Epidermal Sema4A expression is downregulated in psoriasis.

(A) UMAP plots, generated from single-cell RNA-sequencing data (GSE220116), illustrate cell distributions from control (Ctl) and psoriatic lesion (L) samples (n = 10 for Ctl, n = 11 for L). (B) Subclustering of immune cells. (C) SEMA4A expression in keratinocytes, dendritic cells, and macrophages. ****padj < 0.001. NS, not significant. Analyzed using Python and cellxgene VIP. (D) Representative immunohistochemistry and magnified views showing Sema4A expression in Ctl, psoriatic non-lesion (NL), and L. Scale bar = 50 μm. (E) Mean epidermal (Epi) Sema4A intensity in immunohistochemistry (n = 10 per group). Each dot represents the average intensity from 5 unit areas per sample. (F) Relative SEMA4A expression in Epi (n = 10 for Ctl, L, n = 7 for L and NL) and dermis (Derm, n = 6 per group). (G) Proportions of Sema4A- expressing cells in blood CD4 T cells (left), CD8 T cells (middle), and monocytes (right) from Ctl and psoriatic (Pso) patients. (n = 13 per group in CD4 and CD8, n = 11 for Ctl and n = 13 for Pso in Monocytes). (H) Serum Sema4A levels in Ctl (n = 20) and Pso (n = 60). E to H: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant. Figure 1-source data 1 Excel file containing quantitative data for Figure 1.

Imiquimod-induced Psoriasis-like dermatitis is augmented in Sema4AKO mice.

(A) Experimental scheme. Wild-type (WT, green) mice and Sema4A knockout (KO, black) mice were treated with 10 mg/ear of 5% Imiquimod (IMQ) for 4 consecutive days. Samples for flow cytometry analysis were collected on Day 4. (B) Ear thickness of WT mice and KO mice on Day 4 (n = 15 per group). (C) Relative expression of Il17a in epidermis (n = 5 per group). (D and E) The percentages of Vγ3, Vγ2, Vγ2-Vγ3-γδ (DNγδ), CD4, and CD8 T cells (D) and those with IL-17A production (E) in CD3 fraction in the Epi (top) and Derm (bottom) of WT and KO ears (n = 6 per group, each dot represents the average of 4 ear specimens). (F) The percentages of IL-17A- producing γδ, CD4, and CD8 T cells in CD3 fraction in skin-draining lymph nodes (dLN) (n = 9 per group). B-F: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant. Figure 2-source data 1 Excel file containing quantitative data for Figure 2.

Sema4A in keratinocytes may play a role in preventing murine psoriasis- like dermatitis.

(A) Experimental scheme for establishing BM chimeric mice. (B) IMQ Day 4 ear thickness in the mice with the indicated genotypes (n = 14 for WT→WT, n = 13 for WT → KO, n =9 for KO→ WT, n = 9 for KO→ KO). (C) The percentages of IL-17A- producing Vγ3, Vγ2, DNγδ, CD4, and CD8 T cells in CD3 fraction from IMQ Day 4 Epi (top) and Derm (bottom) of the ears from WT→ WT mice and WT→ KO mice (n = 6 per group). Each dot represents the average of 4 ear specimens. B-C: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant. Figure 3-source data 1 Excel file containing quantitative data for Figure 3.

Naïve Sema4AKO epidermis is thicker than WT epidermis with increased γδ T17 infiltration.

(A) Ear thickness of WT mice and KO mice at week (Wk) 8 (n = 15 per group) and representative images. (B) Left: representative Hematoxylin and eosin staining of Day 0 back and Wk 8 back and ear. Scale bar = 50 μm. Right: Epi and Derm thickness in Day 0 back (n = 5) and Wk 8 back (n = 5) and ear (n = 8). (C) Relative Sema4a expression in WT Epi and Derm (n = 5 per group). (D) The percentages of the IL-17A-producing Vγ3, Vγ2, DNγδ, CD4, and CD8 T cells in CD3 fraction (n = 4 per group) in Epi (top) and Derm (bottom). Each dot represents the average of 4 ear specimens. (E) The graphs showing the percentages IL-17A-producing γδ, CD4, and CD8 T cells in CD3 fraction from draining LN (dLN) of WT mice and Sema4AKO mice (n = 6 per group). A-E: *p < 0.05, **p < 0.01, ****p < 0.0001. NS, not significant. Figure 4-source data 1 Excel file containing quantitative data for Figure 4.

Sema4AKO skin shares the features of human psoriatic NL.

(A and B) The volcano plot (A) and Gene ontology (GO) analysis (B), generated from RNA-sequencing data (GSE121212) using RaNA-seq, display changes in gene expression in psoriatic NL compared to Ctl. (C) The difference in the expression of epidermal differentiation markers between Ctl and NL (n = 38 for Ctl, n = 27 for NL) was calculated with the transcripts per million values. **padj < 0.01. NS, not significant. (D) Relative gene expression of epidermal differentiation markers between wk 8 Epi of WT mice and KO mice (n = 5 for Krt14 and Krt16, n = 8 for Krt5, Krt10, Filaggrin, and Loricrin). (E) Left: Representative immunofluorescence pictures of Krt5, Krt10, Krt14, and Krt16 (red) overlapped with DAPI. Scale bar = 50 μm. Right: Accumulated graphs showing the numbers of Krt5, Krt10, Krt14, and Krt16 positive cells per 100 μm width (n = 5 per group) of wk 8 ear (right). Each dot represents the average from 5 unit areas per sample. (F) Transepidermal water loss (TEWL) in back skin of WT mice and KO mice at wk 8 (n = 5 per group). D-F: *p < 0.05, **p < 0.01. NS, not significant. Figure 5-source data 1 Excel file containing quantitative data for Figure 5.

mTOR signaling is upregulated in the epidermis of psoriatic NL and Sema4AKO mice.

(A) Representative results of immunohistochemistry displaying cells positive for p-S6 (Ser235/236), S6, p-Akt (Ser473), and Akt in Ctl, NL, and L. The graphs of accumulated data show the mean intensity of p-S6 and S6 in the upper and lower epidermal layers (n = 9 per group). Scale bar = 100 μm. Each dot represents the average mean intensity from 5 unit areas per sample. (B) The mean intensity of p-S6 (Ser235/236) and p-Akt (Ser473), detected by immunohistochemistry in the epidermis of WT mice and KO mice, were analyzed. Scale bar = 50 μm. Each dot represents the average intensity from 5 unit areas per sample (n = 8 per group). (C and D) Immunoblotting of p-S6 (Ser235/236), S6, p-Akt (Ser473), and Akt in tissue lysates from epidermis without treatment (C) and with IMQ treatment for consecutive 4 days (D) (n = 5 per group, except for p-Akt and Akt in C, for which n = 4). A-D: *p < 0.05, **p < 0.01. NS, not significant. Figure 6-source data 1 Excel file containing quantitative data for Figure 6.

Inhibitors of mTOR signaling modulate the expression of cytokeratins in Sema4AKO mice.

(A and B) Epidermal thickness of Sema4AKO mice treated intraperitoneally with vehicle (Ctl) or rapamycin (A), and Ctl or JR-AB2-011 (B) (n = 5 per group). (C and D) Relative expression of keratinocyte differentiation markers and Il17a in Sema4AKO Epi (C), and the number of T cells in Epi and Derm under Ctl or rapamycin (D) (n = 5 per group). (E and F) Relative expression of keratinocyte differentiation markers and Il17a in Sema4AKO Epi (E), and the number of T cells in Epi and Derm under Ctl or JR- AB2-011 (F) (n = 5 per group). D and F: Each dot represents the sum of numbers from 10 unit areas across 3 specimens. A-F: *p < 0.05, **p < 0.01. NS, not significant. Figure 7-source data 1 Excel file containing quantitative data for Figure 7.

Sema4A is downregulated in the keratinocytes of lesional psoriasis in the single-cell RNA-sequencing data.

(A) Sample information for specimens from Ctl and psoriatic L (GSE220116). (B and C) Clusters of cells were identified by their expression patterns of signature genes. (D) The volcano plot displays changes in gene expression in psoriatic L compared to Ctl. Figure 1-figure supplement 1-source data 1 Excel file containing quantitative data for Figure 1-figure supplement 1.

Gating strategy in flow cytometry.

Gating strategy for human Sema4A expression in blood cells. Large and small cells were distinguished using forward scatter (FCS) and side scatter (SSC) in a dot plot panel, with dead cells being excluded. Monocytes were defined within the live large cell population as CD11c positive. CD4 and CD8 T cells were identified within the live small cell population as CD3 positive CD4 positive and CD3 positive CD8 positive populations, respectively. The empty histogram represents the flow cytometry minus one control for Sema4A.

Gating strategy in flow cytometry.

Gating strategy for murine T cells infiltrating the epidermis and dermis. After excluding dead cells, TCRγδ positive T cells were evaluated for the expression of Vγ2. TCRγδ positive Vγ2 negative population was further assessed the expression of Vγ3. The CD3 positive TCRγδ negative population was evaluated for the expression of CD4 and CD8. Each population was analyzed for cytokine production. The empty histogram represents the isotype control for IL-17A.

IL-23-mediated psoriasis-like dermatitis is augmented in Sema4AKO mice.

(A) An experimental scheme involved intradermally injecting 20 μl of phosphate- buffered saline containing 500 ng of recombinant mouse IL-23 into both ears of WT mice and KO mice for 4 consecutive days. Samples for following analysis were collected on Day 4. (B and C) Ear thickness (B) and Epi and Derm thickness (C) of WT mice and KO mice on Day 4 (n = 12 per group). (D and E) The percentages of Vγ3, Vγ2, DNγδ, CD4, and CD8 T cells (D) and those with IL-17A production (E) in CD3 fraction in the Epi (top) and Derm (bottom) of WT and KO ears (n = 5 per group). Each dot represents the average of 4 ear specimens. B-E: *p < 0.05, **p < 0.01. NS, not significant. Figure 2-figure supplement 2-source data 1 Excel file containing quantitative data for Figure 2-figure supplement 2.

T cells’ fractions infiltrating in the chimeric mice ear.

(A) Sema4a expression in the Epi of WT→ WT mice and WT→ KO mice (n = 8for WT→ WT, n = 7 for WT→ KO). (B) The percentages of Vγ3, Vγ2, DNγδ, CD4, and CD8 T cells in CD3 fraction from IMQ Day 4 Epi (top) and Derm (bottom) of the ears from WT→ WT mice and WT→ KO mice (n = 6 per group). Each dot represents the average of 4 ear specimens. A-B: *p < 0.05, ***p < 0.001. NS, not significant. Figure 3-figure supplement 1-source data 1 Excel file containing quantitative data for Figure 3-figure supplement 1.

Naive Sema4AKO skin shows upregulation of psoriasis related genes and an increase in resident memory T cells.

(A) Relative expression of psoriasis-associated genes in Epi (top) and Derm (bottom) of WT mice and KO mice (n = 5 per group, #: not detected). (B) Representative dot plots showing CD69 and CD103 expression in the indicated T cell fractions from whole skin. The graphs show T cell counts per ear (top) and those with resident memory phenotype (bottom) (n = 7 per group). Each dot represents the average of 4 ear specimens. A-B: *p < 0.05, **p < 0.01. NS, not significant. Figure 4-figure supplement 1-source data 1 Excel file containing quantitative data for Figure 1-figure supplement 1.

Expression of IFNγ and IL-4is comparable between naive WT and Sema4AKO skin.

(A) The percentages of Vγ3, Vγ2, DNγδ, CD4, and CD8 T cells in CD3 fraction from naive WT and KO mice. (B and C) The graphs presenting the percentages of IFNγ (B) and IL-4 (C) -producing Vγ2, DNγδ, CD4, and CD8 T cells in CD3 fraction in the Epi (top) and Derm (bottom) of naive WT mice and KO mice (n = 4 per group). A-C: Each dot represents the average of 4 ear specimens. *p < 0.05. NS, not significant. Figure 4-figure supplement 2-source data 1 Excel file containing quantitative data for Figure 4-figure supplement 2.

Comparable T17differentiation potential under Th17-skewing conditions between WT mice and Sema4AKO mice.

Splenic T cells were cultured for 2 weeks, followed by flow cytometry analysis. The accumulated data display the percentages of IL-17A-producing (right) and IFNγ- producing (left) γδ, CD4, and CD8 T cells within CD3 fraction under various conditions: IL-23 dependent Th17-skewing condition (top), IL-23 only Th17-skewing condition (middle), and IL-23 independent Th17-skewing condition (bottom) . NS, not significant. Figure 4-figure supplement 3-source data 1 Excel file containing quantitative data for Figure 4-figure supplement 3.

The epidermis of psoriatic non-lesion is thicker than that of control skin.

Epidermal thickness of Ctl and psoriatic NL (n = 10 per group). ***p < 0.001. Figure 5-figure supplement1-source data1 Excel file containing quantitative data for Figure 5-figure supplement1.

Upregulation of cytokeratin expression related to psoriasis is not detected at birth in Sema4AKO mice.

Representative immunofluorescence pictures of Krt5, Krt10, Krt14, and Krt16 (red) overlapped with DAPI, and the accumulated graphs showing the numbers of Krt5, Krt10, Krt14, and Krt16 positive cells per 100 μm width (n = 5 per group) in the epidermis of Day 0 back. Scale bar = 50 μm. Each dot represents the average from 5 unit areas per sample. NS, not significant. Figure 5-figure supplement 2-source data 1 Excel file containing quantitative data for Figure 5-figure supplement 2.

Topical application of rapamycin gel yields partially similar results to intraperitoneal treatment.

(A) Comparison of Epi thickness between vehicle (Ctl) gel-treated right ears and rapamycin gel-treated left ears of Sema4AKO mice (n = 10 per group). (B) Relative expression of keratinocyte differentiation markers and Il17a in Sema4AKO Epi under Ctl gel or rapamycin gel treatments (n = 5 per group). (C) The number of T cells in the Epi (left) and Derm (right), under Ctl gel or Rapamycin gel treatments (n = 5 per group). Each dot represents the sum of numbers from 10 unit areas across 3 specimens. A-C: *p < 0.05, **p < 0.01. NS, not significant. Figure 7-figure supplement 1-source data 1 Excel file containing quantitative data for Figure 7-figure supplement 1.

Rapamycin treatment reduced the epidermal swelling observed in IMQ-treated Sema4AKO mice.

(A) Experimental scheme. (B) The Epi thickness on Day 18. (n = 10 for Ctl, n = 12 for Rapamycin). (C) Relative expression of keratinocyte differentiation markers and Il17a in Sema4AKO Epi (n = 10 for Ctl, n = 12 for Rapamycin). (D) The number of T cells in the Epi (left) and Derm (right), under Ctl or rapamycin and IMQ treatments (n = 10 for Ctl, n = 12 for Rapamycin). Each dot represents the sum of numbers from 10 unit areas across 3 specimens. A-C: *p < 0.05, **p < 0.01. NS, not significant. Figure 7-figure supplement 2-source data 1 Excel file containing quantitative data for Figure 7-figure supplement 2.

Patient information

Antibodies used for immunohistochemical, immunofluorescence, and western blot analyses

Primer sequences of real-time quantitative PCR used in human sample experiments

Antibodies used for flow cytometry analysis

Mouse recombinant cytokines