Small EV in plasma of triple negative breast cancer patients induce intrinsic apoptosis in activated T cells

Abstract

Small extracellular vesicles (sEV) in TNBC patients' plasma promote T cell dysfunction and tumor progression. Here we show that tumor cell-derived exosomes (TEX) carrying surface PDL-1, PD-1, Fas, FasL, TRAIL, CTLA-4 and TGF-β1 induce apoptosis of CD8⁺T and CD4⁺T cells but spare B and NK cells. Inhibitors blocking TEX-induce receptor/ligand signals and TEX pretreatments with proteinase K or heat fail to prevent T cell apoptosis. Cytochalasin D, Dynosore or Pit Stop 2, partly inhibit TEX uptake but do not prevent T cell apoptosis. TEX entry into T cells induces cytochrome C and Smac release from mitochondria and caspase-3 and PARP cleavage in the cytosol. Expression of survival proteins is reduced in T cells undergoing apoptosis. Independently of external death receptor signaling, TEX entry into T cells induces mitochondrial stress, initiating relentless intrinsic apoptosis, which is responsible for death of activated T cells in the tumor-bearing hosts. The abundance of TEX in cancer plasma represents a danger for adoptively transferred T cells, limiting their therapeutic potential.

Fig. 1. Characteristics of TEX1 isolated from supernatants of MDA-MB-231, TEX2 isolated from supernatants of MDA-MB-436 and sEV isolated from supernatants of non-malignant HaCat cell line.

a Representative particle distribution images (NTA). b Ratios of TEX numbers/µg protein in SEC fractions #F4. c Representative TEM images; scale bar = 100 nm. d Western blot analysis. e On-bead flow cytometry analysis of immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as relative fluorescence intensity (RFI) values (means ± SD) obtained in three independent experiments. Unpaired T-tests were used to evaluate differences between sEV from different cell lines. Asterisks indicate p values: *p < 0.05; **p < 0.01; ***p < 0.001. f Apoptosis of CD8+ Jurkat cells co-incubated with increasing concentrations of TEX1, TEX2 or HaCaT sEV. Data are presented as means ± SD from 3 independent experiments. Data were analyzed by unpaired t-test. Asterisks indicate p-values: **p < 0.001; **p < 0.01, ns = no significant difference. g Representative flow cytometry images of apoptosis induced in Jurkat cells by increasing levels of TEX1, TEX2 or HaCaT sEV.

Fig. 2. Characterization of sEV isolated from plasma of TNBC-patients or healthy donors (HDs).

a, b Representative particle distribution images (NTA) of sEV derived from plasma of a HD and TNBC Pt #1. c sEV numbers/µg protein in fraction #4 for Pts and HDs. d Representative TEM images of sEV from plasma of a HD and TNBC-Pt #1. e Western blots of sEV (fraction #4) isolated from plasma of a HD and TNBC-Pt #1. f On bead flow cytometry of sEV from plasma of TNBC Pts and HDs. Shown are RFI values for immunosuppressive proteins on the sEV surface (upper row) and for immunostimulatory proteins (lower row). Data are presented as means ± SD. Mann-Whitney tests were used to evaluate differences between TNBC Pts and HDs. ns = no significant difference. g The RFI scores for immunosuppressive or immunostimulatory proteins and the ratios of stim/supp proteins calculated for all the above listed sEV as described in the text. Note the higher suppressive and stimulatory scores for TNBC Pt’s sEV than for HD’s sEV and the significantly higher stim/supp ratio for sEV of HDs than for TNBC Pts. h Apoptosis (%) induced in activated primary human CD8 + T cells by sEV from plasma of HDs (n = 5) and TNBC-Pts (n = 5). Data were analyzed by an unpaired t-test and are presented as means ± SD. i Flow cytometry images of apoptosis induced in CD8 + T cells by increasing doses of sEV from a representative HD or TNBC Pt.

Fig. 3. Uptake of TEX labeled with PKH26 by immune cells.

TEX isolated from supernatants of TNBC cell lines were labeled with the PKH26 dye and were co-incubated with recipient immune cells for various time periods. a Percentages of CD8+ Jurkat cells up-taking labeled TEX during coincubation as measured by flow cytometry. Data are mean values from two independent assays and are shown in the excel table in Supplementary Data 1. b Representative histograms for the uptake of labeled TEX presented as variables of the uptake time. c Microscopic images of CD8+ Jurkat cells co-incubated with PKH26 labeled TEX1. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 20 µm. d Comparison of TEX1 uptake by activated primary CD4 + T and CD8 + T cells at different time points. e Flow cytometry images of apoptosis induced in activated primary CD4 + T, CD8 + T, B and NK cells following co-incubation with TEX1.

Fig. 4. Immunoregulatory proteins expressed on the surface of recipient immune cells and on TEX or sEV of malignant and non-malignant origins.

a The heatmap presenting mean RFI values for proteins expressed on the surface of activated primary CD4 + T, CD8 + T, B and NK cells. b The heatmap presenting mean RFI values for proteins found on the surface of TEX1, TEX2, HaCaT sEV, TNBC Pt-sEV and HD-sEV. c An image of a T cell interacting with various TEX (shown as blue vesicles) that carry immunoregulatory proteins on the surface membrane. TEX binding to complementary receptors expressed by the T cell initiate immunoregulatory signals which result in immune downregulation [(-)Ireg] and/or immune stimulation. The sum of these simultaneously delivered signals will determine whether TEX mediate immune suppression or immune stimulation in a recipient T cell. Note that a single sEV might carry multiple signaling proteins on its surface membrane.

Fig. 5. TEX-induced apoptosis of activated human CD8 + T cells is not blocked by neutralizing Abs or specific inhibitors.

a Apoptosis (%) induced in CD8 + T cells by TEX1 or TEX2 (50 µg/mL) was not significantly blocked by the neutralizing Abs used or the TGF-β inhibitor. b Representative flow cytometry for apoptosis of activated CD8 + T cells co-incubated with TEX1 in the presence of blocking Abs. c Inhibition of apoptosis by various neutralizing Abs of activated CD8 + T cells co-incubated with sEV from TNBC Pts (25 µg/mL). The pretreatment of TNBC-pts sEV with protein kinase (PK) or heat (HI) significantly reduced but did not eliminate T cell apoptosis. d The pretreatment of CD8 + T cells or activated primary CD4 + T cells with the mix of blocking Abs specific for PD1, TRAIL, Fas and anti-CTLA4 (used at the f.c. of 10, 10, 10 and 20 µg/mL, respectively) failed to reduce apoptosis induced by TEX 1 (50 µg/mL). In the blocking assays, primary human T cells were incubated with different blocking Abs and the TGF-β inhibitor. All Abs were used at the f.c. of 10 µg/mL, except for anti-CTLA4 Abs which were used at the f.c. of 20 µg/mL. TGF-β inhibitor was used at the f.c. of 50 nM. Blocking was performed for 30 min before co-incubation for 6 h with TEX or sEV from plasma of TNBC-Pts or HDs. Apoptosis was evaluated by Annexin-V binding assays. The data presented in (a) and (d) are means ± SD from 3 independent experiments.

Fig. 6. Inhibition of TEX uptake by T cells may reduce but does not eliminate apoptosis in recipient T cells.

a Representative images of cellular uptake of PKH26-labeled TEX 1 by Jurkat T cells (upper row) and corresponding images of apoptosis induced by TEX 1 in the recipient T cells (lower row). TEX 1 labeled with the PKH26 dye were pretreated with protein kinase (PK;1 µg) or heat (HI; 80 °C for 1 h) and were co-incubated with CD8+Jurkat T cells for 2 h to measure vesicle uptake and for 6 h to measure T cell apoptosis. CD8+Jurkat T cells were also pre-incubated individually with anti-Fas antibody (10 µg/mL), Cytochalasin-D (20 µM), Dynosore (10 µM) or Pit Stop-2 (10 µM) for 30 min followed by 2 h coincubation with PKH26 labeled TEX 1 (50 µg/mL) to measure uptake and for additional 6 h to measure T cell apoptosis. Images were taken at 40x mag. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 10 µm. To measure T cell apoptosis, Annexin V binding assays were performed following 6 h co-incubation with TEX 1. b Apoptosis (%) induced by TEX 1 co-incubated with CD8+Jurkat T cells following pretreatments of TEX or CD8+Jurkat cells with various blocking agents as described above. Data are means ± SD from 3 independent experiments. Data were analyzed by ANOVA followed by Dunnett’s post hoc analysis. ns = no significant difference.

Fig. 7. Effects of caspase inhibitors on TEX uptake by CD8+Jurkat cells and TEX-induced Jurkat cell intrinsic apoptosis.

a Representative cellular uptake of TEX1 by CD8+Jurkat cells (upper panel) and resulting apoptosis (lower panel). Recipient Jurkat cells were pre-incubated with caspase 8 inhibitor (Z-IETD-FMK, 100 µM) or pan caspase inhibitor (Z-VAD-FMK, 100 µM) for 30 min prior to co-incubation with TEX1 for 2 h to measure TEX uptake by T cells. Then the T cells were incubated for additional 6 h to measure apoptosis. Cellular uptake images were acquired by confocal microscopy at ×40 mag. TEX = Red, Actin = green, DAPI stained nuclei = blue; scale bar = 10 µm. b Apoptosis (%) induced by TEX1 in CD8+Jurkat cells pretreated with caspase inhibitors as in (a) was significantly reduced but not eliminated. Data were analyzed by ANOVA followed by Dunnett’s post hoc analysis and represent means ± SD from 3 independent experiments. c Apoptosis (%) induced by TEX1 in activated CD4+T cells pre-incubated with caspase inhibitors. No reduction in CD4+ T cell apoptosis was noted. d, e Western blots showing expression of pro-apoptotic and anti-apoptotic proteins in the mitochondrial (d) and the cytosol (e) fractions of Jurkat cells which were co-incubated with TEX1 for 3, 6 or 16 h. Mitochondrial and cytosol fractions of Jurkat cells were prepared as described in Methods. β-Actin and Cox IV served as controls for equal loading of the cytosol and mitochondrial fractions, respectively. f, g Western blots of proteins in the mitochondrial (f) and the cytosol (g) fractions of Jurkat cells which were co-incubated with TEX2 or HaCaT sEV for 16 or 24 h. AIF apoptosis-inducing factor. h, i Effects of TEX1 and TEX2 on expression levels of the apoptosis related proteins in CD8+ Jurkat T cells after 6 h co-incubation. Representative histograms are shown in (i) and quantitative mean fluorescence intensity (MFI) values are shown in (h). Data are means ± SD from 3 independent experiments.