Figure 4.
PEVs in blood circulation can reach lymphoid organs and circulate in lymph. (A-C) Fluorescently labeled PEVs generated from activated mouse platelets were IV injected into mice and their presence in blood (A) and different organs (B-C) was monitored after 2, 15, and 60 minutes. Free PEVs (unbound to cells) were identified by flow cytometry for up to 2 minutes in blood, as well as PEVs bound to platelets and to leukocytes (mainly Ly6G+ neutrophils and a few lymphocytes), but were mostly undetectable by 60 minutes. Dashed lines represent the mean of vehicle (n = 9-13); n = 11 (2 minutes), n = 5 (15 minutes) and n = 6 (60 minutes). Data are the mean ± standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001 (Kruskal-Wallis). (B) Representative images of CMFDA-labeled (green) individual PEVs (arrowhead) and PEV aggregates (asterisk) in whole tissue sections (spleen, popliteal LN [PLN], inguinal LN [ILN], bone marrow, lungs, and liver) at 15 and 60 minutes by confocal microscopy; nuclei (Hoechst 33342 ) are blue. Results are representative of observations made in 5 and 6 mice per group. (C) PEVs and aggregates were quantified using 5 different sections for lymph nodes (PLN and ILN; representing a total surface of at least 1.5 mm2), 8 zones of 500 000 µm2 each, randomly assigned on 2 different sections in femurs (total surface, 4 mm2), and 10 zones of 500 000 µm2 each, randomly assigned on 2 sections each of lungs, spleen, kidneys, and brain and 1 section of liver (total surface, 5 mm2) using Zen 3.3 software (n = 6 [phosphate buffered saline, 60 minutes], 5 [15 minutes], and 5-6 [60 minutes]). Data are the mean ± SEM. *P < .05, **P < .01 (Kruskal-Wallis). (D-E) PEVs in lymph were detected by hs-FCM. (D) Gating strategy to analyze expression of MHC-I and proteasome (LWA300) on CD41+ EVs in lymph and representative dot plot of labeled and unlabeled (CD41 only) lymph. (E) Expression of MHC-I and proteasome (LWA300) on CD41+ EVs in lymph was determined. +/+, double positive, and −/−, double negative for MHC-I and proteasome (n = 6). Data are the mean ± SEM.

PEVs in blood circulation can reach lymphoid organs and circulate in lymph. (A-C) Fluorescently labeled PEVs generated from activated mouse platelets were IV injected into mice and their presence in blood (A) and different organs (B-C) was monitored after 2, 15, and 60 minutes. Free PEVs (unbound to cells) were identified by flow cytometry for up to 2 minutes in blood, as well as PEVs bound to platelets and to leukocytes (mainly Ly6G+ neutrophils and a few lymphocytes), but were mostly undetectable by 60 minutes. Dashed lines represent the mean of vehicle (n = 9-13); n = 11 (2 minutes), n = 5 (15 minutes) and n = 6 (60 minutes). Data are the mean ± standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001 (Kruskal-Wallis). (B) Representative images of CMFDA-labeled (green) individual PEVs (arrowhead) and PEV aggregates (asterisk) in whole tissue sections (spleen, popliteal LN [PLN], inguinal LN [ILN], bone marrow, lungs, and liver) at 15 and 60 minutes by confocal microscopy; nuclei (Hoechst 33342 ) are blue. Results are representative of observations made in 5 and 6 mice per group. (C) PEVs and aggregates were quantified using 5 different sections for lymph nodes (PLN and ILN; representing a total surface of at least 1.5 mm2), 8 zones of 500 000 µm2 each, randomly assigned on 2 different sections in femurs (total surface, 4 mm2), and 10 zones of 500 000 µm2 each, randomly assigned on 2 sections each of lungs, spleen, kidneys, and brain and 1 section of liver (total surface, 5 mm2) using Zen 3.3 software (n = 6 [phosphate buffered saline, 60 minutes], 5 [15 minutes], and 5-6 [60 minutes]). Data are the mean ± SEM. *P < .05, **P < .01 (Kruskal-Wallis). (D-E) PEVs in lymph were detected by hs-FCM. (D) Gating strategy to analyze expression of MHC-I and proteasome (LWA300) on CD41+ EVs in lymph and representative dot plot of labeled and unlabeled (CD41 only) lymph. (E) Expression of MHC-I and proteasome (LWA300) on CD41+ EVs in lymph was determined. +/+, double positive, and −/−, double negative for MHC-I and proteasome (n = 6). Data are the mean ± SEM.

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