Analysis of the cytokine microenvironment after DC immunisation.

<p>(A) Histograms from FACS analysis of transcription factors expressed in CD4⁺ T-cells. Grey histograms are T-cells stained with isotype control, the dashed line represents cells from mice that had received WT DCs, the continuous line represents the mice injected with Lcn2^−/− DCs. The graphs below show the percentage of CD4⁺CD25⁺ T-cells used for statistical analysis. Fourteen mice were analysed using the student’s t-test, p value is *p<0.05. (B) The expression levels of cytokines in the spleens of immunised mice were measured 6 and 18 h after target-cell inoculation. The cytokine levels are shown for WT mice that had received 6 h-LPS-treated Lcn2^−/− DCs (black bars) or 6 h-LPS-treated WT DCs (grey bars). These data, shown as mean ± SEM, are the most consistent results of experiments on pooled mice (5 mice/group) from 3 independently performed experiments. (C) IFN-γ, IL-10 and IL-17A expression in CD4⁺ T-cells measured by ELISPOT. Pooled spots from triplicates of OT-II plus Lcn2^−/− or WT DC-OVA calculated after subtracting number of spots per well of the controls. Histograms show mean ± SEM and they were analysed using student’s t-test, p value is *p<0.05. (D) Ranking of cytokines according to the difference of their expression after DC immunisation. The ratio of expression between mice receiving Lcn2^−/− DCs or WT DCs inoculation was calculated. Then the cytokines were ranked based on these differences. For visualization the data were log2 transformed. The extremities represent the highest difference in secretion (mice receiving Lcn2^−/− DCs secreted more IL-5 than mice that received WT DCs, value 0.5). The left side represents reduced secretion by Lcn2^−/− DCs compared to WT DCs, value −1.5. The ranking orders distinguish two distinct environments after DC immunisation: a T_H1 after WT DCs and a T_H2 after Lcn2^−/− DC immunisation.