Humanized mouse models are engineered platforms that reconstitute key components of the human immune system (HIS) in an animal host. By incorporating human immune cells, these models better recapitulate the complexity of the human tumor microenvironment (TME). This is a dynamic ecosystem where tumor cells interact with immune cells, stromal components like fibroblasts and endothelial cells, and therapeutic agents. Humanized mice are particularly valuable for immuno-oncology research, where understanding immune-tumor interactions is critical for developing and evaluating new therapies.
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These models have become essential tools for preclinical testing of immunotherapies, including immune checkpoint inhibitors (ICIs), bispecific and trispecific antibodies, CAR-T cell therapies, and oncolytic viruses. They enable researchers to assess not only therapeutic efficacy but also safety profiles and potential immune-related adverse events in a system that more closely mirrors human biology than conventional immunodeficient models. Humanized mice can be generated using three major approaches: transplantation of peripheral blood mononuclear cells (PBMCs), CD34+ hematopoietic stem cells (HSCs), or bone marrow-liver-thymus (BLT) tissue.
Overview of Humanized Models
Humanized models overview. The three main types of humanized modles are PBMC mice, CD34+ (HSC) mice, and BLT mice. This figure highights key comparisons between the three types, focusing on the functioning immune population, key advantages, and limitations. Created in BioRender. Estipona, D. (2025) https://BioRender.com/ ypw4d9w
Learn how PBMC-humanized models can accelerate the development of next-generation immunotherapies.
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PBMC Humanized Mice
The PBMC humanization approach is the simplest and fastest of the three methods, requiring only the transfer of cells isolated from peripheral blood or, less commonly, from lymphoid tissues such as lymph nodes or spleen. In this model, also known as the Hu-PBL-SCID model, human PBMCs are injected into severely immunodeficient mice. The resulting immune compartment is dominated by CD3+ T cells, along with pre-existing immune cells and cytokines from the engrafted PBMC.
PBMC-humanized mice have proven valuable for studying human immune responses in autoimmunity and for assessing cytokine release syndrome. When the PBMC donor and tumor are HLA-matched, the model can support tumor antigen-specific T cell responses, making it useful for evaluating cancer immunotherapies. The model is particularly well-suited for testing agents that modulate human T cell activity, including therapeutic antibodies, regulatory T cell (Treg) therapies, and cytokine-based interventions.
The primary limitation of PBMC-humanized mice is graft-versus-host disease (GvHD), which occurs when donor T cells attack host tissues. This alloreactivity restricts experiments to 4–6 weeks post-injection and can trigger graft-versus-tumor (GvT) effects that confound therapeutic assessments. Transgenic strains lacking MHC expression, such as NSG-MHC double knockout (DKO) and NOG-dKO mice, can reduce alloreactivity and may extend the experimental window to 12 weeks or longer. Additionally, prescreening donor PBMCs in vitro can identify populations with minimal alloreactivity, improving tumor establishment and therapeutic evaluation.
CD34+ Humanized Mice
Humanized mice generated from CD34+ hematopoietic stem cells (HSCs) develop a more complete and diverse immune system than PBMC-humanized models. HSC engraftment gives rise to both innate and adaptive immune lineages, along with low numbers of red blood cells and platelets, while producing minimal GvHD. HSCs can be isolated from multiple sources, including bone marrow, mobilized peripheral blood, fetal liver, and umbilical cord blood. Cord blood is the most commonly used source due to its high CD34+ cell density and accessibility. Notably, the HSC source may influence the functional properties of the resulting immune system. Fetal-derived HSCs is believed to generate T cells with greater immune tolerance compared to those derived from adult HSCs.
Download this white paper to understand the main benefits and limitations of HSC hCD34+ and hPMBC humanized mouse models and how model selection depends on the specific study.
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Generation of CD34+ humanized mice faces several limitations. B and T cell populations often exhibit incomplete maturation and suboptimal function. Innate immune development is also restricted, and the absence of human HLA expression in the mouse thymus hampers the production of HLA-restricted T cells, which is essential for modeling antigen-specific immune responses. These models also require a longer establishment period compared to the PBMC model. Successful HSC engraftment depends on host preconditioning to deplete endogenous immune cells, typically through irradiation or alkylating agents, although several new immunodeficient mouse strains with targeted genetic alterations have been developed that do not require preconditioning.
BLT Humanized Mice
The bone marrow, liver, and thymus (BLT) model is the most complete humanized mouse platform but also the most technically demanding. It involves the surgical co-implantation of autologous human fetal liver and thymus tissue under the kidney capsule, along with the infusion of fetal liver-derived CD34+ HSCs. This approach generates a functional human thymus-like organ that supports HLA-restricted T cell development, including CD4+, CD8+, and regulatory T cell subsets. BLT mice aims to reconstruct a multilineage hematopoietic immune system with T cells, B cells, dendritic cells, macrophages, and myeloid cells, along with human mucosal and secondary lymphoid tissues that enable complex adaptive immune responses.
The complexity of the BLT mouse model comes with some cost, as several factors limit their clinical applications. The model requires specialized surgical expertise and relies on human fetal tissues, which can be increasingly difficult to access. Even with successful engraftment, B cell and innate immune cell maturation also remains incomplete. Additionally, the human thymic organoid can promote chronic a GvHD-like wasting syndrome, substantially reducing lifespan in some mice. These technical challenges and limited tissue availability make BLT models impractical for large-scale studies, restricting their application in immuno-oncology research.
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Access to Humanized Mouse Models
Humanized mouse models are available through several established repositories and commercial providers, including the Mutant Mouse Resource & Research Centers (MMRRC), European Mouse Mutant Archive (EMMA), The Jackson Laboratory, Charles River Laboratories, Taconic Biosciences, and Crown Bioscience. When selecting a model, researchers should consider factors such as the humanization method (PBMC, CD34+ HSC, or BLT), the specific immune cell populations required for the study, and the type of tumor model (such as PDX or CDX). The experimental timeline and whether GvHD development will interfere with analyses should also be considered. The choice of immunodeficient background strain is also important, as well as any additional genetic modifications, such as MHC knockout or human cytokine knock-in.