Introduction: Humanised mouse models provide a platform to investigate human immune biology in vivo, reducing the need for large animal studies. Importantly they can overcome biological differences between humans and other species, reducing the uncertainty in progressing to clinical studies. In transplantation research, humanised immune system (HIS) models have provided pre-clinical evidence for human-specific therapies such as biologics and regulatory T cell therapy, which are now in clinical trials. Most HIS models lack multilineage leukocyte development, limiting accurate representation of the full alloresponse in transplantation. To address this, we assessed immune reconstitution and human skin transplant rejection in NOD,B6.SCID Il2rγ−/− KitW41/W41 (NBSGW) mice, which are capable of long-term multilineage haemopoiesis without irradiation due to the presence of an inactivating mutation in the stem cell growth factor receptor. Materials and Methods: Human cord blood CD133+ haematopoietic stem cells (HSCs) were injected intravenously into non-irradiated NBSGW mice (n=20). Engraftment in blood and lymphoid organs was assessed over 20 weeks. In a separate group, HSC-engrafted mice (HIS-NBSGW) underwent allogeneic human skin transplantation 10-12 weeks post-injection (Figure 1).At the point of skin transplant rejection (or after 100 days) blood, spleen, bone marrow (BM), thymus and skin were analysed by multiparameter flow cytometry and NanoString gene expression analysis. Results and Discussion: High levels of human CD45+ cell chimerism developed reliably in the blood, BM and spleen after HSC transplantation in a dose-dependent manner (Figure 2).Multilineage engraftment was achieved, consisting of human myeloid cells, B and T lymphocytes at multiple recognised stages of development. Expression of Human Leukocyte Antigen (HLA) Class I and II molecules was identified on CD3- thymocytes. Antigen-presenting cells (APCs), B cells and T cells were found to be functional in in vitro assays. In mice with multilineage human leukocyte reconstitution, allogeneic human skin grafts rejected. Rejection was associated with RNA markers of upregulated immune function and elevated production of IgM, IgG and Th1, Th2 and Th17-related cytokines (Figure 3).Conclusion: The HIS-NBSGW model provides a technically straightforward research system utilising adult mice, which does not require irradiation, co-transplantation of human haematopoietic stromal tissues, or provision of human cytokines. By producing functional multilineage immune cells, it is a useful method for assessing HSC function in vivo and a framework for assessing human transplant responses encompassing the breadth of innate, cellular and humoural immunity. Clarendon Fund, University of Oxford. MRC Discovery Award, Ref: MC_PC_15069.