OMIP‐012: Phenotypic and numeric determination of human leukocyte reconstitution in humanized mice

This panel was developed to determine both the frequency and absolute number of human leukocytes and leukocyte subsets present in the peripheral blood of humanized mice. The panel also provides information concerning the activation state of peripheral leukocytes by cell surface staining for HLA-DR and CD38, relevant to studies of HIV disease pathogenesis (1-3). This panel has been used with EDTA anticoagulated whole blood in conjunction with bead-based enumeration for quantitative assessment of human cell chimerism. This panel also works well for staining of peripheral blood mononuclear cells (PBMCs) prepared by density gradient centrifugation and for dispersed splenocytes. The multicolor panel described here has been used in studies of humanized mouse reconstitution (4) and longitudinal studies of HIV pathogenesis in NSG-BLT mice (5). 1, Classification of human leukocyte subsets in NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ (NSG)-BLT mice and evaluation of lymphocyte activation in HIV-infected mice. A: Left column; samples were first gated for singlet events using a FSC-A by FSC-H plot. Singlet events were then gated for Trucount beads and PBMCs. Trucount tubes were used according to manufacturer's instructions. Briefly, 50 μl of antibody master mix was added to each tube followed by the addition of 50 μl EDTA anticoagulated whole blood and briefly vortexed. After 15 min incubation, 450 μl of BD FACS-Lyse solution was added. Samples were then stored at 4°C until acquisition (within 6 h). The number of Trucount beads is used in conjunction with the number of events within each cell gate to calculate the absolute number of cells per unit volume of whole blood using the formula shown. At right, PBMC were separated by species of origin based on CD45 expression using mouse and human specific anti-CD45 antibodies. Examples are shown for an unmanipulated NSG control mouse (top), a human peripheral blood control (middle), and a humanized NSG-BLT mouse (bottom). Human CD45+ leukocytes were further typed into neutrophils (SSC-A high), monocytes (SSC-A intermediate), and lymphocytes (SSC-A low). Lymphocytes were separated into CD19+ B cells, CD3+ T cells, and a double-negative population. CD3+ T cells were separated into CD4+ and CD8+ T cells. We typically observe most human cells falling in the lymphocyte gate but have also noted that there is an increased frequency and a lower MFI for CD19+ B cells in NSG-BLT mice. B: CD56+ NK cells are enumerated from the CD3−, CD19− (double negative) lymphocyte gate in “A.” A fluorescence minus one (FMO) gating control was used to set the NK cell gate (middle). C: CD4+ T cells (top), CD8+ T cells (middle), and CD19+ B cells (bottom) were evaluated for the activation markers HLA-DR and CD38. Human EDTA anti-coagulated peripheral blood was used as a gating control and for both the FMO (HLA-DR FITC) and isotype (CD38-PE) control. In our experience, an isotype control has been more useful that an FMO for PE staining of whole blood, as there appears to be some nonspecific background binding of PE. (This is not observed when using density gradient separated PBMC or splenocytes). Examples are shown for an NSG-BLT mouse at the time of inoculation (intravaginal with 220,000 TCID50 JR-CSF) when it was HIV-1 negative, and the same mouse 8 weeks later with HIV viremia (HIV RNA 3.86 log10 copies/100 μl plasma), displaying increased expression of HLA-DR and/or CD38 on each of the lymphocyte populations. The development and utilization of humanized mice has accelerated greatly over the past decade as the importance of small animal models for human infectious disease research has become more recognized (6). The need is most apparent in HIV research where there is a strong push to evaluate current and prospective antiretroviral agents for their prophylactic potential to prevent HIV transmission (7, 8). Here, the inability of HIV to infect mouse CD4+ T cells is circumvented by the implantation of human thymus and liver tissue under the kidney capsule of immunodeficient strains of mice, which develops into a Thy/Liv organoid replete with human T cell targets for HIV. The recognition that particular mutations specific to the NOD strain of mice (9), plus knockout of the cytokine common gamma chain (10, 11), allows for peripheral dissemination of human leukocytes throughout the mouse periphery has opened the door to studying the natural routes of HIV transmission in these mice. Unfortunately, varying levels of mouse hematopoietic recovery following irradiative preconditioning makes for an unreliable denominator when ascertaining the percentage of human leukocyte chimerism in mouse peripheral blood. This necessitates the need for quantitative enumeration of human cells on a per unit volume basis for reliable assessment of human cell engraftment. To that end, we have employed a bead enumeration technique using BD Trucount tubes in which the number of events within each cell gate is related to a known number of fluorescent beads to calculate the absolute number of cells per unit volume of whole blood. 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