Journal: Cell Reports Medicine

Article Title: Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

doi: 10.1016/j.xcrm.2024.101441

Figure Lengend Snippet: MED1 is dispensable for T reg cell function across inflammatory contexts (A) Foxp3 YFP−cre (control) and Foxp3 YFP−cre / Med1 fl/fl (Med1 Treg−KO ) mice were aged to 12–14 months and bodyweights were measured. (n = 611 for control and n = 819 for Med1 Treg−KO for male and female, respectively.) (B) Representative hematoxylin and eosin (H&E) staining from organs of mice in (A). (C) Representative photograph of mice aged to 18 months. (D) CD45.1 CD4 + T conv cells were either transferred in alone or with T reg cells isolated from control or Med1 Treg−KO mice to induce colitis. Body weight displayed relative to start (n = 5 per group). (E) Representative H&E staining from colons of mice in (D). (F)EAE was induced by immunization with MOG peptide. Disease incidence was tracked per group during experiment. (G) Clinical scores measured during EAE (n = 10 control and n = 9 Med1 Treg−KO ). (H) Representative flow plots of FOXP3 expression of CD4 T cells within the CNS. (I) Summary of (H) (n = 7 control and n = 7 Med1 Treg−KO ). (J) FOXP3 protein expression in CD4 + FOXP3 + CD25 + T cells in the CNS. Values are normalized to controls. (n = 5 control and n = 5 Med1 Treg−KO .) (K) Percentage of cytokine producing CD4 + FOXP3- Tconv cells in the CNS. (n = 5 control and n = 5 Med1 Treg−KO ). (L) Mice were inoculated with influenza virus and monitored over course of disease. Body weight relative to starting (n = 8 controls and n = 6 Med1 Treg−KO ). (M) Heart rate measured in beats per minute (BPM) (n = 8 controls and n = 6 Med1 Treg−KO ). (N) Oxygen saturation was measured by pulse oximeter (n = 8 controls and n = 6 Med1 Treg−KO ). (A), (D), and (K) used two-way ANOVA with multiple comparisons. (F), (G), (I), (J), and (L–N) use unpaired two-tailed Student’s t test at experiment conclusion. Bars represent ± SEM. Points on graph represent individual mice. (∗p < 0.05, ∗∗p < 0.01.) Related to Figures S1 and .

Article Snippet: CD4 + selection kit II (mouse) , Stem Cell Technologies , #18952.

Techniques: Cell Function Assay, Control, Staining, Isolation, Expressing, Virus, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

doi: 10.1016/j.xcrm.2024.101441

Figure Lengend Snippet: MED1 is required for T reg cell promotion of tumor growth (A) B16 tumors were implanted into flanks of Foxp3 YFP−cre (controls) and Foxp3 YFP−cre / Med1 fl/fl (Med1 Treg−KO ) mice between 8 and 16 weeks of age. and measured for volume. (n = 12 control and n = 11 Med1 Treg−KO ). (B) EG7 tumors were implanted into flanks and measured for volume. (n = 5 control and n = 6 Med1 Treg−KO .) (C) RM1 tumors were implanted into flanks and measured for volume. (n = 4 controls and n = 5 Med1 Treg−KO .) (D) Tumor weights from B16, EG7, and RM1 tumors. (Respectively, for B16, EG7, and RM1 tumors: n = 8, 5, and 4 for controls and n = 11, 6, ad 5 for Med1 Treg−KO .) (E) Experimental setup for tumor experiments using Foxp3 eGFP−CreERT2 , ROSA26Sor CAG-tdTomato (control) Med1 fl , Foxp3 eGFP−CreERT2 , and ROSA26Sor CAG-tdTomato (Med1 Treg-iKO ) with inducible MED1 deletion and FOXP3 lineage trace. (F) RM1 tumors were implanted into flanks and measured for volume. (n = 14 controls and n = 13 Med1 Treg-iKO .) (G) Representative photograph of RM1 tumors. (H) Tumor weights from RM1 tumors (n = 14 controls and n = 13 Med1 Treg-iKO ). (I) Representative flow plots of CD4 and CD8 expression within the CD45 + cells within RM1 tumors. (J) CD45 + CD3ε + CD4 + FOXP3 – , CD45 + CD3ε+ CD8 + , and of CD45 + CD3ε + CD4 + FOXP3 + T cells proportion of total live cells from RM1 tumors (n = 6 controls and n = 7 Med1 Treg-iKO ). (K) Representative flow plots of T conv (FoxP3 – tdTomato – ), ex-FOXP3 (FOXP3 – tdTomato + ), tdT + T reg (FOXP3 + tdTomato + ), tdT – T reg (FOXP3 + tdTomato + ) within the intratumoral CD4 + T cell compartment. (L) Summary data of (K) (n = 4 controls and n = 5 Med1 Treg-iKO ). (M) Representative flow plots of IFN-γ production within intratumoral CD8 + T cell compartment. (N) Summary data of M (n = 6 controls and n = 6 Med1 Treg-iKO ). (O) RM1 tumors were implanted in mice. IgG control or α-CD8 depleting antibody treatment started on day 5. (n = 4 for each group.) (A–C), (F), (H), and (Q–T) use unpaired Student two-tailed t test at experiment conclusion. (D), (J), (L), and (O) used two-way ANOVA with multiple comparisons. Bars represent ± SEM. Points on graph except for (S) represent individual mice. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) Related to Figures S3 , , and Table S1 .

Article Snippet: CD4 + selection kit II (mouse) , Stem Cell Technologies , #18952.

Techniques: Control, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

doi: 10.1016/j.xcrm.2024.101441

Figure Lengend Snippet: Murine and human T reg cells experience divergent paths of differentiation in tumors relative to inflammation (A) Experimental setup for colitis-associated-cancer model and colitis experiments. (B) Integrated UMAP clustering of T reg cells isolated from tumors and colons. (C) UMAP diagrams of T reg cells isolated from tumors and colons. (D) Cluster proportion of total T reg cells from different conditions (n = 2–3 biological replicates per condition, each biological replicate contains cells pooled from 2 to 8 separate mice). p values of less than 0.1 are displayed. (E) Feature plots of cluster 5 enriched genes. (F) Tumor-specific suppressive module score violin plot and UMAP plot. (G) Pseudotime representation portrayed on integrated UMAP plot and specific trajectory toward cluster 5. (H) Graph test results from Monocle 3. Moran’s I plotted for genes from least to greatest. Transcription factors above 0.2 labeled. (I) Experimental setup from (Loo et al. ). CD3 + T cells were isolated from patients, then T reg cells were subset during analysis based on positive CD4 , FOXP3 , and IL2RA expression and negative IL2 expression. (J) UMAP plots of T reg cells from HNSCC and inflamed OM. (K) Cluster proportion of total T reg cells from different conditions (n = 4 biological replicates per condition). p values of less than 0.1 are displayed. (L) Tumor-specific suppressive module score violin plot and UMAP plots for human data. (D) and (K) used two-way ANOVA with multiple comparisons. (F) and (L) used one-way ANOVA with Dunnet’s multiple comparisons test to compare cluster 5 to all clusters. (I) uses the graph test (Moran’s I) to test for genes with multi-directional and multi-dimensional spatial autocorrelation. Related to Figure S8 .

Article Snippet: CD4 + selection kit II (mouse) , Stem Cell Technologies , #18952.

Techniques: Isolation, Labeling, Expressing

Journal: Cell Reports Medicine

Article Title: Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

doi: 10.1016/j.xcrm.2024.101441

Figure Lengend Snippet:

Article Snippet: CD4 + selection kit II (mouse) , Stem Cell Technologies , #18952.

Techniques: Blocking Assay, Control, Virus, Recombinant, Activation Assay, Adjuvant, Selection, Isolation, Staining, Software

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