Figure 4
Functional analysis of hCycT1 transgenesis in T-cell bulk cultures from rats mirrors the subset-specific impact in CD4 T-cells. Activated, spleen-derived T-cell cultures from six hCycT1-tg and two n-tg rats were infected with VSV-G HIV-1NL4-3 GFP. (A-C) Three days later, cells were stained with a rCD4-PE antibody (clone OX-8) and analyzed for GFP expression in CD4-positive or CD4-negative T-cells. Shown are representative flow cytometry dot plots of (A) viable cells identified by FSC/SSC characteristics (R1 gate), or infected, stained T-cells from one (B) n-tg or (C) hCycT1-tg rat with double-positive cells and corresponding MFI values indicated in gates R2 (rCD4-positive cells) and R3 (rCD4-negative cells). The percentage of rCD4-positive cells of all viable, rCD3-positive lymphocytes were (B) 61% (and 39% CD8-positive T-cells), and (C) 27% (and 73% CD8-positive T-cells). The CD4low sub-population in B and C likely reflects residual monocytes. (D-E) Quantitative analysis of early HIV-1 gene expression, represented by the MFI (GFP) in infected T-cell subsets: (D) bulk T-cells, (E) CD4-positive T-cells, (F) CD4-negative T-cells. Histogram bars depict the arithmetic means + SD of triplicates.