Journal: Nature Communications

Article Title: Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

doi: 10.1038/s41467-021-21572-y

Figure Lengend Snippet: a Individualised treatment of melanoma is guided by tumour staging, presence of B-RAF mutations and fitness-for-toxicity. b Colitis, hepatitis and thyroiditis are common immune-related complications of dual therapy with Nivolumab plus Ipilimumab; 31.4% of patients experienced two or more of these immune-related adverse reactions ( n = 89). c Colitis, hepatitis and thyroiditis occurred independently and were not significantly associated with clinical response ( n = 89; F.E). d – h Patients who developed hepatitis of any grade following dual therapy lacked biochemical signs of liver inflammation before treatment. In particular, no clinically meaningful differences in plasma levels of d aspartate transaminase (AST; n = 87; M.W.; Bonferroni-corrected p -value, m = 5), e alanine transaminase (ALT; n = 89; M.W.; Bonferroni-corrected p -value, m = 5), f gamma glutamyl transaminase (γ-GT; n = 89; M.W.; Bonferroni-corrected p -value, m = 5), g total bilirubin ( n = 87; M.W.; Bonferroni-corrected p -value, m = 5), or h C-reactive protein (CRP; n = 85; M.W.; Bonferroni-corrected p -value, m = 5) were observed between patients who developed hepatitis and those who did not. Median values are indicated by a red line. i , j Biochemical markers of tumour burden were not different between patients who developed hepatitis and those who did not. i Pre-treatment levels of lactate dehydrogenase ( n = 89; M.W.; Bonferroni-corrected p -value, m = 4). Median values are indicated by a red line. j Pre-treatment levels of protein S100 ( n = 89; M.W.; Bonferroni-corrected p -value, m = 4). k – m No association was observed between seropositivity for k hepatitis B virus core antigen (HBcAg; n = 85; F.E.; Bonferroni-corrected p -value, m = 3), l hepatitis C virus (HCV; n = 85; F.E.; Bonferroni-corrected p -value, m = 3), or m hepatitis E virus (HEV; n = 67; F.E.; Bonferroni-corrected p -value, m = 3) and development of hepatitis following dual therapy. n No association was observed between rounds of αPD-1/αCTLA-4 administered and development of hepatitis ( n = 89; M.W.).

Article Snippet: Stage IV patients with unresectable metastatic disease who received first- or second-line checkpoint inhibitor therapy were initially treated with Nivolumab (αPD-1; Bristol-Myers Squibb) and Ipilimumab (αCTLA-4; Bristol-Myers Squibb) for four cycles, and thereafter with Nivolumab maintenance therapy (3 mg/kg at 3-week intervals).

Techniques:

Journal: Nature Communications

Article Title: Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

doi: 10.1038/s41467-021-21572-y

Figure Lengend Snippet: a Peripheral blood samples were collected from melanoma patients with metastatic disease receiving αPD-1/αCTLA-4 therapy immediately before administration of the first dose ( n = 89). Leucocyte subsets differentially represented in patients with or without hepatitis were identified in a randomly assigned training set (B.H.-corrected t -tests; n = 44; m = 50; FDR = 0.25). Red dots indicate significantly differently represented subsets. Example gating strategies for analysis of flow cytometry data are provided as Supplementary Figs. – . b CD4 + T EM % in training set patients with or without treatment-related hepatitis ( n = 44; M.W.). Median values are indicated by a red line. c CD4 + T EM % in validation set patients with or without treatment-related hepatitis ( n = 45; M.W.). d ROC analysis of CD4 + T EM % as a discriminatory marker for treatment-related hepatitis in the validation set ( n = 45). e Comparison of the bimodal distribution of CD4 + T EM % in patients with unresectable metastatic disease ( n = 107) and the normal distribution ( n = 49; K2 = 2.79; p = 0.248) of CD4 + T EM % in patients with completely resected tumours. A cut-off of CD4 + T EM ≥ 21% was set (indicated by a dashed red line) below which 99% of completely resected tumour cases should fall. Four pink points represent CD4 + T EM ≥21% patients with metastatic disease who were electively treated with αPD-1 monotherapy. f In the validation set, 68.9% patients were correctly classified using a cut-off of CD4 + T EM ≥ 21 %, whereas 55.6% were correctly classified under the no-information model ( n = 45; F.E.). g CD4 + T EM ≥ 21 % is not a marker of predisposition to αPD-1/αCTLA-4-related colitis ( n = 89; F.E.). h CD4 + T EM ≥21% patients did not experience more severe hepatitis than CD4 + T EM <21% patients ( n = 38; F.E.). Dashed red line indicates a cut-off of CD4 + T EM ≥ 21%. i Time-to-first presentation of hepatitis was not different between CD4 + T EM ≥21% ( n = 12) and T EM <21% ( n = 26) patients (log-rank). j Twelve of 12 patients with unresectable metastatic melanoma and CD4 + T EM ≥ 21% developed hepatitis after αPD-1/αCTLA-4 dual therapy. By contrast, 3 of 4 CD4 + T EM ≥21% patients treated with αPD-1 monotherapy did not develop hepatitis (F.E.; p = 0.007).

Article Snippet: Stage IV patients with unresectable metastatic disease who received first- or second-line checkpoint inhibitor therapy were initially treated with Nivolumab (αPD-1; Bristol-Myers Squibb) and Ipilimumab (αCTLA-4; Bristol-Myers Squibb) for four cycles, and thereafter with Nivolumab maintenance therapy (3 mg/kg at 3-week intervals).

Techniques: Flow Cytometry, Marker

Journal: Nature Communications

Article Title: Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

doi: 10.1038/s41467-021-21572-y

Figure Lengend Snippet: a Seasonal presentation of CD4 + T EM ≥21% patients between 2017 and 2020 ( n = 103; F.E.). Dashed red line indicates a cut-off of CD4 + T EM ≥ 21%. b CD4 + T EM ≥21% status was associated with high serum levels of anti-CMV IgG antibodies ( n = 100; F.E.; p = 1.2 ⨯ 10 −5 ). Dashed red line indicates a cut-off of CD4 + T EM ≥ 21%. c CD4 + T EM ≥21% status was associated with CMV-reactivity in pp65 ELISPOT ( n = 53; F.E.; p = 9.0 ⨯ 10 −5 ). Dashed red line indicates a cut-off of CD4 + T EM ≥ 21%. d Development of hepatitis was associated with CMV-seropositivity and CD4 + T EM ≥21% status ( n = 89). Median values are indicated by a red line. e ROC analysis showing CD4 + T EM % is a superior discriminator of patients at risk of hepatitis when considering only CMV IgG + cases as opposed to all cases. f Classification of patients with unresectable metastatic melanoma who did or did not develop αPD-1/αCTLA-4-related hepatitis according to CMV IgG status and baseline CD4 + T EM cell frequency using a revised cut-off of CD4 + T EM ≥ 16%. Red boxes indicate 34 of 40 (85%) cases correctly classified by our model ( n = 40; F.E.; p = 1.4 ⨯ 10 −5 ). Green box indicates 2 of 17 (11.7%) cases predicted to develop hepatitis who did not. Pink box indicates 4 of 23 (17.4%) cases predicted not to develop hepatitis who did. Blue box indicates 19 of 49 CMV IgG − patients not considered by our model who developed hepatitis.

Article Snippet: Stage IV patients with unresectable metastatic disease who received first- or second-line checkpoint inhibitor therapy were initially treated with Nivolumab (αPD-1; Bristol-Myers Squibb) and Ipilimumab (αCTLA-4; Bristol-Myers Squibb) for four cycles, and thereafter with Nivolumab maintenance therapy (3 mg/kg at 3-week intervals).

Techniques: Enzyme-linked Immunospot

Journal: Nature Communications

Article Title: Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

doi: 10.1038/s41467-021-21572-y

Figure Lengend Snippet: A 54-year-old male patient who received αPD-1 (Nivolumab) plus αCTLA-4 (Ipilimumab) dual therapy for metastatic melanoma presented with late-onset hepatitis. a Course of treatment. b Change in hepatitis-related parameters over time and their association with introduction, withdrawal and re-introduction of valganciclovir treatment. c The patient presented at the end of week 40 with recrudescent hepatitis and was treated with 900 mg/day valganciclovir for 2 days prior to liver biopsy. Histopathological image of the liver biopsy (H&E staining; scale bar 500 µm). d Generally, the liver parenchyma appeared normal (H&E staining; scale bar 100 µm). e Only a few lymphocytes and sparse necrotic hepatocytes were observed in portal areas with small bile plugs (arrows) with no signs of hepatitis (H&E staining; scale bar 100 µm).

Article Snippet: Stage IV patients with unresectable metastatic disease who received first- or second-line checkpoint inhibitor therapy were initially treated with Nivolumab (αPD-1; Bristol-Myers Squibb) and Ipilimumab (αCTLA-4; Bristol-Myers Squibb) for four cycles, and thereafter with Nivolumab maintenance therapy (3 mg/kg at 3-week intervals).

Techniques: Staining

Journal: Nature Communications

Article Title: Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

doi: 10.1038/s41467-021-21572-y

Figure Lengend Snippet: A 49-year-old male patient who received αPD-1 (Nivolumab) plus αCTLA-4 (Ipilimumab) dual therapy for metastatic melanoma presented with late-onset hepatitis. a Course of treatment. b Change in hepatitis-related parameters over time and their association with introduction of valganciclovir treatment. c Histopathological image of a liver biopsy taken at the start of week 72 showing signs consistent with drug toxicity, autoimmunity or viral infection (H&E staining; scale bar 500 µm). d Extensive centrilobular necrosis (arrows) involving 30–40% of hepatocytes was observed (H&E staining; scale bar 100 µm). e Dense inflammatory infiltration of lymphocytes, eosinophils and neutrophils was seen in portal areas (H&E staining; scale bar 100 µm).

Article Snippet: Stage IV patients with unresectable metastatic disease who received first- or second-line checkpoint inhibitor therapy were initially treated with Nivolumab (αPD-1; Bristol-Myers Squibb) and Ipilimumab (αCTLA-4; Bristol-Myers Squibb) for four cycles, and thereafter with Nivolumab maintenance therapy (3 mg/kg at 3-week intervals).

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

doi: 10.1371/journal.ppat.1000313

Figure Lengend Snippet: Flow cytometry plots showing HCV 1073-specific CD8 T cell phenotype directly ex vivo and antigen-specific functions following 7 days of antigenic stimulation in the presence of isotype or blocking antibodies, using liver-derived (A) and blood-derived (B) lymphocytes from chronic patient C57. (Top panels): frequency of HCV 1073-specific CD8 T cells determined by cognate HLA-A2 tetramer staining. (Middle panels) far left: PD-1 and CTLA-4 expression ex vivo in gated tetramer + CD8 T cells (dot plots). Remaining right panels: HCV-specific IFN-γ production and CD107a mobilization in gated tetramer + CD8 T cells on day 7. (Bottom panels): Perforin expression in tetramer + (blue line) and total CD8 T cells (gray shaded) on day 7. (C) Fold increase in the expansion and effector functions of liver-derived (left) and blood-derived (right) HCV-specific CD8 T cells by αPD-L1 alone (white bar), αCTLA-4 alone (gray bar) and combined αPD-L1/αCTLA-4 blockade (black bar) relative to the isotype control for 3 chronic patients. The frequencies of functional tetramer + CD8 T cells in each culture were calculated by multiplying %tetramer + CD8 T cells with %IFN-γ + /tetramer + CD8 T cells, %perforin + /tetramer + CD8 T cells or %CD107a + /tetramer + CD8 T cells. (D) Flow cytometry plots showing CMV-specific CD8 T cells directly ex vivo and their antigen-specific functions following 7 days in vitro cultures from chronic patient C99.

Article Snippet: Of note, PD-1 and CTLA-4 expression in all subjects was examined using FITC-labeled αPD-1 (clone M1H4, BD) and PE-labeled αCTLA-4 (αCD152; clone BNI3, BD).

Techniques: Flow Cytometry, Ex Vivo, Blocking Assay, Derivative Assay, Staining, Expressing, Functional Assay, In Vitro

Journal: bioRxiv

Article Title: Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

doi: 10.1101/2021.05.21.445190

Figure Lengend Snippet: (A) UMAP of scRNA-seq analysis of 22,635 cells isolated from KPF CAF-HIF2 WT tumors (10,703 cells; n = 3 mice) and KPF CAF-HIF2 KO tumors (11,932 cells; n = 3 mice). Cell types were identified through graph-based clustering followed by manual annotation using marker genes. (B) Percentage of myeloid cells in each tumor. (C) M2-polarized TAMs were identified within the myeloid cell population via expression of Arg1 and Mrc1. (D) Immunosuppressive TAMs were identified within the myeloid cell population via expression of Cd274 ( Pdl1 ) and Cd86 ( B7-2 ). (E) Violin plots showing findings on scRNA-seq analysis of Ctla4 in KPF CAF-HIF2 WT and KO tumors in all identified cell types. (F) Left: Representative IHC images of CAF-HIF2 WT and KO tumors stained for FoxP3 (n = 5-6/group); scale bars, 50 µm. Right: Quantification of FoxP3+ Tregs per field. (G) Schematic for administration of PT2399 + αCTLA4 in a syngeneic flank KPC model. i.p., intraperitoneal; o.g., oral gavage; b.i.d., bid in die (twice a day). (H) Tumor growth curve from (A) (n = 10/group). Veh, vehicle; P , by Mann–Whitney U test. (I) Schematic for administration of PT2399 + αCTLA4/αPD1 in a syngeneic orthotopic KPC model. (J) Kaplan-Meier curves showing percentage survival for (C) (n = 10/group); P , by log-rank test. All error bars represent mean ± SEM; P , by Student’s t test unless otherwise noted. See also Supplementary Figure 6 and Supplementary Table 2.

Article Snippet: After 2 weeks of recovery, murine αCTLA4 (clone 9D9, Merck) and murine αPD1 (muDX400, Merck) or isotype control were administered IP every 4 days at 20 μg/mouse, 200 μg/mouse, and 220 μg/mouse, respectively for 2 weeks.

Techniques: Isolation, Marker, Expressing, Staining, MANN-WHITNEY

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: A. 005 GSCs cultured for 24 hr with or without murine IFNγ (mIFNγ: 0 or 3 ng/ml), stained for PD-L1, and analyzed by flow cytometry. B. 005 GSCs infected with G47Δ-E or G47Δ-mIL12 at MOI=1 for 24 hr, stained for PD-L1, and analyzed by flow cytometry. C. Human primary (left) and recurrent (right) GSCs cultured for 24 hr, stained for PD-L1 (cyan), and analyzed by flow cytometry. Percent of PD-L1+ cells indicated in upper right. See also Figure S1.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Cell Culture, Staining, Flow Cytometry, Infection

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: (A–B) Δ-mIL12 treatment. (A–B) 005 GSCs (2 × 104) implanted on day 0, injected intratumorally (IT) with G47Δ-mIL12 (1 × 105 pfu; n=3) or PBS (n=4) on days 18 and 24, animals sacrificed on day 25, and brains collected. Brain tumor sections were stained as indicated. For PD-L1 brain sections, 005 GSCs (2 × 104) implanted on day 0, injected IT with G47Δ-mIL12 (5 × 105 pfu; n=3) or PBS (n=3) on days 17, animals sacrificed on day 24, and brain tumor sections stained with anti-PD-L1 antibody. Representative images are presented; scale bar=100 μm (A). The number of positive cells from 3–9 fields/tumor section (1 section/mouse, except 3 for CD3) were counted (B). Average number of positive cells from each individual mouse is identified by symbol and color. The mean ± SEM of all mice is presented. Data were assessed by Student’s t test; *p<0.05; **p<0.001. See also Figure S1.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Staining

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: (A–B) Mice implanted with 2 × 104 005 GSCs on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 12 (upward arrow) and isotype control IgG (10 mg/kg), anti-(α)PD-L1 antibody (A), or anti-(α)PD-1 antibody (B) injected IP on days 15, 18 and 21 (downward arrows). Values from a single experiment, with Mock (treated with PBS and IgG) and G47Δ-mIL12 groups the same in A and B. Median survival of Mock (33.5 days; n=6) was compared to anti-PD-1 (39 days; n=8, p=0.02), anti-PD-L1 (42 days; n=7, p=0.003), or G47Δ-mIL12 (39 days; n=8, p=0.01) by Log-rank analysis. Similarly, G47Δ-mIL12 was compared to the combination of G47Δ-mIL12 with anti-PD-1 (49 days; n=7, p=0.02) or -PD-L1 (50 days; n=7, p=0.03), and antibodies were compared to the combination of G47Δ-mIL12 with anti-PD-1 (p=0.053) or anti-PD-L1 (p=0.08). Experiment was conducted once (A) or twice (B). C. Mice implanted with 2 × 104 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7). Median survival of Mock (37.5 days) was compared to anti-CTLA-4 (45 days; p=0.002) or G47Δ-mIL12 (40 days; p=0.03) alone by Log-rank analysis. Similarly, combination of G47Δ-mIL12 with anti-CTLA-4 (58 days) was compared to anti-CTLA-4 (p=0.05) or G47Δ-mIL12 (p=0.008). Experiment was conducted 2 times. D. Mice implanted with 2 × 104 005 GSCs on day 0, treated with PBS (right; n=2), rat anti-(α)PD-1 antibody (middle; 200 μg/mouse; n=2), or rat anti-(α)PD-L1 antibody (left; 200 μg/mouse; n=2) injected IP on day 25, and sacrificed 3 hr later. Antibodies were detected with HRP-conjugated anti-(α)rat Ig (brown; right) or control HRP-conjugated anti-(α)rabbit Ig (left). * normal brain adjacent to tumor. Scale bar=100 μm. E. 005 GSCs (1.5 × 105) implanted on day 0, treated with PBS or G47Δ-mIL12 (5 × 105 pfu) IT on day 11 and IgG or anti-CTLA-4 antibody (5 mg/kg) injected IP on days 11, 14, and 17 (n=4/group; Mock=PBS/IgG), and mice sacrificed on day 18. Tumors were harvested, cells stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor FACS performed. Scatter plot (each animal 1 point) of the percentages of live sorted positive cells. The ratio of Teff to Treg ratio is presented in a bar graph. Mean ± SEM. Data were assessed by Student’s t test between indicated groups; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Figure S2 and S3.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Control, Staining

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: A. Mice implanted with 2 × 104 005 GSCs on day 0 and treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 8 (upward arrow) and anti-CTLA-4 (5 mg/kg) and anti-PD-1 (10 mg/kg) or isotype control IgG (5 mg/kg hamster and 10 mg/kg rat IgG) IP on days 8, 11 and 14 (n=9/group; downward arrows). Median survival of Mock (PBS and IgG; 40 days) was compared to the combination of anti-PD-1 and anti-CTLA-4 (55 days; p=0.0002) by Log-rank analysis. Similarly, triple combination treatment with virus (89% of mice surviving long-term) was compared to the combination of anti-PD-1 and anti-CTLA-4 (p<0.0001) or Mock (p<0.0001). Experiment was conducted 3 times. B. Cured mice (n=8) from the triple combination experiment in (A) were re-challenged on day 183 with 5-fold increased number of 005 GSCs (1 × 105) in the contralateral hemisphere (re-challenged). Age matched (8 months) naive mice were implanted as controls (n=5). All re-challenged mice were alive at day 96 post-challenge without tumor; controls had a median survival of 27 days post-challenge (p=0.0001; Log-rank analysis). C. CT-2A cells (1 × 104) implanted in C57Bl/6 mice on day 0, injected IT with G47Δ-mIL12 (1 × 105 pfu) or PBS on day 10 and anti-CTLA-4 antibody and anti-PD-1 antibody or isotype control IgG (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 10, 13 and 16. Median survival of Mock (PBS/IgG) treated mice (20 days; n=9) was compared to G47Δ-mIL12 (21 days; n=8) by Log-rank analysis (p=0.007). Median survival of mice treated with triple combination (66.5 days; 50% of mice surviving long-term) was compared to anti-PD-1 and -CTLA-4 (19 days; n=8, p=0.005) or G47Δ-mIL12 (p=0.01). D. Cured mice (n=4) from the triple combination experiment in (C) were re-challenged on day 109 with 5-fold increased number of CT-2A cells (5 × 104). Similar age naive mice (~6 months) were implanted as controls (n=5). All re-challenged mice were alive at day 46 post-challenge without tumor, and compared to control by Log-rank analysis (p=0.005). See also Figure S4.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Control, Virus

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: (A–C) Mice implanted with 005 GSCs (1.5 × 105) on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 11, and anti-PD-1 and anti-CTLA-4 antibody or rat and hamster IgGs (Mock) (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 11, 14, and 17 (n=4), and sacrificed on day 18. Tumors harvested, dissociated cells stained with fluorochrome-conjugated anti-mouse antibodies, and multicolor FACS was performed. Percentages of live CD3+ cells, CD3+ sorted CD4+ and CD8+ subsets, live CD11b+ (monocytes, macrophages, NK, DC), and live CD45− GFP+ 005 cells from 2 independent experiments (red and black symbols) for M, CPi, V, and CPi+V groups (symbols are individual mice) were analyzed (A). The tumors from the experiment with red symbols in (A) were analyzed for CD3+FoxP3+ subtypes and ratio of CD8+ to CD4+FoxP3+ (B). The tumors from the experiment with black symbols in (A) were analyzed for immune checkpoint expression (PD-L1, CTLA-4, PD-1) on CD45+CD3+CD4+ and CD8+ subsets, CD45−GFP+, and CD11b+CD45hi and lo cells (C). M, Mock (PBS/IgG); V, virus (G47Δ-mIL12); CPi, checkpoint inhibitors (anti-PD-1+anti-CTLA-4); CPi+V, virus + checkpoint inhibitors (G47Δ-mIL12+anti-PD-1+anti-CTLA-4). Data are mean ± SEM and assessed by Student’s t test between indicated groups; *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Staining, Expressing, Virus

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: (A–D) Mice implanted with 005 GSCs (2 × 104) on day 0, treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 17, and checkpoint inhibitors anti-PD-1 antibody and anti-CTLA-4 antibody or rat and hamster IgGs (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 17, 20, and 23 (n=4), sacrificed on day 24, and brain tumor sections stained as indicated (Mock; PBS/IgG). Representative images with positive cells stained brown are presented (A). PD-L1 staining was from a separate experiment, but same treatment as above (n=3 or 4). Scale bars=100 or 200 μm as indicated. Number of positive cells per field in (A) were counted (3–5 fields/section/mouse for CD68 and Ki67; 8–10 fields/section/mouse for pStat1 and iNOS; 4 fields/section and 2 sections/mouse for PD-L1), and individual mice in each group are identified by color (B). Representative images with positive cells stained red (pSTAT1 +, CD3+), blue (CD68+, Ki67+), and red/blue colocalized (examples indicated with arrows) are presented (C). Scale bar=100 μm. Brain sections were incubated sequentially with primary (pSTAT1 or CD3 rabbit antibody) and secondary antibodies (AP-conjugated anti-rabbit Ig), followed by red color development. The same sections were then incubated with primary (CD68 or Ki67; rabbit antibody) and secondary antibodies (AP-conjugated anti-rabbit Ig), followed by blue color development. Number of positive cells per field were counted (8–10 fields/section/mouse) and percent of double positive cells plotted (D) with individual mice identified by color. M, Mock (PBS/IgG); CPi, checkpoint inhibitors (anti-PD-1+anti-CTLA-4); CPi+V, checkpoint inhibitors + virus (G47Δ-mIL12+anti-PD-1+anti-CTLA-4). Data are mean ± SEM, assessed by Student’s t test between indicated groups; *p<0.05, ***p<0.001, ****p<0.0001. See also Figure S6.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Staining, Incubation, Virus

Journal: Cancer cell

Article Title: MACROPHAGE POLARIZATION CONTRIBUTES TO GLIOBLASTOMA ERADICATION BY COMBINATION IMMUNOVIROTHERAPY AND IMMUNE CHECKPOINT BLOCKADE

doi: 10.1016/j.ccell.2017.07.006

Figure Lengend Snippet: A. C57Bl/6 mice implanted with (2 × 104) 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgG (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 8, 11 and 14 (n=6/group; upward arrows). Depletion antibodies against CD4 or CD8 (10 mg/kg) or clodronate liposomes (Clod; first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 4, 7, 10, 13, 20, and 27 (downward arrows), or BLZ945 (BLZ; 200 mg/kg) was gavaged for two cycles from days 6–10 and days 12–16 in triple therapy mice. Median survival of mice was determined: Mock (PBS/IgG/liposome/20% captisol), 35.5 days or triple therapy +αCD4 antibody, 32.5 days; +BLZ, 41.5 days; +αCD8 antibody, 45 days; +Clod, 43 days; +PBS (IgG/liposome/20% captisol). +PBS was compared to +BLZ (p=0.004), +Clod (p=0.02), +αCD4 (p=0.0007), +αCD8 (p=0.02), or Mock (p=0.0006) by Log-rank analysis. Similarly, Mock was compared to +PBS (p=0.0006), +BLZ (p=0.06), +Clod (p=0.01), +αCD4 (p=0.4), or +αCD8 (p=0.0006). B. C57Bl/6 mice implanted with 005 GSCs (2 × 104) on day 0 and treated with G47Δ-mIL12 (5 × 105 pfu) or PBS injected IT on day 18 and anti-CTLA-4 and anti-PD-1 antibodies or isotype control IgGs (5 mg/kg hamster IgG and 10 mg/kg rat IgG) injected IP on days 18, 21 and 24 (n=2/group). Depletion antibodies (αCD4, αCD8; 10 mg/kg) or Clod (first injection 50 mg/kg followed by 25 mg/kg) were injected IP on days 14, 17, 20, and 23, or BLZ (200 mg/kg) gavaged from days 16–20 and 22–25. Twenty-four hr after the last immune checkpoint injection or 8 hr after the last BLZ gavage, animals were sacrificed on day 25 and brains collected. Brain tumor sections (2 sections/mouse, at least 200 μm apart from each other) were stained for CD4, CD8, CD68, and F4/80. Positive cells were counted (5 fields/section for CD4+ and CD8+, 8 fields/section for CD68+, and 6 fields/section for F4/80+) and presented as mean ± SEM. Data were assessed by Student’s t test between indicated groups *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Only significant differences between Mock or triple therapy and other treatments indicated. See also Figure S7.

Article Snippet: C. Mice implanted with 2 × 10 4 005 GSCs on day 0 and treated with G47Δ-mIL12 or PBS injected IT on day 8 and anti-(α)CTLA-4 antibody or isotype control IgG (5 mg/kg) injected IP on days 8, 11 and 14 (n=8/group, except for G47Δ-mIL12 n=7).

Techniques: Injection, Control, Liposomes, Staining