Leukapheresis products or buffy coats obtained from anonymized Sanquin blood donors were used to isolate monocytes and T cells after written informed consent by donors. Citrated whole blood was obtained to isolate fresh platelets and platelet releasate. All procedures were approved by the Sanquin Ethical Advisory Board and are in accordance with the declaration of Helsinki and Dutch regulations.

Monocytes were isolated using the Elutra Cell Separation System (Gambro, Lakewood, CO, USA) from fresh leukapheresis material or from PBMCs isolated from buffy coats using CD14+ magnetic cell separation (Miltenyi Biotech, Leiden, Netherlands) and subsequently frozen until further use as previously described (13, 44). Purity of monocytes was >90% as determined using flow cytometry with APC labeled anti-CD14 (clone MϕP9, BD biosciences). Upon initiation of culture, monocytes were thawed and differentiated into immature dendritic cells (DCs) as previously described (13, 44).

Platelets were isolated from citrated whole blood by centrifugation at 200g for 10 min and supernatant platelet rich plasma was washed (5 min, 1650g) 1:2 with sequestrine buffer (17.5 mM Na2HPO4, 8.9 mM Na2EDTA, 154 mM NaCl, pH 6.9, containing 0.1% [wt/vol] bovine serum albumin, all obtained from Merck Millipore, Amsterdam, Netherlands). After washing, platelets were resuspended in GMP DC serum-free medium (Cellgenix, Freiburg, Germany) containing 100 U/ml penicillin and 100 U/ml streptomycin (Gibco). To collect platelet releasate, platelets were incubated 30 min with 250 µM thrombin receptor-activating peptide 6 amide (TRAP-6) (Bachem, Bubendorf, Switzerland) after which platelets were removed by centrifugation: first 5 min at 1,650 g and subsequently 10 min at 4,000g. If indicated, platelet releasate was hereafter centrifuged to remove microparticles at 100,000g for 1 h in Optima L-100 XP Ultracentrifuge (Beckman Coulter, IN, United States). Experiments that directly compared whole platelets and releasate used releasate and whole platelets from the same donor. Otherwise, releasate was pooled from four or five whole blood donors, aliquoted and stored at −20°C until further use.

Immature moDCs were stimulated in the absence or presence of platelets or platelet releasate with a platelet:DC ratio ranging from 80:1 to 1:1 (or equivalent amount of releasate) in GMP DC serum-free medium (Cellgenix, Freiburg, Germany) containing 100 U/ml penicillin and 100 U/ml streptomycin (Gibco). When indicated, DCs were stimulated using 2.5 µg/ml monophosporyl lipid A (MPLA) (Sigma Aldrich, Zwijndrecht, Netherlands), 5 µg/ml pam3CysSerLys4 (PAM3CSK4) (Invivogen, San Diego, CA, USA), 2.5 µg/ml resiquimod (R848) (Alexis Biochemical, Paris, France) or 0.25 µg/ml flagellin bacillus subtilis (Invivogen) in combination with 1,000 U/ml IFNγ (Boehringer Ingelheim, Alkmaar, Netherlands) or a cytokine maturation cocktail consisting of 10 ng/ml TNFα, 10 ng/ml IL-1β (both Cellgenix, Freiburg, Germany), and 1 µg/ml prostaglandin E2 (PGE₂) (Sigma Aldrich, Zwijndrecht, Netherlands). When indicated, releasate or recombinant human TGF-β (R&D Systems, Minneapolis, MN, United States) was pre-incubated with blocking antibody against TGF-β (Clone 1D11, 10 µg/ml, BioXCell, NH, United States) for 30’ at 37°C, 5% CO₂. For transwell experiments, moDCs were stimulated with MPLA/IFNγ in a 96-well transwell system (Corning, pore size ≤0.3 µM) in absence or presence of TRAP-6 activated platelets (platelet:DC ratio 40:1) that were either below the transwell (i.e. in the same compartment as the DCs) or separated by the transwell. After 24 or 48 h at 37°C, 5% CO₂ culture supernatant was harvested and frozen at −20°C until cytokine measurements.

The levels of IL-6 and IL-10 in culture supernatants were measured using compact PeliKine Cytokine ELISA kits (Sanquin Reagents, Amsterdam, Netherlands); the level of TNF-α was determined with Human TNF-α ELISA set (Diaclone, Besançon, France). To determine production of IL-12p40, a combination of two anti-IL-12p40 antibodies was used (coat: clone C11.79 and detection: clone C8.6, Sanquin Reagents, Amsterdam, Netherlands) and recombinant IL-12p40 was used as calibration. The level of human TGF-β1 was measured using DuoSet ELISA kit (R&D Systems, Minneapolis, MN, United States) either after or without chemical activation of the samples according to manufacturer’s protocol. Absorbance was measured at 540nm with SynergyTM 2 (BioTek, Winooski, VT, USA).

After 48 h of stimulation, DCs were harvested with 0.4% trisodium citrate (Merck Millipore, Amsterdam, Netherlands) in PBS supplemented with 4 mg/ml human albumin (Albuman, Sanquin Reagents, Amsterdam, Netherlands). DCs were stained 30 min at room temperature with LIVE/DEAD Fixable near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) and subsequently with either PE-labeled anti-HLA-DR (Clone G46-6) or with FITC-labeled anti-CD80 (Clone L307.4), PE-labeled anti-CD40 (Clone 5C3), APC-labeled anti-CD83 (Clone HB15e) and BV421-labeled anti-CD86 (Clone 2331) (all from BD Biosciences) in the presence of 3 mg/ml human γ-globulin (Nanogam, Sanquin, Amsterdam, Netherlands). Samples were measured with BD FACSCanto II and analyzed using Flowjo VX (Beckton, Dickinson & Company, Ashland, OR, United States). Gating strategy involved selection of DCs on FSC/SSC, single cells and viable cells.

Total human lymphocytes were isolated using the Elutra Cell Separation System (Gambro, Lakewood, CO, USA) from fresh leukapheresis material and frozen until further use. Naïve human CD4+ T cells were isolated from buffy coats. First, the PBMC fraction was isolated by density gradient centrifugation (Lymphoprep, Axis-shield, Alere Technologies AS, Oslo, Norway). From PBMC fraction, CD4+ cells were isolated by negative selection using magnetic separation followed by negative selection of CD45RA population (both Miltenyi Biotech, Leiden, Netherlands). Purity of isolated naïve CD4+ T cells ≥95% as determined by staining with FITC-labeled anti-CD45RA (Clone HI100, eBioscience) and BV421-labeled anti-CD45RO (Clone UCHL1, BD Bioscience).

Primary blood DCs were isolated from buffy coats as previously described (45). In short, PBMCs were enriched for blood DCs by elutriation with JE-5.0 elutriator (Beckman Coulter, Brea, CA, USA), followed by sorting on FACSAria IIIu or III cell sorter (BD Bioscience, San Jose, CA, USA). For sorting, the cells were stained with APC-labeled anti-CD3 (clone HIT3a), anti-CD14 (clone MϕP9), anti-CD19 (clone SJ25-C1) (all BD Biosciences), PE-Cy7-labeled anti-CD1c (clone L161, Biolegend) and FITC-labeled 6-sulfo LacNAc (Slan)-specific antibody (clone M-DC8, Miltenyi Biotech) and LIVE/DEAD fixable Near-IR Dead Cell Stain Kit (Invitrogen). Different DC subtypes were gated as follows: Slan+ non-classical monocytes as CD3^-CD14^-CD19^-M-DC8⁺ and myeloid cDC2 as CD3^-CD14^-CD19^-CD1c⁺ (see Supplementary Figure S3 for gating strategy). Purity after sort was >90% as tested by flow cytometry. Primary DCs were rested overnight in GMP DC serum-free medium (Cellgenix) containing 100 U/ml penicillin and 100 U/ml streptomycin (Gibco) at 37°C and 5% CO₂. After overnight rest, DCs were stimulated with R848 (Alexis Biochemical, Paris, France) at a concentration of 5 µg/ml in absence or presence of platelets or platelet releasate in a 50:1 platelet:DC ratio.

After thawing and 1 h resting total T cells or naïve CD4+ CD45RA+ T cells were washed twice with PBS and incubated with 5µM CFSE (Invitrogen) for 20 min at room temperature. After washing, T cells were resuspended in IMDM+5%HS medium and incubated 10:1 (T:DC ratio) with autologous (TT-loaded) or allogeneic DCs. If indicated, DCs were first incubated 1 h at 37°C with 5 µg/ml tetanus toxoid (AJVaccines, Copenhagen, Denmark) before stimulation with MPLA/IFNγ. After maturation with MPLA/IFNγ or IL-1β/TNFα/PGE₂, moDCs were washed twice with IMDM + 5%HS medium to remove potential remaining platelets or platelet-derived factors. After 8 days, total T cells were harvested, labeled with LIVE/DEAD Fixable near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) and subsequently with PE-labeled anti-CD4 (Clone SK3) and APC-labeled anti-CD8 (Clone SK1) (both from BD biosciences) in FACS buffer (PBS + 0.5% BSA + 0.02% Azide). After 9-10 days, naïve T cells were stimulated with PMA (100 ng/ml) and ionomycin (1 µg/ml) in presence of Brefeldin A (10 µg/ml) (all from Sigma Aldrich) for 5 h. T cells were harvested and washed, labeled with LIVE/DEAD Fixable near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) at RT and subsequently with BUV395-labeled anti-CD4 (clone SK3, BD Bioscience). Hereafter, cells were fixed for 15 min with 4% paraformaldehyde (Merck, Darmstadt, Germany) after which cells were stained with PECy7-labeled anti-IFNγ (Clone B27, BD Bioscience), APC-labeled anti-TNFα (clone Mab11, Biolegend) in Brilliant Stain buffer (BD Bioscience) in permeabilization buffer containing 0.5% Saponin, 1%BSA, and 0.02% azide. Cells were washed and measured on Canto II (total T cells) or LSR Fortessa (both from BD Bioscience). Data were analyzed in Flowjo VX (Beckton, Dickinson & Company, Ashland, OR, United States), gating involved selection of lymphocytes based on FSC/SSC, single cells, live CD4+ cells, proliferation dye dilution and positivity for specific cytokines.

Graphical presentation and statistical analyses were performed using GraphPad prism v8.02 (GraphPad Software Inc, La Jolla, California, USA). Paired t-tests, repeated measures one-way ANOVA and two-way ANOVA with Tukey post test were used to determine statistical differences in DC and T cell cytokine production, DC co-stimulatory marker expression and viability, and T cell proliferation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

For original data, please contact a.tenbrinke@sanquin.nl

To investigate if and to what extent platelets affect human DC function, monocyte-derived DCs (moDCs) were stimulated with several stimuli to mimic various types of physiological activation, including MPLA (TLR4; Figure 1A ), PAM3CSK4 (TLR1/2; Figure 1C ), R848 (TLR7/8; Figure 1D ) or flagellin (TLR5; Figure 1E ) in the presence of IFNγ or with a cytokine maturation cocktail containing IL-1β, TNF-α; and PGE₂ ( Figure 1B , TNF-α not measured as this was added with stimulation cocktail). After stimulation, moDCs produced pro-inflammatory cytokines TNF-α, IL-12, and IL-6. In the presence of platelets however, pro-inflammatory cytokine production reduced significantly after all stimulations with an average residual cytokine production of 18.9%, 19.7%, and 43.4% for TNFα, IL-12p40, and IL-6 respectively after MPLA/IFNγ stimulation. Platelet-mediated inhibition of cytokine production was not observed for IL-6 production after R848/IFNγ stimulation and IL-12p40 production upon flagellin/IFNγ or cytokine cocktail stimulation, which both failed to induce a strong IL-12p40 response. The decrease in cytokine production was dependent on the number of platelets present ( Supplementary Figure S1A ). Additionally, in presence of platelets anti-inflammatory IL-10 production was induced for R848/IFNγ and IL-1β/TNFα/PGE₂ stimulated moDCs ( Figures 1B, D ). MoDCs stimulated with MPLA/IFNγ and PAM3CSK/IFNγ display a non-significant trend of IL-10 induction in the presence of platelets ( Figures 1A, C ), whereas IL-10 production was not detectable for moDCs stimulated with Flagellin/IFNγ ( Figure 1E ). When immature moDCs were cultured with platelets without additional maturation stimulus, some donors showed an increase in cytokine production. This non-significant trend was most clear for IL-6 and IL-10 production by immature moDCs in presence of platelets ( Supplementary Figure 1B ).

Stimulation with MPLA/IFNγ or IL-1β/TNFα/PGE₂ induced the highest expression of co-stimulatory molecules as measured by flow cytometry. Interestingly, platelets minimally affected the phenotype of MPLA/IFNγ or IL-1β/TNFα/PGE₂ stimulated DCs, with only limited changes in expression of CD83 or CD40 and CD86, respectively ( Supplementary Figure S1C ; for gating strategy see Supplementary Figure S2A ). In summary, platelets reduced moDC pro-inflammatory cytokine production after various DAMP/PAMP stimulations, despite minimal effects on co-stimulatory molecule expression.

Next, the contact dependency of platelet mediated inhibition of DC cytokine production was investigated. When platelets were cultured with DCs in a transwell system to prevent direct cell-cell contact, IL-12p40, IL-6, and TNF-α production by stimulated moDCs was also reduced although to a slightly lower extent, which possibly can be explained by a concentration gradient-induced effect of the transwell itself ( Figure 2A ). Hence platelets inhibited pro-inflammatory cytokine production by moDCs also without direct cell-cell contact.

To further explore the involvement of platelet-derived soluble mediators, the releasate secreted by activated platelets was tested. For this, isolated platelets were activated with the PAR1 agonist thrombin receptor activator peptide-6 (TRAP-6). After activation, the platelet suspension was centrifuged twice to remove whole platelets and cell fragments. Whole platelets and platelet releasate from the same platelet donor induced a comparable decrease of pro-inflammatory cytokine production by MPLA/IFNγ stimulated moDCs ( Figure 2B ), while TRAP-6 itself did not affect moDC viability or cytokine production ( Supplementary Figures S2B, C ). Lastly, to exclude involvement of microvesicles, the releasate was ultracentrifuged at 100,000 g. Releasate with or without ultracentrifugation equally inhibited pro-inflammatory cytokine production by stimulated moDCs ( Figure 2C ). Overall, platelets inhibit MPLA/IFNγ-stimulated moDC pro-inflammatory cytokine production via a soluble factor, and not through release of microvesicles.

As platelets are a major source of DC-tolerizing active TGF-β in plasma (46–50) and platelet-derived TGF-β was shown to inhibit the anti-tumor T cell response in a murine model (29, 51), we investigated the involvement of TGF-β in platelet-mediated DC inhibition. Both platelets and DCs possess machinery to activate latent TGF-β and release the mature 25-kDa dimer (29, 48). Upon chemical activation, inducing dissociation of the mature TGF-β dimer from latent TGF-β binding proteins (LTBPs), we could detect active TGF-β1 in platelet releasate ( Supplementary Figure S3A ), indicating that latent TGF-β is present and may potentially be activated in our culture system. However, blocking TGF-β in platelet releasate could not restore cytokine production by moDCs ( Supplementary Figure S3B-C ), indicating that TGF-β is not the responsible factor in the releasate for the inhibition of cytokine production by moDC.

In human blood, two types of myeloid or conventional DCs (myeloid cDC1 (CD141+) and cDC2 (CD1c+)) can be distinguished (32) and there is a subset of slan+ non-classical monocytes that show DC-like characteristics (52). To investigate to what extent our findings translate to human primary blood DCs, we isolated human myeloid CD1c+ conventional DC2 and slan+ non-classical monocytes from blood ( Supplementary Figure S4 ).

After overnight rest, isolated myeloid cDC2’s and slan+ non-classical monocytes were stimulated with R848 as primary DCs were found to not respond to stimulation with MPLA/IFNγ  (data not shown; (45)). Interestingly, platelet releasate significantly decreased TNF-α production by myeloid cDC2 and a trend for IL-12p40 inhibition was observed ( Figure 3A ), whereas cytokine production by slan+ non-classical monocytes was not affected ( Figure 3B ). Unfortunately, IL-10 production could not be detected for any of the primary DC subsets tested (data not shown). These findings confirm that the platelet-mediated effects found on moDC cytokine production could partly be validated in a subset of primary human blood DCs, namely myeloid cDC2s.

Above we have shown that platelets and platelet releasate affect pro-inflammatory cytokine production by DCs, but the effect on subsequent T cell responses remains largely unresolved. To investigate the effect of platelets on DC-mediated T cell stimulation, moDCs were loaded with tetanus toxoid (TT) and subsequently stimulated with MPLA/IFNγ in presence of platelets. MoDCs stimulated in presence of platelets induced less TT-specific autologous CD4+ and CD8+ T cell proliferation ( Figure 4A and Supplementary Figure S5A ). A similar decrease in the ability of moDCs to induce allogeneic CD4+ but not CD8+ proliferation under influence of platelets or platelet releasate was found ( Figure 4B and Supplementary Figures S5B, C ).

Another interesting aspect of DC biology is their ability to skew naïve T cells toward a specific T helper phenotype upon the initiation of an immune response. MoDCs were cultured with MPLA/IFNγ in presence of whole platelets and platelet releasate. In a subsequent co-culture with naïve CD4+ CD45RA+ T cells, moDCs stimulated in presence of platelets or platelet releasate induced less allogeneic proliferation of naïve T cells ( Figure 4B , left panel, gating strategy in Supplementary Figure S6A ). Additionally, divided T cells (CFSEdim) displayed decreased IFNγ production compared to control co-cultures where moDCs were stimulated in the absence of platelets or platelet releasate ( Figures 4B , middle panel, and 4C ). Furthermore, the divided T cells produced less TNFα compared to control co-cultures where moDCs were stimulated in the absence of platelets but not platelet releasate ( Figures 4B , right panel, and 4C ). No significant production of IL-10 was detected in any of the DC-T cell co-cultures with or without platelet releasate (data not shown).

To determine whether effects on T cell skewing are dependent on the type of stimulation, cultures were performed with moDCs stimulated with TNFα, IL-1β; and PGE₂. We observed an increase in moDC anti-inflammatory IL-10 production when stimulated in presence of platelet releasate ( Supplementary Figure S6B ). Simultaneously, we observed a decrease in IL-6 production ( Supplementary Figure S6B ; TNFα could not be analyzed as it was present in the stimulation cocktail) and also a decreased ability to induce naïve T cell proliferation as measured by CFSE dilution by flow cytometry ( Supplementary Figure S6C , left panel). No effect was found on the IFNγ production of these naïve T cells compared to controls ( Supplementary Figure S6C , right panel). All in all, platelets and platelet releasate can decrease the ability of moDCs to induce both memory and naïve T cell proliferation. Additionally, the ability of MPLA/IFNγ stimulated moDCs to induce IFNγ+ Th1 responses was reduced when stimulated in presence of platelets or platelet releasate.