Elevated serum soluble CD40 ligand in cancer patients may play an immunosuppressive role

Abstract

Tumor cells can induce certain cytokines and soluble receptors that have a suppressive effect on the immune system. In this study, we showed that an extracellular portion of a membrane-bound ligand of CD40 (soluble CD40 ligand; sCD40L) was significantly elevated in the serum of cancer patients compared with healthy donors. In addition, PBMCs from cancer patients had a relatively larger population of myeloid-derived suppressor cells (MDSCs), defined as CD33(+)HLA-DR(-) cells, and these cells expressed higher levels of CD40. T-cell proliferation and IFN-γ production decreased when stimulated T cells were cocultured with an increased amount of autologous MDSCs. The addition of recombinant monomeric sCD40L enriched MDSCs and had an additive inhibitory effect on T-cell proliferation. PBMCs cultured in vitro with sCD40L also showed an expansion of regulatory T cells (CD4(+)CD25(high)Foxp3(+)), as well as induction of cytokines, such as IL-10 and IL-6. Moreover, sCD40L-induced enrichment of programmed death-1-expressing T cells was greater in cancer patients than in healthy donors. Preexisting sCD40L also inhibited IL-12 production from monocytes on activation. These data suggest that the higher levels of sCD40L seen in cancer patients may have an immunosuppressive effect.

Trial registration: ClinicalTrials.gov NCT00019695 NCT00060528 NCT00081848 NCT00179309 NCT00436956 NCT00514072.

Figures

Figure 1

Differential levels of serum sCD40L in cancer patients and healthy donors (HD). (A) Elevated serum sCD40L in cancer patients. Serum sCD40L levels in HDs (n = 16) were compared with levels in patients with metastatic breast cancer (Breast Ca, n = 40), metastatic colon cancer (Colon Ca, n = 17), or first-degree (1°) prostate cancer (Prostate Ca, n = 30) before treatment of any kind. (B) sCD40L levels did not change after vaccination with PSA-TRICOM. Levels of sCD40L in 31 metastatic prostate cancer patients' sera before and after treatment with PSA-TRICOM vaccine were analyzed. (C) Association of pretreatment serum sCD40L levels with overall survival (OS) and Halabi-predicted survival (HPS) in prostate cancer patients vaccinated with PSA-TRICOM. Pretreatment serum samples were evaluated and compared for sCD40L between patients who had longer or shorter survival than their HPS. (D) Decrease in sCD40L in serum of prostate cancer patients after a combination of ketoconazole and alendronate treatments. Levels of serum sCD40L were analyzed by ELISA, and statistical analysis was performed using an unpaired t test. **P < .01. NS indicates not significant.

Figure 2

Differential levels of MDSCs and CD40-expressing MDSCs in PBMCs from cancer patients (CP) and healthy donors (HD). (A) Representative FACS data from 4 HDs and 4 CPs. Age-matched PBMCs from HDs and CPs were stained with antibodies, and propidium iodide (PI) was added 10 minutes before FACS analysis. R1 and PI− cell populations were gated, and the frequency of MDSCs (CD33+HLA-DR− cells) is shown in the right lower quadrant. (B) CPs had significantly higher frequencies of MDSCs in PBMCs than HDs. PBMCs from HDs (n = 30) and untreated patients with metastatic breast cancer (Breast Ca, n = 9) or metastatic prostate cancer (Prostate Ca, n = 11) were evaluated. Statistical analysis was performed using an unpaired t test. (C) Higher frequency of CD40-expressing MDSCs in CPs than in HDs. Samples used in panel B were evaluated for CD40 expression on the MDSC population. The data shown in CPs were from all CPs (9 breast Ca and 11 prostate Ca). The results shown are representative of 2 experiments. Statistical analysis was performed using an unpaired t test. FSC indicates forward scatter; and SSC, side scatter.

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.

Figure 4

sCD40L increased Tregs and CD4+CD25int cells in vitro. (A) FACS analysis of the frequency of the CD4+CD25high population in CD3+ T cells after adding sCD40L. PBMCs from 5 healthy donors (HD) were incubated in medium with or without 2 μg/mL of sCD40L. Both sCD40L− and sCD40L+ cultures contained 25 U/mL of IL-2. Four days later, cells were analyzed by multicolor FACS for CD3, CD4, CD25, and intracellular Foxp3. (B) The cells were analyzed by gating on the CD4+CD25high population. (C) CD4+CD25high cells were mainly Foxp3+. Intracellular staining of Foxp3 was performed, and a comparison of Foxp3 expression between CD4+CD25high and CD4+CD25− cells is shown for 1 sample that is representative of 5 PBMCs that were tested individually. The data were repeated 3 times. Statistical analysis was performed using a paired t test.

Figure 5

sCD40L induced greater PD-1 expression in vitro on T cells from cancer patients (CP) than T cells from healthy donors (HD). (A-B) Induction of CD25 and CD70 on T cells. PBMCs from 6 age-matched HD and 5 metastatic prostate CP were incubated in medium containing 25 U/mL of IL-2 with or without 2 μg/mL of sCD40L for 4 days. Cells were then analyzed by FACS. (C) Comparison of PD-1–expressing T cells in PBMCs between HD and CP after the addition of sCD40L to the culture. The samples were also evaluated for PD-1 expression on CD3+ T cells. (D) Differential enhancement of PD-1 expression on CD4+ and CD8+ T cells after the addition of sCD40L to cell cultures in HD and CP. PD-1 expression on CD4+ and CD8+ T cells was evaluated. All results are representative of 2 individual repeats. Statistical analysis was performed using a paired t test. *P < .05. NS indicates not significant.

Figure 6

Preexisting sCD40L decreased IL-12 production from monocytes after in vitro stimulation. (A) Monocytes were isolated from PBMCs of a healthy donor using CD14 beads. The cells (106/mL) were incubated with 2 μg/mL of sCD40L for 20 hours, after which IFN-γ (300 U/mL) and Golgi Stop (0.8 μL/mL) were added. Two hours later, a second signal (50 ng/mL LPS) was added to the culture. Cells were harvested 6 hours after the initial IFN-γ stimulation, and intracellular staining of IL-12 was performed. (B) Summary of data from 5 individual assays described in panel A of monocytes derived from 5 PBMC samples. The data were repeated 4 times. Statistical analysis was performed using a paired t test. SSC indicates side scatter.