Journal: Nature Communications

Article Title: Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

doi: 10.1038/s41467-019-09884-6

Figure Lengend Snippet: PSA reduces CNS inflammation in HSV-infected WT but not Rag mice. a % and b total numbers (#) of CD45 high leukocytes and CD45 high Ly6C high inflammatory monocytes (IM) infiltrating the brainstem (BS) of Rag mice. c % (left y -axis) and # (right y -axis) infiltrating CD45 high leukocytes in the BS of WT mice. d % CD11b + cells within BS infiltrating CD45 high cells; e % Ly6C high and CD107 + IM within the CD11b + population; f % CD4 + and CD8 + T cells within CD45 high cells in the BS of WT mice. Data compiled from 2 to 4 experiments with n = 6–8/group at day 6 pi. All data show mean ± SEM. *** p < 0.0005, **** p < 0.0001, ns: not significant, as determined by two-tailed Student's t -test

Article Snippet: T cells and B cells were isolated using EasySep mouse CD4, CD8 or CD3 T cell and CD19 B cell immunomagnetic negative selection isolation kits by as per manufacturers recommendations (Stemcell Technologies).

Techniques: Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

doi: 10.1038/s41467-019-09884-6

Figure Lengend Snippet: PSA protection from HSE is independent of induced Tregs. a % FoxP3 + CD4 Tregs and b CD69 + CD4 T cells in spleen and CLN of PSA or PBS-treated WT mice at day 6 pi. c CD25 expression within FoxP3 + CD4 Tregs in WT mice at day 6 pi, % and mean fluorescence intensity (MFI) in () shown in right top quadrant. d % CD25 within FoxP3 + Tregs (left plot) and FoxP3 − CD4 + T cells (right plot), e CD103 expression within FoxP3 + Tregs in WT mice at day 6 pi; % and MFI in () shown in right top quadrant. f CD103 within FoxP3 + Tregs (left plot) and FoxP3 − CD4 + T cells (right plot) in the spleen or CLN of WT mice at day 6 pi. Data from three experiments shown. g PSA-treated Treg depleted and control WT mice were monitored for survival after HSV infection and ACV treatment as in Fig. , ns: not significant determined by log rank Mantel–Cox test ( n = 11–12 mice). After administration of three (1 week) or six doses (2 weeks) of PSA, MLN in uninfected WT mice were monitored for h cellularity, i % CD4 and CD8 T cells, and j # ICOS + , CD39 + , and CD73 + CD4 and CD8 T cells ( n = 3 mice); **** p < 0.0001, ** p < 0.01 as determined by two-way ANOVA or one-way ANOVA with Sidaks or Turkeys correction, respectively, for multiple comparisons tests. All data show mean ± SEM

Article Snippet: T cells and B cells were isolated using EasySep mouse CD4, CD8 or CD3 T cell and CD19 B cell immunomagnetic negative selection isolation kits by as per manufacturers recommendations (Stemcell Technologies).

Techniques: Expressing, Fluorescence, Control, Infection

Journal: Nature Communications

Article Title: Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

doi: 10.1038/s41467-019-09884-6

Figure Lengend Snippet: PSA increases IL-10 and IFNγ-secreting T cells. CD4 and CD8 T cells and B cells in spleens, mesenteric lymph nodes (MLN), and cervical lymph nodes (CLN) of PSA or PBS-treated WT mice at day 6 pi were analyzed for a IL-10 and b IFNγ secretion, n = 2 experiments; * p < 0.05, ** p < 0.01, **** p < 0.0001, as determined by two-way ANOVA with Sidak’s multiple comparisons test. Survival of PSA or PBS treated c IL-10KO mice or d IFN-GKO mice ( n = 8–16 mice); ns: not significant. Bar plots show e % CD45 high leukocytes, f (left y -axis) % Ly6C high IM and (right y -axis) % Ly6G + neutrophils (PMN) within CD45 high CD11b + cells infiltrating the BS of PSA treated 129 WT, IL10KO, and GKO mice at day 6 pi, n = 3 experiments with 2–3 BS/group; * p < 0.05, **** p < 0.0001 as determined by ordinary one-way ANOVA with Turkey’s multiple comparisons tests

Article Snippet: T cells and B cells were isolated using EasySep mouse CD4, CD8 or CD3 T cell and CD19 B cell immunomagnetic negative selection isolation kits by as per manufacturers recommendations (Stemcell Technologies).

Techniques:

Journal: Nature Communications

Article Title: Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

doi: 10.1038/s41467-019-09884-6

Figure Lengend Snippet: PSA protection against HSE requires B and T cells secreting IL-10. a Experimental design for experiments in b and c Donor WT (In black text): Naïve Rag mice were transferred with WT CD4 + or CD8 + T cells or CD19 + B cells 7 days before PSA treatment. Donor IL-10KO (magenta text) and WT (Blue text): four groups of naïve Rag mice were transferred with combinations of donor WT B and T cells, IL-10KO B and T cells, WT B and IL-10KO T cells, IL-10KO B and WT T cells 7-days before PSA treatment. All Rag recipients received six doses of PSA before HSV infection and ACV treatment. b Survival of B cell-depleted mice (BKO, n = 20 mice) and Rag recipients of WT single cell subsets ( n = 6–9 mice/group). B cell depletion in WT mice was initiated 10 days prior to PSA treatment and continued throughout infection, ns: not significant. c Survival of Rag recipients of WT and IL-10KO combination of T and B cells ( n = 10–13/group). *** p < 0.001, * p < 0.05, ns: not significant as determined by log rank (Mantel–Cox) test. FACS plots of BS CD45 high cells (left), Ly6G + PMN (left middle), CD11b + cells within CD45 high cells (right middle), and Ly6C high IM and Ly6C int CD11b + PMN within CD45 high CD11b + cells (right) were analyzed at day 6 pi in the BS of Rag recipients of d IL-10KO B + WT T cells (brown circle) and e WT B + IL-10KO T cells (green circle)

Article Snippet: T cells and B cells were isolated using EasySep mouse CD4, CD8 or CD3 T cell and CD19 B cell immunomagnetic negative selection isolation kits by as per manufacturers recommendations (Stemcell Technologies).

Techniques: Infection

Journal: Nature Communications

Article Title: Bacteroides fragilis polysaccharide A induces IL-10 secreting B and T cells that prevent viral encephalitis

doi: 10.1038/s41467-019-09884-6

Figure Lengend Snippet: Role of the bacterial symbiosis factor PSA in preventing viral encephalitis. HSV infection of susceptible 129 WT mice provokes excessive production of neutrophils (PMN) and Ly6C high inflammatory monocytes (IM) in the bone marrow that invade the brainstem in massive numbers resulting in fatal HSV encephalitis (HSE), despite antiviral treatment from day 4 pi. The bacterial symbiosis factor, PSA given orally is bound by B cells/CD138 + plasmablasts (PB) in the small intestine, which induces IL-10 and IFNγ production by regulatory CD4 and CD8 T cells resulting in the suppression of pathogenic inflammatory myeloid cells concomitant with the induction of IFNγ inducible chemokines in the BS. This novel study reveals the immunomodulatory potential of PSA in protecting from lethal viral infections of the CNS in combination with an antiviral. Cells involved in this protective mechanism are shown in the key. Inhibitory pathways indicated by red blocking arrows

Article Snippet: T cells and B cells were isolated using EasySep mouse CD4, CD8 or CD3 T cell and CD19 B cell immunomagnetic negative selection isolation kits by as per manufacturers recommendations (Stemcell Technologies).

Techniques: Infection, Blocking Assay

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs treatment normalizes markers of senescence in MRL/ lpr mice splenic CD4+ T cells in vivo. (A, B) qPCR analysis showing the expression levels of p21, p16 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. (C) Representative western blot showing the expression levels of p21, p16, p53 and acetylation of p53 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. (D, E) Capillary WES and qPCR analysis showing the expression of Sirt1 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. GAPDH was used as a protein loading control. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: In Vivo, Expressing, Western Blot, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs increase markers of senescence in splenic CD4+ T cells in vitro . (A-C) Splenic CD4+ T cells from MRL /lpr mice were cultured alone or with hUC-MSCs at ratios of 1:1, 10:1, or 50:1 for 48 h. (D-F) In further experiments, MRL/ lpr splenic CD4+ T cells and hUC-MSCs (10:1) were cultured separated by transwells. Sirt1 (A, D), p21 (B, E) and p16 (C, F) RNA levels of CD4+ T cells were quantitated by qPCR. All experimental data were verified in at least two independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s., not significant.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: In Vitro, Cell Culture

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: Sirt1 is a mediator of hUC-MSCs increasing senescence of splenic CD4+ T cells in MRL/ lpr mice. (A) Western blotting showing the expression of Sirt1, p21, p16, p53 and acetylation of p53 in EX527 and DMSO treated MRL/ lpr splenic CD4+ T cells. (B) MRL/ lpr splenic CD4+ T cells were pretreated with SRT1720 12 h before exposed to hUC-MSCs for 24-48 h, western blotting showing the expression of p21, p16, p53 and acetylation of p53. GAPDH was used as a protein loading control. All experimental data were verified in at least three independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Western Blot, Expressing, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs induced miR-199a-5p can increase MRL/ lpr splenic CD4+ T cell senescence. (A) qPCR analysis of the levels of ten potential miRNAs in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice splenic CD4+ T cells. (B-D) qPCR and western blotting analysis of the levels of miR-199a-5p, Sirt1, p21, p16 and acetyl-p53 in vehicle and miR-199a-5p mimic-treated MRL/ lpr splenic CD4+ T cells. (E-F) qPCR and western blotting analysis of the levels of Sirt1, p21, p16 and acetyl-p53 in vehicle and miR-199a-5p inhibitor-treated WT splenic CD4+ T cells. (G-I) MRL/ lpr splenic CD4+ T cells and hUC-MSCs were cultured alone or together in the presence or absence of miR-199a inhibitor using a transwell system. MiR-199a-5p, Sirt1, p21, p16 and acetyl-p53 were quantified in hUC-MSCs (the first bar) or splenic CD4+ T cells (the last three bars). GAPDH was used as a protein loading control. All experimental data were verified in at least two independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Western Blot, Cell Culture, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: MiR-199a-5p agomir treatment increases senescence of MRL/ lpr splenic CD4+ T cells. (A) Experimental outline. CD4+ T cells were harvested to measures miR-199a-5p (B), Sirt1 (C), p21 and p16 (D). GAPDH was used as a protein loading control. (n = 6 per group). * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Control

Journal: PLoS Pathogens

Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection

doi: 10.1371/journal.ppat.1007365

Figure Lengend Snippet: (A) Experimental design. (B) Real-Time PCR analysis of viral genome copies from brain. (C) Number of D b LT359 tetramer + CD8 T cells from brain. (D) Frequency of Ki-67 + D b LT359 tetramer + cells from the brain. (E) Ex vivo staining for IRF4 in D b LT359 tetramer + CD8 T cells. (F) Frequency of IFN-γ + CD44 hi CD8 T cells from brain following ex vivo stimulation with LT359 peptide. (G) Frequency (left) of IFNγ-eYFP + cells and number (right) of D b LT359 tetramer + WT-CD103 + ,WT-CD103 - cells, and αCD4-CD103 - cells from the brains of IFNγ-eYFP mice at day 5 post reinfection. (H) Frequency of IFNγ-eYFP + cells 30 days p.i. Mean ± SD of 9–10 mice per group from 3 independent experiments (A-F) or 8 mice per group from two independent experiments (G, H). ns = not significant, *P<0.05, ***P<0.001, Two-Way ANOVA with Tukey multiple comparisons test (B-F) and one-way ANOVA (G,H).

Article Snippet: CD4 T cells were purified from total brain homogenates using the EasyStep Mouse CD4 Positive Selection Kit II CD4 positive selection kit (Stemcell Technologies, Vancouver, Canada).

Techniques: Real-time Polymerase Chain Reaction, Ex Vivo, Staining

Journal: PLoS Pathogens

Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection

doi: 10.1371/journal.ppat.1007365

Figure Lengend Snippet: (A) Principal component analysis of FACS-sorted D b LT359 tetramer + CD8 T cells pooled from brains of 3–4 MuPyV (each pool designated as TM) i.c. inoculated mice at days 30–40 p.i. Red dots, CD4 T cell-insufficient mice; blue dots, WT mice. (B) Volcano plot representation of differential expression analysis of transcripts of MHCII -/- vs WT-CD103 + (left) MHCII -/- vs CD103 - (middle), and CD103 + vs CD103 - (right). Red represents differentially expressed transcripts over the cut-off of a q-value ≤ 0.05 (y-axis represents -log10[q-value]) and the X-axis represents the fold change. (C) Venn diagram showing the number of differentially expressed genes in MHCII -/- -CD103 - vs CD103 + (top), MHCII -/- -CD103 - vs CD103 - (left bottom), and CD103 + vs CD103 - (right bottom). (D) Ingenuity pathway analysis of differentially expressed transcripts between MHCII -/- -CD103 - and CD103 - . Y-axis represents pathway and X-axis represents enrichment factor. Bubble size represents the number of differentially expressed transcripts and color represents the P-value calculated by Fisher’s Exact test. Blue labels indicate upregulated pathways and red labels indicate downregulated pathways with a cut-off z score ≤ 2.0.

Article Snippet: CD4 T cells were purified from total brain homogenates using the EasyStep Mouse CD4 Positive Selection Kit II CD4 positive selection kit (Stemcell Technologies, Vancouver, Canada).

Techniques: Quantitative Proteomics

Journal: PLoS Pathogens

Article Title: CD4 T cells control development and maintenance of brain-resident CD8 T cells during polyomavirus infection

doi: 10.1371/journal.ppat.1007365

Figure Lengend Snippet: (A) Experimental design. (B) Number of CD4 T cells in brain. (C) Number of virus-specific CD8 T cells from brain at 17 and 30 days p.i. (D) Real-Time PCR analysis of viral genome copies from brain. (E) Frequency of CD103 + D b LT359-specific CD8 T cells in brain at days 17 and 30 p.i. There was a significant increase in CD103 + CD8 T cells (p = 0.0132) in Rat IgG-treated mice between days 8 and 30 p.i. (F) Experimental design. (G) Number of total and virus-specific CD8 T cells (left) and gMFI of CD8 on D b LT359 tetramer + CD8 T cells (right) in brain upon systemic depletion of CD4 and/or CD8 T cells. (H) Experimental design. (I) Number of D b LT359 tetramer + CD8 T cells from brain. (J) Frequency of IFNγ + CD44 hi CD8 T cells from brain upon ex vivo stimulation with LT359 peptide. (K) Real-Time PCR analysis of viral genome copies from brain. Mean ± SD of 8 mice per group from two independent experiments. *P<0.05, ***P<0.001, two-way ANOVA with Sidak’s multiple comparisons test (B-E,G), one-way ANOVA (G), and Mann-Whitney test (I-K).

Article Snippet: CD4 T cells were purified from total brain homogenates using the EasyStep Mouse CD4 Positive Selection Kit II CD4 positive selection kit (Stemcell Technologies, Vancouver, Canada).

Techniques: Virus, Real-time Polymerase Chain Reaction, Ex Vivo, MANN-WHITNEY