Journal: Neurobiology of aging

Article Title: Ethanol withdrawal acts as an age-specific stressor to activate cerebellar P38 kinase

doi: 10.1016/j.neurobiolaging.2010.01.005

Figure Lengend Snippet: Ovariectomized rats underwent repeated EW as described in the Figure 3 legend. At the 14th day of EW, the left hemisphere containing the cerebellar vermis was processed for immunohistochemical analysis to identify pSTAT1-positive Purkinje neurons. Photographs were taken of the cerebellar cortex of lobule II containing Purkinje layers of middle-age rats (15 months). Dark deposits marked with arrows indicate pSTAT1 immunoreactivity in Purkinje neurons, and they distinctively appeared in the EW group. EW rats had a higher number of pSTAT1-positive Purkinje neurons/Purkinje layer (mm) than dextrin (*p = 0.005) or EW+E2 (†p = 0.001) rats. A 20-fold magnification was used to take pictures. The scale bar indicates an actual length of 50 μm. Depicted are mean ± SEM for 6 microscopic fields/rat for 3 rats/group.

Article Snippet: A primary antibody against pP38 (Thr180/Tyr182) or pSTAT1 (Ser727) (Cell signaling, Danvers, MA) was diluted (1:50) in blocking buffer (10% normal goat serum).

Techniques: Immunohistochemical staining

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: T reg cells in VV-resistant tumors have elevated TGFβ and a repressed response to IFNγ. (A) Luminex cytokine analysis of TIF harvested from untreated MEER vvR or MEER vvS implanted in C57BL/6 mice. Three mice per group, three technical repeats per mouse. (B) Active TGFβ 1–3 concentration in the TIF of untreated MEER vvS or MEER vvR tumors as determined by TGFβ reporter assay. (C) TGFβR2 expression on CD8 + , Foxp3− T conv , or Foxp3+ T reg cells in untreated MEER vvR or MEER vvS implanted in Foxp3-reporter mice. Representative histograms from a MEER vvR tumor. 4 repeats, 10 M vvS , 9 M vvR mice. (D) Experimental schema of E–L and Q. Repeated three times. (E–L and Q) Percentage of (E) LAP-TGFβ1+, (F) GARP+, (G) CD103+, (I) TCF1+, (J) CD25 + , (K) CD122+, (L) PD-1+ Tim3+, and percentage and MFI of (H) Nrp1+ and (Q) pSTAT1+ T reg cells by flow cytometry as in D. (M) IFNγ concentration in TIF of MEER vvR and MEER vvS at 7 d after treatment with PBS or VV as in . (N–P) Percentage of pSTAT1 S727 + (N) CD8 + cells, (O) T conv cells, and (P) T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . (R) Representative flow plots and percentage of IFNγ+ T reg cells in MEER vvS or MEER vvR tumors 7 d after treatment with VV as in . Cells were restimulated with PMA/ionomycin direct ex vivo from tumors. M vvS = MEER vvS , M vvR = MEER vvR . Data represent two (A, B, and I–M), three (C, E–H, and Q), or four (N–P and R) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A), unpaired T test (B), one-way ANOVA with Sidak’s multiple comparison test paired T test (C, M–P, and R), or paired t test (E–L and Q). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Luminex, Concentration Assay, Reporter Assay, Expressing, Flow Cytometry, Ex Vivo, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Phenotyping observed in TIL T reg cells is found in dLN and TIL T conv to a lesser extent. (A–I) Quantification of the percentages of dLN T reg cells from paired tumors in Foxp3-Ametrine or Foxp3-RFP mice as in . (A) LAP-TGFβ1+, (B) GARP+, (C) CD103+, (D) Nrp1+, (E) TCF1+, (F) CD25 + , (G) CD122+, (H) PD-1+ Tim3+, and (I) pSTAT1+. (J–Q) Representative flow plots and quantification of T reg phenotyping markers on tumor-infiltrating T conv cells as in . Representative histograms of tumor-infiltrating T conv (Foxp3−) and T reg (Foxp3+) cells are shown for comparison, and quantification axes are scaled for T reg expression. Quantification and flow plots of (J) LAP-TGFβ1+, (K) GARP+, (L) CD103+, (M) Nrp1+, (N) CD25 + , (O) CD122+, (P) Tim3, and PD-1 (Q) pSTAT1+ Tconv cells. (R–U) Representative flow plots and quantification in paired dLN and tumors as in of (R) CD62L and CD44 on T conv cells, (S) CD62L and CD44 on CD8 + cells, (T) TCF1 on T conv cells, and (U) TCF1 on CD8 + cells. CM, central memory CD62L+ CD44 + ; Eff, effector CD62L− CD44 + ; Naïve, CD62L+ CD44 − . Data represent two (R–U) or three (A–Q) independent experiments. Each point represents an individual mouse *P < 0.05, **P < 0.01, ***P < 0.001 by paired T test (A–Q, T, and U) or one way ANOVA with Sidaks multiple comparisons test (R and S). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Comparison, Expressing

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: TGFβ limits IFNγ signaling and increases T reg cell stability. (A) Immunoblot and densitometry of pSTAT1 Y701 , STAT1, and β-actin in T reg cells sorted from spleen and lymph node of a Foxp3 reporter mouse and treated for 30 min with IFNγ, TGFβ1, or both. (B) Quantification and Cell Trace Violet (CTV) plots of the proliferation of stimulated Thy1.1+ CD4 responder cells in the presence of suppressing T reg cells at the 1:8 T reg cell:responder ratio in an in vitro suppression assay. Percent suppression is normalized to the proliferation index of stimulated CD4 + responder control without T reg cells. T reg cells were sorted from spleen and lymph node of a Foxp3-reporter mouse and then cultured for 3 d in IFNγ, TGFβ, or both. Cells were then sorted again to purify Foxp3+ T reg cells and then co-cultured in the suppression assay with CTV-labeled responder CD4 + cells. (C) Surface Nrp1 expression on sorted T reg cells from spleen and lymph node of a Foxp3-reporter mouse cultured in vitro in varying TGFβ concentrations for 48 h (D–F) An EV control and TGFβ 1 overexpressing (TGFβ OE) line were generated from the MEER vvS line. (D) Tumor growth of C57BL/6 mice implanted intradermally with MEER vvS-EV or MEER vvS-TGFβ OE and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (E) Survival of D. (F) Average tumor growth of MEER vvS-EV and MEER vvS-TGFβ OE as in D. Data represent two (C), four (A and D–F), or five (B) independent experiments; each point or line represents an individual mouse (A–D). *P <0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Sidak’s multiple comparison test (A and C), paired T test (B), Mantel-Cox test (E), or mixed effects analysis (F). ns, non-significant. Error bars indicate SEMs. Source data are available for this figure: .

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Western Blot, In Vitro, Suppression Assay, Control, Cell Culture, Labeling, Expressing, Generated, Injection, Comparison

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: VVdnTGFbmm only affects T reg cell phenotype in the tumor. (A) Western blot for TGFβ in EV control and TGFβ 1 overexpressing (TGFβ OE) MEER vvS lines and MEER vvR and MEER vvS as in . (B) Active TGFβ 1–3 levels measured in TIF of LLC, MC38 (colon adenocarcinoma), B16-F10 (melanoma), and C24 ( Pten –/– Braf V600E melanoma) tumors in C57Bl/6 mice as in . The average TGFβ concentration of MEER vvS (light blue) and MEER vvR (gray) from are overlaid as dotted lines. (C) Growth curve of LLC tumors treated with PBS or VV (black arrowhead) as in . (D–N) Representative flow plots and quantification of dLN (D–K) and T reg phenotyping markers on tumor infiltrating T conv cells (L–N) in Foxp3-Ametrine or Foxp3-RFP mice as in . Quantification of dLN (D) percent Foxp3+ of CD4 + and (E) MFI of Foxp3. Quantification and representative flow plots of (F) Nrp1+, (G) pSTAT1+, and (H) LAP-TGFβ1 on dLN T reg cells. Quantification of dLN (I) percent Foxp3− of CD4 + and CD8 + . Quantification and representative flow plots of (J) TNFα and IFNγ in T conv cells with direct ex vivo PMA/ionomycin stimulation and (K) PD-1 and Tim3 on CD8 + cells in dLN. Quantification and representative flow plots of (L) Nrp1+, (M) pSTAT1+, and (N) LAP-TGFβ1 on TIL T conv cells. Data represent two (A–C) or four (D–N) independent experiments. Each dot or line represents a technical repeat (A) or mouse (B–N). *P < 0.05 by one-way ANOVA with Sidak’s multiple comparisons test. ns, non-significant. Source data are available for this figure: .

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Western Blot, Control, Concentration Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: C24 melanoma model has a similar phenotype to MEER vvR after VV treatment. (A) Tumor growth (left, middle) and survival (right) of C57BL/6 mice implanted intradermally with C24 and, when tumors were ∼20 mm 2 , treated with a single IT injection of VV at 2.5 × 10 5 PFU/mouse or PBS control (black arrowhead). Mice were sacrificed when tumors reached 15 mm in any direction. (B) Foxp3-Ametrine or Fpxp3-RFP reporter mice implanted intradermally with C24 were as in A. Tumors and lymph nodes were harvested 7 d after treatment for phenotypic analysis. Percentage and total counts of CD8 + T cells and the ratio of T conv cells to T reg cells in treated tumors. (C) Percentage of Foxp3+ CD4 + T conv cells. (D) PD-1 and Tim3 expression on CD8 + cells. (E and F) (E) Production of granzyme B in CD8 + T cells and (F) MFI of pSTAT1 on T conv , CD8 + , and T reg cells as in B. (G) Production of IFNγ in T reg cells as in B after restimulation with PMA and ionomycin as in B. (H) IFNγ measured by ELISA from the TIL of CL24 untreated and 7 d after VV treatment as in B. Mice were implanted with C24 and treated with VV dnTGFβmm and αPD-1 as in . At day 8 after VV treatment, after three doses of αPD-1 were received, tumors were harvested for TIL analysis. (I–N) Representative flow plots and quantifications of (I) IFNγ+ T reg cells, (J) IFNγ + T conv cells, (K) IFNγ + CD8 + cells, (L) Tim3+ PD-1+ T reg cells, (M) TCF1+ T reg cells, and (N) TCF1+ CD8 + cells. Cytokine analysis was performed direct ex vivo with PMA and ionomycin restimulation. (O) Schematic for P and Q. (P) Tumor growth and survival (bottom) of C57BL/6 mice implanted intradermally with bilateral C24 and, when tumors were ∼20 mm 2 , one was treated with a single IT injection of VV dnTGFbmm (injected) at 2.5 × 10 6 PFU/mouse (black arrowhead). Mice were sacrificed when either tumor reached 15 mm in any direction. Starting at 4 d after VV or PBS treatment, mice were given anti-PD-1 or isotype control IP three times a week. (Q) Average growth of H. Data represent two independent experiments. Each point or line represents an individual mouse (A–P). *P < 0.05, **P < 0.01, ****P < 0.0001 by Welch’s T test (B–H), Mantel-Cox test (A and P), unpaired t Test (I–N), or mixed-effects analysis (Q). ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Injection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: The Journal of Experimental Medicine

Article Title: An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment

doi: 10.1084/jem.20230053

Figure Lengend Snippet: Viral delivery of TGFβ inhibition alleviates immunosuppressive T reg cells in resistant tumors. Foxp3-Ametrine or Foxp3-RFP mice implanted intradermally with MEER vvS or MEER vvR were treated with an IT injection of VV ctrl or VV dnTGFβmm at 2.5 × 10 6 PFU/mouse or PBS control. (A–E) Tumors and lymph nodes were harvested 4 (A) or 7 (B–E) d after treatment for phenotypic analysis. (A) Percentage and total counts of T reg cells at day 4 after treatment. (B) Percentage and MFI of Nrp1+ T reg cells at day 7. (C) Percentage of pSTAT1 Ser727 + T reg cells at day 7. (D) MFI of Foxp3 in T reg cells at day 7. (E) Percentage of LAP-TGFβ1+ T reg cells at day 7. (F) Percentage of T conv cells and CD8 + cells 7 d after treatment. (G) Production of TNFα and IFNγ in T conv cells from treated tumors after restimulation with PMA and ionomycin. (H) Percentage of PD-1- and Tim3-expressing CD8 + cells 7 d after treatment. (I) Percentage of TCF1+ in PD-1 and Tim3 CD8 + populations 7 d after treatment, representative plot of PD-1+Tim3− cells. Data represent three (H and I) or four (A–G) independent experiments. Each point represents an individual mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA (A–G) or two-way ANOVA (H and I) with Tukey’s multiple comparison test. ns, non-significant. Error bars indicate SEMs.

Article Snippet: The following antibody clones were utilized for flow cytometry experiments: CD4 (GK1.5; BioLegend), CD8 (53-6.7; BioLegend), Nrp1 (3E12; BioLegend), IFNγ Receptor 1 (2E2; eBioscience), IFNγ Receptor β chain (MOB-47; BioLegend), PD-1 (29F.1A12; BioLegend), Tim3 (RMT3-23; BioLegend), CD45 (30-F11; Biolegend), LAP-TGFβ1 (TW7-16B4; BioLegend), GARP (F011-5; BioLegend), CD103 (2E7; BioLegend), TCF1/7 (812145; R&D Systems), CD44 (IM7; BioLegend), CD62L (MEL-14; BioLegend), CD25 (PC61; BioLegend), CD122 (TM-β1; BioLegend), Granzyme B (GB11; BioLegend), Foxp3 (FJK-16 s; eBioscience), TNFa (MP6-XT22; BioLegend), IFNγ (XMG1.2; BioLegend), pSTAT1 ser727 (A15158B; BioLegend), and pSTAT1 tyr701 (A17012A; BioLegend).

Techniques: Inhibition, Injection, Control, Expressing, Comparison