Fig 2: Platelet-initiated monocyte signaling and activation pathways.(A) Purified monocytes from donor PBMCs were incubated with platelets (mono+pl+) or without platelets (mono+pl-) for 4 hours. Western blotting was carried out to assess phosphorylation status of Src, Akt, and extracellular signal–regulated kinase (ERK). GRP94 was used as a control to ensure equal protein in each lane. Data are representative of at least two biological replicates. (B) Confocal images (top of rows) and corresponding kymographs (bottom of rows) show calcium fluxing as observed by Fluo-4 (green) fluorescence intensity in freshly isolated monocytes with or without platelets (white). Ionomycin-activated monocytes are shown as positive control. Scale bars, 4.6 µm. mono+pl+, n = 80; mono+pl-, n = 25; mono+pl+ + ionomycin, n = 30. (C) Quantitation of calcium flux in representative mono+pl+ (blue) or mono+pl- (red) monitored at 12-s intervals over 1 hour of imaging. (D) Nuclear translocation of NF?B quantified as MFI within 4',6-diamidino-2-phenylindole (DAPI)–stained nuclei and (E) representative confocal images of mono+pl+ and mono+pl- samples before (day 0) and after (day 1) overnight cultures stained with DAPI (blue), NF?B (green), CD14 (red), and CD62p (white) (n = 67 to 77 per group). Scale bars, 4.20 µm. (F) Illustration of demonstrated signaling pathways involved in platelet-dependent activation of cross-presentation in monocytes. Engagement of PSGL1 on monocytes by P-selectin on platelets initiates Src signaling that is known to activate PI3 kinase (PI3K), which, in turn, activates Akt that leads to the nuclear localization of NF?B. This leads to the up-regulation of MHC I and MHC II and the expression of cytokines. Src and PI3 kinase signaling is known to activate Bruton tyrosine kinase (BTK), which mediates calcium flux, activates MAP kinase pathway, and leads to the activation of transcription factors including NF?B. In the nucleus, NF?B and other activated transcription factors regulate the expression of cytokines, antigen processing and presentation machinery, and other costimulatory molecules. Molecules colored in green (Src, Akt, NF?B, ERK, and Ca2+) were validated by our analyses. PLC?, phospholipase C–?; I?Ba, inhibitor of NF?Ba. All values are means ± SD of at least three independent experiments. One-way ANOVA, ****P < 0.0001.

Fig 3: Monocyte-platelet adhesion synapse formation drives antigen-specific immunity.(A) Top: Confocal imaging of purified human monocytes cultured with platelets overnight. Activated CD61+ (green) platelets are shown forming CD62p+ (yellow) and PSGL1+-mediated junctions (blue) with CD14+ (red) monocytes. Bottom: 3D reconstruction demonstrates punctate monocyte PSGL1 clustering around platelet CD62p. Scale bars, 5.30 µm. (B) Schematic of human experimental setup with soluble antigen (Ag). Platelet-containing (PBMC+pl+) and platelet-depleted (PBMC+pl-) PBMCs were prepared from healthy human donors. PBMC groups were incubated overnight with soluble tumor antigens, melanoma-associated antigen recognized by T cells long peptide (MART1 LP) or E7 LP, followed by washing and 3-day coculture with antigen-specific transgenic human T cells, DMF5 or E7, respectively. Supernatants were analyzed at end of culture (day 4). (C to E) Human IFNg (hIFNg) ELISA of (C) DMF5 cultured with PBMC+pl+ or PBMC+pl- pulsed with MART1 LP at varying concentrations, (D) DMF5 cultured with MART1 LP–pulsed PBMC (left) or monocytes (right) with or without platelets or recombinant human P-selectin stimulation, and (E) E7 T cells cultured with E7 LP–pulsed PBMC+pl+ or PBMC+pl- or E7 T cells only (PBMC-). (F) FACS quantitation of CD86, HLA-DR, HLA-A,B,C, and CD14 in donor PBMCs at throughout incubation (n = 5 to 7). All values are means ± SD of at least three independent experiments. (C to E) One-way ANOVA and (F) two-way ANOVA, ****P < 0.0001, **P < 0.01, *P < 0.05. (F) Each point represents data from an individual healthy blood donor.

Fig 4: Platelets interact with peripheral blood monocytes to activate antigen cross-presentation.(A and B) Platelet-containing (mono+pl+) and platelet-depleted (mono+pl-) monocytes purified from mouse PBMCs were incubated overnight with sOVA, followed by 3-day coculture with CFSE-stained OT1 T cells. (A) Flow cytometry analysis of mono+pl+, mono+pl-, and OT1 only (mono-) cultures in triggering OT1 proliferation. Shown are (left) representative CFSE dilution FACS plots and histogram and (right) quantified proliferation index of dividing OT1 cells. (B) ELISA coculture supernatant quantifications for (left) IL-2 and (right) IFNg. (C to F) Flow cytometry analysis of DC surface marker evolution in PBMCs. Representative FACS plots of (C) (left) CD11b+ cells expressing MHC I–loaded sOVA antigen, SIINFEKL-H2Kb, and (right) quantification (D) (left) SIINFEKL-H2Kb+ CD11b+ cells (orange) overlayed on CD11b and CD11c gated PBMCs (purple), and (right) quantification. (E and F) MFI of (E) (left) CD80 and (right) CD86 expression and (F) MHC II expression on CD11b+ CD11c+ cells in PBMC+pl+ (black) and PBMC+pl- (red). (G) Representative Amnis images of CD11b+ (purple) Ly6G- (blue) monocytes interacting with CD62p-expressing (yellow), CD41+ (red) platelets, quantitated at days 0 and 1 in table above. All values are means ± SD of at least three independent experiments. (A and B) One-way ANOVA and (C to F) two-way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01.