Figure 1

Generation of humanized BLT-NSG (huBLT) mice and human cell reconstitution. (a) Bone marrow-Liver-Thymus (huBLT) mice were generated by transplantation of human fetal liver and thymus under the kidney capsule of an adult NSG mouse. Post-surgical transplant, mice were sublethally irradiated (200 cGy) and intravenously injected with human CD34+ hematopoietic progenitor cells (HPCs) isolated from autologous fetal liver tissue. Post T-cell reconstitution (12–16 weeks following engraftment), huBLT mice were infected with HCMV by intraperitoneal (IP) injection of HCMV-infected fibroblasts or Mock infected by IP injection of uninfected fibroblasts. Beginning at 6 weeks post-infection huBLT mice are screened for HCMV-specific immune responses. Latently-infected huBLT mice are treated with G-CSF to induce viral reactivation at 8 weeks post-infection. (b) Human cell reconstitution was monitored by flow cytometry analysis of peripheral blood for the percentage of human CD45+ leukocytes (out of total human plus murine CD45+ leukocytes) beginning at 8 weeks post-humanization. Human CD45+ leukocytes can be further analyzed using human specific antibodies, including assessment of CD3+ T-cells and CD19+ B-cells (panel 1) and CD3+ T-cells further discriminated into CD4 and CD8 subsets (panel 2). In addition, monocyte subsets, as characterized by CD14 and CD16 staining, are present in the periphery (panel 3). C) Progenitor cell reconstitution was analyzed in the bone marrow using antibodies for human CD45 and CD34 (panel 1). CD34+ HPCs were further analyzed using antibodies for CD117 (c-kit) and CD38 (panel 2), and monocyte subsets cells analyzed using antibodies for CD33 (early) and CD14 (maturing) (panel 3). Data shown in (b and c) is from a huBLT mouse (cohort 3) at 17 weeks post-humanization gated on viable, muCD45- leukocytes. huBLT mice were divided equally into experimental groups based on overall human leukocyte reconstitution (human CD45+) and human T-cell reconstitution (human CD3+) in the periphery. At 8 weeks post-infection, huBLT mice (cohort 2) were reactivated by treatment with G-CSF and AMD3100. Seven days post-reactivation, all mice were euthanized and lymphoid organs collected. Genomic DNA was isolated using DNAzol and viral load determined by qPCR using primers and probe specific for HCMV UL144. Each sample was analyzed in triplicate. Data is shown for the average with standard error of the mean of two spleen tissue sections (d) or four liver tissue sections (e) per mouse (HCMV, n = 7; HCMV + G-CSF, n = 6), normalized to 1 ug of input DNA. Statistical analysis performed by one-way ANOVA.