Figure 3
Figure 3. sCD40L may enhance MDSC suppression of activated T cells. (A) sCD40L expanded MDSCs in vitro. Fresh PBMCs from healthy donors (HD) were enriched for the cell population of CD33+HLA-DR− using the Miltenyi isolation kit, and 0, 0.5, 1, or 2 μg/mL of sCD40L or a single dose of irrelevant protein human serum albumin (2 μg/mL) was added to the culture (2.5 × 105/mL), respectively. Three days later, FACS analysis was performed; the frequency of CD33+HLA-DR− cells is shown. (B) sCD40L further decreased T-cell proliferation in the presence of autologous MDSCs. A uniform number of isolated CD3+ cells (106/mL) from 3 HDs were labeled with CFSE and cocultured with various numbers of autologous MDSCs (106/mL, 5 × 105/mL, and 2.5 × 105/mL, respectively). A total of 2 × 105/mL anti-CD3/CD28 beads were added to the cultures, and 2 μg/mL of sCD40L was present or absent in the cultures. FACS assay was carried out by analyzing CFSE dilution of CD3+ T cells 3 days later. The figure shows representative FACS data from 1 sample. (C) sCD40L inhibited T-cell proliferation in MDSC/T-cell coculture in a dose-dependent manner. Autologous MDSCs and CFSE-labeled T cells were isolated from 4 fresh PBMC samples derived from HDs and cocultured. The MDSC to T-cell ratio was 1:4, and anti-CD3/CD28 beads and the indicated concentration of sCD40L or irrelevant protein were also added to the culture. Three days later, FACS analysis was carried out by looking at CFSE dilution of CD3+ T cells. (D) sCD40L further decreased IFN-γ release in the supernatant of the autologous T-cell/MDSC coculture. The supernatant from panel B was collected 18 hours after coculture, and IFN-γ was tested by ELISA. Each data point represents the mean values of the 3 samples. (E) CD40 blockade reversed the sCD40L inhibitory effect on IFN-γ production. MDSCs and autologous T cells were isolated, and 5 μg/mL of anti-IgG or CD40 blocking antibody was preincubated with the isolated MDSCs for 2-4 hours. The autologous T cells stimulated with CD3/CD28 and 2 μg/mL of sCD40L were then added to the cultures. IFN-γ release in cell culture supernatant was tested by ELISA 18 hours after the coculture. Each data point represents the mean values of 3 samples. (F) CD40 blocking antibody did not alter the IFN-γ release from purified T cells in the presence of sCD40L. T cells (106/mL) from these 3 PBMC samples were isolated and cultured in medium containing 2 × 105/mL anti-CD3/CD28 beads in the presence of either 5 μg/mL IgG or CD40 blocking antibodies and 2 μg/mL of sCD40L. The supernatant was collected 18 hours after treatment and IFN-γ was tested by ELISA. Each data point represents the mean values of 3 T-cell cultures. All results shown are representative of 2-4 individual experiments.

sCD40L may enhance MDSC suppression of activated T cells. (A) sCD40L expanded MDSCs in vitro. Fresh PBMCs from healthy donors (HD) were enriched for the cell population of CD33+HLA-DR− using the Miltenyi isolation kit, and 0, 0.5, 1, or 2 μg/mL of sCD40L or a single dose of irrelevant protein human serum albumin (2 μg/mL) was added to the culture (2.5 × 105/mL), respectively. Three days later, FACS analysis was performed; the frequency of CD33+HLA-DR− cells is shown. (B) sCD40L further decreased T-cell proliferation in the presence of autologous MDSCs. A uniform number of isolated CD3+ cells (106/mL) from 3 HDs were labeled with CFSE and cocultured with various numbers of autologous MDSCs (106/mL, 5 × 105/mL, and 2.5 × 105/mL, respectively). A total of 2 × 105/mL anti-CD3/CD28 beads were added to the cultures, and 2 μg/mL of sCD40L was present or absent in the cultures. FACS assay was carried out by analyzing CFSE dilution of CD3+ T cells 3 days later. The figure shows representative FACS data from 1 sample. (C) sCD40L inhibited T-cell proliferation in MDSC/T-cell coculture in a dose-dependent manner. Autologous MDSCs and CFSE-labeled T cells were isolated from 4 fresh PBMC samples derived from HDs and cocultured. The MDSC to T-cell ratio was 1:4, and anti-CD3/CD28 beads and the indicated concentration of sCD40L or irrelevant protein were also added to the culture. Three days later, FACS analysis was carried out by looking at CFSE dilution of CD3+ T cells. (D) sCD40L further decreased IFN-γ release in the supernatant of the autologous T-cell/MDSC coculture. The supernatant from panel B was collected 18 hours after coculture, and IFN-γ was tested by ELISA. Each data point represents the mean values of the 3 samples. (E) CD40 blockade reversed the sCD40L inhibitory effect on IFN-γ production. MDSCs and autologous T cells were isolated, and 5 μg/mL of anti-IgG or CD40 blocking antibody was preincubated with the isolated MDSCs for 2-4 hours. The autologous T cells stimulated with CD3/CD28 and 2 μg/mL of sCD40L were then added to the cultures. IFN-γ release in cell culture supernatant was tested by ELISA 18 hours after the coculture. Each data point represents the mean values of 3 samples. (F) CD40 blocking antibody did not alter the IFN-γ release from purified T cells in the presence of sCD40L. T cells (106/mL) from these 3 PBMC samples were isolated and cultured in medium containing 2 × 105/mL anti-CD3/CD28 beads in the presence of either 5 μg/mL IgG or CD40 blocking antibodies and 2 μg/mL of sCD40L. The supernatant was collected 18 hours after treatment and IFN-γ was tested by ELISA. Each data point represents the mean values of 3 T-cell cultures. All results shown are representative of 2-4 individual experiments.

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