• CCR8 is a chemokine receptor enrich on regulatory T cells (Tregs) within the tumor microenvironment and plays an important role in immunosuppression, tumor immune evasion, and Treg trafficking. CCL1 is a key ligand for CCR8 and contributes to activation and recruitment of CCR8-positive immune cells in inflammatory and tumor settings.
• CCR8 has emerged as a promising immuno-oncology target because CCR8-positive Tregs are highly enriched in multiple solid tumors, including colorectal cancer, lung cancer, melanoma, and breast cancer. Therapeutic depletion or modulation of CCR8-positive Tregs may enhance antitumor immune responses while minimizing systemic immune toxicity.
• CCR8/CCL1 humanized mice were generated by replacing murine Ccr8 and Ccl1 with their human counterparts under endogenous mouse regulatory control. These mice express human CCR8 and human CCL1 in physiologically relevant immune compartments and tumor microenvironments, enabling translational evaluation of anti-human CCR8 therapeutics.
• This model supports in vivo efficacy studies of anti-human CCR8 antibodies, tumor-infiltrating lymphocyte (TIL) analysis, tumor microenvironment characterization, and immunotherapy combination studies.

Key Advantages

• Dual humanized CCR8/CCL1 axis for physiologically relevant ligand-receptor interactions
• Supports evaluation of anti-human CCR8 antibodies in vivo
• Human CCR8 expression validated in tumor-infiltrating Treg populations
• Preserved immune cell composition and immune homeostasis
• Applicable to tumor microenvironment and Treg biology studies
• Suitable for immuno-oncology drug development and combination therapy evaluation

Validation

• CCL1 Protein Validation: Human CCL1 protein was detectable in homozygous CCR8/CCL1 humanized mice following anti-mCD3ε stimulation, whereas mouse CCL1 was detectable in wild-type mice by ELISA. Human CCL1 was also detected within the tumor microenvironment of MC38 tumor-bearing CCR8/CCL1 humanized mice.
• CCR8 Protein Validation: Human CCR8 protein expression was confirmed in tumor-infiltrating Th cells and Treg cells of CCR8/CCL1 humanized mice by flow cytometry, while mouse CCR8 expression was detected in wild-type controls.
• Immune Homeostasis Validation: Frequencies of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells, and Tregs in spleen, lymph node, and blood remained comparable between CCR8/CCL1 humanized mice and wild-type C57BL/6 mice.
• Functional Validation: Anti-human CCR8 antibody treatment markedly inhibited MC38 tumor growth in CCR8/CCL1 humanized mice, confirming suitability for in vivo efficacy evaluation of anti-human CCR8 therapeutics.

Applications

• Evaluation of CCR8-targeted antibodies and biologics
• Tumor microenvironment and Treg biology research
• Immuno-oncology efficacy evaluation
• Tumor-infiltrating lymphocyte analysis
• Preclinical evaluation of anti-CCR8 immunotherapies
• Combination immunotherapy studies

CCR8

• Exons 2 of the mouse Ccr8 gene, which encode the entire protein (from ATG to stop codon), are replaced with the corresponding human sequences in CCR8 humanized mice.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human CCR8 expression to be driven by the native mouse Ccr8 promoter, while endogenous mouse Ccr8 transcription and translation are abolished.

CCL1

• Exons 1–3 of the mouse Ccl1 gene, which encode the entire protein (from ATG to stop codon), are replaced with the corresponding human sequences.
• The endogenous mouse promoter, 5′ UTR, and 3′ UTR regions are retained, allowing human CCL1 expression to be driven by the native mouse Ccl1 promoter, while endogenous mouse Ccl1 transcription and translation are abolished.

Strain specific CCL1 protein expression analysis in homozygous CCR8/CCL1 humanized mice by ELISA. Serum were collected from wild-type mice (n=2) and homozygous CCR8/CCL1 humanized mice (n=3) that stimulated with 7.5μg anti-mouse CD3ε antibody in vivo. CCL1 protein expression was analyzed by ELISA with species-specific CCL1 ELISA kit.

Strain specific CCL1 protein expression analysis in tumor microenvironment of C57BL/6 and CCR8/CCL1 humanized mice bearing MC38 cells by ELISA. Murine colon cancer MC38 cells were subcutaneously implanted into wild-type C57BL/6 (n=3) and CCR8/CCL1 humanized mice (n=2/3). Tumor tissues were harvested when tumor volume reached approximately 600mm³ and CCL1 protein expression was analyzed by ELISA.

Strain specific CCR8 protein expression analysis in tumor-infiltrating lymphocytes (TILs) from homozygous CCR8/CCL1 humanized mice by flow cytometry. MC38 cells were inoculated into wild-type C57BL/6 (+/+) and homozygous CCR8/CCL1 humanized mice (H/H). Tumor tissues were harvested at the endpoint of experiment, and CCR8 protein expression in tumor-infiltrating lymphocytes (TILs) was analyzed by flow cytometry.

•The percentage of regulatory T cells (Tregs) cells (% in Th cells) in CCR8/CCL1 humanized mice has no significant difference compared with the C57BL/6 mice and CCR8 humanized mice.

Analysis of tumor-infiltrating lymphocytes (TILs) in wild-type C57BL/6 and CCR8/CCL1 humanized mice.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood of CCR8/CCL1 humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and CCR8/CCL1 humanized mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood of CCR8/CCL1 humanized mice. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and CCR8/CCL1 humanized mice (female, 8-week-old, n = 3). Single live cells were gated on the CD3⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Establishment of a MC38 model and in vivo efficacy study of anti-CCR8 antibody in CCR8/CCL1 humanized mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous CCR8/CCL1 humanized mice (female, 7–week-old, n=6). Mice were grouped when tumor volume reached approximately 100 ±50 mm³, at which time they were injected intraperitoneally with anti-human CCR8 antibodies (in house).

Efficacy of anti-human CCR8 antibodies in CCR8/CCL1 humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

MC38 tumor growth curves from individual CCR8/CCL1 humanized mice. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous CCR8/CCL1 humanized mice (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 100-150 mm³, at which time they were intraperitoneally injected with anti-human CCR8 antibody indicated in panel.​​​​​​​

Analysis of tumor-infiltrating lymphocytes (TILs) by flow cytometryin CCR8/CCL1 humanized mice.  Tumor cells were harvested at the endpoint of experiment (n=6). Flow cytometry analysis of the lymphocytes were performed to assess cell number and proportion changes. The proportions of CD3+ T cells, CD4+ T cells, Treg cells, and hCCR8+ Treg cells in the anti-CCR8 antibody treatment group showed no significant changes compared to the control group. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

What are CCR8/CCL1 humanized mice?

CCR8/CCL1 humanized mice are genetically engineered mice expressing human CCR8 and human CCL1, enabling translational studies of CCR8-targeted therapeutics and tumor immunology.

Why is CCR8 an important immuno-oncology target?

CCR8 is highly expressed on tumor-infiltrating regulatory T cells and contributes to immune suppression within tumors. Targeting CCR8 may enhance antitumor immunity.

Can these mice be used for antibody efficacy studies?

Yes. CCR8/CCL1 humanized mice support in vivo efficacy evaluation of anti-human CCR8 antibodies in tumor models.

What tumor models are compatible with CCR8/CCL1 humanized mice?

MC38 colorectal tumor models have been validated, and the model may also support additional syngeneic immuno-oncology studies.

Do CCR8/CCL1 humanized mice maintain normal immune homeostasis?

Yes. Leukocyte subset frequencies in spleen, blood, and lymph nodes are comparable to wild-type C57BL/6 mice.