Abstract A23: Neuropilin-1 expression on regulatory T cells in ovarian cancer
Authors: Cillo Anthony R. , Bruno Tullia , Modugno Francesmary , Edwards Robert , Vignali Dario 
Published: August 2018

Abstract
Introduction: Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that suppress autoimmune responses, but also prevent clearance of tumors and chronic viral infections. In ovarian cancer, a higher frequency of tumor-infiltrating Treg is associated with poor prognosis, but insights into the mechanisms governing the survival and suppressive activity of Treg in this context are limited. Our group has recently described a signaling axis through neurophilin-1 (NRP1) on Treg in murine models of cancer that promotes the survival and suppressive function of Treg. Genetic knockout of NRP1 on murine Treg leads to reduced tumor growth and increased survival, underscoring the importance of NRP1+ Treg in suppressing antitumor immunity. Given the importance of NRP1+ Treg in murine cancer models, we sought to explore the expression pattern of NRP1 on tumor-infiltrating Treg from patients with ovarian cancer.
Materials and Methods: Ovarian tumors were obtained through the health sciences tissue bank at the University of Pittsburgh, and ovarian ascites fluid was obtained through Magee Women’s Research Institute. Peripheral blood was obtained from healthy donors through the Red Cross. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation, and tumor-infiltrating lymphocytes were isolated by first mechanically disrupting the tumor, followed by treatment for 15 minutes at 37°C with 50 ug/mL Liberase DL. For flow cytometric analysis of Treg, cells from blood and tumors were first stained for surface markers, followed by live/dead discrimination, fixation and permeabilization, and staining for intracellular markers. Cells were analyzed on an LSR Fortessa at the Hillman Cancer Center Flow Core. For functional assays, Treg were selected from ascites fluid and assayed for their ability to suppressive naïve CD8+ T cells ex vivo. Wilcoxon rank sum tests were used to assess differences between groups, and a two-sided alpha less than 5% was considered significant.
Results: A total of 12 healthy donors and 20 ovarian cancer patients were studied. Intracellular and surface NRP1 expression was assessed by flow cytometry on CD4+CD25+FOXP3+ Treg from healthy donor peripheral blood and tumor-infiltrating lymphocytes from either ovarian cancer tumors or ascites fluid from patients with ovarian cancer. Intracellular NRP1 was expressed on a median of 1.8% (interquartile range [IQR]: 0.69% to 4.8%) of Treg from healthy donors compared with a median of 66% (IQR: 28% to 90%; p&lt;0.001) and 82% (IQR: 38% to 99%; p=0.004) of Treg from ovarian tumor-infiltrating lymphocytes (TIL) and ascites, respectively. Surface NRP1 was detected on 0.1% (IQR: 0% to 1%) of Treg in PBMC from healthy donors, compared with 11.1% (IQR: 1.1% to 60%; p=0.01) on ovarian TIL and 82% (IQR: 14% to 82%; p=0.0015) on ascites. Comparing expression of intracellular NRP1 on Treg from N=2 benign ovarian tissues versus N=14 malignant ovarian tumors, there was a trend towards increased NRP1 expression (median 9.3% versus 54.5%, respectively; p=0.044). Finally, Treg isolated from ascites fluid were capable of suppressing CD8+ T cells ex vivo proliferation in a dose-dependent manner.
Conclusions and Future Directions: Both intracellular and surface NRP1 are expressed more frequently on tumor-infiltrating Treg and Treg from ascites fluid compared to Treg from healthy donor peripheral blood. Although the number of patients currently included in this study is small, there was also evidence that intracellular NRP1 expression on Treg was higher on malignant compared with benign tissue. Additionally, Treg from ascites fluid were capable of suppressing CD8 cells ex vivo. Future studies will evaluate whether blockade of NRP1 on human Treg limits either the survival or suppressive capacity of Treg.
Note: This abstract was not presented at the conference.
Citation Format: Anthony R. Cillo, Tullia Bruno, Francesmary Modugno, Robert Edwards, Dario Vignali. Neuropilin-1 expression on regulatory T cells in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A23.