoding GPR25 by Vγ4+ γδ T cells, a subset that homes to the lungs and genitourinary tract. This contrasts with selective GPR15 expression by Vγ5+ precursors of "dendritic epidermal T cells" (DETC), cells that populate the skin and transiently occupy the neonatal colon. Figure 3A shows the mutually exclusive expression of Gpr25 by Vγ4+ γδ T cells, and Gpr15 by Vγ5+ cells.

[0151] We used standard transwell assays to ask if Vγ4+ lung homing thymocytes migrate to CXCL17. As initial controls, we compared Vγ4+ cell migration to GPR15L; and we assessed Vγ5+ (Gpr15+, Gpr25-) migration to the two chemoattractants as well. Figure 4 shows that CXCL17 selectively attracts the Gpr25+ lung homing subset, whereas Gpr15L attracts the Vγ5+ cells. These results indicate that GPR25 is a chemoattractant receptor that mediates organotypic lymphocyte homing to the CXCL17+ NLT including lungs and the GU tract.

[0152] CXCL17 activates GPR25 in CHO transfectants. To directly test the ability of CXCL17 to activate GPR25, we co-transfected human GPR25 with the calcium-signaling G protein Ga15 into CHO cells. Control (mock transfected) CHO cells were transfected with Ga15 alone. Cells were loaded with the calcium responsive dye Fura Red, exposed to CXCL17 and monitored for calcium transients by flow cytometry. As shown in FIG.4, CXCL17 triggered a robust, dose-dependent response in GPR25 but not in control mock-transfected cells.

[0153] GPR25+ lymphocytes are selectively enriched in organ sites of CXCL17 expression (CXCL17+ NLT). If Gpr25 contributes significantly to mucosal lymphocyte trafficking, then lymphocytes within CXCL17+ target tissues would be enriched in GPR25 expression compared to circulating cells in blood, or to cells in tissues lacking or low in CXCL17. Analysis of bulk RNAseq data (GTex tissue library) reveals a striking correlation between GPR25 and CXCL17 expression across the organs and tissues of the body (Fig 5). Exceptions are the blood and lymphoid tissues themselves. Available scRNAseq data confirm that GPR25 gene expression is expressed by tissue infiltrating lymphocytes, while CXCL17 is expressed by mucosal epithelial cell subsets in non-intestinal tissues, including alveolar type 2 lining epithelial cells. Fig 5 illustrates GPR25 expression by lymphocytes and CXCL17 by mucosal epithelium in the stomach (scRNAseq). Macrophages are negative.

[0154] GPR25 is a chemoattractant receptor for the orphan chemokine CXCL17, and the GPR25-CXCL17 axis controls lymphocyte homing to the airways and to non-intestinal mucosae (and mucosae-associated exocrine glands) in the body. GPR25 represents a long- sought tissue-selective homing receptor for the tracheo-bronchial tree, upper GI tract and genitourinary mucosae, critical sites of antigen and pathogen exposure. It may also contribute to T cell homing to exocrine and endocrine tissues including the pancreas, with relevance to diabetes.

[0155] We have analyzed data from airways (bronchi, nasal cavity and trachea) and PBMC from a study of 9 healthy donor children and adults, and 3 COVID patients. Airway GPR25 is highly expressed by CD8 T and innate lymphocytes in airways, and by significant subsets of Th17 and Treg CD4 cells (Fig 6). T naive and T follicular helper (Tfh) T cells are largely negative for GPR25; and airway lymphocytes lack the organotypic homing receptors for colonand SI. Data for nasal cavity and lower airway (bronchial) lymphocytes are shown, and are similar. COVID patient airway immune cell data in the same study showed overall similar features with possible alterations in GPR25 in some subsets.

[0156] Airway epithelial cells express CXCL17 but not colon (GPR15L) or SI (CCL25) chemokines (Fig 7). CXCL17 is not significantly altered in COVID patients, consistent with its proposed role as a homeostatic chemokine. In contrast, GPR15L is highly expressed by epithelial cells in the colon (data from Teichmann Gut Cell Atlas), while CXCL17 expression is low.

[0157] In PBMC from the same cohort, GPR25 is most highly expressed by dividing CD4 and CD8 blasts (Fig 8), but GPR25 on blasts was variable, and blasts were not present in all patients. These observations are consistent with an important role for GPR25 in trafficking programs of cells engaged in active immune responses. Minor subsets of circulating CD4 and CD8 Teff and Tregs also express GPR25 at levels comparable to airway T cells, as expected. Monocytes and dendritic cells are negative, supporting that GPR25 is unlikely to contribute directly to CXCL17-dependent myeloid responses.

[0158] Analysis of tonsil CD4 T cells reveals "upregulation" of GPR25 in Th17 and Treg cells arising along pseudotime trajectories from naive and activated precursors (Fig 9). In this experiment, we enriched for activated and transitional T cells during CD4 T cell sorting, yielding increased visibility of dividing and differentiating subsets.

[0159] Chemotaxis: Studies show dose-dependent migration of blood effector and regulatory CD4 cells, CD56+ CD16- NK, CD56+ CD16- NKT and antibody-secreting IgA and IgG plasmablasts to CXCL17 (Figures 10 and 11). Example 2

[0160] A synthetic 4Cys CXCL17 with an N terminal Ybbr tag is fully active, ruling out a critical role for the N terminal amino acids. Moreover, we find that a 26 AA C terminal peptide is sufficient to mediate chemotaxis of lymphocytes, albeit with reduced potency. Together these data confirm the predicted role of the C terminus in of CXCL17 in receptor engagement and activation.

[0161] CXCL17-binding flow cytometry assay: A fluorescent conjugate of 4CysCXCL17 bound human lymphocytes including, as illustrated in Fig 12, subsets of memory CD4 T cells: Consistent with predictions from our scRNAseq and T cell chemotaxis data, incubation with 4CysCXCL17 (AF488 conjugated) stains a small population of CD4 T cells in PBMC. In the donor tested (recent i.m. Flu+COVID vaccinee), CD4 cells labeled with CXCL17 comprised both mem / effector and regulatory T cells, and were enriched in Treg compared to CXCL17 nonbinding T cells. Moreover, CXCL17 binding was largely mutually exclusive with GPR15,suggesting segregation of T cell trafficking to colon (GPR15) vs the non-intestinal mucosae (GPR25).

[0162] IgG1 Fc-4CysCXCL17 chimeric proteins: We designed and validated a functional bivalent IgG1 Fc chimera with 4Cys human CXCL17 at the C terminus. The CXCL17 chimera stains GPR25-transfected but not control CHO cells, and binding is blocked by preincubation with cold CXCL17 (Fig 13). We have designed an IgG1 Fc-mouse4CysCXCL17 as well. When directly conjugated (e.g. site-specific labeling with GlyClikTM) these Fc-CXCL17 chimeras allow staining of lymphocytes for CXCL17 receptor / GPR25 expression and are useful in monitoring GPR25 expression in patients and animal models. Example 3

[0163] Role of GPR25 in T cell homing to CXCL17 target tissues. Homing to NLT in wild type host mice. To determine if GPR25 mediates T cell recruitment into CXCL17+ target tissues, short-term competitive homing assays are performed by co-injecting T cells transduced with a control or a GPR25-encoding retrovirus into young adult (6-week) B6 hosts. Mouse Gpr25 is cloned into MSCV-IRES-Thy1.1, and high titer virus prepared (Vectorbuilder). Spleen and lymph node CD4 and CD8 CD62L+ T naïve cells are sorted and stimulated with αCD3ε and αCD28 antibodies cross-linked by plate-bound αhamster IgG. At day 1 and day 2 after stimulation, naive cells from B6.CD45.1 mice are transduced with Gpr25, and B6(CD45.2) cells transduced with empty vector to provide an internal control. Cells are re-plated with IL-2 on day 3 and harvested on day 5. Two x107Gpr25+ T cells and the same number of control transduced cells are injected i.v. into recipient WT B6 mice.8-10 hours later and 3 minutes prior to sacrifice, recipient mice are injected i.v. with anti-CD45 (in a distinct 'color') or anti- CD31 antibody to label intravascular cells. Lymphocytes are harvested from the target tissues using standard mincing / collagenase / dissociation methods, optimizing digestion times for each tissue. Nasal lymphoid tissue (NALT) lymphocytes are accessible as well. Homing is quantified by flow cytometry of harvested lymphocytes: Transduced donor cells are identified by Thy1.1 expression, Gpr25 transductants by CD45.1, and recruited cells are distinguished from intravascular cells labeled by the i.v. antibody injected right before sacrifice.

[0164] Homing to NLT in CXCL17-deficient host mice. To test whether GPR25-dependent homing is CXCL17-dependent as expected homing of GPR25- and control vector- transduced T cells in CXCL17 deficient vs littermate CXCL17+ / - and + / + mice id determined.

[0165] GPR25-dependent homing to chronically inflamed NLT: homing in an established model of multi-organ T cell dependent autoimmune inflammation is determined. Transfer of Foxp3-deficient B6.scurfy (B6.Cg-Foxp3sf / J) mice into B6.Rag1- / - mice results in widespread infiltration of autoimmune T cells into NLT within 5 weeks of cell transfer. This "scurfy T cell transfer" model has a high penetrance of lymphocyte-predominant sialadenitis,dacryoadenitis, pancreatitis, gastritis, intestinal inflammation, colitis, and myositis, rendering it an excellent model for studying organotypic lymphocyte recruitment mechanisms in the setting of established chronic inflammation. Methods: 2.5 x 106lymph node cells from B6.scurfy are injected i.v. into b6.RAG- / - recipients. 3-5 weeks later, after inflammation of NLT is well established, retrovirally transduced GPR25+ or control T cells are injected and tissues harvested 8-10 hours later for analysis by flow cytometry as outlined above.

[0166] GPR25 is differentially expressed by T cell subsets and is regulated during lymphocyte differentiation. In particular, CXCL17 attracts myeloid cells, and CXCL17 deficiency impacts the representation of macrophage subsets in the lungs. To critically test the role of GPR25 in the homeostasis of innate and adaptive immune cells mixed bone marrow chimeras in which Gpr25- / - and Gpr25+ / - or WT cells compete for their niches in WT mice are generated. To test the GPR25-dependent and -independent roles of CXCL17, we evaluate WT vs Gpr25- / - competition in CXCL17-deficient hosts.

[0167] Methods: Sublethally irradiated WT and Cxcl17- / - CD45.2 mice (or Rag1- / - mice) are injected with a 1:1 ratio of CD45.1+Gpr25- / - and (CD45.2 / CD45.1) F1 Gpr25+ / + bone marrow cells. After 8-10 weeks, tissues from recipients are harvested and the accumulation of specific T cell and other immune subsets in NFT and control sites are analyzed by FACS. The ratio of CD45.1+ (mutant) to F1 (WT) among reconstituted immune subsets from blood, immune organs and NLT (including lungs and other CXCL17+ NLT, as well as blood, thymus, lymph nodes, spleen, SI and colon) is calculated, particularly to the effects of Gpr25 sufficiency vs deficiency (as assessed by the CD45.2 / CD45.1 ratio) on T cells in NFT, including CD4+ Tregs, Teff cells, effector CD8 subsets, and γδ T cells. Given the reported effect of CXCL17 deficiency on lung macrophages (reduction in alveolar but not interstitial macrophages, and an increase in F4 / 80+CDllcmidcells), markers to evaluate these subsets are incorporated as well.

[0168] Chemoattractant receptors are regulated during naive T cell differentiation in response to local cytokines. Organotypic chemokine receptors are additionally controlled by factors associated with specific regions or tissues in the body. In vitro studies include GPR25 regulation by environmental factors that program other organotypic chemokine receptors: retinoic acid enhances CCR9 (SI homing); Vitamin D3 upregulates CCR10 (epidermotropism); aryl hydrocarbon receptor (Ahr) ligands induce GPR15 (colon homing).

[0169] In vitro polarization: Magnetic bead (Miltenyi)-enriched naive CD4 (or CD8) T cells are isolated from the spleen of normal WT C57BL6 mice, stimulated with IL-2 and plate bound anti-CD3 / anti-CD28, and subjected to standard polarizing conditions. Th0 (media alone), Th1 (IL-12 + anti-IL-4), Th2 (IL-4 + anti-IFN-γ), Th17 (IL-6, TGF-β, IL-1β, IL-23 + anti-IFN-γ andanti-IL-4), Tregs (TGF-β). CD8 naive cells are also polarized. Cells are cultured for 5–6 d and analyzed by flow cytometry.

[0170] To assess effects of known environmental regulators, 1,25OH3 vitamin D3, retinoic acid or Ahr ligands (TCDD and FICZ) are added during initial polarizing cultures and again at day 3. FACS contrasts effects on GPR25 vs CCR9, CCR10, GPR15 and other chemokine receptors such as CCR6 and CXCR3.

[0171] GPR25 surface expression by immune cells: GPR25 expression and regulation in vivo is surveyed at the protein level by flow cytometry of T and B cells (and other immune cells) from the NLT's and from lymphoid organs (lymph nodes draining diverse NLT, spleen, bone marrow, thymus). GPR25 in pre-and perinatal organotypic γδ T thymocytes are examined.

[0172] Organotypic induction of GPR25 during T cell activation in vivo: To assess organotypic regulation of GPR25 during T cell activation, we use the methods we developed previously. In systemically immunized mice, antigen-activated naive T cells are rapidly programmed within secondary lymphoid organs (SLO) to express tissue-specific homing receptors that target them to distinct NLT. Programming occurs within the first few cell divisions after activation, while antigen-specific cells remain sequestered within lymphoid organs and experience organotypic environmental signals. Young adult B6 mice receive 10 million splenocytes or LN cells from sex-matched OTI or OTII (ovalbumin / OVA-specific T cell transgenic) RAG2 KO donors by retroorbital injection 24 h before immunization. In some instances, lymphocytes are labeled with CFSE, a dye which by dilution in proliferating cells allows enumeration of cell divisions. Mice are immunized by i.p injection of 100 ug LPS alone or with 500 ug OVA. Lymph nodes (LN) draining specific NLT (e.g. hilar, gastric, pancreatic, mesenteric, salivary LN) are harvested 2-3 d after immunization, and expression of GPR25, a4b7 (DATK32), GPR15 and CCR9 by Ag-activated OTI or OTII T cells is assessed by flow cytometry.

[0173] Public datasets are used to identify GPR25 and CXCL17 expression at the single cell level in different lymphoid and nonlymphoid tissues, developmental stages, and disease states. Data sets will be processed, batch corrected and integrated using our in-house computational pipeline to ensure standardization. Raw normalized gene expression values are analyzed. To enhance interpretability, minimally imputed (t2 magic) expression is used, calculated for each sample to preserve patient / donor variation.

[0174] Chemotaxis of human lymphocyte subsets in standard transwell assays functionally assesses migratory responses to CXCL17, and to GPR15L for comparison. T and B cell subsets and innate populations from PBMC, lymph nodes and selected NLT are compared. For this purpose, airway (nasal cavity) lymphocytes are readily available from surgical specimens, as are tonsils. Other NLT cells can be isolated from organ donors if needed.

[0175] In addition to transcriptomic surveys, anti-GPR25 MAbs or tagged CXCL17 constructs are applied for a global flow cytometric survey of GPR25 expression by immune cells.

[0176] Induction of GPR25 on immunoblasts during tissue-specific immune challenge: After acute infection or in settings of active immune responses, circulating plasmablasts (PB) and dividing T cell blasts increase dramatically, peaking 7 to 10 days after immunization or the onset of illness. PBMC from donors 7-10 days after onset of localized infectious or inflammatory symptoms (e.g. sore throat, URI or GU symptoms, etc.) or vaccination (e.g. influenza i.m. vs nasal Flumist); or from patients with active inflammatory disease (e.g. colitis, pancreatitis, gastric ulcer, upper respiratory infection) are profiled by high dimensional CyTOF or spectral flow cytometry using anti-GPR25 or tagged CXCL17 and a broad panel of MAbs including MAbs to T and B subset markers, activation antigens (CD38, HLA-DR), anti- intracellular Ki67 (dividing cells to define responding T cell blasts) and homing receptors (to define GPR25 in the context of overall trafficking programs). Example 4 A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and brain

[0177] Tissue-selective chemotactic mechanisms direct lymphocytes to epithelial surfaces of the body to establish local immune environments, to regulate immune responses to food antigens and commensal organisms, and to protect the body from pathogens. Chemoattractants for small intestines, colon, and skin are known, but chemotropic mechanisms for the respiratory tract and other non-intestinal mucosal tissues (NIMT) 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 NIMT and distinguishes them from intestinal mucosae. Analyses of single cell transcriptomic data shows that GPR25 is induced on innate lymphocytes prior to 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 and regulatory T lymphocytes in NIMT and lungs in human and we show that it mediates lymphocyte homing to the respiratory tract, stomach, and genitourinary tract in mouse models. GPR25 is also expressed by T cells in cerebrospinal fluid and CXCL17 by neurons, providing a link to CNS immune regulation. We reveal widespead imprinting and expression of GPR25 on regulatory T cells, suggesting a mechanistic link to population genetic evidence that GPR25 is protective in autoimmunity. Our results define a GPR25-CXCL17 chemoaffinity axis with the potential to integrate immunity and tolerance at non-intestinal mucosae and the CNS.

[0178] GPR25 is expressed by lymphocytes in the respiratory tract and non-intestinal mucosae. Leukocyte chemoattractant receptors are class A G protein-coupled receptors (GPCRs); thus we initially asked if lymphocytes infiltrating the airways can be differentiated from intestinal lymphocytes on the basis of their GPCR expression profiles. Using single-cell transciptomic data from 63 patient samples derived from the nasal cavity, trachea, bronchus, lung, colon, caecum, and small intestines (duodenum, jejunum, and ileum), we evaluated the expression of 510 class A GPCRs.2x2 plots of mean mRNA expression highlighted selective expression of the orphan receptor GPR25 in airways compared to both intestinal compartments, while CCR9 was prominently expressed in the small intestines and GPR15 in the colon. Differences in airway vs gut T cell expression were consistent across patient samples, and particularly prominent for CD8 T cells.

[0179] GPR25 is a poorly studied GPCR of unknown function. It has been implicated in autoimmunity through genome-wide association studies (GWAS). A compendium of diverse cell type and tissue gene expression data indicate that GPR25 expression is largely restricted to lymphocytes and NK cells, suggesting a unique role for the receptor in lymphocyte biology. Violin plots reveal that CD4 and CD8 effector / memory cells, Tregs, and CD8 T resident memory cells (CD8 TRM, identified as ITGAE high) in lungs and airways express GPR25 but not the chemoattractant receptors for colon (GPR15) or small intenstines (CCR9). Conversely, GPR25 is relatively poorly expressed by conventionallymphocytes in the intestines. GPR25 is also selectively expressed (compared to CCR9 and GPR15) by T cells in the stomach, pancreas and salivary glands, while GPR25 and GPR15 are both expressed by T cells in the female genitourinary (GU) tract (data from cervix). In contrast, CCR9 expression is largely restricted to the small intestines. Surprisingly, T cells in the cerebral spinal fluid (CSF) of human donors also express GPR25. Table 1 SNP Variant Disease or p- Odds Ratio p- value varian Normalize ct orrs2297909 1_200991179_G_A Ankylosing 5.00E NA 3.56E-06 Up 0.16 spondylitis -12rs35730213 1_200905101_G_ Crohn's 1.17E 0.8899411 2.90E-10 Up 0.24 C disease -10rs296543 1_200920705_T_C Primary 1.60E 1.11 7.50E-08 Down -0.19 biliary -03 5 isa single exon gene. The SNPs are all up or downstream in the untranslated regions.

[0180] Among B cells, GPR25 is highly expressed by barrier tissue-homing FCRL4+ B cells in the respiratory tract and lung, and by circulating plasma cells. Innate lymphocyte subsets including γδT cells, NK cells, NK T cells, mucosae-associated invariant T cells (MAIT), innatelymphoid cells (ILC) in the fetal respiratory tract and lung, and ILC progenitors in the fetal SI express GPR25, while the gene is less well expressed by innate populations in adult lung as a whole, and by intestines. Myeloid cells from blood and tissues evaluated lacked GPR25 expression.

[0181] Homeostatic chemoattractant receptors display characteristic patterns of regulation reflecting their dual role in seeding mucosal surfaces with innate lymphocyte populations and in directing the migration of antigen- activated T and B cells. To elucidate the regulation of GPR25 during T cell development, we evaluated gene expression profiles of thymocytes. GPR25 is upregulated during thymic T cell differentiation in innate populations including NKT, CD8aa unconventional T cell lineages and subsets of TCRγδ cells at a late developmental stage, suggesting the potential to contribute to migration of innate T cells to target tissues. Highest expression of GPR25 defines CD8aa(II) cells, a population that includes TCRαβ and TCRγδ T cells and comprises thymic precursors of mucosa-homing CD8αα unconventional intraepithelial cells.

[0182] To assess regulation during lymphocyte activation and differentiation in an immunologically active peripheral lymphoid organ, we evaluated T cells in the inflamed tonsil. The tonsil processes antigens in the oral pharynx contiguous with the trachea and airways. To focus on immunologically responding CD4 T cells, we enriched for T cells expressing activation antigens by fluorescence activated cell sorting (FACS) prior to cell capture for scRNAseq. A UMAP of tSpace trajectories (tUMAP) reconstructed developmental branches from naive T cells to T follicular helper (Tfh) cells, and to Th17 (IL17A+ and / or IL17F+) and Treg (FOXP3+, IL2RA+) phenotypes. GPR25 expression emerges during the transition from naive to Treg and Th17 branches, and on dividing T cells, and expression is enhanced in magnitude and frequency as cells mature, presumably in preparation for exit and migration to target tissues. Naive T cells and most Tfh cells lack the receptor gene. The selective expression of GPR25 by lymphocytes and its patterns of induction on innate cells and on antigen-reactive T cells as they mature to tissue migratory phenotypes are consistent with a role in lymphocyte trafficking.

[0183] We similarly evaluated GPR25 induction in activated tonsil B cells. Activated B cells and plasma cells were enriched by cell sorting (see methods) and processed for scRNAseq as above. tUMAP projection revealed GPR25 upregulation in the transition of naive and immature dividing cells to plasma cells and memory B cells (MBC). GPR25 expression is particularly enriched among the tissue homing FCRL4+ B cell subset, a subset identified as tissue resident sentinel B cells within mucosal epithelia. Thus GPR25 is imprinted in tonsils during effector differentiation of activated B and T cells.

[0184] GPR25 is induced on Tregs in gut- and lung-draining lymph nodes. Tissue-targeting homing receptors are imprinted during lymphocyte activation in LNs, conferring targeted migration of antigen-specific T cells. To assess the tissue-specific imprinting of GPR25, we examined the transcriptomes of T cells from thoracic lymph nodes (TLN, lung-draining) and mesenteric lymph nodes (MLN, intestine-draining). We compared GPR25 with the gene for the small intestinal homing receptor CCR9, which in mice is selectively imprinted in MLN. As expected, CCR9 was expressed by T cells in MLN and nearly absent in TLN. In contrast, T cells in both tissues expressed GPR25.

[0185] In TLN, T effector and regulatory CD4 cells express GPR25 similarly. In contrast, MLN mature Tregs (identified by high expression of FOXP3 and IL2RA; "Treg hi" cells) more prominently express GPR25 than Teff. On average, ~80% of Treg-hi cells in MLN were GPR25+ with mean expression levels similar to or greater than that of their Treg-hi counterparts in TLN. Moreover, while cells with high GPR25 expression in TLN were enriched for Th1, Th17 and Treg, in MLN cells with high GPR25 expression were principally enriched for Treg-hi cells. Almost all GPR25+ Treg-hi cells in MLN co-expressed CCR9, suggesting local induction or imprinting in response to gut antigens and metabolites. Consistent with this, GPR25+ Treg-hi cells align late along a candidate developmental (pseudotime) trajectory from naive CD4 cells; and in MLN, GPR25 expression arises 'later' than CCR9. Most GPR25+ Tregs and Teff identified in the small intestinal lamina propria also co-expressed CCR9. Co- expression of CCR9 likely explains the presence of GPR25+ Tregs in the lamina propria, since the GPR25 ligand (identified below) is poorly expressed in the normal intestines. Together, these data show that GPR25 is imprinted on T cells in gut as well as lung-draining lymphoid tissues. Imprinting of GPR25 on Tregs in gut-associated lymphoid tissues may position Tregs to contribute to the systemic dissemination of tolerance to gut microbes and food antigens.

[0186] Bioinformatic identification of CXCL17 as a ligand for GPR25. We considered potential characteristics of a ligand for GPR25 based on structural features of the receptor. GPR25 is well conserved in vertebrate evolution and contains a canonical DRYLAVV motif in the third transmembrane sequence. It has a conserved negatively charged N-terminal extracellular peptide comprising a DY candidate sulfation site characteristic of many leukocyte chemoattractant receptors for positively charged polypeptides of the chemokine family. The closest relative of GPR25 is the colon homing receptor GPR15. CoINPocket analysis indicates that the receptor ligand binding pocket of GPR25 shares similarity to those of GPR15 and other chemotactic GPCRs that, like GPR15, are activated by C-terminal peptides of chemoattractants including Apelin, Bradykinin, and Chemerin (ligand for CMKLR1 and GPR1). Based on these features we postulated that a physiologic attractant for GPR25 would be a chemokine-like protein (molecular weight < 25 kD) with a net positive charge (pI > 8, facilitating charge-based receptor and GAG binding), and that it would comprise an evolutionarilyconserved C-terminal peptide sequence for receptor insertion and activation. We identified 455 genes encoding known or predicted secreted peptides within the expected pI and MW range. Of these, 142 had homologs in multiple species and displayed well conserved (BLAST bit-score score > 4) C-terminal peptide sequences. To further enrich likely candidates, we assessed the pattern of tissue expression of potential ligands: As GPR25 expression is limited to lymphocytes, we reasoned that expression of a physiologic ligand within tissues might correlate with infiltration of GPR25+ cells and thus with GPR25 message detection. We evaluated the correlation of GPR25 with genes for ligand candidates across 49 tissues with bulk RNAseq gene profiles from the Human Protein Atlas (lymphoid organs were excluded). Tissue expression profiles of GPR25 and CXCL17 are illustrated.

[0187] These criteria converged to identify the orphan chemokine CXCL17 as a candidate GPR25 ligand. The full length CXCL17 protein comprises 6 cysteines but may be cleaved in vivo to release a C-terminal chemoattractant peptide with 4 cysteines ("4C" CXCL17) and a predicted MW of 6.6 kD. The peptide displays anti-microbial activity at micromolar concentrations and is implicated in angiogenesis and tumor progression. It has been reported to attract myeloid cells but not bulk lymphocyte populations, although published data have been inconsistent and the chemotactic potency of the ligand for myeloid cells has been questioned. An initial suggestion that GPR35 is a receptor for CXCL17 is now considered incorrect. In silico modeling of CXCL17 indicates that it does not assume a classical chemokine conformation. Moreover, CXCL17 evolved independently of the classical chemokine family: it is first found in the genome after divergence of the mammalian lineages from other tetropods, and may have emerged as a response to the immune challenges associated with the evolution of the mammalian lung. It has a predicted MW of ~11 kD and a pI of ~11, and a highly conserved C terminus. Consistent with our hypothesis, unsupervised AlphaFold modeling predicts insertion of the conserved C-terminal FALPL peptide of CXCL17 into the signaling pocket of GPR25.

[0188] GPR25 is a lymphocyte chemoattractant receptor for CXCL17. To determine if GPR25 is activated by CXCL17 and to evaluate its chemotactic function, we expressed the receptor in L1.2 cells, a mouse pre-B cell lymphocyte line. We established stable transfectants expressing hGPR25 (L1.2GPR25) alone or with a serum response factor response element (SRF-RE) luciferase reporter (L1.2hGPR25-SRF-RE). We used the reporter system to quantify activation of the small GTPase RhoA pathway (Promega), which regulates cytoskeletal dynamics and integrins during leukocyte chemotaxis. CXCL17 dose-dependently activated L1.2hGPR25-SRF-REcells with an EC50 of ~ 60 nM. A peptide comprising the C- terminal 26 amino acids of mouse CXCL17 (with Cys to Asn substitutions to avoid crosslinking) retained signaling activity, albeit with reduced potency (EC50 ~ 220 nM), whereas truncation of the four C-terminal amino acids "ALPL" rendered the chemoattractant inactive. The human GPR15 ligand, GPR15LG, failed to trigger the reporter as well.

[0189] L1.2GPR25cells migrated efficiently in transwell chemotaxis assays to 6Cys-CXCL17 and to 4Cys- CXCL17, less well to the 26 amino acid C-terminal peptide, while migration towards the C-terminal truncated chemokine or GPR15LG was similar to vehicle control. GPR25 shows similarity in its ligand binding pocket to the paralogous receptors GPR15, BDKRB2, CMKLR1, and the mammalian Apelin receptor, APLNR. The ligands for these receptors (Apelin, Apela, Bradykinin, GPR15LG and Chemerin) failed to induce L1.2GPR25migration. Host L1.2 cells and L1.2 cells transfected with GPR15 or CMKLR1, which migrated to their respective cognate ligands, failed to respond to CXCL17. GPR25-dependent migration was inhibited by PTX indicating theinvolvement of chemotaxis-associated Gαi proteins, andrequired a CXCL17 gradient as shown in acheckerboard assay. Together these resultsidentify CXCL17 as a conserved and selective chemoattractant ligand for GPR25.

[0190] Epithelial and neuronal expression of CXCL17. CXCL17 is expressed by mucosal tissues, but a comprehensive picture of cell type expression across organs is missing. A pseudobulk analysis of CXCL17 expression by cell types annotated under the Human Protein Atlas project. A log scale illustrates expression by diverse epithelial cell types including respiratory epithelium, epithelial cells of the esophagus, stomach, biliary tract (liver cholangiocytes), prostate, pancreas, salivary glands and squamous mucosae. Excitatory and inhibitory neurons in the human brain and spinal cord neurons in mouse express CXCL17, contrasting with microglia. A linear scale highlights that respiratory epithelia have the highest expression of CXCL17. scRNAseq data from the respiratory tract illustrate similar patterns of expression by epithelial cell subtypes in trachea, bronchial and nasal tissues. In a comparison of airway cells from COVID-19 patients vs healthy controls, expression was upregulated with disease. In a mouse model of spinal cord injury, neurons displayed a transient reduction in Cxcl17. Together these data are consistent with roles for the chemoattractant in immunity and immune homeostasis.

[0191] RNAscope analyses reveal histologic patterns of CXCL17 expression by epithelial cell subsets. In the stomach CXCL17 is highly expressed by glandular epithelium, less so by the surface epithelium. Expression in salivary gland and pancreas is limited to exocrine cells and varies dramatically from one glandular cluster to the next. In the duodenum, scattered cells of the Brunner's gland express the chemokine. Squamous mucosae of the oral cavity, esophagus, vagina and conjunctivae express CXCL17. Consistent with the scRNA data, RNAscope confirms that CXCL17 is poorly expressed in small and large intestines. In comparison GPR15LG is robustly expressed by colonocytes and microfold cells in the colon but poorly expressed in respiratory epithelium and digestive glands. Interestingly, GPR15LGis expressed with CXCL17 in most squamous mucosae, including sites of squamous metaplasia in the respiratory tract; although GPR15LG is enriched in basal epithelial cells while CXCL17 characterizes suprabasal layers. These cell-type specific data are consistent with and extend prior bulk RNA and immunohistochemical studies of the mucosal expression of CXCL17. As predicted from scRNAseq data, RNAscope identifies CXCL17 expression by granule neurons of the cerebellum and immunohistochemistry shows immunoreacitivity for CXCL17 in neuronal bodies and axonal projections in white matter in human. Together with expression of GPR25 by CSF lymphocytes, these results suggest a role for the GPR25- CXCR7 axis in neuroimmune function.

[0192] GPR25 and CXCL17 support lymphocyte homing to lungs and NIMT. We used mouse models to test whether GPR25 mediates lymphocyte localization and accumulation in mucosal target tissues. We first injected mixed GPR25 and control transduced lymphocytes (1:1 ratio) into Rag1- / - mice and examined tissues 1 or 7 weeks later, and 5 min after i.v. injection of PE- anti-CD45 antibody to label and exclude intravascular cells. In these recipients, which lack endogenous T and B cells, adoptively transferred T cells localize and proliferate to occupy empty niches. Compared to vector control transduced cells, mGpr25-transduced T cells preferentially populated the GU, stomach, and trachea. Gpr25 transduced cells showed significant enrichment vs control cells in whole lung isolates after 7 weeks but not at 1 week after infusion, potentially reflecting maturation or repositioning within the lung over time (Figure 18A). To ask if GPR25 mediates homing from blood into NIMT, we examined in vivo localization of GPR25-transduced and co-injected control vector transduced T cells 10-12 hours after i.v. transfer into WT C57BL / 6N and Cxcl17- / - recipients (Figure 18B-F). Recipient mice received anti-CD31 20 minutes prior to sacrifice to delineate blood vessels and discriminate intravascular from extravasated cells. In WT recipients, GPR25 expression conferred advantage in homing to trachea, stomach, tongue, gallbladder and uterine mucosae, but not to the intestines or control lymph nodes or spleen (Figure 18B, and confocal images, Figure 18C-D). The ratio of mGPR25 transduced to control cells was high for extravasated cells, but the ratio was also elevated for cells still within the vasculature bound to endothelium in trachea, stomach, tongue and uterus, suggesting a role for the receptor in endothelial recognition in CXCL17-expressing target sites. (Venous endothelial cells transcytose epithelium-derived chemoattractants for presentation to circulating lymphocytes, triggering their initial arrest). The GPR25 advantage disappeared in Cxcl17- / - recipients, suggesting that CXCL17 is the principal functional ligand for GPR25 in these organs. Insufficient cells localized to the eye, salivary gland, pancreas, bladder, and CNS (spinal cord, cerebellum or choroid plexus) for analysis.

[0193] Within the peribronchovascular interstitium of the lungs, many mGPR25-transduced cells localized to bronchi (Figure 18E and F). In contrast, control cells were enriched nearveins, suggesting GPR25-dependent repositioning of transduced donor cells from venous sites of entry to the bronchial submucosa. GPR25-transduced and control cells were equally represented in alveolar interstitium away from the peribronchovascular sheath. GPR25- transduced cells showed no affinity for bronchioles and failed to segregate from control cells in Cxcl17- / - recipients. The results suggest that, within the steady state lung, GPR25 chemoaffinity for CXCL17 drives localization to pulmonary bronchioles, while initial extravasation may be largely GPR25-independent and mediated by alternative mechanisms and chemoattractant receptors and ligands. CXCL17 deficiency also limits endogenous T cell localization: immunohistologic analyses show that bronchiolar and gastric intraepithelial CD8, and to a lesser extent CD4 cells, are reduced in frequency and number in Cxcl17- / - vs WT mice (Figure 18G and H).

[0194] We conclude that GPR25-CXCL17 axis mediates lymphocyte recruitment into the respiratory, upper GI, biliary and GU tracts. In Figure 18G and H, we show that mice deficient in CXCL17 have reduced T cells in the respiratory and gastric epithelium. Compared to WT mice, Cxcl17- / - have an ~85% reduction in CD8 T cells within the epithelium of bronchioles in the lungs and in the stomach intraepithelial compartment. CD4 T cells are also significantly reduced in the stomach epithelium. Thus, in spite of redundant mechanisms and the known capacity of T cells to expand to fill available niches, CXCL17 has a non-redundant role in T cell population of barrier mucosae.

[0195] Cxcl17- / - mice recruit fewer CD8 TRM and suffer increased viral replication in the vaginal mucosae in herpes virus infection, consistent with involvement of the GPR25-CXCL17 axis in effector T cell recruitment and function in NIMT barrier immune responses.

[0196] Our studies identify GPR25 as a lymphocyte chemottractant receptor for the NIMT / CNS-expressed chemoattractant, CXCL17, and show that GPR25 mediates lymphocyte homing to the respiratory tract, upper GI, biliary and GU tracts. We provide an atlas of GPR25 regulation in human lymphocyte development and of GPR25 and CXCL17 expression in target tissues: Innate lymphocytes upregulate GPR25 in the thymus prior to migration to barrier tissues, and effector and regulatory B and T cells are imprinted with GPR25 expression during peripheral immune responses. Our analyses show that GPR25 characterizes tissue resident CD8 T cells and FCRL4+ B cells, populations that localize to mucosal epithelia and serve as immune sentinels and modulators in barrier tissues. CXCL17 is most highly expressed in the airways, showing that the respiratory tract may serve as a central hub for a GPR25-CXCL17 defined immune network. The data also show a prominent role for GPR25 in regulatory T cell dissemination. CD4+ conventional Tregs in each of the CXCL17-expressing target tissues including the CNS express GPR25. More surprising is the imprinting of GPR25 on regulatory T cells in GALT, cells thought to play an important role in oral tolerance and prevention of autoimmune responses to benign microbiota. GPR25-dependent tropism to the respiratorysystem, NIMT and CNS may ensure control of systemic immune responses to benign food and gut antigens or microbes. IFNG+ Th1 cells and IL17-expressing Th17 cells can express GPR25 in MLN as well, although less frequently than Tregs, suggesting that the GPR25 / CXCL17 axis may disseminate gut effector T cells in pathogenic settings. Development of animal models will be required to elucidate the protective and pathogenic roles of this pathway in infectious diseases, cancer and autoimmune pathologies.

[0197] Discovery of the NIMT / CNS chemoaffinity program presents opportunities to understand unexplained aspects of immune system integration and dysregulation in disease. GPR25 induction and directed migration of T cells may explain the ability of intranasal vaccination to provide effective protection in the genitourinary tract. As discussed above, GPR25 / CXCL17-dependent dissemination of Tregs may help prevent autoimmunity to self- antigens cross-recognized through “molecular mimicry” by T cells responding to intestinal microbes and food antigens. Poor dissemination of regulatory T cells could explain the association observed in population studies between reduced GPR25 expression and susceptibility to autoimmune diseases including MS, diabetes, uveitis and Sjogren’s disease. Recent studies in rat models of EAE suggest that pathogenic T cells undergo ‘licensing’ in the respiratory tract before they can migrate to the CNS and induce disease: GPR25 could mediate respiratory tract localization for licensing. Alternatively, induction of GPR25 during activation or re-activation of effectors in respiratory lymphoid tissues could trigger migration to the CNS for disease initiation. GPR25-dependent recruitment of regulatory or effector T cells could contribute to reported pro- and anti-tumor effects of CXCL17 in cancers, and to CD8 T cell-dependent neurodegeneration in models of Alzheimers.

[0198] Antibodies to GPR25 allow monitoring of vaccine responses and identification of cells involved in pulmonary or NIMT tolerance or inflammation in disease settings. Engineered expression of GPR25 enhances the efficacy of adoptive regulatory T-cell therapies for autoimmune diseases. Thus GPR25 and its ligand CXCL17 define a core axis of the integrated mucosal immune system with broad implications for understanding and manipulating immunity and inflammation. Our data represent a valuable resource for the biology of respiratory / NIMT / CNS lymphocyte traffic and its role in systemic immunity and immune pathogenesis. Materials and Methods

[0199] Mice and human subjects. All mice in this study were maintained in specific pathogen- free (SPF) facilities at the Veterans Affairs Palo Alto Health Care System (VAPAHCS). B6 / SJL Prprc Pep3BoyJ (CD45.1), C57B6 / J CD45.2 and Rag1- / - mice were purchased from Jackson laboratories. The Cxcl17- / - mouse strain (C57BL / 6NAtm1BrdCxcl17tm1b(EUCOMM)Wtsi / MbpMmucd) RRID:MMRRC_047263-UCD, was obtained from the Mutant Mouse Resource and Research Center (MMRRC) at University of California at Davis, an NIH-funded strain repository, andwas donated to the MMRRC by The KOMP Repository, University of California, Davis. Originating from Kent Lloyd, UC Davis Mouse Biology Program mice were purchased from the MMRRC at UC Davis. C57B6 / N controls were purchased from the MMRRC as well. Animals were maintained in accordance to US National Institutes of Health guidelines, and experiments were approved by Stanford University Institutional Animal Care and Use Committee. Mice were used at 8-12 weeks of age unless otherwise stated and both sexes were included in experiments, except for homing experiments where the female GU tract was required.

[0200] Human peripheral mononuclear cells (PBMC) were obtained from healthy donors and surgically removed tonsils from Stanford University, Department of Pathology. Some tissues for RNAscope and IHC were collected sterilely from rapid research autopsies by the Research Autopsy Center at Stanford (RACS), consented under Stanford IRB 63818 and autopsy consent. In general, this study was carried out in accordance with the recommendations of the US National Institutes of Health guidelines, with written informed consent from all subjects. The protocol was approved by the Stanford University Institutional Review Board.

[0201] Leukocyte isolation from organs and flow cytometry cell phenotyping. Intestinal lamina propria cells were isolated as previously described with minor modifications. To digest the intestines we used the gentleMACS octo dissociator with heaters and C-tubes (Miltenyi). Lung, salivary gland, GU tract, stomach and esophagus were processed and cells isolated as previously described. Brain leukocytes were isolated following a modified version of the following protocol. Following advice from Dr. Gabriela Constatin (University of Verona, Italy), the digestion step after the mechanical dissociation was performed with Liberase TL (Roche) and we applied a percoll gradient-based isolation spinning the cell preparation at 4C to enhance cell viability.

[0202] Isolated mouse leukocytes were surface-stained with monoclonal anti-mouse antibodies: BV421 anti mouse Ki67 (16A8), BV650 anti mouse CD69 (H1-2F3), PercpCy5.5 anti mouse TCRγδ (GL3), AF700, anti mouse NK1-1 (S17016D), APCCy7 anti mouse CD3 (145-2c11), BV421 anti mouse CD8β (Ly-3), AF647 anti mouse CD90.1-Thy1.1 (OX-7) and BV711 anti mouse CD4 (RM4-5) were purchased from Biolegend. FITC anti mouse CD45.2 (104), BV605 anti mouse IgA (C10-1), BUV395 anti mouse CD45.1 (A20), BV786 anti mouse CD103 (M290) and BUV737 anti mouse B220 (RA3-6B2) were purchased from BD. PercpCy5.5 anti mouse CD11a (M17 / 4) and PECy7 anti mouse TCRβ (H57-597) were purchased from Tonbo. PercpCy5.5 anti mouse CD45.1 (A20) was purchased from eBiosciences. AF700 anti mouse CXCR6 (221002) was purchased from R&D.

[0203] Isolated human leukocytes were surface-stained with monoclonal anti-human antibodies: PE anti human CD161 (HP-3G10), PE-Cy7 anti human CD103 (Ber-ACT8), PercpCy5.5 anti human CD45RA (HI100), AF700 anti human CD69 (FN50), APC-Cy7 antihuman CD25 (M-A251), BV421 and PercpCy5.5 anti human CD3ε (UCHT1), BV785 anti human CD4 (RPA-T4), BV650 anti human CD19 (HIB19), APC anti human CD14 (HCD14), BV605 anti human CD45RO (UCHL1), BV650 anti human CD62L (DREG-56), PE anti human CD123 (S18016C), BV421 anti human HLA / DR (L243) and BV650 anti human CD11c (Bu15) were purchased from Biolegend. BUV395 anti human CD8β (2ST8.5H7), BUV737 anti human TCRαβ (T10B91A-31), BV711 anti human TCR Vα7.2 (OF 5A-12), BUV395 anti human CD56 (NCAM16.2) and FITC anti human CD16 (3G8) were purchased from BD. APC anti human TCRγδ (11F2) was purchased from Miltenyi.

[0204] Staining for intracellular Foxp3 was performed using the transcription factor staining kit (eBioscience) and the anti-mouse Foxp3 antibody (150D) from Biolegend or anti-human Foxp3 antibody (PCH101) from eBioscience. Cell viability was always determined using fixable Aqua (510) live / dead cell stain (1:250) (Thermoscientific).

[0205] Retroviral transduction for tropism and short-term homing experiments.1 or 7 week long cell tropism experiment. The coding region of mouse GPR25 or a stuffer sequence (empty vector) was cloned into the MSCV-IRES-Thy1.1 retroviral construct (purchased from Vectorbuilder). Retroviruses were generated by transient transfection of Platinum-E (Plate-E) cells. Mouse T CD4 naive or total T cells were isolated from the spleen of B6 / SJL Prprc Pep3BoyJ CD45.1 and C57B6 / J CD45.2 mice (Jackson laboratories) by magnetic negative selection using the Mouse T CD4 isolation kit (STEMCELL technologies). T CD4 cells were stimulated with plate bound 0.5 µg / ml of anti-mouse CD28 (37.51, eBiosciences) and 1 µg / ml of anti-mouse CD3 (145-2c11, eBiosciences) in RPMI-10. On day 1 and 2 after activation, cells were transduced with retroviruses with the following spinfection conditions (850g, 32C, 4 hours with 8 ug / ml of polybrene). On day 3, the cells were washed and transferred to a different plate under 130 U / ml of mouse IL2 and 10 ng / ml of mouse IL7 (Peprotech). On day 5 cells were harvested and purified with a hystopaque 1.077 density gradient. The CD45.1 for mouse GPR25 and CD45.2 for empty vector (and viceversa) transduced cells, were mixed at 1:1 ratio and 20-30 million total cells were injected i.v. into the recipient mice (Rag1- / -).1 week after cell transfer, 2µg PE-CD45 (30-F11) antibody from Biolegend was injected i.v. 5 minutes before sacrifice to identify and exclude intravascular cells. Cells were then recovered from organs and analyzed by flow cytometry. Only TCRβ+ CD4+ Thy1.1+ cells were considered for analysis and GPR25 / empty vector ratios were calculated for each organ.

[0206] Short term competitive homing. Culture and transduction conditions were the same as above. However, here only C57B6 / J CD45.2 mice were used as donors and on day 5 of culture only transduced (Thy1.1+) cells were magnetically isolated using CD90.1 microbeads (Miltenyi). Importantly, only transduced cells (>95% purity) were maintained in culture after this point. Mouse GPR25 and empty vector cultures were maintained for 2 days under IL2 andIL7 as indicated above. On day 7 mouse GPR25 cells were labeled with CFSE and empty vector with Yellow fluorescent dye (Thermoscientific), washed and subsequently mixed at a 1:1 ratio. Dyes were switched in one of the 4 independent experiments shown, with comparable results. A total of 20-30 million total cells were injected i.v. into the recipient mice (WT C57B6 / N or Cxcl17- / -).10-12 hours later the mice were injected with anti-CD31 (far red, clone 390, InvivoMab) antibody 30 minutes before sacrifice to identify blood vessels. Subsequently, the mice were sacrificed and organs analyzed by flow cytometry (PLN, spleen) and confocal microscopy (whole mount (trachea, uterine horns) or frozen sections (all the rest)). Results are shown as the ratio of GPR25 transduced to control empty vector transduced cells in either whole organs or specific organ compartments. For analyses of microenvironmental localization in the lungs, the distance of cells to histological landmarks (bronchial basement membranes or veins in the lungs) were measured using the Imaris software.

[0207] RPMI-10: RPMI 1640 with L-glutamine, 10% FBS, Pen / Strep, 1X MEM Non-essential aminoacids, 1mM sodium pyruvate, 50µM b-mercaptoethanol and 1mM HEPES.

[0208] In vitro chemotaxis assays. L1-2 cells were stably transfected by electroporation with a pcDNA3.1 encoding human GPR15, human GPR25 or human CMKLR1 as described. L1-2 transfectants were routinely maintained under G418500 µg / ml and stimulated with 5 mM butyrate 16 hours before the assay.

[0209] Human PBMCs were isolated from heparinized peripheral blood (10–40 ml) that was obtained via venipuncture from healthy donors. Blood samples were processed using ficoll density gradient centrifugation (Histopaque-1077, Sigma-Aldrich). The resulting interface containing the PBMC layer was extracted, washed twice with PBS, and resuspended in RPMI- 10. Tonsil cells were mechanically isolated from the surgical specimens and purified with a ficoll density gradient centrifugation as well. Cells were stored frozen in FBS with 10% DMSO until assayed. Frozen PBMC and tonsil cells were incubated for 2 hours at 37C in chemotaxis medium (RPMI-10) to recover. Transwell chemotaxis assays were performed as previously described, with the exception of the checkerboard assay where in some wells human 4CysCXCL17 was placed at 250nM both at the top and bottom chambers of the transwell insert. Cells were migrated for 3 hours, and migrated and input cells analyzed by flow cytometry to assess the percent of input cells that migrated to the bottom well.

[0210] GIG. 16J and L subsets were defined with the following immunophenotyping: Naive (CD45RO- CD45RA+) or indicated effector / memory (CD45RO+ CD45RA-) TCR⍺β+ CD4+ subsets were defined with MAbs to intracellular Foxp3 and CD25 (Tregs), and CD161, a marker of mucosal tissue homing T cells. 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+. Monocytes were defined as CD3- CD19- CD14+, HLA / DR+.

[0211] Human CCL25, GPR15LG and CXCL12 were purchased from Peprotech. Human chemerin mouse 6CysCXCL17 and human 6CysCXCL17 were purchased from R&D. Human 4CysCXCL17 and human 4CysCXCL17 truncated in the C-terminus (lacking the last 4 aminoacids, ALPL) were provided by Dr. Handel. The following peptides, were purchased from Phoenix Pharmaceuticals: synthesized human 4CysCXCL17, mCXCL17 26aa C-term, bradykinin, apelin-36 and apelin-32. Fortessa (BD) flow cytometer and FlowJo software were used for analyses of input and migrated cells.

[0212] Antibody secreting cells were quantified by ELISPOT for IgG and IgA as previously described.

[0213] Human blood T CD4 cells in vitro chemotaxis analyzed by scRNAseq. Blood was collected by venipuncture from a healthy donor and PBMC were isolated with a ficoll gradient. T cells were isolated by negative magnetic selection using the total T cell isolation kit (STEMCELL technologies). T cells were then rested for 2 hours in RPMI-10 at 37C and subsequently used as input in a transwell-based chemotaxis assay using h4CysCXCL17 300nM as chemoattractant as described above. Migrated cells were recovered and along with the corresponding input samples, were separately hash tagged and finally mixed together in preparation for a single 5’ 10x Genomics scRNAseq capture. The captured sample was subsequently processed with the 10x genomics v3 kit and sequenced on the Illumina NovaSeq X platform. CD4 Treg and Tem cells were identified as indicated in the “Single-cell RNA-seq data analysis” section, and % migration of subsets defined by GPR25 expression levels were determined as for flow cytometry based assays above.

[0214] Human tonsil B and T cell scRNAseq experiment. A human tonsil specimen obtained by surgical resection was mechanically processed and cells were preserved frozen. Cells were later thawed and stained with the following flow cytometry panel: anti human CD45RA-FITC (HI100), anti human HLA -PercpCy5.5 (L243), anti human TCRαβ-BUV737 (IP26) and anti human IgD-BUV395 (IA6-2) antibodies from BD: anti human CD69-PECy7 (FN50), anti human CD45RO-BV605 (UCHL1), anti human CD8-BV421 (SK1), anti human CD4-BV786 (SK3), anti human CD38-APCCy7 (HIT2) and anti human CD19-AF700 (HIB19) antibodies from Biolegend. Next, cells were FACS sorted as follows using an Aria lll sorter (BD) and the indicated number of cells from each subset were combined in preparation of two separate 5’ 10x genomics scRNAseq captures.

[0215] T cell sample: CD45RA+CD45RO+ CD4+ T Cells (50K), HLADR+CD38+ / - CD4 T Cells (20K) and total TCRαβ+ CD4+ cells (10K).

[0216] B cell sample: TCRαβ- CD4- CD8- (20K), TCRαβ- CD4- CD8- IgD- (20K), TCRαβ- CD4- CD8- IgD- CD38+ (20K), TCRαβ- CD4- CD8- IgD- CD38+++ (15K). The captured samples were subsequently processed with the 10x genomics v2 kit and sequenced on the Illumina NovaSeq platform. Data was analyzed as indicated in the “Single-cell RNA-seq data analysis” section below.

[0217] Single-cell RNA-seq data analysis

[0218] Data integration and processing. Published 5’ 10x Genomics scRNAseq data from the human airway, lung, stomach, pancreas, salivary gland, CSF, cervix, small intestine, colon, skin, blood, thymus, and lymph node were processed to evaluate leukocyte chemoattractant receptor expression. 5’ 10X Genomics scRNAseq data from the Human Protein Atlas was analyzed for cell type-specific GPR25 expression, and 3’ or 5’ 10X data from the human airway, gut, brain and the mouse brain and spinal cord were assessed for CXCL17 expression. Per cell normalization of raw count data was performed using the deconvolution method implemented in the computeSumFactors function from the “scran” package. Batch correction was performed using the multibatchNorm function from the “batchelor” package. Count data was log transformed using the logNormCounts function from the “scater” package with a pseudo count of 1. When published UMAP coordinates were unavailable, data was integrated using the fastMNN function from the “batchelor” package (v1.0.1) and UMAPs were computed using the R implementation from the umap package. Gene imputation was performed on log- normalized count data within each batch using the original implementation of the MAGIC (Markov Affinity-based Graph Imputation of Cells) algorithm with optimized parameters (t = 2, k = 9, ka = 3). Imputed data was used in all figures and for trajectory analysis using tSpace.

[0219] Cell type identification. Gating for T and B cells were performed on the integrated datasets. For T cells, only cells that were CD45+, CD14- and CD3E-high were included for downstream analysis. Innate T cells including NK, NKT, MAIT, γδT and ILC were defined according to the original labels of the respective datasets. CD4 T cells were defined as CD4- high, CD8A-low and CD8B-low. Subsets of CD4 T cells were defined sequentially as follows: regulatory T cells (Tregs) were defined as FOXP3+ and IL2RA+; T helper 17 cells (Th17) were defined as FOXP3- and IL17A / F+; CD4 naïve cells were defined as FOXP3-, IL2RA-, CCR7- high, SELL-high, and negative for peripherally induced chemoattractant receptors (CXCR3 / 5 / 6, CX3CR1, and CCR1 / 2 / 3 / 4 / 5 / 6 / 8 / 10); T follicular helper cells (Tfh) were defined as FOXP3-, IL2RA-, TOX+ and CXCR5+; from the remaining cells CD4 central memory cells were defined as FOXP3-, CCR7-high and SELL-high; and Type 1 T helper cells (Th1) were defined as FOXP3-, TBX21+ and IFNG+. CD8 T cells were gated as follows: CD8 naïve cells were defined as CCR7-high, SELL-high and negative for the chemoattractant receptors as listed above; CD8 central memory cells were defined as CCR7-high and SELL-high; amongthe remaining cells tissue resident memory cells were defined as ITGAE+; and effector memory cells were defined as ITGAE-. For B cells, plasma cells were defined as per the original labels of the respective datasets. Target tissues lack naïve cells, therefore non-plasma cells were defined as B memory cells, among which cells that were FCRL4-high were defined as FCRL4+ MBC. Tonsil B cell subsets were identified by a combination of Leiden clustering and expression of markers as described. For scRNAseq data of the thymus and tissues analyzed for CXCL17 expression, as well as the bulk RNA-seq data of the PBMC, cell types from the original publications were used for the analyses.

[0220] GPCR binding site similarity analysis. Pairwise GPCR binding site similarity data was obtained from Supplementary Dataset 1 of Ngo et al.The GPCR-CoINPocket score was used to quantify the similarity between GPCR binding sites based on their contact strength profiles. The pairwise similarity data was compiled into a matrix format, and the GPCR labels were matched to official HGNC gene symbols, with any outdated symbols manually updated. Briefly, GPCR-CoINPocket performs a sequence alignment for the transmembrane domains of all GPCRs analyzed, and using the ligand interaction patterns across all Pocketome entries, the pairwise similarities of GPCR TM domain sequences were calculated and normalized to control for evolutionarily-conserved TM domain regions. The normalized similarity scores were averaged to generate a final binding site similarity score.

[0221] Protein analysis for GPR25 ligand search. Isoelectric point (pI) and molecular weight (MW) of human secreted proteins were calculated using the “Peptides” package. Proteins with pI > 8 and MW < 25 kD were identified, and amino acid sequences of the human, mouse, rat, rabbit, dog, cow orthologs were obtained from Ensembl BioMart. To evaluate C-terminal conservation of candidate ligands, sequences of the C-terminal 6 amino acids of all proteins were extracted, and the human C-terminal peptide was aligned with that of each of the other 5 species, where orthologue is present, and sequence similarity was calculated by Protein BLAST. The BLAST bit-scores from all pairwise alignments were averaged for each protein. To determine correlation of tissue expression profiles between C-terminally conserved candidate ligands and GPR25, bulk RNA-seq data for 49 human tissues (excluding lymphoid tissues) were obtained from Human Protein Atlas, and Pearson correlation was performed for the cross-tissue gene expression profiles of GPR25 and the candidate ligands with mean BLAST bit-score > 4.

[0222] Cold competitive binding assay. CHO-K1 cells transfected for human GPR25 expression were from DiscoverX, Inc. (Fremont, CA, USA). GPR25 transfectants or normal CHO-K1 cells were incubated in binding buffer [HBSS Ca2+ / Mg2+with FBS 2% (v / v)] for 30 min at 4C with 100 nM of either human IgG-Fc or a chimeric protein comprising the human 4CysCXCL17 linked to human Fc through the 4CysCXCL17 N-terminus (“tracer”) (Curia Inc., Belmont, CA, USA).

[0223] For “cold competition binding assay,” GPR25 transfectants were pre-incubated for 30 min with control media or with several concentrations (0.3nM to 1µM) of the native h4CysCXCL17 (Phoenix) in binding buffer. Then the “tracer,” here human 4CysCXCL17 linked to human Fc, was added to the cells at a concentration of 100 nM and incubated for 30 min.

[0224] Finally, the cells were washed in staining buffer (PBS BSA 0.5%), then stained with PE-Goat F(ab’)2 anti human IgG (Invitrogen), and acquired on a Fortessa (BD), using FACS Diva Software.

[0225] GPCR activation assay. L1.2 hGPR25-SRF-RE stable transfectants were established by electroporation of pcDNA3.1(+)-N-HA-hGPR25 (Gene script) and pGL4.34[luc2P / SRF- RE / Hygro] (Promega), followed by the selection with G418 (500 µg / ml) and Hygromycin B (400 µg / ml). Cell transfectants were maintained in RPMI-10 with G418 (500 µg / ml) and Hygromycin B (400 µg / ml). The day before the reporter assay, cells were stimulated with 5 mM butyrate for 16 hours and subsequently starved for 2 hours in 2% FBS RPMI complete medium. Next, cells were plated in 12-well plates at 1.5 x 105cells per well and treated with ligands for 4 hours at 37°C / 5% CO2 in RPMI-10. The cells were pelleted and lysed using Lysis Reagent (E1531, Promega). Finally, the luminescence was measured using the Promega ONE-Glo EX Luciferase Assay System using the Turner Biosystems Photometer 20 / 20n luminometer. Results were normalized by protein quantification using the Pierce BCA Protein Assay Kit (Prod #23225) ThermoScientific.

[0226] RNAscope studies. From archival formalin fixed paraffin embedded material, sections were cut at 5 μm. Single molecule in situ hybridization was performed using a proprietary high sensitivity RNA amplification and detection technology (RNAscope, Advanced Cell Diagnostics), according to the manufacturer’s instructions using the indicated proprietary probes and the RNAscope 2.5 HD Duplex Reagent Kit. Following in situ hybridization, sections were counterstained with hematoxylin before analysis and imaging with a light microscope (Olympus BX53) and attached camera (Olympus DP73). Proprietary (Advanced Cell Diagnostics) probes used were: Human, Hs-CXCL17 (513241) and Hs-C10orf99-C2 (437401- C2).

[0227] Production of recombinant human 4CysCXCL17 and its C-term mutant in E. coli. Recombinant human CXCL17-4C (containing residues 64-119 of human CXCL17) or its C- term truncated mutant (containing residues 64-115) were cloned onto the 3’ end of His8-tagged ubiquitin-3D3 in a pHUI3D3 vector. The plasmids were transformed into One Shot™ BL21(DE3)pLysS Chemically Competent E. coli cells (Invitrogen™). Cells were grown to an optical density of 0.6-0.7 in Luria-Bertani medium at 37C, and protein expression was induced by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside. After 6 hours of induction at 37C, cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris, pH 7.5, 150mM NaCl) with addition of benzonase (New England Biolabs), and lysed by sonication. The cell pellet containing CXCL17-4C inclusion body was spun down at 12,000x g, 4C for 15 minutes. The pellet was then solubilized and sonicated in 50 mM Tris, 6 M guanidine-HCl at pH 8, and centrifuged at 12,000x g, 4C for 15 minutes. The resulting supernatant was passed over a gravity column filled by Ni-NTA agarose (Invitrogen™). The resin was washed with 50 mM MES, 6 M guanidine-HCl at pH 6, and protein was eluted with 50 mM sodium acetate, 6 M guanidine-HCl at pH 4. The eluate was adjusted to pH 7 and reduced by 4 mM DTT at room temperature for 2 hours. Then it was diluted 25-fold with refolding buffer (50 mM Tris, 500 mM arginine-HCl, 1 mM EDTA, 1 mM glutathione disulfide, pH 7.5) and slowly stirred at 4 ℃ overnight. The refolded protein was dialyzed in 20 mM Tris, pH 8.0 and 200 mM NaCl. After dialysis, His8-tagged ubiquitin-3D3 was cleaved by home-made deubiquitylating enzyme Usp2cc for approximately 48 hours. The cleaved product was loaded to a reversed-phase Vydac™ 218TP C18 HPLC Column (Grace™) (buffer A: 0.1% trifluoracetic acid; buffer B: 0.1% trifluoroacetic acid; 90% acetonitrile). With linearly increasing buffer B concentration from 33 to 45%, pure CXCL17-4C and the C-term truncated mutant were eluted at around 37% and 35% of buffer B, respectively. Formation of two disulfide bonds in each protein was confirmed through conducting intact protein analysis by MALDI-TOF and ESI-TOF mass spectrometry in Center for Metabolomics and Mass Spectrometry, The Scripps Research Institute. The pure proteins were lyophilized and stored at −80 °C until usage.

[0228] Immunofluorescence studies. Tissues were harvested and fixed in 4% PFA for one hour, washed in PBS, and incubated in 30% sucrose for 24 hours at 4°C slowly shaking. Tissues were then embedded in OCT, flash frozen on dry ice, cut into 10 μm cryosections and stored at -80°C. On the day of staining, slides were thawed, washed consecutively in PBST (0.4% TWEEN® 20, 4% BSA in PBS). Slides were blocked in blocking buffer (2% goat serum, 0.3 % triton in PBS). Slides were then incubated overnight at 4°C with conjugated antibodies APC-anti mouse TCRβ (clone H57-597) 1:50, from Biolegend; PE-anti mouse CD103 (clone 2E7) 1:50 from Biolegend, as well as the following primary antibodies, the anti mouse CD8α (clone 4SM15) from Invitrogen or the anti mouse CD4 (clone RM4-5) from Biolegend (1:100 dilution for both) CD8α Invitrogen 14-0808-82; 1:100, CD4 Biolegend100506). Slides were then washed in PBST 3 times for 10 mins followed with blocking with 5% of mouse serum for 10 mins. Slides were then incubated with goat anti-rat IgG cross-adsorbed AF488 secondary antibody (1:50) from invitrogen for 45 mins at room temperature. Slides were then washed in PBST 3 times for 10 mins and closed with Fluoromount-G (Southern Biotech). Imaging was performed with a Nikon or Zeiss 880 confocal microscope. Imaris and Image J software were used for image processing.

[0229] Immunohistochemistry studies. Human cerebella were from post-mortem tissue, formalin fixed and embedded in paraffin via standard methods. After deparaffinization and re- hydration to water, slides were placed in 3% hydrogen peroxide for 15 minutes to quench endogenous peroxidase.10mM citric acid (pH6) was used for antigen retrieval for 25 minutes at 95C. Normal goat serum was used for 30min at room temperature to block unspecific binding before overnight incubation at 4C with the primary anti-human CXCL17 (clone 422204, R&D) or isotype control (clone G3A1 mouse IgG1, Cell Signaling) antibodies. Next, the secondary polymerized goat anti mouse IgG ImmPRESS (peroxidase) kit from Vectorlabs was used. Finally, sections were rehydrated in ethanols, and cleared in xylenes before coverslipping with synthetic mounting media. In figure supplementary figure 9 DAB is shown in images without counterstain. Images were captured with an slide digital scanner Aperio AT2 operated with the v12.4.3.5008 software.

[0230] Data and statistical analysis. Results are expressed as mean ± SEM and individual data points represent biological replicates. Statistics were calculated using Fisher's exact test or multivariate regression in R, or using GraphPad Prism as indicated in the figure legends. Differences were considered significant at P < 0.05. Example 5 A T cell-neuronal chemoaffinity axis in brain resilience

[0231] Recent studies implicate T-cells in cognition, neurodegenerative diseases, brain resilience in aging. The mechanisms involved are poorly understood. Here we highlight GWAS and eQTL analyses that indicate that GPR25 regulatory variants that enhance GPR25 expression reduce susceptibility to neuroinflammation (multiple sclerosis, MS) and autoimmune neural degeneration (narcolepsy). In the CNS, GPR25 is expressed by subsets implicated in both CNS resilience and neurodegeneration, including CD4 effector and regulatory cells (Tregs) and CD8 T cells including CD8 resident memory cells (TRM), a population implicated in context-dependent neuronal maintenance and neurodegeneration. Moreover, neurons express CXCL17, suggesting GPR25 involvement in T-cell recruitment, targeted migration and / or motility for immune surveillance of neurons. To elucidate roles of GPR25 in the brain, GPR25 expression by CNS T-cell subsets is quantified during aging and neurodegeneration in humans, and in blood of patients with GPR25 regulatory variants.

[0232] CXCL17 is protective in EAE, and has a role in inhibiting viral latency in the dorsal root ganglia in the spinal cord. Compared to Cxcl17- / -mice, mice with normal CXCL17 expression have reduced latent herpes virus in the dorsal root ganglia after experimental infection and have reduced susceptibility to experimental allergic encephalomyelitis, implicating CXCL17 in neuroprotection. These data support the physiologic role of the GPR25-CXCL17 axis in endogenous lymphocyte localization and function in barrier immunity.

[0233] GPR25 is expressed by CSF lymphocytes, and its ligand by neurons. These results show a role for the GPR25-CXCL17 axis in neuroimmune interaction, whether in recruitment, motility and surveillance or other functions.

[0234] scRNAseq data (from mouse and human CNS) further confirm selective neuronal expression of CXCL17 (FIG. 26). More importantly, population genetic studies associate blood GPR25 expression levels with CNS traits. The table inserted here provides a compendium of relevant SNPs and associations which together reveal a consistent association of variants that enhance blood GPR25 expression with reduced diagnosis of Multiple Sclerosis and Schizophrenia, and reduced measures of cognition (Cognitive Performance, measures of Educational Attainment).database reports Summary-data-based Mendelian Randomization (SMR) for GPR25 eQTLs for CNS related conditions. SMR leverages genetic variants that affect gene expression as instrumental variables to assess whether gene expression may mediate a disease or trait. Whole blood GPR25 expression correlated negatively with Multiple Sclerosis (SMR p value=1.5e-9), Schizophrenia (p=1.67e-5), Educational Attainment (p=2.2e-5) and Cognitive Performance (p=1.06e-4)."

[0236] The genetic association of GPR25 expression variants with multiple sclerosis and measures of cognition, along with the mouse studies showing that CXCL17 protects against EAE and against viral infection of neurons of the dorsal root ganglia, provides a strong rationale for emphasizing the surprising CNS connection.

[0237] GPR25-defined T cell subsets and GPR25 expression levels in aging and neurodegeneration. GPR25 gene expression is evaluated in public 5' scRNA datasets from brain, spinal cord and CSF immune cells. (These analyses are limited to 5' 10x protocols, as GPR25 is essentially undetectable in 3 prime scRNA datasets, likely due to a short polyA tail) When indicated to complement existing data, lymphocytes are isolated from these tissues from old and young donors for scRNAseq. This allows characterization of GPR25+ T cell subsets in normal subjects at different ages, different brain regions, and correlation of CNS and blood phenotypes.

[0238] Preliminary analysis of a CSF dataset reveal differential GPR25 expression by CD4 and CD8 T cell subsets within a context of patient variation. GPR25 is highly expressed by Foxp3+ CD8 TRM in healthy donors compared to donors with reduced cognition, while Foxp3- CD8 TRM shows the reverse pattern. Foxp3+ CD8 T cells share immune suppressive / regulatory features with conventional CD4 Foxp3+ Treg; which display similarly higher GPR25 levels in healthy donors. Plots of GPR25 vs age suggests that GPR25 may be progressively downregulated on T cells with age.

[0239] Identification of the T cell subsets affected by cognition / MS-associated regulatory variants in GPR25 expression. The GTEX consortium GWAS / eQTL database includes patients with single nucleotide variants (SNVs) reported to link GPR25 expression with disease and cognition. Patients with the SNVs that originally defined these association are identified. Variant GPR25 expression on specific subsets (e.g. Tregs vs CD8 TRM or NK T cells) is correlated with metadata measures of cognition (cognitive performance, educational attainment) and neuroinflammation (e.g. MS, Narcolepsy). Aliquots of cells are frozen for future studies with anti-GPR25 antibodies, and for chemotaxis assays to correlate protein expression and function to altered gene levels.

[0240] Competitive assays with Gpr25 transduced T cells: GPR25 or control vectors are retrovirally transduced into activated T-cells using a retroviral MSCV vector with an IRES Thy1.1 or IRES EGFP or mScarlet3 reporter. For short term homing, transduced cells expressing the Thy1.1 constructs are purified with anti-Thy1.1 beads (Miltenyi), labeled with CellTrace dyes (e.g. CFSE or CellTrace FarRed) and injected iv. To discriminate intravascular vs extravasated T cells, anti-CD31 is injected just prior to sacrifice, highlighting endothelium. Localization of co-injected GPR25 and control cells to the meningeal, CNS and choroid plexus vasculature, and entry into the brain is evaluated at various times (10-72 hr) by whole mount and fixed section confocal microscopy. We compare localization in young adult vs aged recipients (NIH aged rodent resource, or in house), in 3xTAD mice, (McNerney lab) and if indicated in mice with active CNS inflammation (e.g. in experimental autoimmune encephalitis (EAE) models). For longer term studies (1 week-3 months), cells are quantified by flow cytometry (using CD45.1 vs .2 allotype markers to distinguish the transductants). Use of EGFP and Scarlet reporter vectors improve quantification of CNS GPR25 and control transduced T cells, and allow visualization of microenvironmental localization using whole brain imaging / light sheet or brain slice / 2 photon microscopy. In some assays, lymphocyte-deficient (RAG KO) recipients are used to reduce competition with endogenous T cells. These assays will directly assess the effects of GPR25 expression on lymphocyte recruitment and subsequent population of CNS compartments.

[0241] To ask if endogenous GPR25 affects CNS T cell localization, mixed Gpr25 WT (CD45.1) / KO(CD45.2) bone marrow chimeras are generated in which WT and GPR25deficient cells compete for tissue repopulation. Improved C57BL / 6-CD45.1 knockin mice are used as WT donors. WT(CD45.1) / WT(CD45.2) chimeras are generated as additional controls. The ratio of WT to KO cells in CNS, assayed by flow cytometry is compared with that in spleen and lymph nodes as control organs that lack CXCL17. References

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[0297] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed thatperform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. Sequences (SEQ ID NO:1) Human GPR25 protein Residues 40-60; 76-96; 127-147; 156-176; 201-220; 243-263 and 290-310 are annotated as transmembrane regions. MAPTEPWSPSPGSAPWDYSGLDGLEELELCPAGDLPYGYVYIPALYLAAFAVGLLGNAFVV WLLAGRRGPRRLVDTFVLHLAAADLGFVLTLPLWAAAAALGGRWPFGDGLCKLSSFALAG TRCAGALLLAGMSVDRYLAVVKLLEARPLRTPRCALASCCGVWAVALLAGLPSLVYRGLQP LPGGQDSQCGEEPSHAFQGLSLLLLLLTFVLPLVVTLFCYCRISRRLRRPPHVGRARRNSLR IIFAIESTFVGSWLPFSALRAVFHLARLGALPLPCPLLLALRWGLTIATCLAFVNSCANPLIYLL LDRSFRARALDGACGRTGRLARRISSASSLSRDDSSVFRCRAQAANTASASW (SEQ ID NO:2) Human C-X-C motif chemokine 17 precursor Residues 1-21 are annotated as a signal sequence. The mature peptide is annotated as residues 64-119. MKVLISSLLLLLPLMLMSMVSSSLNPGVARGHRDRGQASRRWLQEGGQECECKDWFLRA PRRKFMTVSGLPKKQCPCDHFKGNVKKTRHQRHHRKPNKHSRACQQFLKQCQLRSFALP L

Claims

WHAT IS CLAIMED IS:

1. A method of modulating localization of immune cells to CXCL17-expressing tissues, the method comprising: modifying the interaction between CXCL17 and an immune cell expressing GPR25.

2. The method of claim 1, wherein the CXCL17-expressing tissues are lungs or airways of a mammal.

3. The method of claim 1, wherein the CXCL17-expressing tissues are non-intestinal mucosal tissues (NIMT) or exocrine glands.

4. The method of claim 3, wherein the CXCL17-expressing tissues are selected from upper GI tract, conjunctiva, exocrine glands, stomach, esophagus, pancreas, urinary tract, serous, mucous and sebaceous glands, prostate, oral and vaginal squamous mucosae.

5. The method of claim 1, wherein the CXCL17-expressing tissue is the central nervous system.

6. The method of claim 1, wherein the CXCL17-expressing tissue is small or large intestines.

7. The method of claim 1, wherein the CXCL17-expressing tissue is skin.

8. The method of any of claims 1-7, wherein the mammal is a human.

9. The method of any of claims 1-8, wherein the interaction between CXCL17 and its receptor GPR25 is inhibited by administering an inhibitor of the interaction.

10. The method of claim 9, wherein the inhibitor is an antibody, or a chimeric antigen receptor, specific for CXCL17 or GPR25.

11. The method of claim 10, wherein the inhibitor is a soluble, non-activating fragment of CXCL17 or GPR25.

12. The method of claim 10, wherein the inhibitor is a soluble, activating fragment of CXCL17 or GPR25 that can desensitize or altenatively disrupt gradient perception.

13. The method of any of claims 1-12, wherein lymphocyte localization is inhibited.

14. The method of any of claims 1-8, wherein the interaction between CXCL17 and its receptor GPR25 is increased by upregulating expression of GPR25 in a cell.

15. The method of any of claims 1-8, wherein the interaction between CXCL17 and its receptor GPR25 is modulated by administering an activator of the interaction.

16. The method of any of claims 1-8, wherein the interaction between CXCL17 and its receptor GPR25 is modulated by increasing local availability of CXCL17 in a tissue of interest.

17. The method of any of claims 1-16, wherein the immune cell is a lymphocyte.

18. The method of claim 15, wherein the lymphocyte is a T cell or a B cell.

19. The method of claim 16, wherein the T cell is an effector T cell, helper T cell, regulatory T cell, or CAR T cell.

20. A method of monitoring or quantitating immune cell trafficking and activity in CXCL17 expressing tissues, the method comprising determining the presence of immune cells expressing GPR25 in blood or biopsies.

21. A method of screening candidate agents for activity in modulating lymphocyte trafficking to tissues expressing CXCL17, the methods comprising determining the activity of a candidate agent in altering the interaction between CXCL17 and GPR25.

22. A method of screening candidate agents for activity in modulating lymphocyte trafficking of cells expressing GPR25, the methods comprising determining the activity of a candidate agent in modulating GPR25 activity.

23. The method of claim 22, wherein the candidate alters the interaction between CXCL17 and GPR25.

24. The method of any of claims 21-23, wherein the candidate agent is an antibody, polypeptide, aptamer, or small molecule.

25. An immunogenic composition comprising a binding moiety specific for GPR25 for use in modulating lymphocyte trafficking.

26. A method to increase localization of engineered immune cells to CXCL17 expressing tissues, the method comprising expressing GPR25 or a variant thereof in the engineered cells prior to in vivo transfer.

27. The method of claim 26, wherein the engineered cells are CAR T cells.

28. A method to increase localization of immune cells a tissue of interest, the method comprising inducing expression of GPR25 on the immune cells to increase interaction with a ligand for localization.

29. The method of claim 28, wherein the ligand is CXCL17.

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