Enhanced human immune system engraftment in a novel triple-engineered NCG-hIL6-mTSLP-mFlt3-KOmouse model

Robust humanized mouse models are critical for evaluating human hematopoietic stem cell (HSC) biology, immune system development, and therapeutic efficacy. The NCG strain provides a superior platform for human HSC engraftment due to severe immunodeficiency. To further enhance human immune system reconstitution, we engineered a next generation strain NCG-mTSLP-hIL6-mFlt3 KO by: 1. knocking in human IL6 (hIL6) to support human myeloid cell survival and inflammation, 2. transgenic (Tg) expression of mouse TSLP (mTSLP) to enhance human T and B cell development and lymphoid organogenesis, and 3. knocked-out (KO) mouse Flt3 (mFlt3) to eliminate cytokine competition for FLT3 ligand (FLT3L) and promote the expansion of FLT3-expressing human dendritic cells (DCs) and natural killer (NK) cells. Human CD34+ HSCs were transplanted into NCG and NCG-mTSLP-hIL6-mFlt3 KO mice. Human immune cell reconstitution was longitudinally monitored in the peripheral blood of mice by flow cytometry (up to 18 weeks post-transplantation). Human immunoglobulin (IgG and IgM) levels were quantified by ELISA. Selected cohorts of HSC-NCG-mTSLP-hIL6-mFlt3 KO mice received repeated intraperitoneal injections of recombinant human FLT3L protein at 18 weeks post-transplantation. Lymphoid tissue development was assessed anatomically. HSC-NCG-mTSLP-hIL6-mFlt3 KO mice exhibited improved engraftment and reconstitution of human immune cells compared to HSC-NCG controls. Flow cytometry revealed significantly higher levels of human T cells, NK cells, plasmacytoid DCs (pDCs), and conventional DCs (cDCs) in peripheral blood at multiple time points. Furthermore, functional human B cell maturation was confirmed by elevated serum levels of human IgG and IgM. Anatomical analysis revealed advanced development of various lymph nodes, which are key sites for antigen presentation and the initiation of adaptive immunity. Administration of exogenous human FLT3L further amplified reconstitution levels of human NK cells, pDCs, and cDCs, validating the responsiveness to human cytokine signaling. The NCG-mTSLP-hIL6-mFlt3 KO mouse model offers a powerful platform for human HSC engraftment and development of a functional, multi-lineage human immune system, including lymphoid (T, B), myeloid (DC subsets), and NK cells. Together with enhanced lymphoid organ development, this model is positioned to be a valuable tool for translational immunology and preclinical evaluation of immunotherapies.