Survival of Ovarian Cancer Patients Is Independent of the Presence of DC and T Cell Subsets in Ascites

Abstract

Ascites is a prominent feature of ovarian cancer and could serve as liquid biopsy to assess the immune status of patients. Tumor-infiltrating T lymphocytes are correlated with improved survival in ovarian cancer. To investigate whether immune cells in ascites are associated with patient outcome, we analyzed the amount of dendritic cell (DC) and T cell subsets in ascites from ovarian cancer patients diagnosed with high-grade serous cancer (HGSC). Ascites was collected from 62 HGSC patients prior to chemotherapy. Clinicopathological, histological and follow-up data from patients were collected. Ascites-derived immune cells were isolated using density-gradient centrifugation. The presence of myeloid DCs (BDCA-1⁺, BDCA-3⁺, CD16⁺), pDCs (CD123⁺BDCA-2⁺), and T cells (CD4⁺, CD8⁺) was analyzed using flow cytometry. Complete cytoreduction, response to primary treatment and chemosensitivity were associated with improved patient outcome. In contrast, immune cells in ascites did not significantly correlate with patient survival. However, we observed a trend toward improved outcome for patients having low percentages of CD4⁺ T cells. Furthermore, we assessed the expression of co-stimulatory and co-inhibitory molecules on T cells and non-immune cells in 10 ascites samples. PD-1 was expressed by 30% of ascites-derived T cells and PD-L1 by 50% of non-immune cells. However, the percentage of DC and T cell subsets in ascites was not directly correlated to the survival of HGSC patients.

Keywords: T cells; ascites; dendritic cells; immune environment; ovarian cancer.

Figure 1

T cell and DC subsets in ascites. DC en T cells subsets were measured in ascites of 62 high-grade serous ovarian cancer patients using flowcytometry. (A) Gating strategy for myeloid dendritic cells. Lymphocytes were excluded based on FSC/SSC. BDCA-1⁺, BDCA-3⁺, and CD16⁺ mDCs were gated in the CD45⁺CD14⁻ cell population. (B) pDCs were identified based on co-expression of BDCA-2 and CD123. (C) T cell gating strategy. CD4⁺ and CD8⁺ T cells were gated in the CD3⁺ lymphocyte population. (D) Percentage of DC subsets in ascites. (E) Percentage of T cells in ascites. Red line indicates mean.

Figure 2

Kaplan-Meier curves for progression-free survival of HGSC patients. (A) Progression-free survival curves for clinical characteristics. (B) Progression-free survival for patients stratified as having low or high percentages of immune cells in ascites. Cut-off values based on median. BDCA-1: 1.8%; BDCA-3: 0.9%; CD16: 2.8%; pDC: 2.1%; CD4: 45.5%; CD8: 33.0%; (C) Progression-free survival for patients stratified as having low or high CD4/CD8 ratios. Cut-off at 1.3, based on population median. P-values for significant differences are given. P-values < 0.05 were considered significant.

Figure 3

Kaplan-Meier curves for overall survival of HGSC patients. (A) Overall survival curves for clinical characteristics. (B) Overall survival for patients stratified as having low or high percentages of immune cells in ascites. Cut-off values based on median. BDCA-1: 1.8%; BDCA-3: 0.9%; CD16: 2.8%; pDC: 2.1%; CD4: 45.5%; CD8: 33.0%; (C) Overall survival for patients stratified as having low or high CD4/CD8 ratios. Cut-off at 1.3, based on population median. P-values for significant differences are given. P-values < 0.05 were considered significant.

Figure 4

Expression of MHC, co-stimulatory and co-inhibitory molecules on CD3⁺ T cells and non-immune cells in ascites. Flow cytometry gating of immune checkpoint and MHC on (A) CD3⁺ positive cells and (C) CD45⁻ cells. Gray line represents control; black line represents CD3⁺ T cells or CD45⁻ cells. (B) CD3⁺ T cells and (D) CD45⁻ cells positive for co-stimulatory, co-inhibitory, and MHC molecules. Red lines indicate mean.