• Programmed cell death protein 1 (PDCD1) is an immune-inhibitory receptor expressed in activated T cells; it is involved in the regulation of T-cell functions, including those of effector CD8+ T cells. The interleukin-2 (IL2) protein, encoded by the IL2 gene, is a critical cytokine secreted by activated CD4+ and CD8+ T lymphocytes, playing a pivotal role in the proliferation of T and B lymphocytes. The expression of IL2 in mature thymocytes is characterized by monoallelic regulation, representing a distinctive mechanism for the precise control of gene expression. The high-affinity IL2 receptor is composed of the interleukin 2 receptor alpha (IL2RA) and beta (IL2RB) chains, in conjunction with the common gamma chain (IL2RG). Homodimeric configurations of the alpha chains (IL2RA) yield a low-affinity receptor, while the beta chains (IL2RB) in a homodimeric state produce a receptor with medium affinity. Typically, IL2RA is an integral membrane protein; however, a soluble form has been identified and is known to arise from extracellular proteolytic processes. The interleukin-15 (IL15) protein, encoded by the IL15 gene, is a crucial cytokine involved in the regulation of T and natural killer cell activation and proliferation, sharing numerous biological activities with interleukin-2 (IL2). The population of CD8+ memory cells is modulated by a delicate balance between IL15 and IL2. IL15 activates Janus kinase (JAK) kinases and facilitates the phosphorylation and activation of signal transducers and activators of transcription 3, 5, and 6 (STAT3, STAT5, and STAT6). The IL15 receptor alpha (IL15RA) gene encodes a receptor that binds specifically to IL15 with high affinity. The receptors for IL15 and IL2 share two common subunits, IL2R beta and IL2R gamma, underpinning the overlapping biological activities of these two cytokines. The protein encoded by this gene exhibits structural similarity to IL2R alpha, an additional IL2-specific subunit required for high-affinity IL2 binding. Notably, IL15RA can bind IL15 with high affinity independently of other subunits, implying distinct functional roles for IL15 and IL2.
• Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene. The mouse Il2 gene that encodes the full coding sequence was replaced by human IL2 full coding sequence. The mouse Il2ra gene that encodes the extracellular domain was replaced by human IL2RA counterpart gene sequences. The mouse Il2rb gene that encodes the extracellular domain was replaced by human IL2RB counterpart gene sequences. The mouse Il2rg gene that encodes the full coding region sequences was replaced by human IL2RG counterpart gene sequences. The mouse Il15 gene that encodes the full coding sequence was replaced by human IL15 counterpart gene sequences. The mouse Il15ra gene that encodes the extracellular region was replaced by human IL15RA counterpart gene sequences.
• Human PD-1, IL2RA, IL2RB, and IL2RG were exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, but not in wild-type C57BL/6 mice.
• B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice provide a powerful preclinical model for in vivo evaluation of the tumor disease.
• Application: This product is used for pharmacodynamics and safety evaluation of the tumor diseases.

Gene targeting strategy for B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

• Human PD-1 gene encoding the extracellular region and mouse PD-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.
• The exons 1-4 of mouse Il2 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human IL2 expression is driven by endogenous mouse Il2 promoter, while mouse Il2 gene transcription and translation will be disrupted.
• The exons 2-6 of mouse Il2ra gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il2ra gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL2RA expression is driven by endogenous mouse Il2ra promoter, while mouse Il2ra gene transcription and translation will be disrupted.
• The exons 2-8 of mouse Il2rb gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il2rb gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL2RB expression is driven by endogenous mouse Il2rb promoter, while mouse Il2rb gene transcription and translation will be disrupted.
• The exons 1-7 of mouse Il2rg gene that encode signal peptide, extracellular domain, transmembrane domain, cytoplasmic region  were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter and 5’UTR region of the mouse gene are retained. The human IL2RG expression is driven by endogenous mouse Il2rg promoter, while mouse Il2rg gene transcription and translation will be disrupted.
• The exons 3-8 of mouse Il15 gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The promoter, 5’UTR and 3’UTR region of the mouse gene are retained. The human IL15 expression is driven by endogenous mouse Il15 promoter, while mouse Il15 gene transcription and translation will be disrupted.
• The exons 2-6 of mouse Il15ra gene that encode extracellular domain were replaced by human counterparts in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. The genomic region of mouse Il15ra gene that encodes cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric IL15RA expression is driven by endogenous mouse Il15ra promoter, while mouse Il15ra gene transcription and translation will be disrupted.

Strain specific PD-1 expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Mouse PD-1 was only detectable in wild-type mice.

Strain specific PD-1 expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908). Human PD-1 was only detectable in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RA expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RA antibody (Biolegend, 102008) and anti-human IL2RA antibody (Biolegend, 302610). Mouse IL2RA was only detectable in wild-type mice.

Strain specific IL2RA expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RA antibody (Biolegend, 102008) and anti-human IL2RA antibody (Biolegend, 302610). Human IL2RA was only detectable in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RB expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RB antibody (Biolegend, 105912) and anti-human IL2RB antibody (Biolegend, 339005). Mouse IL2RB was only detectable in wild-type mice.

Strain specific IL2RB expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RB antibody (Biolegend, 105912) and anti-human IL2RB antibody (Biolegend, 339005). Human IL2RB was only detectable in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL2RG expression analysis in wild-type C57BL/6JNifdc mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RG antibody (Biolegend, 132307) and anti-human IL2RB antibody (Biolegend, 338605). Mouse IL2RB was only detectable in wild-type mice.

Strain specific IL2RG expression analysis in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Splenocytes were collected from homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (male, 7-week-old, n=3/group) stimulated with or without anti-CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 h,  and analyzed by flow cytometry with species-specific anti-mouse IL2RG antibody (Biolegend, 132307) and anti-human IL2RG antibody (Biolegend, 338605). Human IL2RG was only detectable in homozygous B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Strain specific IL15 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by ELISA. Serum was collected from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (Female, 6-week-old, n=3/group,) stimulated with 350mg/kg APAP in vivo for 18 hrs. Expression level of human IL15 was analyzed by ELISA (anti-human IL15 ELISA kit: R&D D1500).Human IL15 was exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. Values are expressed as mean ± SEM.

Strain specific IL15RA expression analysis in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice by flow cytometry. Bone marrow cells were collected from wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, and cultured in 6-well plates stimulated with GM-CSF and IL-4 for 6 days and LPS (1 μg/mL) for 18 h to induce BMDCs. Then BMDCs were collected and analyzed by flow cytometry with anti-mIL15RA antibody (BD, 568235) and anti-hIL15RA antibody (Biolegend, 330207). The mIL15RA was only detectable in wild-type mice. The hIL15RA was exclusively detectable in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of NK cells, dendritic cells, monocytes, macrophages, neutrophils and Tregs in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were similar to those in C57BL/6JNifdc mice. The frequency of T cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were lower than in C57BL/6JNifdc mice, while the frequency of B cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were higher than in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood were isolated from wild-type C57BL/6JNifdc mice and B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the blood was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of NK cells, dendritic cells, monocytes, macrophages, neutrophils and Tregs in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were similar to those in C57BL/6JNifdc mice. The frequency of T cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice was lower than in C57BL/6JNifdc mice, while the frequency of B cells in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were higher than in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice and B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 8-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of B cells, NK cells and Tregs in B-hPD-1 plus/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (24h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry. T cell activation in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (48h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry. T cell activation in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

In vitro T cell activation by anti-mCD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (72h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry. T cell activation in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG /hIL15/hIL15RA mice was significantly up-regulated by anti-mCD3ε antibody and anti-mCD28 antibody.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (24h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 24h. T cell proliferation was tested by flow cytometry.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (48h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 48h. T cell proliferation was tested by flow cytometry.

Quantification of T cell proliferation in vitro by anti-CD3ε antibody with or without anti-mCD28 antibody in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (72h). T cells were isolated from splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female,13-week-old, n=3), and incubated in the presence of anti-mCD3ε antibody (2ug/ml, BioXcell, BE0001-2), with or witnout anti-mCD28 antibody (5ug/ml, BioXcell, BE0015-1) for 72h. T cell proliferation was tested by flow cytometry.

In vitro cytokine production (IFN-γ and IL-2) in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice. T cells (2×10⁵) were isolated from the splenocytes of C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), incubated in the presence of anti-mouse CD3ε antibody (BioXCell, BE0001-1, clone 145-2C11, 2ug/ml) and anti-mCD28 antibody (BioXCell, BE0015-1, clone 37.51, 5ug/ml) for 24h, 48h and 72h. IFN-γ and IL-2 productions were then tested using ELISA method.

Subcutaneous tumor growth of B-hPD-L1 B16-F10 cells. B-hPD-L1 B16-F10 cells (1×10⁵) and wild-type B16-F10 cells (2×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 7-week-old, n=6). Tumor volume and body weight were measured three times a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hPD-L1 B16-F10 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression evaluated on B-hPD-L1 B16-F10 tumor cells by flow cytometry. B-hPD-L1 B16-F10 cells were subcutaneously transplanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 7-week-old, n=6). At the end of the experiment, tumor cells were harvested and assessed for mouse PD-L1(BioLegend, 124312) and human PD-L1(BioLegend, 329706) expression by flow cytometry. As shown, human PD-L1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 B16-F10 cells can be used for in vivo efficacy studies evaluating novel PD-L1 therapeutics.

Subcutaneous tumor growth of B-hPD-L1 MC38 plus cells. B-hPD-L1 MC38 cells (2×10⁵ and 5×10⁵) and wild-type MC38 cells (5×10⁵) were subcutaneously implanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 9-week-old, n=6). Tumor volume and body weight were measured three times a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-hPD-L1 MC38 plus cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression evaluated on B-hPD-L1 MC38 plus tumor cells by flow cytometry. B-hPD-L1 MC38 plus cells were subcutaneously transplanted into B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 9-week-old, n=6). At the end of the experiment, tumor cells were harvested and assessed for mouse PD-L1(BioLegend, 124312) and human PD-L1(BioLegend, 329706) expression by flow cytometry. As shown, human PD-L1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 MC38 plus cells can be used for in vivo efficacy studies evaluating novel PD-L1 therapeutics.