Fig 2: CTLA4+ and PD-L1+ expression on Treg. (A) CTLA4+ and PD-L1+Treg percentage from culture. Healthy donors, n=12, RA patients, n=15. (B) MFI of CTLA-4 and PD-L1 expression on Treg in culture. Healthy donors, n=12, RA patients, n=15. (C) Gating strategy for CTLA-4 and PD-L1. The data are presented as the median and 25-75% interquartile range, and was analyzed using the Mann-Whitney test for independent groups and with the Wilcoxon test for related groups. *P<0.01. w/o, without; a, anti; stim, stimulated; Treg, regulatory T cells; HC, healthy controls; RA, rheumatoid arthritis; MFI, mean fluorescence intensity; FMO, fluorescence minus one.

Fig 3: Changes in CD3(+) and CD3(-) exosome subsets carrying PD-L1 or CTLA4. (a): Changes in CD3(+)PD-L1 + T cell-derived exosomes. Results for all patients showed no changes during therapy. In patients who had a recurrence, levels of CD3(+)PD-L1+ exosomes were elevated at baseline (p < 0.05) but decreased at week 5 (p < 0.005). (b): Changes in CD3(+)CTLA4+ exosomes. Only patients with NED after therapy had elevated levels of CD3(+)CTLA4+ exosomes at baseline and showed a decrease in levels of these exosomes during therapy. Note that four-fifths patients with recurrence had low baseline levels of CD3(+)CTLA4+ exosomes. *p < 0.05, **p < 0.005; NSD: no significant difference for overall comparison of changes from baseline.

Fig 4: Changes in CD3(-)PD-L1+ TEX during therapy. (a): Patients who did not have recurrence had significantly increased levels of these exosomes at baseline relative to patients with recurrence with a significant decrease at week 5. Also, in patients with recurrence, there was a significant increase of CD3(-)PD-L1+ TEX at weeks 5 and 14 relative to baseline. (b): Changes in CD3(-)CTLA4+ TEX during therapy. Note that in patients with recurrence, the baseline levels of these exosomes were significantly lower than those in patients with NED and remained low throughout therapy, whereas in patients with no evident disease decreasing levels of CD3(-)CTLA4+ exosomes are visible during therapy. *p < 0.05, **p < 0.005; NSD: no significant difference for overall comparison of changes from baseline.

Fig 5: Surface values of immune checkpoints on exosomes isolated from plasma of HNSCC patients.Exosomes from n = 17 HNSCC patients obtained at defined time-points were captured using biotinylated CD63-antibodies and stained with fluorochrome-conjugated antibodies against PD-L1 (a), CTLA-4 (b), OX40 (c) and OX40-L (d). Surface values as determined by on-bead flow cytometry are shown as RFI compared to an appropriate isotype control. Results are plotted as box-and-whisker blots representing the median value, the 25th and 75th quartiles and the range. Wilcoxon related samples tests were applied to each compare pre-OP, post-OP, end (C)RT and follow-up. Mann–Whitney test was applied to compare recurrence with follow-up. * or # and ** or ## correspond to p = 0.05 and p = 0.01, respectively. Asterisks and values above boxes indicate the significance compared to pre-OP. Hashtags above boxes indicate the significance compared to follow-up. c RT (chemo)radiotherapy