Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects - PubMed (original) (raw)

Comparative Study

Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects

D D Taylor et al. Br J Cancer. 2005.

Abstract

Dendritic and lymphoid 'exosomes' regulate immune activation. Tumours release membranous material mimicking these 'exosomes,' resulting in deletion of reactive lymphocytes. Tumour-derived 'exosomes' have recently been explored as vaccines, without analysis of their immunologic consequences. This investigation examines the composition of tumour-derived 'exosomes' and their effects on T lymphocytes. Membranous materials were isolated from ascites of ovarian cancer patients (n=6) and Western immunoblotting was performed for markers associated with 'exosomes.' Using cultured T cells, 'exosomes' were evaluated for suppression of CD3-zeta and JAK 3 expressions and induction of apoptosis, measured by DNA fragmentation. 'Exosome' components mediating suppression of CD3-zeta were isolated by continuous eluting electrophoresis and examined by Western immunoblotting. 'Exosomes' were shown to be identical with previously characterised shed membrane vesicles by protein staining and TSG101 expression. 'Exosomes' expressed class I MHC, placental alkaline phosphatase, B23/nucleophosmin, and FasL. 'Exosomes' suppressed expression of T-cell activation signalling components, CD3-zeta and JAK 3 and induced apoptosis. CD3-zeta suppression was mediated by two components: 26 and 42 kDa. Only the 42 kDa component reacted with anti-FasL antibody. These results indicate that, while 'exosomes' express tumour antigens, leading to their proposed utility as tumour vaccines, they also can suppress T-cell signalling molecules and induce apoptosis.

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Figures

Figure 1

Figure 1

SDS–PAGE separation of chromatographically isolated membrane vesicles (designated lanes A) and centrifugally isolated ‘exosomes’ (designated lanes B) from the ascites of the same ovarian cancer patients, visualised by silver staining.

Figure 2

Figure 2

(A) Representative Western immunoblots of SDS–PAGE separation of chromatographically isolated membrane vesicles (designated lanes A) and centrifugally isolated ‘exosomes’ (designated lanes B) from the ascites of the same ovarian cancer patients, incubated with anti-TSG101 antibody and visualised by ECL. (B) Densitometric analysis of TSG101 expression on these membrane vesicle isolates.

Figure 3

Figure 3

Western immunoblots demonstrating presence of HLA-A, Fas ligand, B23/nucleophosmin, and placental alkaline phosphatase on centrifugally isolated ‘exosomes’ from the ascites of ovarian cancer patients.

Figure 4

Figure 4

(A) Western immunoblots indicating the expression of CD3-ζ protein and JAK 3 by Jurkat cells, following incubation with 400 _μ_g ml−1 of the centrifugally isolated ‘exosomes’ or analogous gradient material (Control, C) for 48 h. (B) Densitometric quantitation of CD3-ζ and JAK 3 expression by treated Jurkat cells.

Figure 5

Figure 5

Induction of apoptosis in Jurkat T cells by 400 _μ_g ml−1 of the centrifugally isolated ‘exosomes’ vs the analogous fraction from control female controls for 24 h, as defined by DNA fragmentation.

Figure 6

Figure 6

Identification of the ‘exosomes’-associated inhibitors of CD3-ζ expression. ‘Exosomes’ were fractionated by continuously eluting electrophoresis and fractions were subsequently assayed for ζ suppression in a Jurkat bioassay. The fractions suppressing ζ expression were examined by SDS–PAGE with silver staining (A). Western immunoblotting of the two fractions with anti-FasL demonstrated reactivity with the 42 kDa component, but not with the 26 kDa (B).

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