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