Fig 1: Activation of DC and T lymphocytes by Nano-DOX-treated 4T1 cells. a–d Immunostaining of CD40, CD80, CD83 and MHC-II (markers of DC activation) in bone marrow-derived DC (BMDC) assayed by FACS. Representative FACS histograms are presented in Additional file 1: Fig. S6. e Spleen-derived CD4+ and CD8+ T lymphocytes characterized by immunofluorescent staining and FACS.f, g Proliferation of CD4+ and CD8+ T lymphocytes, assayed by CFSE staining and FACS. Representative FACS histograms are presented in Additional file 1: Fig. S7. h, i Immunostaining of CD69 (a marker of T lymphocyte activation) in CD4+ and CD8+ T lymphocytes, assayed by FACS. Representative FACS dot plots are presented in Additional file 1: Fig. S8. j Immunohistochemical staining of CD4, CD8, CD69 and foxp3 (a marker of Treg) in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. In the ex vivo cell experiments, BMDC were first treated with Nano-DOX (2 µg/mL) or culture medium conditioned by Nano-DOX (2 µg/mL)-treated 4T1 cells (ND-CM) for 24 h. Spleen-derived lymphocytes were then co-cultured with the BMDC in the same system for another 24 h. In FACS analysis, geometric means were used to quantify fluorescence intensity. Values were mean ± SD (n = 3, *p < 0.05, **p < 0.01)
Fig 2: Primed enhancers are pre-existing, but gain activation markers with treatment.(A) Comparing primed enhancers marked by H3K4me2 peaks reveals strong correlation between untreated and treated samples. Normalized tag counts in the No Peptide condition are plotted against those in a 1 µM PCC condition, with red dots coloring those that are more than two-fold up-regulated in the 1 µM PCC condition. The up-regulated enhancers are both few in number and low in tag count. (B) De novo motif finding identifies lineage-determining transcription factor (LDTF) motifs among primed enhancers shared by the five conditions. An ETS motif is most prominent, and a RUNX motif is likewise highly enriched over the randomly selected background. Both ETS and RUNX factors play important roles in T cell development. (C) Among primed enhancers shared by all five conditions, including the untreated condition, pro-inflammatory transcription factor motifs are enriched. An IRF family motif, AP-1 family motif (represented by BATF), and NF-?B motif (represented by REL) are all significantly enriched among shared enhancers marked by H3K4me2. (D). Comparing H3K27Ac tag counts at enhancers in No Peptide as compared to 1 µM PCC treatment reveals that many enhancers see increasing H3K27Ac deposition upon stimulation. Points in red indicate greater than two-fold increase in tags upon treatment. (E) Enhancers that are more active upon stimulation, as determined by greater than two-fold H3K27Ac tags in 1 µM PCC treatment as compared to No Peptide, are enriched for pro-inflammatory transcription factor motifs. BATF, an AP-1 family member, and NF-?B are most prominent. (F) Enhancers that are more active with stimulation are enriched near activation signature genes, as can be seen with this enhancer upstream of the activation signature gene Il2ra (CD25). (G) Enhancers upstream of the activation signature gene Cd69 show an increase in H3K27Ac deposition upon treatment with 1 µM PCC. (H) Genome-wide, deposition of H3K27Ac, a marker of transcription factor activity, reflects increasing TCR signal strength at the binding sites of AP-1 family members, including BATF.DOI: http://dx.doi.org/10.7554/eLife.10134.012
Fig 3: aPV immunization impairs TRM cell induction in the noses of BALB/c mice.BALB/c mice were immunized twice with aPV or wPV or left unvaccinated (Ctrl) prior to challenge with 106 CFU of B1917GR and the induction of CD4+ TRM cells in the noses of mice was measured at the indicated time points. a Absolute numbers of nasal CD103- (upper left panel) and CD103+ (upper right panel) CD4+ TRM cells and representative graph illustrating differential enrichment of CD44+ CD103+ among the CD69+ CD4+ resident T cells 7 (in black) and 28 d.p.c. (in red) in the nose of control and vaccinated BALB/c mice (lower panels). Numbers indicate percentages of events in each square. b Representative graphs showing the increase in IL-17- and/or IFN-?-producing nasal CD103- (left panels) and CD103+ (right panels) CD4+ TRM cells between 7 (in black) and 28 d.p.c. (in red). Numbers indicate percentages of events in each square. c Absolute numbers of IL-17+ CD103- (left panel) and IL-17+ CD103+ (right panel) nasal CD4+ TRM cells at indicated time points after Bp challenge. d Absolute numbers of nasal IFN-?+ CD103- (left panel) and IFN-?+ CD103+ (right panel) CD4+ TRM cells at indicated time points after Bp challenge. Results shown are geometric means ± SD. n = 6 per group. Kruskal–Wallis tests were performed to compare aPV (#) and wPV (*) immunized mice to control mice. Only significant differences are indicated. *P < 0.05; ###p < 0.001.
Fig 4: Bp-infected BALB/c mice develop Th17 TRM cells in the nose.Six-week-old C57BL/6 mice and BALB/c mice were infected with 106 CFU of B1917GR. Nasal immune cells were collected at indicated time points post challenge (D0–D28). Ten minutes before euthanasia, mice were intravenously injected with anti-CD45-PE antibody enabling us to discriminate TRM cells from circulating cells. TRM cells express CD44, CD69, and/or CD103. Cell activation was assessed by nonspecific ex vivo stimulation and intracellular staining of IL-17 and IFN-?. Cells were fixed and permeabilized to perform intracellular staining of IL-17 and IFN-?. Numbers indicate percentages of events in each square. a Representative graph showing the accumulation of CD103+ CD44+ TRM cells in the nose of BALB/c mice at indicated time points after Bp challenge. b Representative graph comparing the pattern of expression of CD44 (left panels) and CD103 (right panels) in the nose of C57BL/6 (black) and BALB/c mice (gray) at indicated time points after Bp challenge. Numbers indicate mean fluorescence intensities and dotted lines indicate the cut-off between positive and negative cells. c Representative graph showing the proportion of IL-17- and/or IFN-?-producing CD4+ TRM cells in the nose of C57BL/6 (left panels) and BALB/c mice (right panels) at indicated time points after Bp challenge. Numbers indicate percentages of events in each square.
Fig 5: Both iMSC and ASC increase differentiation of T cells into Tregs and suppress CD69 expression. (A) Timeline and experimental details of iMSC/ASC coculture with mouse splenocytes (B) Representative flow cytometry dot plots showing increased CD25+Foxp3+ Tregs in iMSC/ASC cocultures with activated splenocytes. (C) Quantification of a percent increase in CD25+Foxp3+ cells with (D) a reduction in CD69+ cells in cocultures. Data shown as mean ± SEM from a single experiment repeated independently with similar results (*** p < 0.0001 one-way ANOVA).
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