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. 2015 Nov;3(11):1269-78.
doi: 10.1158/2326-6066.CIR-15-0086. Epub 2015 Jun 25.

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T-cell Signaling Cascade

Raymond J Kelleher Jr  1 , Sathy Balu-Iyer  2 , Jenni Loyall  1 , Anthony J Sacca  1 , Gautam N Shenoy  1 , Peng Peng  1 , Vandana Iyer  2 , Anas M Fathallah  2 , Charles S Berenson  3 , Paul K Wallace  4 , Joseph Tario  4 , Kunle Odunsi  5 , Richard B Bankert  6
Affiliations

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T-cell Signaling Cascade

Raymond J Kelleher Jr et al. Cancer Immunol Res. 2015 Nov.

Abstract

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients' antitumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T-cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80 nm. The T-cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS-expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle-induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immunosuppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T-cell function represent a potential therapeutic target for patients with ovarian cancer.

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Conflict of interest statement

Conflict of Interest: No conflicts to disclose.

Figures

Fig 1
Fig 1. Lipids present in tumor ascites fluid and phosphatidylserine (PS) containing liposomes induce T cell signaling arrest
Human normal donor peripheral blood lymphocytes (NDPBL) were incubated in medium only (UN) or with immobilized anti-CD3/CD28 in medium only (Act), A. in medium with 50% ovarian tumor ascites fluid (Act + AF), or in medium with the aqueous phase of delipidated 50% ovarian tumor ascites fluid (Act + D-AF) (n=4, mean ± SEM) B. in medium with PC liposomes or with PS/PC liposomes (n=7, mean ± SEM). Activation was monitored by confocal microscopy to quantify the percentage of CD3+ T cells with nuclear NFκB. The numbers in parentheses indicate percent inhibition. NS = Not Significant; * = p<0.05; ** = p<0.01; *** = p<0.005 – Same for all figures.
Fig 2
Fig 2. Tumor ascites fluid directly inhibits CD4+ and CD8+ T cell activation
NDPBL sorted for CD4+ or CD8+ were incubated in media only (UN), with immobilized anti-CD3/CD28 in medium only (Act), or in medium with 50% ascites fluid (Act + AF) (n=3, mean ± SEM). * = p<0.05. See also Figure S2.
Fig 3
Fig 3. Extracellular vesicles derived from ovarian tumor ascites fluid by ultracentrifugation inhibit the activation of T cells in a dose dependent manner
NDPBL were incubated in medium only (UN) or with immobilized anti-CD3/CD28 in medium only (Act), in medium with 50% ovarian tumor ascites fluid (Act + AF), A. in medium with extracellular vesicles derived from 50% ovarian tumor ascites fluid (Act + EV) (n=8, mean ± SEM), B. in medium with the supernatant of 50% ovarian tumor ascites fluid after ultracentrifugation (Act + UC-AF) (n=11, mean ± SEM). C. NDPBL were incubated with increasing concentrations of extracellular vesicles based on EV protein weight (n=3, mean ± SEM). D. NDPBL were incubated with PMA and Ionomycin in the presence or absence of extracellular vesicles (n=8, mean ± SEM).
Fig 4
Fig 4. Flow cytometric analysis demonstrates that extracellular vesicles derived from ascites fluids bind to fluorescently labeled Annexin V
A. Unlabeled EV. B. EV labeled with R-phycoerythrin (PE) conjugated Annexin V (PE-Annexin V). C. EV labeled with PE-Annexin V, competed with 100× unlabeled Annexin V. D. EV labeled with PE-Annexin V, competed with 100× unlabeled bovine serum albumin. E. EV stained with PE-Annexin V and labeled CD90 (Endothelial cell marker), CD326 (EpCAM), or CD45 (Leukocyte marker) antibodies. F. EV stained with labeled CD41 (platelet marker), CD71 (transferin), CD235 (erythrocyte marker), or MHC Class I antibodies.
Fig 5
Fig 5. Antibodies to phosphatidylserine block the extracellular vesicle induced T cell signaling arrest
NDPBL were incubated in medium only (UN) or with immobilized anti-CD3/CD28 in media only (Act), A. in media containing extracellular vesicles derived from 50% ovarian tumor ascites fluid with (Act + EV + αPS) or without (Act + EV) phosphatidylserine antibody (n=3, mean ± SEM), B. in extracellular vesicles derived from solid ovarian tumor with (Act + TEV + αPS) or without phosphatidylserine antibody (Act + TEV) (n=3, mean ± SEM). C. Human NDPBL were incubated in medium only (UN) or with immobilized antibodies to CD3 and CD28 in media only (Act) or in media containing extracellular vesicles derived from 50% ovarian tumor ascites fluid without (Act+ EV) or with depletion using anti-phosphatidylserine antibody coupled magnetic beads (Act + Depl. EV) or in a negative control depletion using isotype labeled magnetic beads (Act + Iso. EV) (n=3, mean ± SEM).
Fig 6
Fig 6. Inhibition of diacylglycerol kinase reverses the tumor ascites fluid- and extracellular vesicle-induced T cell signaling arrest
NDPBL were incubated in medium only (UN) or with immobilized anti-CD3/CD28 in media only (Act), A. in media containing 25% ovarian ascites fluid with (Act + AF + DGKi) or without (Act + AF) 100µM DGK inhibitors (n=3, mean ± SEM), B. in ascites fluid-derived extracellular vesicles with or without each of the DGK inhibitors. (n=3, mean ± SEM). [DGK inhibitors used are a combination of R59949 and R59022].
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