Fig 1: Isolated platelets from liquid platelet rich fibrin (iPRF) induce a regulatory phenotype in CD4+ T cells. CD4+ T cells and platelets (Plt.) were cocultured at a ratio of 1 to 50 and were treated with 10 µg/mL anti-GARP antibody (Ab). CD4+ T cells were prelabled with carboxyfluorescein succinimidyl ester (CFSE) to track their proliferation and stimulated with 0.5 µg/mL anti-CD3 and 1.0 µg/mL anti-CD28 Abs in the presence or absence of platelets. (A–C) The expression of Foxp3 (A), GARP (B), and proliferation (C) of CD4+ T cells was measured on day 3 of coincubation via flow cytometry. (D,E) After 6 days of coincubation, cells were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and monesin for 5 h. IL-2 and IFN-γ production was quantified via intracellular flow cytometry. Representative dot plots of three independent experiments are shown. Graphs show CD4+ T cells cocultured with platelets normalized to untreated CD4+ T cells alone control (n = 3, means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and ns determined by one-way ANOVA).
Fig 2: Liquid platelet rich fibrin (iPRF) polarizes M0-macrophages in a more “M0/M2-like” phenotype through a GARP independent mechanism. Human monocyte derived macrophages were polarized, treated with 400 µL iPRF/well, 10 μg/mL anti-GARP antibody (Ab) or were left untreated for 2 days as indicated. Surface marker expression was analyzed via flow cytometry. Bar diagram shows the mean fluorescence intensity (MFI) of each surface marker normalized to the respective M0 control. Representative histograms of 3 independent experiments are shown (n = 5 donors, bar means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 determined by two-way ANOVA).
Fig 3: Glycoprotein A repetitions predominant (GARP) is expressed on the surface of platelets and exists as a soluble factor found in liquid platelet rich fibrin (iPRF). (A,B) iPRF was stained directly for the platelet marker, CD41a, and GARP and underwent flow cytometric analysis. Isotype antibodies were used as a control. (A) Dot blots show one representative result. (B) Bar diagrams of GARP expression show the pooled data of percentages (%) of GARP positive platelets in iPRF and the mean fluorescence intensity (MFI) of GARP (n = 3 ± SD, analyzed by paired Students T-tests). (C) Expression of GARP in iPRF was analyzed via Western blot. iPRF lysate was loaded in increasing amounts. Recombinant soluble GARP (sGARP) was loaded at 1 ng/lane and served as a positive control, whereas β-actin acted as the loading control. (D) Bar graph shows the soluble GARP (sGARP) levels in peripheral blood and iPRF analyzed via ELISA (n = 3 ± SD, ns determined by paired Students T-tests).
Fig 4: External beam radiation therapy increases [111In]In-GARP-DOTAGA uptake in vivo by SPECT/CT imaging. A. 14 days after 4T1 triple negative murine breast cancer, mice were assigned in 2 groups: external beam radiation at the dose of 8 Gy or control without irradiation. 7 days post irradiation, they underwent SPECT/CT imaging at 24h and 48h post-injection with [111In]In-GARP-DOTAGA. Mice were sacrificed after the last imaging and gamma counting was performed ex vivo on tumours. B. Representative SPECT/CT images of 4T1 tumour-bearing mice irradiated (n = 17) or not (n = 18) at 24h and 48h post-injection of 15MBq/100 μL of [111In]In-DOTAGA-GARP. Tumours are highlighted with circles. C. The scatter dot plot represents the percentage of the injected dose of [111In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (n = 17) or not (n = 18) at 24h and 48h post-injection of 15 MBq/100 μL of [111In]In-DOTAGA-GARP. Results are presented as the median with the interquartile range, (∗∗∗∗p < 0.0001 ∗∗∗p = 0.0001). D. The scatter dot plot represents the percentage of injected dose of 111In-DOTAGA-GARP per gram of tumour of 4T1 tumour-bearing mice irradiated (n = 11) or not (n = 12) at 48h post-injection of 15 MBq/100 μL of [111In]In-GARP-DOTAGA measured by gamma counting. Results are presented as the median with the interquartile range, (∗∗p = 0.0056). E. Representative SPECT/CT images of 4T1 tumour-bearing mice irradiated (8Gy) at 24h and 48h post-injection of 15MBq/100 μL of [111In]In-DOTAGA-GARP (n = 6) or [111In]In-DOTAGA-GARP + 100x unlabelled DOTAGA-GARP (blocking, n = 3). The scatter dot plot represents the percentage of the injected dose of [111In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (8Gy) at 24h and 48h post-injection of 15 MBq/100 μL of [111In]In-DOTAGA-GARP or blocking. Results are presented as the median with the interquartile range, (∗p < 0.05). F. The scatter dot plot represents the percentage of the injected dose of [111In]In-DOTAGA-GARP per gram of tumour (%ID/g) of 4T1 tumour-bearing mice irradiated (8Gy) at 48h post-injection of 15 MBq/100 μL of [111In]In-DOTAGA-GARP or blocking measured by gamma-counting. Results are presented as the median with the interquartile range, (∗p < 0.05).
Fig 5: GARP expression with or without external beam radiation therapy in 4T1 tumour model. A. In vitro detection of membrane GARP expression in 4T1 cells with or without external beam radiation therapy at the dose of 8 Gy (48h post irradiation) by flow cytometry. Results are presented as the median with the interquartile range, p=0.0002. B. Representative immunostaining and quantification of GARP (red) in 4T1 tumour from mice 7 days after 8 Gy irradiation (n=6) or not (n=7). Results are presented as the median with the interquartile range, p=0.0082. C. Schematic representation of the experimental design: ex vivo detection of GARP expression in 4T1 tumours by flow cytometry. 14 days after 4T1 cells injection mice were divided into two groups: control or 8 Gy irradiation. 7 days after irradiation, flow cytometry was performed on dissociated tumours. 1. Percentage of GARP + cells in alive cells in tumours (p=0.0093). 2. Percentage of CD3+CD4+CD25+FoxP3 + cells expressing GARP in the tumour (p=0.018). 3. Percentage of GARP + cells within CD3+CD4+CD25+FoxP3+ regulatory T cells (p=0.0067).
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