Stripecke, R. | bioRxiv Science

bioRxiv2024

T-cell development and activation in humanized mice lacking mouse major histocompatibility complexes

Humanized mice transplanted with CD34+ hematopoietic progenitor cells (HPCs) are used to study human immune responses in vivo. However, the mismatch between the mouse major histocompatibility complexes (MHCs) and the human leukocyte antigens (HLAs) is not optimal for T-cell development and can trigger xenograft reactivity. We evaluated human T-cell development in NOD.Scid.Gamma mice lacking expression of MHC class I and II (NSG-DKO). Human leukocyte engraftment was detectable at 8 weeks post-transplantation. Human CD4+ and CD8+ T-cells were detectable in blood, thymus, bone marrow and spleen of humanized NSG-DKO mice for up to 20 weeks post-transplantation. Further, we evaluated the effects of lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN- and the human cytomegalovirus gB antigen. LV delivery promoted development and activation of human central memory, {beta} and {gamma}{delta} T-cells with amplifications of the T-cell repertoire. LV administration unleashed multiple reactome pathways such as type-I interferon responses, cell cycle and metabolic processes. In summary, development of human T-cells in humanized mice does not rely on mouse MHCs and can be boosted systemically via LV administration.

immunology↗

bioRxiv2023

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells

I.Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3{zeta} (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3{zeta} gene as the CARs activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCR{gamma}/{delta} T cells, regulatory T cells, and NK cells. In T cells, CD3{zeta} in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3{zeta}-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3{zeta}-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC-edited CAR-T cells, integration of a Her2-CAR into CD3{zeta} conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3{zeta} gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3{zeta} gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key pointsO_LIIntegration of {zeta}-deficient CARs into CD3{zeta} gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use C_LIO_LICD3{zeta}-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions C_LI

immunology↗