Il17a ^Cre and ROSA26 eYFP were kindly provided by Britta Stockinger (The Francis Crick Institute, UK); S1P₁ ^loxP (RRID: IMSR_JAX:019141) were kindly provided from the Cardiovascular Research Center (CVRC) UKE Hamburg. Il17a ^Cre mice were described and characterized previously (22). Moreover, we successfully created Il17a ^Cre x ROSA26 ^eYFP x S1P₁ ^loxP mice (in cooperation with the transgenic core facility of the University Medical Center Hamburg-Eppendorf). All mice were on the C57BL/6J background. For cre-mediated recombination, we generated Il17a ^Cre/+ heterozygous mice (Il17a ^Cre/+ x ROSA26 ^eYFP and Il17a ^Cre/+ x ROSA26 ^eYFPx S1P₁ ^loxP), so that IL-17A production was maintained. In all experiments, we used littermate controls (Il17a ^Cre/+ x ROSA26 ^eYFPx S1P₁ ^loxP/loxP and Il17a ^Cre/+ x ROSA26 ^eYFPx S1P₁ ^wt/wt). All mice were raised under specific pathogen-free conditions. All animal experiments were performed according to national and institutional animal care and ethical guidelines and were approved by the local authorities.

For the induction of imiquimod (IMQ)-induced, psoriasis-like skin inflammation, one ear from 8- to 12-week-old male mice was treated daily with 5 mg IMQ cream (5% IMQ; MEDA Pharma, Bad Homburg, Germany). Control groups received vaseline applications. Thickness of ears was measured daily with a caliper. Based on this measurement, thickness score was calculated with: 0% - 20%: 0, 20% - 35%: 1, 35% - 50%: 2, 50% - 65%: 3, > 65%: 4. In addition, ears were daily scored for erythema and desquamation. Both parameters were independently scored with: 0: no, 1: mild, 2: moderate, 3: strong and 4: severe symptoms. The psoriasis score represents the mean of all three scores. H&E Staining was performed using routine laboratory methods.

Total RNA of the skin (ear) was prepared according to standard laboratory methods. Real-time PCR was performed for 40 cycles on a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA) as previously described (23). All samples were run in duplicate and normalized to 18S rRNA. The following TaqMan probes (Thermo Fischer) were used: Il17a (Mm00439618), S1pr1 (Mm00514644) and 18S rRNA (Mm03928990).

The leukocytes from murine thymus, lung, lymph nodes, spleen, kidneys, skin, and gut were isolated as previously described (23). Briefly, the various tissues were digested for up to 45 minutes at 37°C by adding 0.4 mg/ml collagenase D (Roche, Mannheim, Germany) and 0.01 mg/mL DNase I (Roche) to RPMI 1640 medium (Life Technologies, Karlsruhe, Germany) supplemented with 10% heat-inactivated FCS (Gibco, Eggenstein, Germany). Subsequently, the tissue was finely minced using the gentleMACS Dissociator (Miltenyi Biotec, Teterow, Germany). Single-cell suspensions were separated using Percoll density gradient centrifugation.

Cells were stained with fluorochrome-labeled antibodies directed against CD45, CD3, CD4, CD8, γδTCR, IL-17A, IFN-γ, CD11b, and Ly6G (Biolegend, BD Biosciences, eBioscience, or R&D Systems), as previously described (24). Flow cytometry measurements were performed using the BD FACS LSR II. Data were analyzed by using the FlowJo software (Tree Star).

The Seurat package (version 5.1.0) was used for unsupervised clustering. For normalization, scaling, and principal component analysis, default parameters were used. Uniform Manifold Approximation and Projection (UMAP) was used to visualize clustering results. Differentially expressed genes in each cluster were calculated by the FindMarkers function (min.pct = 0.1), which included Wilcoxon rank sum tests. To calculate the Th17 score in scRNA-seq data, Seurat function AddModuleScore was used. Thymus scRNA-seq data were obtained from E-MTAB-8581 (only postnatal datasets were used) (https://developmental.cellatlas.io). The spatial transcriptome datasets of the healthy and psoriasis skin were obtained from https://zenodo.org/record/7562864.

The results are shown as the mean ± SEM when presented as a bar graph, or as single data points with the mean in a scatter dot plot. Differences between two individual experimental groups were compared using a two-tailed t test. When three or more groups were analyzed, ANOVA was used. p<0.05 was considered statistically significant.

To explore the development of T cells, including Th17 cells, in lymphoid and barrier organs, we analyzed bulk RNA-sequencing data from the Human Protein Atlas (www.proteinatlas.org) (25). Analysis of key transcription factors in T cell differentiation revealed that RORC, which encodes the transcription factors RORγ and RORγt – crucial for T cell survival in the thymus and the development of Th17 cells, respectively (26–28) – exhibited the highest expression levels in the thymus ( Figure 1A ). The expression of RORC was also observed in barrier organs, including the skin. The predominant expression of RORC in the thymus prompted us to investigate the potential differentiation of T cells into Th17 cells within this organ.

To this end, we examined publicly available single-cell RNA-sequencing (scRNA-seq) data of thymic T cells (29). This dataset encompassed various maturation stages, including double-negative and double-positive cells, alongside more mature T cell subsets ( Figure 1B ). Notably, RORC expression was detectable across most thymic T cells, corroborating its established role in thymic T cell viability ( Figure 1C ). Within the mature T cells, we identified a Th17 cell cluster, characterized by the expression of markers such as CCR6, IL6R, IL23R, IL17A, and IL22 ( Figures 1C–F ). Remarkably, these Th17 cells showed the highest levels of the receptor S1PR1 ( Figure 1G ), pivotal for the egress of lymphocytes from the thymus and secondary lymphoid organs into the bloodstream and peripheral organs (21). The pronounced expression of S1PR1 in thymic Th17 cells suggested their readiness for migration towards peripheral organs.

To delineate the distribution and functional capacity of Th17 cells across various organs, including the thymus and barrier organs, we employed a fate reporter system utilizing Il17a ^Cre/+ x Rosa26 ^eYFP transgenic mice ( Figure 2A ). In this genetically engineered model, T cells expressing Il17a are irreversibly marked with enhanced yellow fluorescent protein (eYFP), enabling the precise identification of T cells which underwent Th17 cell differentiation. Consistent with our prior observations in Figure 1 , CD4⁺ YFP⁺ T cells were detected not only in the expected barrier sites such as the skin, intestine, and mesenteric lymph nodes (mLN), where they showed the highest abundance, but also within the thymus, albeit at lower frequencies ( Figure 2B and Supplementary Figure 1 ).

Prompted by our observation of the highest expression of S1PR1 in Th17 cells in the human thymus, we sought to investigate the receptor’s role in mediating Th17 cell migration to peripheral organs. To this end, we utilized I17a ^Cre/+ x Rosa26 ^eYFP x S1P₁ ^loxP/loxP mice (hereafter, S1pr1 conditional knockout (cKO) mice) ( Figure 3A ). In these mice, cells expressing Il17a are permanently marked as eYFP⁺, while concurrently losing S1pr1 expression, allowing us to specifically examine the impact of S1PR1 deficiency on Th17 cells. For a control group, we used I17a ^Cre/+ x Rosa26 ^eYFP x S1P₁ ^wt/wt mice. We observed a marked reduction in S1pr1 expression in the S1pr1 cKO mice CD4⁺ YFP⁺ T cells compared to the control group, without affecting Il17a expression levels ( Figures 3B, C ). Intriguingly, the frequency of CD4⁺ YFP⁺ T cells was significantly elevated in the thymus of the S1pr1 cKO mice ( Figure 3D ), suggesting that Th17 cells were unable to egress from the thymus. While the prevalence of CD4⁺ YFP⁺ cells in other organs remained unchanged, a notable decline was observed in the skin ( Figure 3D ).

We further examined whether Th17 cells accumulated in the thymus retained their ability to produce IL-17A. In the control group, approximately half of the CD4⁺ YFP⁺ T cells produced IL-17A. However, the S1pr1 cKO mice displayed a significantly reduced proportion of IL-17A production in CD4⁺ YFP⁺ T cells ( Figure 3E ), suggesting a shift away from the Th17 phenotype while they were retained in the thymus. In contrast, the capacity for IL-17A production in the skin and other organs remained unchanged between the control and S1pr1 cKO mice ( Figure 3E ). Collectively, these findings suggest the role of S1PR1 in facilitating Th17 cell migration from the thymus to the skin under physiological conditions.

S1PR1-expressing T cells are recruited by S1P, a ligand for S1PR1. S1P is produced by SPHK1 and SPHK2, which are key enzymes responsible for converting sphingosine to sphingosine-1-phosphate (S1P). These enzymes have redundant functions; therefore, the expression of either enzyme is sufficient for producing S1P and recruiting T cells. To investigate the expression of these enzymes in the skin, we examined gene expression data from the Human Protein Atlas. Notably, SPHK1 was highly expressed in the skin as well as in the appendix, lymph nodes, and bone marrow ( Figure 4A ). The expression of SPHK2 was mainly observed in the intestine. To further analyze the expression of these enzymes in different tissue compartments, we analyzed a publicly available spatial transcriptomics dataset of human skin. Both SPHK1 and SPHK2 were expressed in the epidermal and dermal compartments ( Figures 4B–D ). Once again, SPHK1 showed more dominant expression than SPHK2 in healthy skin.

Th17 cells play key roles in the skin not only in homeostasis but also in autoimmune diseases. In particular, the pathogenic involvement of Th17 cells in psoriasis is well-documented (30–32). Moreover, elevated levels of S1P have been reported in the blood of patients, particularly those with severe forms of the disease (33, 34). We therefore investigated whether the expression of S1P-producing enzymes in the skin increases in psoriasis. By analyzing spatial transcriptome data of healthy control skin and inflamed and non-inflamed skin from psoriasis patients, we found predominant expression of SPHK1 in both the epidermal and dermal compartments ( Figures 5A–C ). Quantification of SPHK1 and SPHK2 expression levels across 17 compartments showed the highest expression in the epidermis 2 compartment compared to other compartments ( Figure 5D ). Notably, the expression of SPHK1 and SPHK2 increased in the inflamed psoriatic skin compared to healthy control skin and non-inflamed skin from psoriasis patients ( Figure 5E ). These observations suggest that Th17 cells might be more efficiently recruited to the skin during inflammation.

To investigate the role of S1PR1-mediated Th17 cell migration to inflamed skin, we utilized an imiquimod (IMQ)-induced murine psoriasis model and assessed the impact of Th17 cell-specific S1pr1 knockout on disease progression ( Figure 6A ). Remarkably, in S1pr1 cKO mice, we observed a significant reduction in Th17 cells within the skin and an increase in the number of Th17 cells in the thymus ( Figures 6B, C ), indicating that S1PR1 plays a key role in Th17 cell trafficking to inflamed skin. Furthermore, the S1pr1 cKO mice exhibited a markedly less severe psoriatic phenotype, as evidenced by histopathological analysis ( Figure 6D ) and reductions in skin desquamation, thickness, erythema, and combined clinical scores compared to controls ( Figures 6E, F ). Taken together, these findings highlight the critical role of S1PR1 in Th17 cell migration during skin inflammation and psoriasis development.