Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyers patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Maturation of antibody responses entails somatic hypermutation (SHM), class-switch DNA recombination (CSR), plasma cell differentiation, and generation of memory B cells, and it is thought to require T cell help. We showed that B cell Toll-like receptor 4 (TLR4)-B cell receptor (BCR) (receptor for antigen) coengagement by 4-hydroxy-3-nitrophenyl acetyl (NP)-lipopolysaccharide (LPS) (Escherichia coli lipid A polysaccharide O-antigen) or TLR5-BCR coengagement by Salmonella flagellin induces mature antibody responses to NP and flagellin in Tcrβ^-/-Tcrδ^-/- and NSG/B mice. TLR-BCR coengagement required linkage of TLR and BCR ligands, "linked coengagement." This induced B cell CSR/SHM, germinal center-like differentiation, clonal expansion, intraconal diversification, plasma cell differentiation, and an anamnestic antibody response. In Tcrβ^-/-Tcrδ^-/- mice, linked coengagement of TLR4-BCR by LPS or TLR5-BCR by flagellin induced protective antibodies against E. coli or Salmonella Typhimurium. Our findings unveiled a critical role of B cell TLRs in inducing neutralizing antibody responses, including those to microbial pathogens, without T cell help.