Fig 2: Tr1 cells suppress immune responses via IL-10 signaling.(A) Representative histogram of surface MHC-II, CD40, CD80, and CD86 on BMDCs in each group (n = 4 for each group). (B) Quantitative data of the MFI of surface MHC-II, CD40, CD80, and CD86 on BMDCs in each group (n = 4 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) Representative histogram of CFSE dilution of CD8+ T cells in each group (n = 4 for each group). (D) Quantitative data of the proportion of CFSEdim proliferating CD8+ T cells in each group (n = 4 for each group). Values are shown as the mean ± SEM; ***P < 0.001 and ****P < 0.0001.

Fig 3: MB49 EVs contain key immunoregulatory cargo proteins and activate BMDCs. (A) Historam graph of number of released MB49 EVs per cell and their size (nm) estimated by Nano-particle Tracking Analysis. (B) Graph for the identified pathway hits using Panther database of MB49 EV protein cargo identified by Mass Spectrometry Analyses. Table listing identified immune system-related pathways and the number of proteins present on MB49 EVs for each of them. (C,D) Western blotting analysis of MB49 EVs targeting common EV markers and validating immune-related cargo proteins presence. (E) Gating strategy to show purity level of Bone Marrow-derived DCs in vitro differentiation and activation level changes (MHC-II and CD86) after MB49-EV treatment in vitro; quantification shown in bar graph. Error bars show standard deviation. Statistical significance was calculated by paired independent Student t-test.

Fig 4: DC maturation induces an actin-dependent increase in cortical stiffness.(A) BMDCs were untreated or matured by treatment with LPS, and cortical stiffness was measured by AFM microindentation. Fluorescent anti-CD86 labeling was used to select immature (CD86-negative) or mature (CD86 high) cells. (B) Ex vivo DCs were purified from spleens of untreated mice or from mice injected with lipopolysaccharide 24 hr before harvesting the spleen, and analyzed as in A. (C) Immature or LPS-matured BMDCs either left untreated or treated with 10 µm of the actin depolymerizing agents latrunculin-B or cytochalasin-D before AFM measurements. (D) Immature or LPS-matured BMDCs were plated on substrates of different stiffness before AFM measurements. Data are pooled from 2 to 3 independent experiments. Each data point represents an average of two stiffness measurements at different locations around a single cell nucleus. Error bars denote standard deviation. n.s non-significant, **p<0.01, ***p<0.001, ****p<0.0001 calculated by an unpaired one-way ANOVA, post-hoc Tukey corrected test.Figure 1—source data 1.Figure 1 - data table.

Fig 5: MC-mediated immune stimulatory properties of fibroblasts and keratinocytes.(A) NIH3T3 or (B) MPEK stable cells lines expressing either GFP alone (neg. control), OVA or MC.OVA were incubated for 60 hours. MC.OVA +rim had 10 nM rim present in the culture. After 60 hours, cells were harvested and MHC-I expression was analyzed by flow cytometry. (C-E) MPEK stable cells surface expression of CD80, CD86, and CD40. MFI values of live cells are reported relative to the mean MFI of negative control cells, transduced with an empty-vector, in each respective sample. (F) CD3+CD8+ OT-1 or WT T cells were purified by MACs and labeled with CellTrace violet (CTV) dye. Then 5 x 104 CD3+CD8+ CTV dye-labeled OT-1 or WT T cells were incubated with 5 x 104 B6-MPEK cells for 60 hours. By flow cytometry, live CTV+ cells were analyzed for CTV dye dilution as a measure of cell proliferation. (G) Representative histograms of live CTV+ cells. Percentages represent the percent of cells proliferating. n = 3, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons.