Fig 2: CCL27a-knockout has little effect on the establishment of CCR10+ lymphocytes in the skin(A) Representative FC analysis of gated skin CD45+ immune cells for T cell subsets and ILCs in adult CCL27a-/- and CCL27a+/+ mice.(B) Percentages of T cell and ILC populations from CCL27a-/- versus CCL27a+/+ or CCL27a+/- adult mouse skin. N = 7 mice each for CCL27a-/- and control groups. Unpaired one-tailed Student’s t-test.(C) Representative FC analysis of skin T cell subsets and ILCs from adult CCL27-/-CCR10+/EGFP and CCL27a+/+CCR10+/EGFP mice gated for their CCR10(EGFP)+ subpopulations. CD8+ and CD4+ T cells are gated on CD8+ and CD4+ CD45+Thy1.2+CD3int+ populations respectively. ILCs are CD45+Th1.2+CD3-. ?dT cells are CD45+Thy1.2+CD3int+TCRd+ (excluding CD3high dendritic epidermal ?dT cells).(D) Comparative percentages of CCR10+ subsets of indicated skin lymphocyte populations from CCL27a-/-CCR10+/EGFP versus CCL27a+/+CCR10+/EGFP or CCL27a+/-CCR10+/EGFP adult mice. N = 6 mice each for CCL27a-/- and control group. Unpaired one-tailed Student’s t-test.

Fig 3: Severely impaired migration of CCR10+ lymphocytes into the skin of total CCL27-knockout mice(A) Diagram of CRISPR targeting in total CCL27-knockout mice.(B) Genomic PCR identifying wild-type (+/+), heterozygous (+/-), and homozygous (-/-) total CCL27-KO mice. Band sizes of wild-type and knockout CCL27 alleles are 484 and 607bp, respectively.(C) Immunofluorescent a-CCL27 antibody staining images on 10µm skin sections from CCL27+/+ and CCL27-/- mice. Staining with a nonspecific goat IgG antibody or the secondary antibody (2' Ab) only is included as controls. The dashed lines mark the surface of the skin. N = 3 mice for each genotype.(D) FC analysis of CD45+ cells from the skin and spleen of CCL27+/+ and CCL27-/- recipient mice two days after receiving transfer of sLN cells of CCR10+/EGFP mice. Donor-derived CCR10(EGFP)+ lymphocytes are gated on CD45.1+ cells. N = 6 mice for each group.(E) Total numbers (#) of donor cells and percentages (%) of donor cells that express CCR10(EGFP) in the skin and spleen of CCL27+/+ and CCL27-/- recipient mice. N = 6 mice for each group. *p < 0.05; **p < 0.01; ns: no significant difference. Paired one-tailed t-test.

Fig 4: Preferential localization of CCR10+ lymphocytes around growing hair follicles correlates with high CCL27 expression by follicular keratinocytes(A) Representative immunofluorescent skin sections (14µm) stained with anti-CCL27 antibody. The dashed lines mark the epidermal surface of the skin. N = 7 mice for D0, 1 week, and adult ages, six mice for 2 weeks.(B) Immunofluorescent microscopic images of anti-CCL27 antibody-stained skin sections (14µm) at resting (telogen) and growing (anagen) phases of HF cycling in adult mice. N = 3 mice for each phase.(C) Fluorescent images (7µm) representative of skin at telogen and anagen phases of hair follicle cycling stained by in situ hybridization with an antisense CCL27 RNA probe or a nonspecific RNA probe as the control. The control probe recognizes the DapB gene (accession # EF191515) of a soil bacterial strain Bacillus subtilis SMY. N = 3 mice per HF cycle phase. N = 3 mice for each phase.(D) Anti-CCL27 antibody immunofluorescent staining images of 14µm skin sections of SPF and GF mice.(E) Real-time RT-PCR analysis of CCL27 expression in the skin of SPF and GF mice, normalized to ß-actin. N = 5 mice for 2-week-old GF, seven for 2-week-old SPF, seven for adult GF, and five for adult SPF. Unpaired one-tailed t-test.

Fig 5: Increased skin inflammation in CCL27-/- mice in response to topical imiquimod stimulation(A) Images of ears 5 days after topical treatment with IMQ.(B) Microscopic images of H&E-stained ear sections of CCL27+/+ and CCL27-/- mice 8 days after the IMQ treatment. The average epidermal thickness of IMQ-treated ears of CCL27+/+ and CCL27-/- mice was shown in the graph on the right. One dot is of one mouse.(C) FC analysis of gated CD45+CD3int+ and CD45+CD3int+ ?dTCR+ skin T cells of IMQ-treated (day 8) mice for IL-17A and IL-22 expression.(D) Percentages of CD3+, ?d, CD4+, and CD8+ T cells that express IL-17A in IMQ-treated skin of CCL27+/+ and CCL27-/- mice. N = 5 mice for both CCL27+/+ and CCL27-/- categories. Paired one tailed t-test. *p < 0.05; **p < 0.01; ***p < 0.001.