Development of a human immune system experimental model to investigate immune cells activation and targeting for CVD

Aim Human immune system (HIS) animal models have emerged as powerful tool for immunotherapy research, enabling the direct investigation of human immune-cell contribution to disease mechanisms and therapeutic outcomes. Here we present a dyslipidemic mouse models reconstituted with human hematopoietic stem cells (CD34) to investigate the role of human leukocytes as drivers and targets of the impaired immune-inflammatory response in atherosclerosis. Our aim is to exploit engineered iPSC-derived CD34 as a source of human cells to reproduce pathological defects in immune response and their effects on disease development. Methods Humanized TKO-LDLr KO mice (by crossing LDLr-KO with immunodeficient Rag2-KO/IL2rg-KO/CD47-KO mice, HuTKOL) were generated by hepatic injection of commercial or iPSCs-derived CD34+ cells, following 250cGy irradiation. Engraftment was checked through flow-cytometry and after 12weeks HuTKOL were fed 12-week high-cholesterol diet (WTD) to investigate immune-metabolic phenotype and atherosclerosis compared to normocholesterolemic humanized models. Results HuTKOL presented human leukocytes (%hCD45+cells/total number of live leukocytes: 34,55%, SE7,02%) in the circulation after 12wks from the engraftment with commercial hCD34+cells. CD19-B-lymphocytes are the most abundant population at 12 wks and decrease after 24wk; in contrast, CD3-T-lymphocytes increase from 12 to 24 wks resembling human profile. Characterization of the bone marrow shows engraftment of human CD34, myeloid and lymphoid lineages, while in the thymus T cell maturation recapitulates human phenotype. After 12 wks WTD, HuTKOL developed dyslipidemia (cholesterol:1211,96mg/dl,SE±63,72) and atherosclerosis (%aortic plaque occlusion: 18,35%,SE±1,90%), with predominantly hCD4-T-cell infiltration into the plaques. 12-weeks of WTD induced the expansion of hCD4-memory-Tcells and the production of IgM against atherosclerosis-related-antigens. Characterization and engineering of iPSCs-derived CD34+cells, coupled with their engraftment in TKO-L, are ongoing. Conclusions HuTKOL mice represent a valuable platform to investigate human adaptive response under dyslipidemia and to test immuno-modulation for CVD. iPCS-derived CD34+cells would allow to investigate the pathological and pharmacological editing of immune cells for CVD prevention.