CD4(+) T cell activation promotes the differential release of distinct populations of nanosized vesicles - PubMed (original) (raw)

CD4(+) T cell activation promotes the differential release of distinct populations of nanosized vesicles

Els J van der Vlist et al. J Extracell Vesicles. 2012.

Abstract

Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4(+) T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4(+) T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.10-1.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.10-1.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status.

Keywords: NTA; T cells; exosomes; extracellular vesicles; flow cytometry; immune regulation; microparticles; microvesicles; nanosized vesicles.

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Figures

Fig. 1

Fig. 1

Analysis of T cell activation after different levels of TCR- and co-stimulation triggering. CD4+ T cells (KO4C1) were activated by TCR-triggering (0.1 or 10 µg/ml plate-bound anti-CD3), with or without additional co-stimulation (0.5 or 5 µg/ml plate-bound anti-CD28) for 20 hours and compared to non-activated T cells. Cells were analysed for CD69 upregulation (A), TCR downregulation (B) and IFN-gamma production (C) by flow cytometry. Histograms (top panels) show the expression of indicated markers on non-activated T cells (grey line) or on T cells activated with anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml) (black line) or isotype control stainings (filled histograms). The bar graphs (bottom panels) show the geometric means expressed as percentages of maximal expression of the indicated markers (set to 100%) of at least 3 independent experiments.

Fig. 2

Fig. 2

Quantitative analysis of nanosized vesicles released by T cells. KO4C1 T cells were activated as described in Fig. 1. Vesicles released by T cells were isolated from culture supernatants, labelled with the fluorescent membrane dye PKH67 and floated to equilibrium density into a sucrose gradient. The vesicles in the collected sucrose gradient fractions were analysed by fluorescence-based high-resolution flow cytometry. (A) Dot plot of reduced wide-angle FSC versus PKH67 fluorescence representing fluorescent vesicles derived from non-activated T cells (from pooled fractions with densities of 1.12–1.17 g/ml). (B) Fluorescently labelled vesicles from differently activated T cells were quantified using our time-based quantification method. Indicated are the average and standard deviation of the number of vesicles released of at least 3 independent experiments (from fractions with densities of 1.10–1.19 g/ml). The number of vesicles derived from non-activated T cells was set to 1. (C) Fluorescently labelled vesicles from non-activated or activated (10 µg/ml anti-CD3 + 5 µg/ml anti-CD28) T cells were quantified using our time-based quantification method. Indicated is the number of events measured in 30 seconds in the indicated sucrose gradient density fractions. One representative out of 6 experiments is shown (D) Fold increase in the number of released vesicles upon activation (10 µg/ml anti-CD3 + 5 µg/ml anti-CD28) of T cells per density fraction. Indicated are the averages and standard deviations of 9 independent experiments. The number of vesicles released by non-activated T cells was set to 1 for each density fraction. Asterisks denote significant differences (* p<0.05, ** p≤0.01, *** p≤0.001).

Fig. 3

Fig. 3

Heterogeneity in T cell-derived vesicle populations with different buoyant densities. Vesicles released by non-activated T cells were isolated and analysed as described in Fig. 2. Histograms indicating size as determined by NTA (A) or reduced wide-angle FSC as determined by high-resolution flow cytometry (B) of 100 nm beads (filled histograms, light grey) and T cell-derived vesicles in fractions with densities of 1.17 g/ml (black line) or 1.14 g/ml (dark grey line). (C) Reduced wide-angle FSC geometric means of vesicles from non-activated T cells with densities of 1.17 or 1.14 g/ml. Indicated are the averages and standard deviation of geometric mean values of 6 independent experiments. Asterisks denote significant differences (**p ≤ 0.01). Histograms in (A) and (B) are from the same experiment, representative of 6 (A) or 5 (B) experiments. (D) Dot plots representing wide-angle FSC and PKH67 fluorescence of vesicles from non-activated T cells floating at 1.17 (left) and 1.14 g/ml (right). Gates were set around vesicles subpopulations with low reduced wide-angle FSC levels and low fluorescence (FSClowFLlow), high(er) reduced wide-angle FSC levels and high(er) fluorescence (FSChighFLhigh) or high(er) reduced wide-angle FSC levels and low fluorescence (FSChighFLlow).

Fig. 4

Fig. 4

T cells differentially regulate the release of distinct vesicle subpopulations upon different activation signals. Vesicles from activated (10 µg/ml anti-CD3 + 5 µg/ml anti-CD28) and non-activated T cells were isolated and analysed as described in Fig. 2. (A) Time-based quantification of T cell-derived vesicles in different density fractions in the 3 different vesicle subpopulation gates, as described in Fig. 3D. Indicated are the numbers of vesicles per gate measured in 30 seconds. One representative experiment out of 4 is shown. (B) Indicated is the average fold increase ± SD in number of vesicles released by T cells, per FSC-FL gate, activated by TCR-triggering alone or with additional co-stimulation, relative to the number of vesicles from non-activated T cells (set to 1). Averages and standard deviations are displayed for vesicles with a density of 1.17 (left graph) or 1.14 g/ml (right graph) of 4 independent experiments. Asterisks denote significant differences (** p≤0.01, *** p≤0.001).

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