Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Synthesis of anti-CTLA4-IR700 and cell-killing specificity of CTLA4-targeted NIR-PIT. A) Evaluation of anti-CTLA4-IR700 by SDS-PAGE (left: Colloidal Blue staining, right: 700 nm fluorescence). Unconjugated anti-CTLA4 antibody was used as a control. B) Evaluation of anti-CTLA4-IR700 by SEC. The APC demonstrated light absorption at 280 and 689 nm and corresponding fluorescence at 700nm. C) Flow-cytometric analysis of CTLA4 expression on cancer cell lines. Representative histograms are shown. D) PI-stained dead cell % after in vitro NIR-PIT on cancer cell lines (n = 4; unpaired t test; ns, not significant). E) Flow-cytometric analysis of CTLA4 expression of splenocytes from non-tumor bearing mouse. Total (surface and intracellular) CTLA4 was stained. F) Flow-cytometric analysis of CTLA4 expression in spleen T cells. Representative histograms and RFI are shown (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ***P < 0.001, ****P < 0.0001). RFI, Relative fluorescence intensity. G) Treg percentages of T cells after ex vivo NIR-PIT for splenocytes analyzed by flow-cytometry (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ****P < 0.0001).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: SDS Page, Staining, Fluorescence, Control, Expressing, In Vitro, Ex Vivo, Flow Cytometry

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: CTLA4 was expressed by Treg cells more than other T cells in tumor tissue. A) CTLA4 expression in T lymphocyte populations in MC38-luc, LL/2-luc, and MOC2-luc tumors were analyzed by flow cytometry. Representative histograms for surface or total (surface + intracellular) CTLA4 expression in CD8+ T cells (CD3+CD8+), CD4+Fopx3−T cells (CD3+CD4+Foxp3−), and CD4+Foxp3+ Tregs (CD3+CD4+Foxp3+) and relative fluorescence intensity (RFI) of CTLA4 are shown (n = 4; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ****P < 0.0001). B) CTLA4hi percentage was calculated based on the total expression. The CTLA4hi gates are shown in histograms in A (n = 4; one-way ANOVA followed by Tukey’s test; ****P < 0.0001).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Selective CTLA4hi cell depletion by CTLA4-targeted NIR-PIT. CTLA4hi cells within MC38-luc tumor, tumor draining lymph node, and spleen were assessed with flow cytometry 3 hours after the light exposure. CTLA4 was stained with anti-CTLA4 (clone UC10-4B9) after fixation and permeabilization. A) Representative dot plots to show CTLA4 expression in live cells, and CTLA4 and CD3 expressions among CD45+ live cells. B) The percentages of CTLA4hi cells among total live cells, CD45+CD3+ T cells and CD45+CD3− non-T hematopoietic cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Flow Cytometry, Staining, Expressing

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Efficacy of in vivo CTLA4-targeted NIR-PIT. CTLA4-targeted NIR-PIT was performed on MC38-luc, LL/2-luc and MOC2-luc mouse tumor models. A) Treatment schedule. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e., NIR-light was exposed only to the tumor. C) Fluorescent imaging before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 6) and after (day 8-11) NIR-PIT in MC38-luc tumor bearing mice. E) Luciferase activity calculated from BLI (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; vs. control group). F) Tumor volume curves (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, ****P < 0.0001). G) Survival curves (n = 10; log-rank test with Bonferroni correction; **P < 0.01, ***P < 0.001; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: In Vivo, Irradiation, Imaging, Luciferase, Activity Assay, Control

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Selective cell depletion immediately after the CTLA4-targeted NIR-PIT against MC38-luc tumors. T cell populations after the therapy were analyzed by flow cytometry. A) Treg population in tumors and spleens 3 hours after light exposure. The dot plots show the representative examples of CD4 and Foxp3 expressions in the CD3+ T cells. Scatter plots show the percentage of Tregs in CD3+ cells and ratios of non-regulatory/regulatory CD4+ T cells (CD4+Foxp3−/CD4+Foxp3+) (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***, P < 0.001; ns, not significant). B) CD8+ T cell populations in the tumors 3 hours after light exposure. The dot plots show the representative examples of CD8 and IFN-γ expressions in the CD3+ T cells. Scatter plots show the percentage of intra-tumoral CD8+ T cells among total cells and IFN-γ+ cells among CD8+ T cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Flow Cytometry

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: Immune cell response after the CTLA4-targeted NIR-PIT. A) Early phase activation shows increased percentages of CD69 positive intratumoral CD8+ T cells and NK cells by flow cytometry 1 day after therapy (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant). B) The expression of activation markers was analyzed in the regional lymph nodes 3 days after therapy. CD69+ and CD25+ percentages among CD8+ T cells were calculated (n = 5-6; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***P < 0.001; ns, not significant). C) Multiplex immunohistochemical staining of tumors 5 days after the therapy. Representative pictures are shown. Scatter plots show the CD8+ cell density within stroma or tumor and the CD8+/CD4+Foxp3+ cell ratio within tumor (n = 3; one-way ANOVA followed by Tukey’s test; *, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, not significant). White dashed line represents tumor border; Scale bar = 100 μm; pCK, pan cytokeratin.

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Activation Assay, Flow Cytometry, Expressing, Multiplex Assay, Immunohistochemical staining, Staining

Journal: Advanced therapeutics

Article Title: Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

doi: 10.1002/adtp.202000269

Figure Lengend Snippet: In vivo efficacy of CTLA4-targeted NIR-PIT against MC38-luc tumor in athymic nude mice. A) Treatment regimen. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e. only to the tumor. C) Fluorescent imaging demonstrates 700-nm fluorescence before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 5) and after (day 7-10) NIR-PIT. E) Luciferase activity calculated from BLI (n = 13; repeated measures two-way ANOVA; ns, not significant). F) Tumor volume curves (n = 13; repeated measures two-way ANOVA; ns, not significant). G) Survival curves (n = 13; log-rank test with Bonferroni correction; ns, not significant).

Article Snippet: Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: In Vivo, Irradiation, Imaging, Fluorescence, Luciferase, Activity Assay

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) Left: Total somatic mutation burden in ectopic Apobec3 overexpressing lines and parental control lines. Right: somatic mutation burden from parental lines and lines exposed to short-wave ultra violet radiation. (B) Survival and 10 day acute response to anti-PD1/anti-CTLA4 immune checkpoint therapy in mice bearing tumors from parental T11 cell line and KPB25L cell lines. (C) Survival and acute response in T11-Apobec and KPB25Luv lines. (D) Immune cell gene expression signature expression levels. (E) Immune checkpoint gene mRNA expression levels. (F) Left, interferon gamma signature expression levels. Right, serum interferon gamma as measured by ELISA. In boxplots, bars mark the average and standard deviation. The p-values mark are two-tailed from unmatched T-tests. In Kaplan-Meier plots, p-values are from Log-rank (Mantel-Cox) tests. Signature levels are calculated as median value of genes within and mRNA is the median centered Log2 expression level.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Mutagenesis, Control, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) Signature development pipeline. The immune activity signature is noted by the blue bar near the heatmap. The B cell/T cell co-cluster is marked by the purple bar and featured. (B) Boxplot for the B cell/T cell co-cluster in pretreatment samples from a human melanoma study of anti-CTLA4 therapy(Van Allen et al., 2015). (C) Boxplot of the B cell/T cell co-cluster in pretreatment samples from a human melanoma study of anti-PD1/ anti-CTLA4 therapy (Sade-Feldman et al., 2018). (D) Boxplot of the B cell/T cell co-cluster in pretreatment breast cancer samples from CALGB40601, trastuzumab arm (Tanioka et al., 2018). (E) Boxplot of the B cell/T cell co-cluster in pretreatment samples from the human breast cancer dataset GSE32646, P-FEC = neoadjuvant paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide(Miyake et al., 2012). (F) Boxplot of the B cell/T cell co-cluster in pretreatment samples from the human breast cancer iSPY clinical trial; A/C/T arm = Doxorubicin hydrochloride and cyclophosphamide, followed by treatment with paclitaxel(Esserman et al., 2012). (G) Boxplot of the B cell/T cell co-cluster in pretreatment samples from the TNBC NCT 01560663 clinical trial(Echavarria et al., 2018). Boxplots mark the mean and standard deviation. All panels except C, the p-values show two-tailed p-value from standard T-tests; in panel C the data is non-gaussian and thus a Mann-Whitney test was used.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Activity Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) RNA-seq signatures for sensitive tumors at 7 days (5mm= day 0/ treatment initiation) without or with anti-PD1/anti-CTLA4 therapy. (B) Flow cytometry results for CD8+ cells and CD4+ using memory markers (Cd44, Cd62L). (C) Flow cytometry of tumor infiltrating B cells with or without ICI therapy. On the right shows staining for B cells gated for activation markers. (D) Quantification of flow cytometry for activated B cells (B220+, Cd19+ or Cd20+, MHC II +, Cd80+ or Cd86+). (E) IHC staining for IgG-kappa chain in KPB25Luv tumors. (F) IgG binding assay showing serum-IgG binding (Fitc+) to KPB25Luv cells. (G) Quantification of Fitc+ cells in IgG binding assay for KP25Luv cells and off-target binding. (H) Quantification of Fitc+ IgG binding assay for T11-Apobec cells following reabsorption on off-target cells. In boxplots, bars signify the mean and standard deviation. The p-values are two-tailed from unmatched T-tests. All tumors collected after 7days of treatment or non-treatment.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: RNA Sequencing, Flow Cytometry, Staining, Activation Assay, Immunohistochemistry, Binding Assay, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) TSNE analysis of cells that passed quality checks in KPB25Luv tumors. Cells/clusters are color coded by the major cell type found.(B) The distribution of cell types between treated and non-treated tumor cells. (C) Heatmap of mRNA variance between treated and non-treated tumor cells. (D) Violin plot of Cd8a mRNA levels. (E) Heatmap of significant genes (plus Pdcd1, Ctla4) in clusters of ICI treated CD8+ T cells. (F) Classification of ICI treated CD8+ T cell clusters. Classes are coded to the heatmap in E. (G) Feature plot showing expression of key genes across CD8+ T cell clusters. (H) Violin plot of Cd4 mRNA levels. (I) Heatmap of significant genes(plus Ctla4) in clusters of ICI treated CD4+ T cells (n=20) (J) Classification of ICI treated CD4+ T cell clusters. Classes are coded to the heatmap in I. (K) Feature plot showing expression of key genes across CD4+ T cell clusters. (L) Violin plot of Cd20 mRNA levels.(M) Heatmap of significant genes in clusters of ICI treated B cells (n=20). (N) Classification of ICI treated B cell clusters. Classes are coded to the heatmap in M. (O) Feature plot showing expression of key markers in B cell clusters. (P) Results of 5’ TCR/BCR sequencing. In bar-plots, read counts for each clone is shown along with the calculated Shannon entropy (where higher values indicate high diversity/low clonality). Above each bar, the percent of all reads occupied by a clone(s). Heatmap values are depicted in the legend. Violin plots mark the mean and SEM. Markers were identified using Seurat and Wilcoxon rank sum testing.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Expressing, Sequencing

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) Survival for mice given anti-PD1/anti-CTLA4 therapy with anti-Cd4 or anti-Cd8 antibodies (B) 21 day acute response for mice given anti-PD1/anti-CTLA4 with anti-Cd4 or anti-Cd8 antibodies. (C) Survival for mice given anti-PD1/anti-CTLA4 therapy with anti-Cd19 or anti-Cd20 antibodies (D) 21 day acute response for mice given anti-PD1/anti-CTLA4 with anti-Cd19 or anti-Cd20 antibodies. (E) Flow cytometry results for T cell subsets after 7days of aPD1/aCTLA4 therapy with/without anti-CD19 based B cell inhibition. In Kaplan-Meier plots, p-value show results of Log-rank (Mantel-Cox) tests. Boxplots show the mean and standard deviation. The p-values are two-tailed from unmatched T tests.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Flow Cytometry, Inhibition, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: (A) Flow cytometry for B cells in KPB25Luv tumors after 7 days of ICI and CD4+ T cell depletion. (B) Quantification of results from A. (C) X-Y plot of IgG and CIBERSORT Tfh T cell signatures in mRNA-seq of sensitive tumors at day 7. (D) Boxplot of CIBERSORT Tfh T cell signature levels in sensitive tumors (mRNA-seq) at day 7. (E) X-Y plot of IgG signature and Il21 mRNA in mRNA-seq of sensitive tumors at day 7. (F) Boxplot of Il21 mRNA levels in sensitive tumors (mRNA-seq) at day 7. (G) Flow cytometry results for activated B cells in T11-Apobec & KPB25Luv tumors during Tfh/IL21 blockade. (H) IHC staining for IgG-kappa chain in KPB25Luv tumors during Tfh/IL21 blockade. (I) Survival for T11-Apobec bearing mice during ICI therapy and Tfh/IL21 blockade. (J) Survival for KPB25Luv bearing mice during ICI therapy and Tfh/IL21 blockade. (K) Western blot for serum IgG in Igmi and Balbc mice with T11-Apobec tumors. The blue bars mark Igmi mouse sera, purple note Balbc sera. (L) 21 day acute response in Igmi and Balbc control mice with T11-Apobec tumors. (M) Survival of Igmi mice withT11-Apobec tumors and treated with anti-PD1/anti-CTLA4 therapy in contrast to Balbc controls. (N) Survival in KPB25Luv tumor bearing mice treated with ICI therapy or ICI therapy with CD16/32 blockade . In Kaplan-Meier plots, p-values are from Log-rank (Mantel-Cox) tests. Boxplots show the mean and standard deviation. The p-values are two-tailed from standard T-tests. In X-Y plots, p-values were determined by linear regression analysis. The asterisks denote significance (***, p<0.0001, *, P<0.05).

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Flow Cytometry, Immunohistochemistry, Western Blot, Control, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Control, Virus, Recombinant, Staining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Microarray, Plasmid Preparation, Software, Membrane, Transfection

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: Gene expression patterns of tumor and immune cell features. The triangles mark the position of major tumor models in the heatmaps. Black bars mark tumor lines from each model. Blue bars to the side note models in the treatment study. Below this, blue bars show samples getting anti-PD1/anti-CTLA4 therapy. The heatmaps show median expression values for subtype and immune cell signatures. The lower heatmap is expression values of immune checkpoint mRNAs as shown by the color bar.

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Gene Expression, Expressing

Journal: Cell

Article Title: B cells and T follicular helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer.

doi: 10.1016/j.cell.2019.10.028

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse CTLA4 (clone 9D9) , Bioxcel , Cat: BE0164; RRID: AB_10949609.

Techniques: Control, Virus, Recombinant, Staining, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Microarray, Plasmid Preparation, Software, Membrane, Transfection

Journal: Molecular Therapy

Article Title: Oncolytic VSV Primes Differential Responses to Immuno-oncology Therapy

doi: 10.1016/j.ymthe.2017.05.006

Figure Lengend Snippet: α-CTLA-4 Antagonist mAb Combines with VSV-mIFNβ Treatment to Elicit Increased Therapeutic Responses to CT26 Tumors

Article Snippet: Anti-mouse CTLA-4 antibody (9D9) was cloned and reformatted into a mouse IgG1 isotype at MedImmune.

Techniques: