Journal: Vaccines

Article Title: β-Catenin in Dendritic Cells Negatively Regulates CD8 T Cell Immune Responses through the Immune Checkpoint Molecule Tim-3

doi: 10.3390/vaccines12050460

Figure Lengend Snippet: β-catenin in DCs negatively regulates DC vaccine-induced CD8 T cell responses against tumor antigens. WT and CD11c-β-catenin active mice were immunized with anti-DEC-205-hgp100 plus CpG, and cross-priming of adoptively transferred Thy1.1 + Pmel-1 CD8 T cells was examined on day 5 following a 5-hour in vitro stimulation with hgp100 25–33 with Brefeldin A (BFA) ( A , B ), recall responses were examined on day 26 after recall with hgp100 25–33 peptide on day 21 ( C , D ). ( A , B ) CD11c-β-catenin active mice exhibit impaired cross-priming of adoptively transferred hgp100-specific (Pmel-1) CD8 T cells upon DC-targeted vaccination compared with WT mice ( n = 4–5). ( A ) The percentages of Thy1.1 + Pmel-1 cells in total CD8 T cells (95% intervals: spleen, 3.0 ± 0.2% vs. 1.4 ± 0.2%; LN, 2.7 ± 0.3% vs. 2 ± 0.2%) and ( B ) the percentages of IFN-γ + effectors out of total Thy1.1 + Pmel-1 cells (95% intervals: spleen, 43.4 ± 1.8% vs. 31.8 ± 1.7%; LN, 53.8 ± 4% vs. 41 ± 3.2%) on day 5 following a 5-hour in vitro stimulation with hgp100 25–33 with Brefeldin A (BFA) are shown. Student’s t -test, *** p < 0.001. ( C , D ) CD11c-β-catenin active mice exhibit impaired recall responses compared with WT mice ( n = 5). ( C ) The percentages of Thy1.1 + Pmel-1 cells in total CD8 T cells (95% intervals: spleen, 3.2 ± 0.7% vs. 0.9 ± 0.3%; LN, 2.4 ± 0.6% vs. 0.4 ± 0.3%) and ( D ) the percentages of IFN-γ + effectors out of total Thy1.1 + Pmel-1 cells (95% intervals: spleen, 42.8 ± 4.7% vs. 28 ± 5.3%; LN, 46.7 ± 4.8% vs. 29.6 ± 3.6%) on day 5 after recall are shown. Student’s t -test, ** p < 0.01 and *** p < 0.001. The data shown are representative of two or more experiments.

Article Snippet: In brief, cells from the spleen or pooled draining LNs were stimulated for 5 hours with hgp100 25–33 peptide (4 μg/mL, AnaSpec, Fremont, CA, USA) in the presence of Brefeldin A (BFA, 5 μg/mL, Biolegend), stained for surface and intracellular (e.g. IL-2, TNFα and IFN-γ) antigens, and evaluated by flow cytometry.

Techniques: In Vitro

Journal: Vaccines

Article Title: β-Catenin in Dendritic Cells Negatively Regulates CD8 T Cell Immune Responses through the Immune Checkpoint Molecule Tim-3

doi: 10.3390/vaccines12050460

Figure Lengend Snippet: scRNA-seq of gp100-specific CD8 T cells identifies distinct populations and reveals differences in CD8 differentiation between CD8 T cells primed in WT and CD11c-β-catenin active mice. WT and CD11c-β-catenin active mice adoptively transferred Thy1.1 + Pmel-1 CD8 T cells were immunized with anti-DEC-205-hgp100 plus CpG. Spleen cells were harvested on day 10 after immunization, and FACS-sorted Pmel-1 cells were subjected toscRNA-seq as described. ( A ) Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction mapping analysis of single-cell gene expression data of Pmel-1 cells isolated 10 days after vaccination with ant-DEC-205-hgp100. Each dot represents one single cell. A total of 9 clusters were identified and color-coded as indicated. UMAP visualization of single cells from combined Pmel-1 cells (left), Pmel-1 cells from WT mice (middle), and Pmel-1 cells from CD11c-β-catenin active mice (right). ( B ) Distribution of Pmel-1 cells from either WT or CD11c-β-catenin active mice within each of the 9 clusters as depicted in ( A ). ( C ) Bubble plots depicting the expression of top DEGs for UMAP clusters are shown in ( A ). ( D ) Violin plot visualizing expression of the top 3 DGEs associated with clusters 2, 3, and 4 among the UMAP clusters. ( E ) UMAPs depicting the module score of gene sets associated with effector or memory, and expression of selected gene markers for CD8 effector ( Gzmb , Ifng , and Prf1 ) and memory ( Sell , Tcf7 and Id3 ) cells. UMAPs for Pmel-1 cells from WT (upper UMAPs) and CD11c-β-catenin active mice (lower UMAPs) are shown. Gradient expression levels are color-coded as indicated. ( F ) GO enrichment analysis identifies multiple GO Biological Processes (BP), Molecular Function (MF), and Cellular Component (CC) that are downregulated in Pmel-1 cells primed in CD11c-β-catenin active mice.

Article Snippet: In brief, cells from the spleen or pooled draining LNs were stimulated for 5 hours with hgp100 25–33 peptide (4 μg/mL, AnaSpec, Fremont, CA, USA) in the presence of Brefeldin A (BFA, 5 μg/mL, Biolegend), stained for surface and intracellular (e.g. IL-2, TNFα and IFN-γ) antigens, and evaluated by flow cytometry.

Techniques: Gene Expression, Isolation, Expressing

Journal: Vaccines

Article Title: β-Catenin in Dendritic Cells Negatively Regulates CD8 T Cell Immune Responses through the Immune Checkpoint Molecule Tim-3

doi: 10.3390/vaccines12050460

Figure Lengend Snippet: Anti-Tim-3 treatment reverses β-catenin-mediated inhibition of cross-priming. WT and CD11c-β-catenin active mice were immunized with anti-DEC-205-hgp100 plus CpG, with half of the CD11c-β-catenin active mice ( n = 5) also being treated with anti-Tim-3 antibody. Cross-priming of adoptively transferred Thy1.1 + Pmel-1 CD8 T cells was examined on day 5 after immunization. ( A ) The percentages of Thy1.1 + Pmel-1 cells in total CD8 T cells (95% intervals: spleen, WT 3.2 ± 0.4%, CD11c-β-catenin active 1.3 ± 0.2%, CD11c-β-catenin active + αTim3 2.8 ± 0.5%; LN, 3.5 ± 0.6%, 2.4 ± 0.2%, 4.8 ± 1.1%), ( B ) the percentages of IFN-γ + effectors out of total Thy1.1 + Pmel-1 cells (95% intervals: spleen, 46.9 ± 2.6%, 29.4 ± 4.8%, 47.7 ± 4.3%; LN, 53.8 ± 4%, 41.4 ± 3.2%, 50 ± 3.4%), and ( C ) the percentages of IFN-γ + IL-2 + effectors out of total Thy1.1 + Pmel-1 cells (95% intervals: spleen, 33.1 ± 2%, 23.2 ± 4.8%, 41.2 ± 3.4%; LN, 35.6 ± 1.4%, 22.8 ± 4%, 32.1 ± 3.6%) are shown. Data are representative of two experiments. One-way ANOVA and post hoc T-tests with Bonferroni correction were used. *** p < 0.001, ** p < 0.01, and * p < 0.05.

Article Snippet: In brief, cells from the spleen or pooled draining LNs were stimulated for 5 hours with hgp100 25–33 peptide (4 μg/mL, AnaSpec, Fremont, CA, USA) in the presence of Brefeldin A (BFA, 5 μg/mL, Biolegend), stained for surface and intracellular (e.g. IL-2, TNFα and IFN-γ) antigens, and evaluated by flow cytometry.

Techniques: Inhibition

Journal: Vaccines

Article Title: β-Catenin in Dendritic Cells Negatively Regulates CD8 T Cell Immune Responses through the Immune Checkpoint Molecule Tim-3

doi: 10.3390/vaccines12050460

Figure Lengend Snippet: Anti-Tim-3 treatment reverses β-catenin-mediated inhibition of memory CD8 T cell responses. WT and CD11c-β-catenin active mice were immunized with anti-DEC-205-hgp100 plus CpG, with half of the CD11c-β-catenin active mice ( n = 5) also being treated with anti-Tim-3 antibody. Recalled memory responses of adoptively transferred gp100-specific CD8 T (Pmel-1) cells were examined 5 days after recall with hgp100 25–33 peptide on day 21. ( A ) The percentages of Thy1.1 + Pmel-1 cells in total CD8 T cells (95% intervals: spleen, 3.6 ± 0.8%, 1.2 ± 0.3%, 4.1 ± 1%; LN, 3.9 ± 1%, 1 ± 0.4%, 4.7 ± 1.2%), ( B ) the percentages of IFN-γ + effectors out of total Thy1.1 + Pmel-1 cells (95% intervals: spleen, 45.3 ± 6.9%, 24 ± 4.9%, 44 ± 4.6%; LN, 48.7 ± 3.2%, 28.3 ± 2.3%, 52.4 ± 4%) following a 5-hour in vitro stimulation are shown. Data are representative of two experiments. One-way ANOVA and post hoc T-tests with Bonferroni correction were used. *** p < 0.001, ** p < 0.01.

Article Snippet: In brief, cells from the spleen or pooled draining LNs were stimulated for 5 hours with hgp100 25–33 peptide (4 μg/mL, AnaSpec, Fremont, CA, USA) in the presence of Brefeldin A (BFA, 5 μg/mL, Biolegend), stained for surface and intracellular (e.g. IL-2, TNFα and IFN-γ) antigens, and evaluated by flow cytometry.

Techniques: Inhibition, In Vitro

Journal: Vaccines

Article Title: β-Catenin in Dendritic Cells Negatively Regulates CD8 T Cell Immune Responses through the Immune Checkpoint Molecule Tim-3

doi: 10.3390/vaccines12050460

Figure Lengend Snippet: Anti-Tim-3 treatment improves the anti-tumor efficacy of DC-targeted vaccines using anti-DEC-205-hgp100. B16F10-bearing WT mice ( n = 7–9) were not treated or were immunized with anti-DEC-205-hgp100 plus CpG with or without anti-Tim-3 treatment (tumor sizes around 3–6 mm), following Thy1.1 + Pmel-1 CD8 T cell transfer. ( A , B ) Anti-Tim-3 treatment improves the anti-tumor efficacy of DC vaccines. Increased tumor sizes from the day of treatment are shown in ( A ) and tumor weight on day 19 in ( B ). A linear mixed model (Lme4) was fitted to the data in ( A ), and ANOVA for the fitted linear mixed model was then performed to determine the difference between groups. One-way ANOVA and post hoc T-tests with Bonferroni correction were used for ( B ). *** p < 0.001. ( C ) Photo of the tumors on day 19 after tumor inoculation. Data are representative of two experiments.

Article Snippet: In brief, cells from the spleen or pooled draining LNs were stimulated for 5 hours with hgp100 25–33 peptide (4 μg/mL, AnaSpec, Fremont, CA, USA) in the presence of Brefeldin A (BFA, 5 μg/mL, Biolegend), stained for surface and intracellular (e.g. IL-2, TNFα and IFN-γ) antigens, and evaluated by flow cytometry.

Techniques: Vaccines