Memory T Cells in MHC-Deficient Humanized Mice

Major histocompatibility complexes (MHC) govern antigen presentation and T-cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC–TCR interactions. Humanized NOD-scid-IL2Rγc ^null (NSG) mice engrafted with human CD34⁺ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T-cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T-cell maturation in vivo . CD34⁺ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC-deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single-cell TCR sequencing revealed marked differences in T-cell differentiation across models. Busulfan-conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naïve, NKT, and regulatory T-cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft-versus-host disease and represent a refined enabling platform for human T-cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.