Figure 4

Transduction of CD4+ T cells from line 18 mice does not affect proliferation or cytokine production. CD4+ T cells were isolated from spleen and lymph nodes of line DO11.hCARΔcyt.18 double transgenic mice, transduced for 30' with Ad5.UbP.GFP at a MOI of 10 and incubated for 24 h at 37°C/5%CO₂. The GFP positive and GFP negative population were then sorted on a FACSvantage cell sorter before assay. A. Proliferative responses of the GFP- and GFP+ population stimulated for 72 h with increasing amount of OVA peptide in the presence of irradiated BALB/c splenocytes. 50 μCi/ml [³H]thymidine was added to the cultures for 48 h before harvesting the cells and count of the incorporated radioactivity. The curves represent the dose-dependant proliferative responses (as indicated by the [³H]thymidine incorporation in CPM) of the GFP⁺ (closed circles) and the GFP- (open diamond) populations. B. Cytokine production of the transduced CD4+ cells. The GFP+ and GFP- sorted population were stimulated with irradiated BALB/c splenocytes and 5 μg/ml ovalbumin peptide in conditions driving them toward a Th1 or a Th2 phenotype. One week later, the culture supernatant were collected and analyzed by ELISA for the production of IFN-γ, IL-4 and IL-10. The histogram indicates the concentration of the different cytokines produced by the indicated populations. The relative concentrations were calculated using serial dilution of a known standard for each cytokine as a reference curve.