Abstract

B cells in the human immune system (HIS) mice exhibit weak responses to external antigens, characterized by insufficient antigen-specific B cell proliferation, low antibody titers, and a lack of differentiation into effector B cell subsets. We hypothesize that this failure is due to the absence of second signals provided by T cells following BCR stimulation. To address this, we developed an organoid screening system using HIS mouse splenocytes to identify missing signals. A combination of IL-4, IL-10, IL-21 together with CD40L was found to drive potent B cell proliferation and differentiation. Further organoid-based screening revealed that TNF-α and CpG synergistically promoted IgG class-switch, and that temporal separation of expansion and differentiation signals enhanced B cell responses. Translating these findings in vivo, CpG-adjuvanted vaccination followed by sequential i.v. delivery of expansion and differentiation cytokine mixtures induced antigen-specific B cell expansion, B cell differentiation, and IgG class-switch in HIS mice without affecting non-specific B cells. Sorting RBD-specific B cells from immunized mice yielded recombinant antibodies with high binding affinity and neutralizing activity against SARS-CoV-2 pseudovirus. Our study establishes a spleen organoid platform for screening factors that influence B cell responses in HIS mice and provides a generalizable strategy to obtain fully human antibodies for therapeutic development.