CXCR6: Highly expressed in tissue-resident memory T cells (TRM)

• Gene Information: C-X-C Motif Chemokine Receptor 6 (CXCR6) is a protein-coding gene located on chromosome 3p21.31, which belongs to the CXC chemokine receptor family.
• Protein Expression: CXCL16 expressed in immune system cells, keratinocytes, and endothelial cells is the only known natural ligand of chemokine receptor CXCR6. This receptor is found on various immune system cells, including natural killer cells, CD4 and CD8 T lymphocytes, innate lymphoid cells (ILCs), as well as on fibroblasts, smooth muscle cells, and endothelial cells.
• Signaling Pathway: CXCR6 and its unique ligand CXCL16 are components of a signaling pathway that regulates the migration of T lymphocytes to various peripheral tissues, including the liver, spleen red pulp, intestine, lungs, and skin. This pathway also facilitates interactions between T lymphocytes and dendritic cells, as well as fibroblastic reticular cells. CXCR6/CXCL16 also controls the localization of resident memory T lymphocytes.
• Therapeutic Inhibition: TRM cells, show increased expression of CXCR6 in recurrent psoriatic lesions and contribute to inflammation. The knockout of CXCR6 significantly improves the psoriatic immune microenvironment by decreasing the retention of TRM cells and reducing the likelihood of disease relapse.

CXCR6

• The exons 2 of mouse Cxcr6 gene that encode the whole molecule (ATG to STOP codon were replaced by human counterparts in B-hCXCL16/hCXCR6 mice.
• The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human CXCR6 expression is driven by endogenous mouse Cxcr6 promoter, while mouse Cxcr6 gene transcription and translation will be disrupted.

CXCL16

• The exons 2-5 of mouse Cxcl16 gene that encode extracellular domain and transmembrane domain is replaced by human counterparts in B-hCXCL16/hCXCR6 mice.
• The genomic region of mouse Cxcl16 gene that encodes signal peptide is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric Cxcl16 expression is driven by endogenous mouse Cxcl16 promoter, while mouse Cxcl16 gene transcription and translation will be disrupted.

Note: B-hCXCL16/hCXCR6 mice were obtained by mating B-hCXCR6 mice and B-hCXCL16 mice.

• Mouse CXCR6 was detected exclusively in wild-type C57BL/6N mice
• Human CXCR6 was detected in homozygous B-hCXCL16/hCXCR6 mice, but not in wild-type mice.

Strain specific CXCR6 expression analysis in homozygous B-hCXCL16/hCXCR6 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H) and analyzed by flow cytometry with species-specific anti-CXCR6 antibody(Biolegend, 151105; Biolegend, 356003). mCXCR6 was detectable in T cells and NK cells of wild-type mice, hCXCR6 was detectable in T cells and NK cells of homozygous B-hCXCL16/hCXCR6 mice.

• Mouse CXCR6 was detected exclusively in wild-type C57BL/6N mice
• Human CXCR6 was detected in homozygous B-hCXCL16/hCXCR6 mice, but not in wild-type mice.

Strain specific CXCR6 expression analysis in homozygous B-hCXCL16/hCXCR6 mice by flow cytometry. Liver cells were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H) and analyzed by flow cytometry with species-specific anti-CXCR6 antibody(Biolegend, 151105; Biolegend, 356003). mCXCR6 was detectable in T cells and NK cells of wild-type mice, hCXCR6 was detectable in T cells of homozygous B-hCXCL16/hCXCR6 mice.

• Mouse CXCR6 was detected exclusively in wild-type C57BL/6N mice
• Human CXCR6 was detected in homozygous B-hCXCL16/hCXCR6 mice, but not in wild-type mice.

Strain specific CXCR6 expression analysis in homozygous B-hCXCL16/hCXCR6 mice by flow cytometry. Lung cells were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H) and analyzed by flow cytometry with species-specific anti-CXCR6 antibody(Biolegend, 151105; Biolegend, 356003). mCXCR6 was detectable in T cells and NK cells(weak expression) of wild-type mice, hCXCR6 was detectable in T cells and NK cells (weak expression)  of homozygous B-hCXCL16/hCXCR6 mice.

• Human CXCL16 was detectable in serum from B-hCXCL16/hCXCR6 mice but not on those cells of wild-type C57BL/6 mice.

Strain specific CXCL16 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCXCL16/hCXCR6 mice by ELISA. Serum was collected from wild-type C57BL/6N mice (+/+) (male, n=3, 6-week-old) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H)  (male, n=3, 6-week-old). Expression level of mouse and human CXCL16 were analyzed by ELISA (Human CXCL16 ELISA Kit : Abcam, ab187397; Mouse CXCL16 ELISA Kit : Abcam, ab197744). Mouse CXCL16 was exclusively detectable in wild-type C57BL/6N mice (n=3). Human CXCL16 was exclusively detectable in homozygous B-hCXCL16/hCXCR6 mice (n=3). Values are expressed as mean ± SEM.

• Human CXCL16 was detectable in skin of  psoriasis model induced by IMQ from B-hCXCL16/hCXCR6 mice but not on those cells of wild-type C57BL/6 mice.

Immunohistochemical (IHC) analysis of CXCR6 protein expression in wild-type C57BL/6 mice and homozygous humanized B-hCXCL16/hCXCR6 mice. Mouse skins were collected from wild-type C57BL/6 mice (+/+) (male, 8-week-old, n=1) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H)(male, 8-week-old, n=1). Psoriasis model was induced by IMQ. Protein expression was analyzed with anti-human CXCR6 antibody(Abcam, ab273116) by IHC. Human CXCR6 was detected in the skin of IMQ induced psoriasis model in B-hCXCL16/hCXCR6 mice.

• Human CXCL16 was detectable in skin of  psoriasis model induced by IMQ from B-hCXCL16/hCXCR6 mice but not on those cells of wild-type C57BL/6 mice.

Immunohistochemical (IHC) analysis of CXCR6 protein expression in wild-type C57BL/6 mice and homozygous humanized B-hCXCL16/hCXCR6 mice. Mouse skins were collected from wild-type C57BL/6 mice (+/+) (male, 8-week-old, n=1) and homozygous B-hCXCL16/hCXCR6 mice (H/H;H/H)(male, 8-week-old, n=1). Psoriasis model was induced by IMQ. Protein expression was analyzed with anti-human CXCR6 antibody(Abcam, ab273116) by IHC. Human CXCR6 was detected in the skin of IMQ induced psoriasis model in B-hCXCL16/hCXCR6 mice.

IMQ-induced skin inflammation in B-hCXCL16/hCXCR6 mice phenotypically resembles psoriasis. (A) Experimental schedule for induction of psoriasis-like skin lesions in B-hCXCL16/hCXCR6 mice. B-hCXCL16/hCXCR6 mice (Female, 8 week-old, n=5) were scored daily for up to 6 days for body weight and clinical signs of skin inflammation following treatment with imiquimod (IMQ) cream, Control mice were treated similarly with Vaseline cream. Mice in group 3 were treated with the monoclonal antibody TA targeting human CXCR6(Provided by the client). (B) Phenotypical presentation of mouse back skin after 6 days of treatment. (C) Body weight changes during treatment. (D-E) Erythema and scaling score of the back was scored daily on a scale from 0 to 4. Additionally, the cumulative score (F) (erythema plus scaling) is depicted. (G)H&E staining of the back skin of mice. Values are expressed as mean ± SEM.