• Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on activated T cells and is widely targeted in immuno-oncology drug development.
• IL2, IL2RA, IL2RB, and IL2RG form the interleukin-2 cytokine and receptor system that regulates T cell expansion, immune activation, and lymphocyte biology.
• IL15 and IL15RA regulate NK cell and memory CD8+ T cell survival and share IL2RB/IL2RG signaling components with the IL2 pathway.
• B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice combine humanized PD-1 plus with humanized IL2/IL15 cytokine and receptor pathways in a C57BL/6 background.
• This model supports pharmacodynamic, safety, T cell activation, cytokine-response, and tumor efficacy studies involving PD-1, IL2, IL15, and related immunotherapy pathways.

Key Advantages

• Multi-target humanized PD-1 plus/IL2/IL2RA/IL2RB/IL2RG/IL15/IL15RA model for immuno-oncology research
• Human PD-1, IL2RA, IL2RB, IL2RG, IL15, and IL15RA expression validated by flow cytometry or ELISA TNFα
• Supports T cell activation and proliferation assays after anti-CD3ε and anti-CD28 stimulation
• Supports IFN-λ and IL-2 cytokine production analysis in stimulated T cells
• Spleen, blood, and lymph node leukocyte subpopulation profiles characterized by flow cytometry
• Compatible with B-hPD-L1 B16-F10 and B-hPD-L1 MC38 plus tumor models for in vivo efficacy studies

Validation

• Protein Validation: Human PD-1, IL2RA, IL2RB, IL2RG, IL15, and IL15RA were detected in homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice using species-specific flow cytometry or ELISA assays.
• Phenotypic Validation: Spleen, blood, and lymph node leukocyte subpopulations were characterized by flow cytometry, with key immune populations compared against C57BL/6JNifdc controls.
• Functional Validation: T cell activation, T cell proliferation, IFN-λ production, and IL-2 production were evaluated after anti-CD3ε and anti-CD28 stimulation.
• Tumor Model Validation: B-hPD-L1 B16-F10 and B-hPD-L1 MC38 plus cells established tumors in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice and expressed human PD-L1 in tumor tissue.

Application

• PD-1/PD-L1 immunotherapy efficacy studies
• IL2 and IL15 cytokine pathway pharmacology
• T cell activation, proliferation, and cytokine-response assays
• Pharmacodynamic and safety evaluation of tumor immunotherapies
• Humanized B-hPD-L1 B16-F10 and B-hPD-L1 MC38 plus tumor studies
• Combination immunotherapy research involving PD-1, IL2, IL15, and common gamma-chain receptor biology

In B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice, the human PD-1 extracellular region with mouse PD-1 transmembrane and cytoplasmic regions was inserted after the mouse Pdcd1 initiation codon. Mouse Il2 exons 1-4, Il2rg exons 1-7, and Il15 exons 3-8 were replaced by human counterparts to drive human cytokine or receptor expression from endogenous mouse regulatory regions.
For receptor humanization, mouse Il2ra exons 2-6, Il2rb exons 2-8, and Il15ra exons 2-6 encoding extracellular domains were replaced by the corresponding human sequences while retaining mouse cytoplasmic regions where applicable. This strategy generates a PD-1 plus/IL2/IL15 pathway humanized model for translational immuno-oncology studies.

Strain-specific PD-1 expression was analyzed 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 ug/mouse, i.p.) for 24 h and stained with species-specific anti-mouse PD-1 and anti-human PD-1 antibodies. Mouse PD-1 was detectable in wild-type mice.

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

Strain-specific IL2RA expression was analyzed 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-CD3epsilon antibody for 24 h, and stained with species-specific anti-mouse IL2RA and anti-human IL2RA antibodies. Mouse IL2RA was detectable in wild-type mice.

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

Strain-specific IL2RB expression was analyzed 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-CD3epsilon antibody for 24 h, and stained with species-specific anti-mouse IL2RB and anti-human IL2RB antibodies. Mouse IL2RB was detectable in wild-type mice.

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

Strain-specific IL2RG expression was analyzed 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-CD3epsilon antibody for 24 h, and stained with species-specific anti-mouse IL2RG and anti-human IL2RG antibodies. Mouse IL2RG was detectable in wild-type mice.

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

Strain-specific IL15 protein expression was analyzed 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 female 6-week-old mice (n=3/group) stimulated with 350 mg/kg APAP in vivo for 18 h. Human IL15 was measured using an anti-human IL15 ELISA kit (R&D, D1500) and was detectable in homozygous humanized mice. Values are expressed as mean ± SEM.

Strain-specific IL15RA expression was analyzed in BMDCs by flow cytometry. Bone marrow cells from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were cultured with GM-CSF and IL-4 for 6 days and LPS (1 ug/mL) for 18 h to induce BMDCs. Mouse IL15RA was detectable in wild-type mice, while human IL15RA was detectable in homozygous humanized mice.

Frequency of leukocyte subpopulations in spleen was analyzed 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). NK cells, dendritic cells, monocytes, macrophages, neutrophils, and Tregs were similar between strains. T cell frequency was lower and B cell frequency was higher in humanized mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood was analyzed by flow cytometry.
Blood 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). NK cells, dendritic cells, monocytes, macrophages, neutrophils, and Tregs were similar between strains. T cell frequency was lower and B cell frequency was higher in humanized mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph node was analyzed 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). B cells, NK cells, and Tregs in humanized mice were similar to those in C57BL/6JNifdc mice. Values are expressed as mean ± SEM.

In vitro T cell activation was evaluated after anti-mCD3ε stimulation with or without anti-mCD28 antibody for 24 h.
T cells were isolated from splenocytes of wild-type C57BL/6JNifdc and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3). Cells were cultured with anti-mCD3ε antibody (2 ug/mL, BioXCell BE0001-2) with or without anti-mCD28 antibody (5 ug/mL, BioXCell BE0015-1). T cell proliferation was measured by flow cytometry, showing increased activation after CD3/CD28 stimulation.

In vitro T cell activation was evaluated after anti-mCD3ε stimulation with or without anti-mCD28 antibody for 48 h.
T cells from wild-type C57BL/6JNifdc and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3) were cultured with anti-mCD3ε antibody (2 ug/mL) with or without anti-mCD28 antibody (5 ug/mL). T cell proliferation was tested by flow cytometry, and activation in humanized mice was significantly up-regulated by anti-mCD3ε plus anti-mCD28 stimulation.

In vitro T cell activation was evaluated after anti-mCD3ε stimulation with or without anti-mCD28 antibody for 72 h.
T cells from wild-type C57BL/6JNifdc and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3) were cultured with anti-mCD3epsilon antibody (2 ug/mL) with or without anti-mCD28 antibody (5 ug/mL). T cell proliferation was tested by flow cytometry, confirming activation after CD3/CD28 costimulation.

T cell proliferation was quantified after 24 h in vitro stimulation.
T cells were isolated from splenocytes of wild-type C57BL/6JNifdc and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice (female, 13-week-old, n=3), then cultured with anti-mCD3ε antibody (2 ug/mL) with or without anti-mCD28 antibody (5 ug/mL). Flow cytometry was used to quantify proliferation, supporting functional T cell activation in the humanized cytokine-receptor background.

T cell proliferation was quantified after 48 h in vitro stimulation.
T cells from wild-type and homozygous B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were cultured with anti-mCD3ε antibody with or without anti-mCD28 antibody. Flow cytometry analysis showed increased proliferation after TCR/CD28 stimulation, supporting functional immune-cell responsiveness in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.

T cell proliferation was quantified after 72 h in vitro stimulation.
T cells isolated from wild-type C57BL/6JNifdc and B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice were cultured with anti-mCD3ε antibody with or without anti-mCD28 antibody. Flow cytometry demonstrated sustained proliferation after CD3/CD28 stimulation, confirming the utility of the model for T cell activation studies.

IFN-λ and IL-2 production were measured after in vitro T cell stimulation.
T cells (2 x 10^5) 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), then incubated with anti-mouse CD3ε antibody and anti-mCD28 antibody for 24 h, 48 h, and 72 h. IFN-λ and IL-2 production were measured by ELISA, supporting cytokine-response evaluation in this model.

Subcutaneous tumor growth of B-hPD-L1 B16-F10 cells was evaluated in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.
B-hPD-L1 B16-F10 cells (1 x 10^5) and wild-type B16-F10 cells (2 x 10^5) were implanted into female 7-week-old humanized mice (n=6). Tumor volume and body weight were measured three times per week. Tumor volume was calculated as V=0.5 x long diameter x short diameter^2. B-hPD-L1 B16-F10 cells established tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression on B-hPD-L1 B16-F10 tumor cells was evaluated 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 with anti-mouse PD-L1 and anti-human PD-L1 antibodies. Human PD-L1 was highly expressed on tumor cells, supporting use of B-hPD-L1 B16-F10 tumors for PD-L1 therapeutic efficacy studies.

Subcutaneous tumor growth of B-hPD-L1 MC38 plus cells was evaluated in B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice.
B-hPD-L1 MC38 cells (2 x 10^5 and 5 x 10^5) and wild-type MC38 cells (5 x 10^5) were implanted into female 9-week-old humanized mice (n=6). Tumor volume and body weight were measured three times per week. Tumor volume was calculated as V=0.5 x long diameter x short diameter^2. B-hPD-L1 MC38 plus cells established tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

PD-L1 expression on B-hPD-L1 MC38 plus tumor cells was evaluated 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 endpoint, tumor cells were harvested and assessed with anti-mouse PD-L1 and anti-human PD-L1 antibodies. Human PD-L1 was highly expressed on tumor cells, supporting use of B-hPD-L1 MC38 plus tumors for PD-L1 therapeutic efficacy studies.

Q1: What are B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice?

B-hPD-1 plus/hIL2/hIL2RA/hIL2RB/hIL2RG/hIL15/hIL15RA mice are multi-gene humanized mice carrying humanized PD-1 plus, IL2, IL2RA, IL2RB, IL2RG, IL15, and IL15RA for immuno-oncology and cytokine-pathway research.

Q2: Why are IL2 and IL15 pathways important in this model?

IL2 and IL15 regulate T cell, NK cell, and memory CD8+ T cell biology through shared IL2RB and IL2RG signaling components, making the model useful for studying cytokine-driven immune activation.

Q3: How was target expression validated?

Human PD-1, IL2RA, IL2RB, IL2RG, IL15, and IL15RA were validated in homozygous humanized mice by species-specific flow cytometry or ELISA assays.

Q4: Can this model be used for functional T cell studies?

Yes. T cell activation, T cell proliferation, IFN-λ production, and IL-2 production were assessed after anti-CD3ε and anti-CD28 stimulation.

Q5: What are the main applications of this model?

Applications include PD-1/PD-L1 immunotherapy studies, IL2/IL15 pathway pharmacology, T cell functional assays, cytokine-response analysis, safety evaluation, and B-hPD-L1 B16-F10 or B-hPD-L1 MC38 plus tumor efficacy studies.