Abstract Introduction: Allogeneic transplantation of “off-the-shelf” cell therapies offers a transformative approach for treating a wide range of diseases, including various oncological diseases. However, host-mediated immune rejection ‘’Allogeneic rejection’’ remains a critical barrier to therapeutic success. To address this, immune shielding strategies have been developed to evade immune detection, necessitating robust in vivo models for evaluating their protection from immune cells. Many in vivo models fall short in mimicking the human host immune milieu. We have established a model that incorporates multiple transplantation sites for therapeutic cells, such as under the kidney capsule of the mice. Additionally, we have characterized relevant readouts for the evaluation of the survival of immune-shielded therapeutic cells and monitoring allogeneic rejection. Methods: We utilized the immunodeficient BRGSF mice. These mice were humanized using CD34+ from distinct human donors, resulting in the BRGSF-HIS mice (purchased from Genoway). We transplanted these mice with therapeutic stem cell-derived cells. Post-transplantation, mice received human FLT3 ligand to enhance human myeloid cell development. At the end of the study, blood, spleen, and kidney tissues are collected for flow cytometry, MSD cytokine profiling, and histology. Other functional readouts were performed during the in-life phase. Results: The model was validated by transplanting wild-type (non-immune shielded) stem cell-derived therapeutic cells under the kidney capsule of BRGSF-HIS mice. In addition, a subset of BRGSF-HIS mice serves as a control group and does not undergo transplantation. The BRGSF-HIS were tested by flow cytometry of blood and spleen to confirm the presence of all the human immune cells essential for allogeneic rejection, including CD4/CD8 T-cells, NK cells, B-cells, monocytes, and M1/M2 macrophages. A comparative analysis was performed on both transplanted and non-transplanted BRGSF-HIS, focusing on the assessment of immune cells and cytokines in the blood and spleen. The transplanted mice exhibited a pronounced pro-inflammatory phenotype in T-cells, characterized by increased CD4+ and CD8+ TEMRA and CD69+ populations, elevated Th1 subsets, and reduced Th2 cells. We observed a tendency for a shift of splenic M2 to M1 macrophages in the transplanted mice. Systemic immune activation was further demonstrated by increased levels of pro-inflammatory cytokines found in both plasma and spleen samples. Histological assessment of immune infiltration at the transplantation site revealed a high abundance of human T-cells, accompanied by macrophages. Conclusion: We developed a model to evaluate allogeneic rejection of immune-shielded therapeutic cells, assess their survival, and analyze immunophenotyping resulting from transplantation, as well as human host immune infiltration. Citation Format: Mino Yakoub, Katrin Ridders, Henning Jelten, Audrey Holtzinger, Matthias Austen, Claudia Wrzos, . Adapting and validating a humanized mouse model for testing of immune-shielded genetic modifications in therapeutic cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3710.
Yakoub et al. (Fri,) studied this question.