Fig 2: Characterization of cell membrane protein differences between cell types using quantitative proteomicsMSCs and fibroblasts were grown in standard culture conditions, and membrane proteins were analyzed using LC-MS/MS(A) PCA plot depicting membrane protein profiles of MSC and fibroblast cell types (n = 3 biological replicates for MSCs and n = 2 biological replicates for MEF and MDF).(B) Heatmap utilizing hierarchical clustering showing expression level differences of membrane proteins.(C) GO-term enrichment analysis of differentially expressed membrane proteins between C57-MSCs and BALB-MSCs. MSC membrane proteins were quantified using LC-MS/MS and analyzed for differential protein expression using the DEP R package (Zhang et al., 2018).(D) Volcano plot highlighting significantly differentially expressed proteins between C57-MSCs and BALB-MSCs and notable proteins of interest (blue dots represent proteins with fold change >2 and adjusted p-value < 10-4). (E) Protein expression levels of the LPS-receptor CD14 across the cell types examined in this study (n = 3 biological replicates for C57- and BALB-MSCs and two biological replicates for MDFs and MEFs; data are represented as mean +/- SD).

Fig 3: Antibacterial and single-cell transcriptional analyses of BALB-MSCs overexpressing endogenous CD14 via CRISPRa(A) E. coli CFUs after 6 h co-cultured with wild-type or BALB-MSC-CRISPRa cells. E. coli CFUs from co-cultures were normalized to E. coli monoculture CFU controls performed alongside each replicate. Three different sgRNAs were tested in CRISPRa MSCs, and the data from distinct sgRNAs are depicted by different shapes (for scrambled sgRNAs: circle = nontargeting sgRNA 1, square = nontargeting sgRNA 2, triangle = nontargeting sgRNA 3. For CD14 sgRNAs: circle = CD14-83, square = CD14-105, triangle = CD14-13). Lines are drawn at mean, and statistical significance was determined by t test; ***, p < 0.001.(B) Nuclear translocation of NF-?B during LPS-exposure using p65 immunostaining and quantitative microscopy in BALB-MSC-CRISPRa cells expressing a scrambled sgRNA or CD14 sgRNAs (scrambled sgRNA = nontargeting control one; n = at least three biological replicates per sample).(C) UMAP plot depicting single-cell transcriptional profiles of MSCs during LPS-exposure (scrambled sgRNA = nontargeting control one; CD14 sgRNA = CD14-83). Single-cell RNA-seq of BALB-MSC overexpressing CD14 during LPS-exposure was prepared using the 10x Genomics Chromium platform followed by Illumina sequencing.(D) Heatmap of early LPS response gene expression in 100 individual BALB-MSC-CRISPRa cells expressing either scrambled control sgRNA (top) or CD14 sgRNA (bottom). Each sub-column represents gene expression level in an individual cell.(E) GO-terms enriched from significantly upregulated genes identified BALB-MSC-CRISPRa-CD14 when compared with BALB-MSC-CRISPRa-Scrambled in the absence of LPS-exposure.

Fig 4: 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 5: 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.