Journal: Science immunology

Article Title: The tumor-intrinsic role of the m 6 A reader YTHDF2 in regulating immune evasion

doi: 10.1126/sciimmunol.adl2171

Figure Lengend Snippet: ( A and B ) The tumor growth of WT and Ythdf2 -KO MC38 tumors ( n = 5) and B16-OVA tumors ( n = 6) in C57BL/6 mice. C57BL/6 mice were subcutaneously injected with either 1 × 10 6 WT and Ythdf2 -KO MC38 cells (A) or 1 × 10 6 WT and Ythdf2 -KO B16-OVA cells (B). Tumor sizes were measured every other day starting from the 12th or 14th day after tumor inoculation. ( C and D ) Overall survival of immunocompetent mice subcutaneously implanted with WT or Ythdf2 -KO B16-OVA tumor cells ( n = 14 for WT and n = 15 for KO) (C) or MC38 tumor cells ( n = 8 for WT and n = 10 for KO) (D). ( E and F ) WT or Ythdf2 -KO B16-OVA tumors were transplanted from Rag1 −/− to C57BL/6 mice ( n = 4). Tumor volume (E) and tumor weight (F) were measured on the 14th day after the tumor was transplanted. ( G ) A total of 1 × 10 6 WT or Ythdf2 -KO B16-OVA tumor cells were subcutaneously implanted in C57BL/6 mice pretreated with anti–immunoglobulin G (IgG), anti-NK1.1, anti-CD4, and anti-CD8 antibodies ( n = 5 or 6). Tumor growth was monitored by measuring tumor sizes every other day starting from the eighth day after tumor inoculation. ( H and I ) WT or Ythdf2 -KO MC38-OVA (H) and B16-OVA (I) tumor growth in Rag1 −/− mice that were adoptively transferred with OT1 CD8 + T cells. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). ( J ) WT or Ythdf2 -KO B16F10 tumor growth in Rag1 −/− mice that were adoptively transferred with gp100-stimulated pmel-1 splenocytes. The mice were subcutaneously injected with either 1 × 10 6 WT or Ythdf2 -KO tumor cells. Tumor sizes were measured every other day starting from the sixth day after tumor inoculation ( n = 5). Data are represented as means ± SD. Statistical analysis was performed using two-way ANOVA with a mixed-effects model with P values adjusted by a Holm-Šídák method for multiple comparisons (A, B, G, H, I, and J), unpaired two-tailed t test (E and F), or Kaplan-Meier survival analysis and log-rank test (C and D). The data presented represent one of two or three independent experiments. * P < 0.05, ** P < 0.01, and **** P < 0.001.

Article Snippet: Splenocytes isolated from pmel-1 T cell receptor transgenic mice were stimulated with gp100 25–33 (5 μg/ml; MedChemExpress) and mouse recombinant IL-2 (100 IU/ml) in RPMI 1640 complete medium at 37°C for 24 hours.

Techniques: Injection, Two Tailed Test

Journal: Japanese Journal of Radiology

Article Title: Targeted theranostic nanomedicine using targeted CT-imageable particles that release tebentafusp.

doi: 10.1007/s11604-025-01782-w

Figure Lengend Snippet: Graphical abstract. Two componenents of this theranostics. A : Imaging of malignancy using CT-detectable nanoparticles. B : Targeted immunotherapy using particles that release their contents upon irradiation, consisted of following two sub-components. B-1: induction of HLA-A*02–01-GP100 and B-2:formation of immunological synapse and releasing of perforin/granzymes from CD3 + T-cells

Article Snippet: After heat-mediated antigen retrieval with citrate buffer (pH 6), the samples were incubated with the primary antibody for GP-100 (PMEL/Melanoma gp100 (HMB45) Mouse mAb #38,815, Cell Signaling Technology, Danvers, MA, USA) at a 1/250 dilution, or anti-CD3 + antibody (Anti-Mouse CD3 antibody MAB4841-100, R&D Systems Inc., Minneapolis, MN, USA) at a 1:6400 dilution for 30 min at room temperature.

Techniques: Imaging, Irradiation

Journal: International journal of cancer

Article Title: Antitumor immune response of dendritic cells (DCs) expressing tumor-associated antigens derived from induced pluripotent stem cells: in comparison to bone marrow-derived DCs.

doi: 10.1002/ijc.28367

Figure Lengend Snippet: Figure 3. Expression of hgp100 in the genetically modified DCs. The intracellular expression of hgp100 in the genetically modified BMDCs and iPSDCs. DCs were analyzed using intracellular staining flow cytometry. The staining patterns of hgp100 (black) and FITC- matched controls (thin lines) are shown in histograms.

Article Snippet: Intracellular staining with anti-melanoma (hgp100) polyclonal antibodies (Acris Antibodies, Herford, Germany) and FITC-conjugated anti-goat IgG polyclonal antibodies (Vector Laboratories) was performed using a Fixation and Permeabilization Solution Kit (BD Biosciences, San Jose, CA).

Techniques: Expressing, Staining, Cytometry

Journal: International journal of cancer

Article Title: Antitumor immune response of dendritic cells (DCs) expressing tumor-associated antigens derived from induced pluripotent stem cells: in comparison to bone marrow-derived DCs.

doi: 10.1002/ijc.28367

Figure Lengend Snippet: Figure 5. Therapeutic efficacy of genetically modified DCs in the subcutaneous tumor model. Tumor growth suppression in the mice immunized with genetically modified DCs in the subcutaneous tumor model (n 5 5 for each group). The genetically modified DCs were as follows: PBS (———), BMDCs-AxCALacz (—— ), BMDCs- AxCAhgp100 (), iPSDCs-AxCALacZ (– –) and iPSDCs- AxCAhgp100 (——). The results are presented as the mean tumor volume 6 SD of the mice that developed tumors in each group. *Significantly higher therapeutic efficacy than that observed in the other cells (Day 20, p < 0.0001). **No significant differences com- pared to the genetically modified DCs expressing hgp100 in terms of therapeutic efficacy (Day 20, p > 0.05).

Article Snippet: Intracellular staining with anti-melanoma (hgp100) polyclonal antibodies (Acris Antibodies, Herford, Germany) and FITC-conjugated anti-goat IgG polyclonal antibodies (Vector Laboratories) was performed using a Fixation and Permeabilization Solution Kit (BD Biosciences, San Jose, CA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD10 , REAL318 , 50 , 130-118-368 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: Cell Reports Medicine

Article Title: Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function

doi: 10.1016/j.xcrm.2022.100621

Figure Lengend Snippet:

Article Snippet: anti Melanoma Ag AF488 , Novus Biologicals , cat#NBP2-34681AF488.

Techniques: Recombinant, RNA HS Assay, Isolation, Sequencing, Software

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Palmitic acid-conjugated peptides induce enhanced T cell activation in vitro and in vivo (A) moDCs were pulsed with different concentrations of gp100 280–288/45–59 or C16:0 gp100 280–288/45–59 for 3 h and co-cultured o/n with gp100-specific CD8 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data are presented as mean ± SEM of six individual experiments, measured in triplicate. Statistical analyses were performed using a two-way ANOVA with a Sidak post hoc multiple comparison test. ∗∗∗p < 0.001. (B) OVA 257–264/323–339 - and C16:0 OVA 257–264/323–339 -loaded BMDCs were co-cultured with OT-I T cells for 3 days. Mean ± SEM measured in triplicate. Data are representative of three individual experiments. Statistical analyses were performed using a two-way ANOVA with a Sidak post hoc multiple comparison test. ∗∗p < 0.01, ∗∗∗p < 0.001. (C) moDCs were pulsed with different concentrations of gp100 280–288/45–59 or C16:0 gp100 280–288/45–59 for 3 h and co-cultured o/n with gp100-specific CD4 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data are presented as mean ± SEM measured in triplicate and representative of three individual experiments (D) gp100 280–288/45–59 and C16:0 gp100 280–288/45–59 (30 μM) were injected intradermally into human skin with (black line) or without (blue line) GM-CSF/IL-4. At the site of injection punch biopsies were taken and cultured for 2 days. The migrated skin APCs were collected and co-cultured o/n with gp100-specific CD8 + T cells. IFNɣ secretion was used as a measure for T cell activation. Data of two individual donors per condition are shown.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Activation Assay, In Vitro, In Vivo, Cell Culture, Comparison, Injection

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: The addition of a single palmitic acid affects the processing pathway of antigens moDCs were treated with different inhibitors or DMSO control (ctrl) 30 min prior to and during the 3 h antigen pulse. Thereafter, moDCs were washed and co-cultured o/n with gp100-specific CD8 + T cells. IFNγ secretion was determined by ELISA as a measure of T cell activation. (A) Illustration of interference in antigen processing by the different inhibitors. (B–F) Relative IFNγ secretion induced by co-culture of gp100-specific CD8 + T cells with gp100 280–288/45–59 - or C16:0 gp100 280–288/45–59 -pulsed moDCs that were pre-treated with (B) bortezomib, (C) cathepsin S inhibitor, (D) chloroquine, (E) primaquine, and (F) brefeldin A. Data are represented as relative to the short peptide control after subtraction of background (IFNγ levels of unpulsed moDCs). Symbols represent the mean of triplicates of seven donors. Statistical analyses were performed with a paired Student’s t test. ∗p < 0.05, ∗∗∗p < 0.001. (G) Overview of experimental setup for antigen presentation with DCs from Batf3 KO mice. (H) Proliferation of OT-I cells after co-culture with C16:0 or unmodified peptide in WT and Batf3 KO BMDCs. Statistical analysis was performed with a Student’s t test. ∗∗p < 0.01.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Co-Culture Assay, Immunopeptidomics

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Palmitoylation of peptides is a membrane-targeting moiety that allows for continuous loading and processing (A) HA signal at different time points in fixed and permeabilized moDCs that were pulsed with 10 μM gp100 280–288/45–59 (black circles) or C16:0 gp100 280–288/45–59 (black squares) for 45 min at 4°C, after which moDCs were transferred to a 37°C incubator without washing away the product. Data are shown as mean ± SEM of three individual experiments. (B) Membrane-bound HA signal (AF647, upper panel) and intracellular HA signal (AF488, lower panel) of moDCs that were pulsed with C16:0 peptide or left untreated for 45 min at 4°C with and without permeabilization. (C) Visualization of the membrane localization of the C16:0 gp100 280–288/45–59 -HA peptide in BMDCs after a 45-min incubation at 4°C determined by STED confocal microscopy. (D) Mannose receptor (MR) and C16:0 peptide HA detection in moDCs treated with glucose depletion medium prior to the antigen pulse (45 min at 4°C). Data are shown as mean ± SEM of four individual experiments. Statistical analyses were performed with a paired Student’s t test. ∗∗p < 0.01. (E) Decay of membrane and total (membrane + intracellular) HA signal at different time points in moDCs that were pulsed with 10 μM C16:0 gp100 280–288/45–59 . Graphs are presented as mean ± SEM of three individual experiments.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Membrane, Incubation, Confocal Microscopy

Journal: Molecular Therapy Oncolytics

Article Title: Palmitoylated antigens for the induction of anti-tumor CD8 + T cells and enhanced tumor recognition

doi: 10.1016/j.omto.2021.04.009

Figure Lengend Snippet: Exploiting the membrane-integrating capability of C16:0 peptides as a tool for antigen enrichment in tumor cells and tumor-derived apoptotic vesicles (A) HA signal in DCs (humans and mice) and different tumor cell lines (JY, THP-1, Mel-JuSo, and Mel-BRO cells) that were pulsed with 10 μM gp100 280–288/45–59 or 16:0 gp100 280–288/45–59 for 45 min at 4°C. Data shown are representative of multiple individual experiments. (B) Viability of human THP-1 cells (leukemia) and (C) murine GL261 (glioblastoma) tumor cells that were pulsed with 10 μM C16:0 gp100 280–288/45–59 or C16:0 OVA 257–264/323–339 and co-cultured with antigen-specific T cells (gp100 CD8 + T cells/CD8 + T cells from OVA-immunized mice). Data show representative experiment measured in triplicate. (D) Overview of experimental setup for antigen enrichment of apoptotic extracellular vesicles. (E) IFNγ secretion by gp100-specific CD8 + T cells after co-culture with moDCs that were incubated with apoptotic cell-derived extracellular vesicles (ApoEVs) which were first loaded with 10 μM C16:0 gp100 280–288/45–59 . Data are presented as mean ± SEM of three individual experiments, measured in triplicate.

Article Snippet: gp100 280–288/45–59 (VTHTYLEPGPVTANRQLYPEWTEAQRLDC) and OVA 257–264/323–339 (CEEKSIINFEKLISQAVHAAHAEINEAGRKEEK) peptides were produced by solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry on a Symphony peptide synthesizer (Protein Technologies).

Techniques: Membrane, Derivative Assay, Cell Culture, Co-Culture Assay, Incubation