These last mentioned (IC IgL-high+) cells were then gated for high expression of CD38 (bottom plots)

These last mentioned (IC IgL-high+) cells were then gated for high expression of CD38 (bottom plots). investigate this hypothesis, we created a genetically engineered mouse strain in which a complementary DNA (cDNA) encoding full-length hBAFF replaces Emr4 the mBAFF-encoding gene. Expression of hBAFF in the endogenous mouse locus did not lead to higher numbers of mature and effector human B cells in hu-mice. Instead, B cells from hBAFF knock-in (hBAFFKI) hu-mice were in proportion more immature than those of hu-mice expressing mBAFF. Memory B cells, plasmablasts, and plasma cells were also significantly reduced, a phenotype that associated with diminished levels of immunoglobulin G and T-cellCindependent antibody responses. Although the reasons for these findings are still unclear, our data suggest that the inefficient B-cell maturation in hu-mice is not due to suboptimal bioactivity of mBAFF on human B cells. Visual Abstract Open in a separate window Introduction Hematopoietic humanized mice (hu-mice) have been developed to study the human immune system in an experimental in vivo model.1-3 These mice bear a transplanted human immune system that can be manipulated and studied with methodologies similar to those used in mice. Major advances in engrafting a human immune system in mice have been achieved using mice with genetic manipulations that lead to severe immunodeficiency and, consequently, minimal rejection of human hematopoietic stem cells (hHSCs) and their differentiated progeny.4-8 One example of these recipients is the BALB/c-(BRG) strain that, when transplanted with hHSCs, develop human B cells, T cells, and, with varying frequencies, other human hematopoietic cell types.9-17 This strain has been recently modified into BRGS with the introduction of the NOD-derived allele ((BRG) and BALB/c-(BRGS) have been DRI-C21045 previously described.9,13,14 Human BAFFKI mice (described in supplemental Methods) were backcrossed into BRG and BRGS genetic backgrounds. All BRG(S) mice were bred and maintained on a diet enriched with Septra under specific pathogen-free and biosafety level 2 conditions at the Biological Resource Center at National Jewish Health (NJH; Denver, CO) or at the University of Colorado Denver Anschutz Medical Center (UCD-AMC) Vivarium (Aurora, CO). To generate hu-PBL mice, peripheral blood mononuclear cells (PBMCs) collected from healthy adult donors in the Clinical Division of NJH were isolated over Ficoll-density gradients. PBMCs were enriched for B cells by depleting CD2+ cells with an Automacs (Miltenyi Biotec) to reach a cell mixture in which B cells were 15% of DRI-C21045 total. Approximately 20 106 of these B-cellCenriched PBMCs were injected per mouse intraperitoneally or IV into adult BRGS and BAFFKI-BRGS mice, 3 to 5 5 hours after sublethal irradiation (250 rad). Hu-PBL mice were analyzed 2 to 3 3 weeks after transplant and before any visible onset of graft-versus-host disease, which is known to occur in this model.25 For the generation of hu-mice, a model that is not affected by graft-versus-host disease, human umbilical cord blood (CB) samples were obtained from the University of Colorado Cord Blood Bank at ClinImmune Labs (Aurora, CO) as samples that were rejected due to low volume or other reasons. CB CD34+ cells were prepared using the CD34+ selection kit (Miltenyi Biotec) and expanded in culture as previously described.13,16 DRI-C21045 Approximately 100?000 DRI-C21045 to 400?000 in vitroCexpanded CD34+ cells were injected IV (typically) or intrahepaticly (less frequently) into 1- to 3-day-old BRG(S) or hBAFFKI-BRG(S) that were previously irradiated with 350 rad. Hu-mice were analyzed 23 to 24 weeks after CD34+ cell transplant. Investigators in this study were blinded from donor identities, and the studies were performed in compliance with NJH and University of Colorado Institutional Review Boards and in accordance with the Declaration of Helsinki. Animal procedures were approved by the NJH Animal Care and Use Committee or the UCD-AMC Institutional Animal Care and Use Committee. Analyses of BAFF expression Analyses of mBAFF and hBAFF by DRI-C21045 enzyme-linked immunosorbent assay (ELISA) and of and by quantitative polymerase chain reaction are described in supplemental Methods. Cell staining, flow cytometry, and cell sorting Cells were stained in staining buffer (phosphate-buffered saline, 1% bovine serum albumin, 0.1% sodium azide) for 15 minutes at 4C and washed 2 times with the same buffer. For intracellular staining, cells were fixed in 2% formaldehyde and permeabilized and stained in 0.5% saponin (Sigma) as previously described.17 Stained cells were run on a Cyan analyzer (Beckman Coulter) either at NJH or UCD Cancer Center flow cytometry cores, and analyses were performed with FlowJo software (TreeStar). Cell sorting was performed on a FACSAria Fusion (BD Biosciences) at UCD, and purity of the sorted populations was at least 97%. Antibodies used in.