Highly Efficient Human NK Cell Immune Reconstitution Mouse Models: NKG-hIL15 & huHSC-NKG-hIL15 Mice

Immunotherapy works by mobilizing immune cells within the body to enhance the immune system's ability to recognize, kill, and eliminate tumor cells. Given the differences between the immune systems of mice and humans, immunotherapies are typically evaluated in preclinical studies using humanized immune system (HIS) mouse models.^[1-2] The classical approach to humanization involves transplanting human hematopoietic stem cells (HSCs) into severely immunodeficient mice, such as NKG mice. Early immunotherapies, like CAR-T and tumor-infiltrating lymphocyte (TIL) therapy, primarily functioned through T cells. In recent years, immunotherapies relying on natural killer (NK) cells – such as CAR-NK and ADCC – have gained attention. Simultaneously, NK cell-related targets have become popular in the development of immune checkpoint therapies. These therapies require the involvement of human NK cells; however, commonly used humanized immune system models have a low proportion of human NK cells, which is unfavorable for evaluating these therapies.^[2-3] Consequently, models that efficiently reconstitute human NK cells have become a key focus in this area of research.

Figure 1. Development methods of different humanized immune system mouse models and their cell reconstitution types.^[3]

Efficient Reconstitution of NK Cells Requires Humanized IL-15

NK cells can rapidly recognize and initiate immune defense mechanisms, swiftly eliminating diseased and cancerous cells. Compared to other immune cells, NK cells are more efficient and effective in killing tumor and virus-infected cells. However, conventional severely immunodeficient mice lack the necessary factors to support the development of human NK cells, leading to a low proportion of NK cells in human immune system reconstitution in these mice.

Interleukin-15 (IL-15) is a cytokine that regulates the activation and proliferation of T cells and NK cells. Research has shown that IL-15 plays an essential role in the differentiation, function, and survival of NK cells. Providing sufficient human IL-15 can maintain the stable function of human NK cells within mice.^[4-5] Therefore, humanizing the IL15 gene in severely immunodeficient mice helps in the differentiation of HSCs into the NK cell lineage during reconstitution and further promotes T cell proliferation.^[6]

Figure 2. Interleukin-15 (IL-15) is an important cytokine for regulating the differentiation of hematopoietic stem/progenitor cells into NK cells. ^[4]

The NKG-hIL15 mouse (Product Code: C001513) is a second-generation severely immunodeficient mouse model developed by Cyagen by knocking in the human IL15 gene into NKG mice, enabling human stem cell xenotransplants to effectively reconstitute human NK cells. To this aim, the huHSC-NKG-hIL15 humanized immune system (HIS) mouse model (Product Code: C001526) is developed by transplanting human hematopoietic stem cells (CD34+ HSC) into NKG-hIL15 mice. Compared to huHSC-NKG mice, the huHSC-NKG-hIL15 mouse model can reconstitute various immune cells and significantly increases the reconstitution proportion of human NK cells. Therefore, both the NKG-hIL15 and huHSC-NKG-hIL15 mouse models provide a powerful tool for the research and development of NK cell-targeted immunotherapies and preclinical drug evaluations. Detailed phenotype information of these models is provided below.

1. Detection of Human IL-15 Protein Expression in NKG-hIL15 Mice

Compared to NKG mice, NKG-hIL15 mice exhibit significant expression of human IL-15 protein.

Figure 3. ELISA detection of human IL-15 protein in 5-week-old female homozygous NKG-hIL15 mice and NKG mice.

2. huHSC-NKG-hIL15 Mice Maintain Normal Growth And Survival Rates

The huHSC-NKG-hIL15 human immune reconstitution model is developed by transplanting human CD34+ (huCD34+) hematopoietic stem cells (HSCs) into NKG-hIL15 mice. huHSC-NKG-hIL15 mice maintain normal growth, with a gradual increase in body weight, and approximately 78% survival rate at 225 days post-transplantation.

Figure 4. Survival of 4-week-old female homozygous NKG-hIL15 mice after transplantation with huCD34+ HSCs.

3. huHSC-NKG-hIL15 Mice Provide Efficient Reconstitution of Various Immune Cell Types

In huHSC-NKG-hIL15 mice, human leukocytes are rapidly reconstituted in peripheral blood by the third week. Human T cells begin reconstitution by the eleventh week, with their proportion gradually increasing and stabilizing around 25% by week 27. The proportion of human B cells starts to fluctuate and rise around the third week and stabilizes after week 17. Notably, human NK cells are rapidly reconstituted between the third and fifth weeks, stabilize after week 17, and maintain a reconstitution proportion of about 20% by week 27.

Figure 5. Reconstitution of various immune cells in the peripheral blood of huHSC-NKG-hIL15 mice.

4. huHSC-NKG-hIL15 Mice Achieve Reconstitution of NK Cells at Different Developmental Stages

Throughout the reconstitution process, huHSC-NKG-hIL15 mice exhibit NK cells at various developmental stages in their peripheral blood. These include NKG2D+ NK cells (NKG2D is expressed during the early developmental stages of NK cells and remains active in mature NK cells) and KIR3DL+ NK cells (KIR3DL is expressed during the mid-to-late developmental stages of NK cells). The huHSC-NKG-hIL15 mouse model successfully achieves the reconstitution of mature CD57+ NK cells, as CD57 is expressed during the final maturation stage of NK cells.

Figure 6. Reconstitution of NK cells at different developmental stages in huHSC-NKG-hIL15 mice.

Summary

The NKG-hIL15 mouse (Product Code: C001513), developed by knocking in the human IL15 gene into NKG mice, is a second-generation severely immunodeficient model which successfully expresses high levels of human IL15 protein. The expression of human Il-15 in NKG-hIL15 mice enables the effective reconstitution of a variety of human immune cells, including NK cells, from human stem cell transplants. 

To this aim, the huHSC-NKG-hIL15 humanized immune system (HIS) mouse model (Product Code: C001526) is developed by transplanting human hematopoietic stem cells (CD34+ HSC) into NKG-hIL15 mice. The huHSC-NKG-hIL15 mice can maintain normal growth and survival, reconstituting various immune cells while significantly increasing the proportion of reconstituted human NK cells. Therefore, the NKG-hIL15 and huHSC-NKG-hIL15 mouse models provide a powerful tool for the research and development of NK cell-targeted immunotherapies and preclinical drug evaluations.

Additionally, Cyagen offers various other types of immunodeficient and humanized immune system reconstitution models. Cyagen also provides a wide range of tumor cell lines and related transplantation models to meet the experimental needs of researchers in different areas of cancer therapy research.

Cyagen’s Immunodeficient Mice

Cyagen’s Humanized Immune System Mouse Models

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References:
[1]Chuprin J, Buettner H, Seedhom MO, Greiner DL, Keck JG, Ishikawa F, Shultz LD, Brehm MA. Humanized mouse models for immuno-oncology research. Nat Rev Clin Oncol. 2023 Mar;20(3):192-206.
[2]Walsh NC, Kenney LL, Jangalwe S, Aryee KE, Greiner DL, Brehm MA, Shultz LD. Humanized Mouse Models of Clinical Disease. Annu Rev Pathol. 2017 Jan 24;12:187-215.
[3]Genomab Biotech. “Human immune system (HIS) mice.”, Retrieved May 23, 2024, from https://www.genomab.com/the-mouse-with-human-immune-system
[4]Liquitaya-Montiel AJ, Mendoza L. Dynamical Analysis of the Regulatory Network Controlling Natural Killer Cells Differentiation. Front Physiol. 2018 Aug 2;9:1029.
[5]Huntington ND, Legrand N, Alves NL, Jaron B, Weijer K, Plet A, Corcuff E, Mortier E, Jacques Y, Spits H, Di Santo JP. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. J Exp Med. 2009 Jan 16;206(1):25-34.
[6]Katano I, Nishime C, Ito R, Kamisako T, Mizusawa T, Ka Y, Ogura T, Suemizu H, Kawakami Y, Ito M, Takahashi T. Long-term maintenance of peripheral blood derived human NK cells in a novel human IL-15- transgenic NOG mouse. Sci Rep. 2017 Dec 8;7(1):17230.