CELL INTELLIGENCE · 肿瘤细胞治疗研究

The oncogenic Epstein Barr virus (EBV) is an exclusively human pathogen with related lymphocryptoviruses (γ1-herpesviruses) only present in monkeys. Therefore, experimentation with EBV infection in a small animal model requires reconstitution or adoptive transfer of human lymphocyte populations, primarily EBV's main host cell, the human B cell. In this protocol we describe human immune system reconstitution after neonatal transfer of CD34 + hematopoietic progenitor cells in lymphodeplete immune compromised mouse strains, using NOD-scid γ c -/- (NSG) mice as a commonly used example. Such reconstituted humanized mice allow intraperitoneal and intranasal infection with EBV and we describe injection of 10 5 infectious particles of the prototypic EBV strain B95-8 that can be produced from a recombinant bacmid (p2089) in HEK293 cells. Infection with this dose mimics symptomatic primary EBV infection, infectious mononucleosis (IM), with high viral loads plateauing 4 weeks after infection, with CD8 + T-cell lymphocytosis at week 5 and 6 after infection. This IM-like primary EBV infection in humanized mice leads to clonal EBV-induced B-cell lymphoproliferations that resemble large B-cell lymphomas with the latency III program of EBV infection. We describe Basic Protocols to monitor viral loads, immunohistochemistry of infected tissues and spectral flow cytometry to characterize protective T-cell expansion. The described mouse model has been used by us and others to characterize mutant EBV infections, cell-mediated immune control of EBV, modulation of EBV pathogenesis by co-infections with human immunodeficiency virus (HIV) and Kaposi sarcoma associated herpesvirus (KSHV), as well as passive transfer of vaccine elicited antibodies to test their protection against EBV infection. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CD34 + human hematopoietic progenitor cell isolation and characterization Basic Protocol 2: Human immune system reconstitution and characterization Basic Protocol 3: Recombinant EBV production and humanized mouse infection Basic Protocol 4: Viral load quantification, lymphoma assessment, and immunohistochemistry after EBV infection of humanized mice Basic Protocol 5: Human T-cell response analysis after EBV infection of humanized mice.