Figure 4

SLPI reduces generation of regulatory T cells in vitro. A: Human naïve CD4+ T cells were stimulated with anti-CD3- and anti-CD28-coated beads in serum-free medium in the absence or presence of 500 ng/mL SLPI. After six days, cells were analyzed by flow cytometry for CD4 (DAPI channel (not shown)), CD25 (PE channel) and FoxP3 (APC channel). Left side: Dot blot representing staining with an antibody of the same isotype as the FoxP3 antibody (AlexaFluor 647 mouse IgG1, k, BioLegend), middle: representative dot blot of control treated naïve human CD4+ T cell cultures, right side: representative dot blot of T cell cultures incubated with 500 ng/mL SLPI. B: Column chart shows the average concentration of FoxP3+ CD25^hi CD4+ T cells in CD4+ T cell cultures. p = 0.011 comparing FoxP3+ CD25^hi CD4+ T cells incubated with and without 500 ng/mL SLPI. C: Human naïve CD4+ T cells were stimulated with anti-CD3- and anti-CD28-coated beads in RPMI medium in the absence or presence of 500 ng/mL SLPI. After four days, the cells were cocultured at a ratio of 1:5 with naïve CFSE labeled CD4+ T cells from the same donor. The CFSE profiles of the labeled cells were determined after 4 days. The number of divisions was determined by counting the number of peaks. Representative histogram of CSFE labeled T cells. D: Bars represent the proportion of CFSE labeled CD4+ T cells which proliferated and the total number of CFSE labeled cells. For every number of divisions, the differences between cell cultures containing SLPI-treated T cells and control treated T cells were tested for their statistical significance using a Suet' t-test (*, p < 0.05; **, p < 0.01). E: Human naïve CD4+ T cells were stimulated with anti-CD3- and anti-CD28-coated beads in RPMI medium in the absence or presence of 500 ng/mL SLPI and various concentrations of TGF-α. After four days, the cells were analyzed by flow cytometry for CD4 (DAPI channel), CD25 (PE channel) and FoxP3 (APC channel). The columns represent the average density of FoxP3+ CD4+ T cells in the in vitro cultures. *: p < 0.05 comparing FoxP3+ CD4 T cells incubated with and without SLPI