Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP induces immune microenvironment changes and synergizes with α-PD-L1 in CT26-FL3 tumor therapy. a CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of the PBS and OxP-treated groups on day 28, analyzed by flow cytometry ( n = 4). b Relative mRNA expressions of various cytokines in tumors of the OxP-treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). c Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP treatment using DAPI (blue), anti-CD3 (red), anti-CD11c (red), anti-CD274 (red), and anti-IL-10 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. d Treatment scheme and tumor growth curves of orthotopic CT26-FL3 tumors in PBS, α-PD-L1, OxP, and OxP + α-PD-L1 treated groups ( n = 5 mice per group). e Th17 cell ratios in the splenocytes of the mice after various treatments on day 28 ( n = 4). Significant differences were assessed in d using two-way ANOVA with multiple comparisons and in a , b , and e using t test. Results are presented as mean (SD). ns not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Combination of OxP and LPD-PD-L1 trap gene therapy on orthotopic CT26-FL3 tumor model. a OxP and PD-L1 trap combination treatment scheme. b Tumor growth curves of orthotopic CT26-FL3 tumors in PBS, LPD-pGFP, PD-L1 trap, OxP and OxP+PD-L1 trap gene treated groups ( n = 5 mice per group). c TSR% results on day 35. d Mice survival curves. e CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of mice after various treatments, analyzed by flow cytometry ( n = 4). f Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP+PD-L1 trap treatment using DAPI (blue) and anti-CD3 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. g Relative mRNA expressions of cytokines in tumors of the OxP+PD-L1 trap treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). h Th17 cell ratios in the splenocytes of mice after various treatments on day 28 ( n = 4). Significant differences were assessed in b using two-way ANOVA with multiple comparisons, in d using log rank test and in c , e , g , and h using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP and LPD-PD-L1 trap gene therapy on B16F10 and 4T1 tumor models. a Treatment scheme and tumor growth curves of B16F10 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). b Treatment scheme and tumor growth curves of 4T1 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). c CD4 + and CD8 + T cell ratios in the B16F10 tumors after various treatments, analyzed by flow cytometry ( n = 4). d CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the B16F10 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). e CD4 + and CD8 + T cell ratios in the 4T1 tumors after various treatments, analyzed by flow cytometry ( n = 4). f CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the 4T1 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). Significant differences were assessed in a and b using two-way ANOVA with multiple comparisons and in c , d , e , and f using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: IM + anti-VEGF prolongs survival of GBM mice and is immunostimulatory (A) Schematic of the long-term therapeutic trials in the lentiviral-induced mouse model of glioma. (B) Representative images of H&E-stained tissue sections from a tumor that developed in an end-stage LVRshp53 animal. Scale bar, 30 μm. Representative of whole-slide images of three tumors. (C) Survival of tumor-bearing LVRshp53 animals subjected to the indicated treatments. Control (Ctrl) (n = 10), anti-VEGF (n = 7), IM + anti-VEGF (n = 7), IM + anti-VEGF + CDL (n = 8). (D) Normalized bioluminescence in LVRshp53 animals treated as indicated for 2 weeks. (E) Survival of PDG animals subjected to the indicated treatments. Ctrl (n = 9), IM + anti-VEGF (n = 10). (F) Representative images of CD8 (green) and DAPI nuclear staining (blue). Scale bar, 50 μm. Image is illustrative of the analysis shown in (H). (G) High-magnification images of CD8 T cells in a Ctrl versus an IM + anti-VEGF-treated tumor. Scale bar, 50 mm (H) Quantification of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Each dot indicates the average of 8–12 immuno-stained tumor tissue sections from one mouse. (I) Flow cytometry analysis of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Cells were gated as CD45 + CD3 + CD8 + . Ctrl (n = 15), anti-VEGF (n = 8), IM (n = 10), IM + anti-VEGF (n = 10). (J and K) Representative images (J) and quantification (K) of CD4 T cells in whole LVRshp53 tumor tissue section. Animals were treated for 12 days. Scale bar, 50 μm. Ctrl (n = 6), anti-VEGF (n = 4), IM (n = 4), IM + anti-VEGF (n = 6). (L) Assessment of the functional contributions of CD8 and CD4 T cells to survival benefit. Ctrl (n = 6), αCD8 + αCD4 (n = 4), IM + anti-VEGF (n = 5), IM + anti-VEGF + αCD8 + αCD4 (n = 5), IM + anti-VEGF + αCD8 (n = 5). (M) Normalized bioluminescence in LVRshp53 mice treated as indicated in (L). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. For survival analyses, Mantel-Cox test was performed. Other analyses by Mann-Whitney or one-way ANOVA tests.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Staining, Control, Flow Cytometry, Functional Assay, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: CD8 and CD4 T cells are activated upon IM + anti-VEGF treatment (A) Flow cytometry analysis of effector T cells (CD62L-CD44 + ). Ctrl (n = 6), IM (n = 4), anti-VEGF (n = 7), and IM + anti-VEGF (n = 7). (B) FACS analysis of IFNγ intracellular staining in fixed and permeabilized CD8+T cells. Ctrl (n = 16), IM (n = 10), anti-VEGF (n = 12), IM + anti-VEGF (n = 12). (C and D) Flow cytometry analysis of GzB (C) and TNFα (D) intracellular staining in CD8 T cells. Ctrl (n = 15), IM (n = 10), anti-VEGF (n = 8), IM + anti-VEGF (n = 8). (E) Functional importance of IFNγ for the survival of LVRshp53 animals subjected to the indicated treatments. Ctrl (n = 9), anti-IFNγ (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + anti-IFNγ (n = 6). Statistical analysis by Mantel-Cox test. (F, G, and H) Flow cytometry analysis of Ki67 (F), pSTAT5 (G), and TCF1 (H) in CD8 T cells. Ctrl (n = 7), IM (n = 4), anti-VEGF (n = 9), and IM + anti-VEGF (n = 10). (I) Representative image of HIF-1α (red) and CD8 (green) in the anti-VEGF-treated tumor. Image is illustrative of the analysis performed in (J). Scale bar, 50 mm (J) Quantification of the proximity of CD8 T cells to hypoxic regions in the entire area of full sections of GBM tumors. The zones were divided into 0 μm (i.e., within the HIF-1α+ zone), >0 and <5 μm, and >5 μm separating T cells and HIF-1α+ regions. Ctrl (n = 11), IM (n = 4), anti-VEGF (n = 4), IM + anti-VEGF (n = 5). (K) Flow cytometry analysis of intracellular HIF-1α expression in fixed and permeabilized CD8 T cells. Ctrl (n = 5), IM (n = 4), anti-VEGF (n = 8), anti-VEGFR2 (n = 5). (L) Flow cytometry analysis of intracellular FOXP3 expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 8). (M) Flow cytometry analysis of intracellular TGFβ expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 7). (N and O) Flow cytometry analysis of SLAMF7 (N) and GzB (O) expression in CD4 T cells. Ctrl (n = 8), IM (n = 5), anti-VEGF (n = 5), IM + anti-VEGF (n = 7). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by one-way ANOVA, unless otherwise indicated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Flow Cytometry, Staining, Functional Assay, Expressing

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Imipramine downregulates an M2-like program in TAMs (A) Western blot analysis of ARG1 and IL-10 in single tumors treated or not with IM or anti-VEGF. (B) Flow cytometry analysis of ARG1 and IL-10 expression in GBM tumors treated for 1 week. Ctrl (n = 9 tumors), IM (n = 6), anti-VEGF (n = 6), anti-VEGFR2 (n = 6), Axitinib (n = 6). Macrophages were gated as CD45 + CD11b+Ly6C-Ly6G−. (C) Expression of ARG1 and IL-10 in microglia (CD49d−) and MDMs (CD49d+) assessed by FACS in untreated tumors (n = 5). (D) Expression of MHC-II within microglia as assessed by flow cytometry. Ctrl (n = 4 tumors) and IM + anti-VEGF (n = 4). (E) Ex vivo co-cultures of tumoral CD11b cells and activated splenic CFSE-labeled CD8 or CD4 T cells. Each dot represents the average of two or three technical replicates. T cells alone (n = 4), Ctrl co-culture (n = 5), anti-VEGF (n = 3), IM (n = 4), IM + anti-VEGF (n = 4). (F) Analysis of the M2-like program in cytokine-polarized macrophages as assessed by qRT-PCR analysis of Ctrl and IM-treated M2-like BMDMs. Expression is normalized to 18S statistics by Welch’s t test. Each dot represents an individual sample. Data are representative of three independent experiments. (G) Analysis of the M1-like program in BMDMs assessed by FACS. Each dot represents an individual replicate. (H) Expression of Hrh1 mRNA normalized to 18S in ex vivo M1-and M2-polarized BMDMs, either untreated or IM treated for 24 h. Each dot represents an individual sample. Data are representative of three independent experiments. (I) mRNA expression of Hrh1 in FACS-sorted microglia or MDMs from Ctrl and IM-treated tumors. (J) mRNA expression of Arg1 , Chil3 , and Il10 in M2-polarized macrophages that were transfected with si Ctrl or two different si Hrh1 constructs. Cells were treated with 40 μm IM for 24 h. Data are representative of two independent experiments. (K) Western blot analysis of MRC1 and ARG1 expression of siRNA-transfected M2 BMDMs. Data are representative of two independent experiments. (L) CD8 and CD4 T cell proliferation during co-culture with tumoral CD11b cells isolated from untreated (n = 2) or TFP-treated tumors (n = 4). (M) mRNA expression of Hrh1 , Arg1 , and MMP2 in CD11b cells isolated from tumors treated with IM (n = 4), TFP (n = 4), or untreated Ctrl (n = 5). (N) Phagocytosis assay involving sorted microglia and MDMs from untreated or IM-treated tumors assayed with green pHrodo S. aureus bioparticles. Data presented as mean fluorescence intensity (MFI) of pHrodo/live cells. (Para break) Data in all quantitative panels are presented as mean ± SEM ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Western Blot, Flow Cytometry, Expressing, Ex Vivo, Labeling, Co-Culture Assay, Quantitative RT-PCR, Transfection, Construct, Isolation, Phagocytosis Assay, Fluorescence, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Macrophage-derived CXCR3 ligands are required for the therapeutic benefit conveyed by the combinatorial regimen of IM + anti-VEGF (A) Cxcl10 and Cxcl9 expression in bulk tumors. mRNA expression is shown relative to Gapdh . Ctrl (n = 6), anti-VEGF (n = 9), IM (n = 6), IM + anti-VEGF (n = 8). (B) Representative image of CXCL10 (magenta), F4/80 (red), and DAPI (blue) staining of LVRshp53 tumors treated with IM + anti-VEGF. Scale bar, 50 μm. Images are illustrative of five to six fields in tissue sections from three different tumors. (C) CXCL9 expression in TAMs in untreated (n = 6) or tumors treated with anti-VEGF (n = 4), IM (n = 4), or IM + anti-VEGF (n = 8) revealed by flow cytometry. (D) CXCL9 expression in MDMs and microglia, evaluated as in (C). (E) mRNA Cxcl9 and Cxcl10 expression assessed in bulk tumors treated with IM + anti-VEGF (n = 10) ± αCD49d (n = 7) to selectively deplete MDMs but not microglia. Expression is normalized to Gapdh housekeeping gene. (F) Assessing the contribution of CXCR3 function to the survival of LVRshp53 animals subjected to the indicated treatments. Treatment cohorts: αCXCR3 (n = 6), IM + anti-VEGF + αCXCR3 (n = 6), IM + anti-VEGF (n = 5). (G) Representative images of CD8 T cells aimed to assess the effects of αCXCR3. Representative of whole-slide image analysis of three tumors per treatment. Scale bar, 50 mm (H) Flow cytometry analysis of CD8 T cells in tumors subjected to indicated treatments. (I) Ex vivo co-culture of tumor-derived CD11b cells and CFSE-labeled CD8 or CD4 T cells. Myeloid cells were isolated from tumors treated with IM + anti-VEGF (n = 4), IM + anti-VEGF + αCXCR3 (n = 4), or untreated Ctrl (n = 5). Each dot represents an average of two or three technical replicates. (J) Minimal effect on IFNγ secretion by CD8 T cells in tumors treated with αCXCR3, IM + anti-VEGF, or the triple combination. (K and L) No effect of αCXCR3 on (K) GzB or (L) TNFα secretion by CD8 T cells co-treated with IM + anti-VEGF. (M) Quantification of immunostaining for HEVs in tumors treated with IM + anti-VEGF (n = 8) or IM + anti-VEGF + αCXCR3 (n = 4). The data are shown as number of HEVs per square millimeter of tumor tissue. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, Ex Vivo, Co-Culture Assay, Labeling, Isolation, Immunostaining, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: PD-L1 is induced in relapsing tumors and its blockade potentiates T cell function to prolong survival benefit in GBM mice (A) FACS analysis of PD-L1 in the live cell compartment of tumors treated as indicated. Ctrl (n = 4 tumors), responding to IM + anti-VEGF (n = 4), and relapsing from IM + anti-VEGF (n = 8). Responding tumors were collected after 12 days of treatment. Relapsing tumors were collected when mice became symptomatic or when tumors started to re-grow following a stable phase. (B) Representative image of immunostaining to reveal PD-L1 (red), CD45 (green), and DAPI nuclei (blue) in relapsing tumors after IM + anti-VEGF. Scale bar, 50 μm. Assessed in four relapsing tumors, n = 8–10 fields imaged per tumor. (C) Percentage of PD-L1-positive live cells comparing the CD45 − and CD45 + compartments of n = 4 relapsing GBM tumors as revealed by flow cytometry. (D) PD-L1 expression in the CD11b− and CD11b+ compartments of CD45 + cells in n = 8 tumors assessed by flow cytometry. (E) Percentage of PD-L1-positive TAMs assessed by FACS. Ctrl (n = 4), responding tumor (n = 4), relapsing tumor (n = 8). (F) Representative immunostaining to reveal PD-L1 expression in TAMs. CD206 (magenta), PD-L1 (red), and DAPI in n = 3 relapsing tumors, 8–10 images per tumor. Scale bar, 50 μm. (G) Expression of PD-L1 in MDMs and microglia of n = 8 relapsing tumors assessed by flow cytometry. (H) MHC-II expression in microglia comparing responding (n = 4) and non-responding tumors (n = 4), assessed by flow cytometry. (I and J) Representative images (I) and quantification (J) of CD8 T cells in untreated (n = 3), responding (n = 4), and relapsing (n = 3) tumors under IM + anti-VEGF treatment. CD8 (magenta) and DAPI-stained nuclei. Scale bar, 50 μm. Each dot indicates the total number of CD8 T cells in an entire tissue section from a tumor. (K) Abundance of CD8 T cells from tumors treated short term with IM + anti-VEGF (n = 5) or IM + anti-VEGF + αPD-L1 (n = 5), assessed by flow cytometry. (L–N) GzB (L), IFNγ (M), and TNFα (N) expression in CD8 T cells from tumors treated as in (K). (O) Assessment of the benefits of early versus late incorporation of anti-PD-L1. Ctrl (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + late anti-PD-L1 (n = 4), IM + anti-VEGF + early anti-PD-L1 (n = 7), anti-VEGF + anti-PD-L1 (n = 6). (P) The combination of a TCA (e.g., imipramine) and VEGF/VEGFR inhibitors induces autophagy in cancer cells and remodels the tumor vasculature, conveying survival benefit for mice bearing GBM. Imipramine reprograms M2-like TAMs to more pro-inflammatory phenotype, via inhibition of histamine receptor signaling. Consequent to the dual treatment, CD8 and CD4 T cells are recruited and activated to evoke their cytotoxic effects. The inclusion of anti-PD-L1 in the therapeutic regimen helps sustain the immune response and increases survival benefit. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Cell Function Assay, Immunostaining, Flow Cytometry, Expressing, Staining, Inhibition, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet:

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Purification, Plasmid Preparation, Virus, Recombinant, Activation Assay, Staining, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Fluorescence, cAMP Assay, Competitive ELISA, Selection, Cell Isolation, Derivative Assay, Negative Control, shRNA, Software

Journal: Melanoma Research

Article Title: Potential clinical and immunotherapeutic utility of talimogene laherparepvec for patients with melanoma after disease progression on immune checkpoint inhibitors and BRAF inhibitors

doi: 10.1097/CMR.0000000000000444

Figure Lengend Snippet: CD4 + and CD8 + T cell infiltration of in-transit melanoma metastasis after 1 year of intralesional administration of talimogene laherparepvec. A solitary in-transit melanoma metastasis was biopsied and analyzed by IHC for the presence of HMB45 + melanoma cells, CD4 + T cells, and CD8 + T cells. Infiltration of CD4 + T cells and CD8 + T cells was noted in the peripheral aspects of the mass, which was largely devoid of melanoma cells determined by HMB45 staining.

Article Snippet: Antibodies (Cell Marque Corporation, Rocklin, California, USA) used included mouse anti-HMB45 antibody (clone HMB-45, 282M-96; Cell Marque Corporation), mouse anti-CD4 antibody (4B12, PA0427; Cell Marque Corporation), and mouse anti-CD8 (4B11, PA0183; Cell Marque Corporation).

Techniques: Staining

Journal: Nature Communications

Article Title: Vaccine-induced protection against SARS-CoV-2 requires IFN-γ-driven cellular immune response

doi: 10.1038/s41467-023-39096-y

Figure Lengend Snippet: a Schematic diagram of vaccination, CD4 + or CD8 + T cell depletion, viral challenge, and pathological studies. b The viral loads in the lung and NT of BNT162b2 vaccinated and unvaccinated C57BL/6 J μMT mice at 2 d.p.i. with CD4 + , CD8 + , or both T cell depletion ( n = 10). (aCD4, anti-CD4 monoclonal antibody; aCD8, anti-CD8 monoclonal antibody). c Representative images of the H&E-stained lung tissues of BNT162b2 vaccinated μMT mice treated with aCD4, aCD8, both depleting antibodies or PBS. Scale bar = 200μm. d Schematic diagram of flow cytometry analysis of CD4 + and CD8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection. e Percentage of CXCR6 + and CD69 + CD4/8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection ( n = 5). f Percentage of CD44 + IFN-γ + CD4/8 + and CD44 + Granzyme B + CD8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection ( n = 5). g Representative dot plots showing IFN-γ- and Granzyme B-producing CD44 + CD4/8 + T cells in lung tissues of vaccinated and unvaccinated C57BL/6 J μMT mice after Alpha infection. Data are presented as mean ± SD. Statistical significance was calculated using one-way ANOVA test or unpaired two-tailed Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant). Figure 7a, d were created with BioRender.com.

Article Snippet: Indicated mice were injected intraperitoneally with PBS or 250 μg in 200 μL of diluted in PBS of either anti-mouse CD4 (BioXcell InVivoMab Clone GK 1.5), anti-mouse CD8 (BioXcell InVivoMab Clone 2.43), or both at indicated time points.

Techniques: Staining, Flow Cytometry, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas

doi: 10.1038/s41467-024-44932-w

Figure Lengend Snippet: a Flow cytometry analysis of CT2A CRISPR control, Atrx KO-A or KO-B tumor-bearing hemispheres harvested 14 days post-intracranial implant showing percent live cell density of CD3 + , CD4+ and CD8 + T-cells and macrophages (CD45 + , CD3/19neg, NK1.1neg, CD11bhi, CD45hi). Gating strategy is provided in Supplementary Fig. . n = 10 for CRISPR control – CD3 + , CD4 + , CD8+ markers; n = 9 for CRISPR control - macrophage markers; n = 7 for Atrx KO-A - all markers; n = 5 for Atrx KO-B – all markers; n = 9 for normal brain - CD3 + , CD4 + , CD8+ markers; n = 8 for normal brain – macrophage markers. Data are presented as mean ± SEM. Asterisks denote significant one-way ANOVA with Dunnett’s post-hoc test comparing CRISPR control with Atrx KO tumor-bearing hemispheres (* p < 0.05, ** p < 0.01). CD3 + : Control vs Atrx KO-A – p = 0.0296; CD4 + : Control vs Atrx KO-A – p = 0.0117; Macrophages: Control vs Atrx KO-A – p = 0.0167, Control vs Atrx KO-B – p = 0.0086. b Representative IHC images for CD45 (A, B, C) and F4/80 (D, E, F) expression (brown) in CT2A CRISPR Control ( Atrx WT ) (A, D), Atrx KO-A (B, E) and KO-B (C, F) tumors, with hematoxylin counter-staining (blue). Scale bar: 50μm. Number of brains subjected to IHC per group: CRISPR Control – n = 3; Atrx KO-A – n = 2; Atrx KO-B – n = 3. c Representative IHC images for CD45 (A, B) and CD3 (C, D) expression (brown) in RCAS/ Ntv-a control (A, C) and Atrx -KO (B, D), with hematoxylin counter-staining. Scale bar: 50μm. Number of brains subjected to IHC per group: RCAS/ Ntv-a control – n = 6; Atrx KO – n = 4. d , e Kaplan–Meir survival curves for C57BL/6 mice bearing intracranial CT2A CRISPR ctrl (Control) or Atrx KO-B tumors treated with 250 μg of isotype control IgG, anti-CD4 antibody ( d ) or anti-CD8 antibody ( e ) as per schema in Supplementary Fig. . N Number of mice per group; MS median survival in days. P -values represent group comparisons calculated using log-rank test. Source data are provided as a Source Data file.

Article Snippet: For CD4+ and CD8 + T-cell depletion experiments, C57BL/6 mice ( n = 10 mice per group) received 250 μg per dose of rat IgG2B isotype control (BioXcell, Cat. no. BE0090), anti-mouse CD4 antibody (BioXcell, Cat. no. BE00003) or anti-mouse CD8 antibody (BioXcell, Cat no. BE0061) intraperitoneally 4 days prior to implant.

Techniques: Flow Cytometry, CRISPR, Control, Expressing, Staining