The hematopoietic humanized mouse (hu-mouse) model is a powerful resource to study and manipulate the human immune system. However, a major and recurrent issue with this model has been the poor maturation of B cells that fail to progress beyond the transitional B cell stage. Interestingly, a similar problem has been reported in transplant patients that receive cord blood stem cells. In this study, we characterize the development of human B and T cells in the lymph nodes (LNs) and spleen of BALB/c-Rag2nullIl2rγnull hu-mice. We find a dominant population of immature B cells in the blood and spleen early, followed by a population of human T cells, coincident with the detection of LNs. Notably, in older mice we observe a major population of mature B cells in LNs and in the spleens of mice with higher T cell frequencies. Moreover, we demonstrate that the T cells are necessary for B cell maturation, as introduction of autologous human T cells expedites the appearance of mature B cells, while in vivo depletion of T cells retards B cell maturation. The presence of the mature B cell population correlates with enhanced IgG and Ag-specific responses to both T-dependent and T-independent challenges, indicating their functionality. These findings enhance our understanding of human B cell development, provide increased details of the reconstitution dynamics of hu-mice, and validates the use of this animal model to study mechanisms and treatments for the similar delay of functional B cells associated with cord blood transplantations.

Lang et al. show in a humanized mouse model that human B cells undergo central tolerance via a combination of receptor editing and clonal deletion.

Key Points• Expression of full-length hBAFF from cDNA in the endogenous murine locus does not improve maturation of human B cells in hu-mice.• mBAFF is not limiting the maturation of human B cells in hu-mice.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of “B cells that are naïve IgD+” (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.