Figure 1.

AEP is required for normal IFN-γ production in human and mouse CD4⁺ T cells. (A,B) CD46 drives AEP expression and nuclear translocation. Human CD4⁺ T cells were left non-activated (NA) or activated with the depicted antibody combinations and AEP expression assessed 36 h post activation by (Ai) FACS with (Aii) statistical analyses and (Bi) Western blotting of the cytoplasmic and nuclear fractions with (Bii) respective statistical analyses of the signals by densitometry. Shown are one representative FACS and two Western blot experiments of n = 3 using a different donor each time. (C) AEP inhibition suppresses human Th1 induction. T cells were activated as described under “A” with or without 25 or 50 μM of a specific AEP inhibitor and IFN-γ and IL-10 (co)secretion measured 36 h post activation. (Ci) shows FACS data derived from a representative donor whilst (Cii) summarizes the analyses for the shown activation conditions of n = 6 donors. (D) AEP inhibition does not affect cell proliferation. Cell trace violet-labeled CD4⁺ T cells were CD3+CD46-activated in the presence or absence of 50 μM AEP inhibitor and cell proliferation measured at 6 d post activation. (Di) Shows a representative FACS profile and (Dii) the accompanying statistical analysis from four different experiments (n = 4). (E) AEP is also required for normal Th1 induction in mice. Naïve CD4⁺ T cells isolated from wild type (WT) or AEP-deficient (Lgmn^−/−) mice (n = 5) were activated for 6 days under Th1, Th2, or Th17 skewing conditions and the total numbers of IFN-γ (Th1), IL-4 (Th2), or IL-17-positive (Th17) cells assessed by intracellular cytokine staining. The number of FoxP3-positive natural regulatory T cells (nTregs) was assessed in activated cell cultures without addition of skewing cytokines/antibodies. Error bar graphs represent mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns, not significant.