Sialic Acid-Dependent Inhibition of T Cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments

Abstract

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. In this study, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their TCR. This arrest is inhibited by Ab blockade of exosomal GD3 or by the removal of GD3⁺ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

Figure 1.

Exosomes derived from ascites fluid of ovarian cancer patients express GD3 on their surface. (A) 2-D TLC was used to identify gangliosides GD3 and GM3 in ascites fluids. Arrows and numbers (right lower corner) indicate the directions of first and second solvent runs. Origin is in the lower right. Ganglioside standards, noted along the top (solvent 1) and left (solvent 2) margins, are: GM1 (II³NeuAc-GgOse₄Cer); GD1a (IV3NeuAc,II³NeuAc-GgOse₄Cer); GD1b (II³(NeuAc)₂-GgOse₄Cer; GT1b (IV³NeuAc-II³(NeuAc)₂-GgOse₄Cer). Standards on the right margin include GM3 (II³NeuAc-LacCer); GM2 (II³NeuAc-GgOse3Cer); GM1; GD3 (II³(NeuAc)₂-LacCer); GD1a; GD1b. (B) Exosomes were attached to latex beads and either left unstained (filled histogram), labeled with secondary antibody only (dotted line) or with anti-GD3 antibody (solid line) and data acquired using a flow cytometer. Data are representative of 3 independent experiments.

Figure 2.

Immunodepletion of GD3+ exosomes diminishes exosome-mediated T cell arrest. (A) NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with total exosomes (Exo) or exosomes subjected to depletion using either anti-GD3 antibody coupled magnetic beads (GD3 Dep) or isotype control coupled magnetic beads (Iso Dep). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. (C) Calculated % knockdown of inhibition is shown. Mean ± SEM. NS = Not significant (p > 0.05). * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 3.

Antibody-mediated blockade of ganglioside GD3 diminishes exosome-mediated immunosuppression. NDPBL were either left unactivated (Unact) or activated with immobilized antibodies to CD3 and CD28 in media without (Act) or with exosomes (Exo) derived from ovarian tumor ascites fluid in the presence or absence of 10 mg of anti-GD3 antibody. Activation was monitored by determining nuclear translocation of NFkB in CD3+ cells after 2 hours by confocal microscopy (A) or intracellular IFN-g or IL-2 expression in CD3+ cells after 6 hours by flow cytometry (B-E). Representative experiment shown for IFN-g (B) and IL-2 (D). Mean ± SEM from 3 independent experiments shown for IFN-g (C) and IL-2 (E). Percentage inhibition is shown in parentheses. * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Figure 4.

GD3 liposomes arrest activation of T cell through the TCR in a dose dependent manner. (A) NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 with various doses of PC or GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. (B) Percentage inhibition is shown. (C) NDPBL were either left unactivated or activated for 2h with PMA and ionomycin with 1 mM GD3/PC on liposomes. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Percentage inhibition is shown in parentheses. Mean ± SEM; n =3. NS = Not significant (p > 0.05) Mean ± SEM; n =3. ** p ≤ 0.01

Figure 5.

Sialidase treatment of exosomes knocks down T cell arrest. NDPBL were either left unactivated (Unact) or activated for 2h with immobilized antibodies to CD3 and CD28 in media only (Act) or in media with exosomes that were untreated (Exo) or treated with 0.8 U/mL sialidase (Exo + Sia). A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any exosomes present (Act + Sia). Activation was determined by monitoring the upregulation of CD69 on live T cells by flow cytometry following overnight incubation. (A) Representative experiment shown. (B) Compiled data from 5 experiments is shown. Numbers in parentheses indicate % inhibition. There was a 42% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), **p ≤ 0.01.

Figure 6.

Sialidase treatment of GD3 liposomes knocks down T cell arrest. NDPBL were either left unactivated or activated for 2h with immobilized antibodies to CD3 and CD28 in media only or in the presence of 1 mM PC or GD3/PC on liposomes that were untreated or treated with 0.8 U/mL sialidase. A control group was also included where the cells were activated in the presence of the same concentration of sialidase without any liposomes present. Activation was determined by counting the number of CD3+ cells with nuclear NFκB using confocal microscopy. Compiled data from 3 experiments is shown. There was a 97% knockdown of the T cell arrest. Mean ± SEM. n = 3. NS = Not significant (p > 0.05), ***p ≤ 0.001.