Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing balb/c nude mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). B Double staining of CD8 and αSMA in AB1-HA tumors from balb/c mice treated with or without KL001 (studied in Fig. ). Scale bar, 200 μm. C Representative images and D quantitative evaluation of sections from KL001-treated AB1-HA tumors stained for CD8, CD4, Foxp3, and F4/80 ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Quantitative evaluation of sections from CLK8-treated AB1-HA tumors stained for CD8, CD4, Foxp3 ( n = 15 fields from five mice per group). * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Injection, Double Staining, Staining, MANN-WHITNEY

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: CD8 + and CD4 + T cells, macrophages, and Tregs were identified using a rat anti-CD8a monoclonal antibody (1:150, 53-6.7; BD Pharmingen, Franklin Lakes, NJ, USA), rat anti-mouse CD4 (1:150, H129.19; BD Pharmingen), rat anti-mouse F4/80 monoclonal antibody (1:100 dilution, CI:A3-1; Abcam, Cambridge, MA, USA), and rabbit anti-Foxp3 polyclonal antibody (1:400 dilution; Novus Biologicals, Centennial, CO, USA), respectively.

Techniques: Staining

Journal: Translational Oncology

Article Title: Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

doi: 10.1016/j.tranon.2021.101205

Figure Lengend Snippet: cEGFR p527 immunization mediates CD8 infiltration and antibody deposition into canine tumors. Postmortem bladder cancer tissue from a cEGFR p527 immune dog and tissue from healthy, non-cancerous bladder was examined for infiltration of CD8 T cells and endogenous antibody. A. CD8 isotype control. B. CD8 T cell staining (green). Arrows illustrate presence of CD8+ cells within the tumor tissue. C. & D. Antibody deposition in canine bladder tumor tissue from a cEGFR p527 immunized dog (two different fields). E. & F. Canine IgG deposition in normal canine bladder tissue (two different fields). Magnification is 40x (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Article Snippet: CD8 T cells were stained in tissues using mouse anti-dog CD8 (MAB6709; R&D Systems, Minneapolis, MN), followed by goat anti-mouse IgG ( H + L ) Alexa Fluor 488 (A11017; Life Technologies, Eugene, OR) and counterstaining with DAPI (Molecular Probes; Eugene, OR).

Techniques: Control, Staining

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Oncolytic adenovirus in treating malignant ascites: A phase II trial and longitudinal single-cell study.

doi: 10.1016/j.ymthe.2024.04.029

Figure Lengend Snippet: Figure 4. Characteristics and dynamics of CD8+ and CD4+ T cell subsets during H101 treatment (A) Distinct cell subtypes of CD8+ T cells demonstrated by UMAP plot. (B) Cell proportion of each CD8+ T cell subtype. Black asterisks represent the statistical significantly increased proportion on day 7 compared with that at baseline. (C) Fraction of CD8_MKI67 cells in two response groups on days 0, 7, and 14. Wilcoxon test. *p < 0.05. (D) Representative double-color immunofluorescence staining of CD8+Ki67+ cells in MA samples (P04 [T-long patient] and P07 [T-short patient]) on days 0, 7, and 14, with

Article Snippet: For double-color immunofluorescence of CD8A and Ki67, mouse anti-human CD8a (CST, Cat#70306) and rabbit anti-human Ki67 (Abcam, Cat#15580), were applied.

Techniques: Staining

Journal: Research in Veterinary Science

Article Title: Relationship between rate of infection and markers of inflammation/immunity in Holy Birman cats with feline coronavirus

doi: 10.1016/j.rvsc.2014.08.009

Figure Lengend Snippet: Results recorded in Birman cats and in cats from other breeds.

Article Snippet: Immunophenotyping by flow cytometry was performed on aliquots of 50 μL of the cell suspension to identify lymphocyte subpopulations as previously described ( ) using the following panel of antibodies for feline surface antigen: 2.5 μL of mouse anti feline CD4 (specific for T helper cells, clone MCA1350, Serotec, Oxford, UK), 1 μL of mouse anti feline CD8 alpha/beta (specific for T cytotoxic cells, clone MCA1347G, Serotec, Oxford, UK), 50 μL of mouse anti feline CD5 (specific for T cells, clone MCA2038S, Serotec, Oxford, UK), and 1 μL of mouse anti canine CD21, specific for B cells, that cross reacts with feline species (clone MCA1781R, Serotec).

Techniques:

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic individuals. ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes were sorted from HLA-A*0201-positive SYMP and ASYMP individuals, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 25,638 genes. ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes detected by FACS in SYMP vs. ASYMP individuals. ( c ) Heatmap is showing 772 differentially expressed genes among SYMP and ASYMP individuals. ( d ) Heatmap showing statistically significant pathways that are affected in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals. Parametric Gene Set Enrichment Analysis (PSGEA) method was applied based on data curated in Gene Ontology and KEGG. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied. ( e ) Bulk RNA heatmap comparing differentially expressed CAM pathway associated T cell co-stimulatory and T cell exhaustion genes in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Gene Expression, Infection, Biomarker Discovery, RNA Sequencing, Expressing

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Single-cell RNA sequencing of trigeminal ganglia-resident CD45 + leukocytes from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Illustration of the experimental design and validation of differentially expressed genes in CD45 + leukocytes sorted on day 15 p.i. from the trigeminal ganglia (TG) of SYMP and ASYMP HLA Tg rabbits. ( b ) Heatmap expression of the most significant 140 differentially expressed genes among eight different clusters detected in TG-resident CD45 + leukocytes from HSV-1 infected SYMP and ASYMP HLA Tg rabbits (top two heatmap panels). Each cluster represents an individual immune cell population, determined on the basis of specific molecular markers: CD8 + T cells (CD8A), CD4 + T cells (CD4), NK cells (NKG7), B cells (CD19), macrophages (CD68), monocytes (CD14), granulocytes (FUT4) and dendritic cells (CD1c). The t-SNE dimensionality reduction, applied to single-cell RNA sequencing data revealed eight distinct clusters of immune cell populations among CD45 + leukocytes for the TG of HSV-1 infected ASYMP HLA Tg rabbits (middle panels). The total number of differentially expressed genes within each immune cell clusters (nCount) (lower panels). ( c ) Average frequencies of different immune cell populations detected within TG-resident CD45 + leukocytes of SYMP and ASYMP HLA Tg rabbits. ( d ) Volcano plot illustrates the total copy number reads observed for all the genes within one single cell (nFeature).

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: RNA Sequencing, Infection, Biomarker Discovery, Expressing

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from trigeminal ganglia of HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP HLA Tg rabbits. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes were sorted from TG of HLA-A*0201-positive SYMP and ASYMP HLA Tg rabbits, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 23,669 rabbit genes (OryCun2.0 (GCA_000003625.1). ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes detected by FACS in TG of HLA-Tg rabbits. ( c ) The heatmap is showing the most significant 2,879 differentially expressed genes among SYMP and ASYMP HLA Tg rabbits. Genes with minimum count per million (CPM) ≥ 0.5 were used for obtaining the transformed counts data for clustering using regularized log (rlog). ( d ) Bulk RNA heatmap shows the pathways that are different among ASYMP and SYMP HLA Tg rabbits. Genes differentially expressed in both single-cell RNA sequencing and bulk RNA sequencing were considered for pathway analyses. Parametric gene set enrichment analysis (PSGEA) method based on data curated in Gene Ontology and KEGG was applied. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Gene Expression, Infection, Biomarker Discovery, RNA Sequencing, Expressing, Transformation Assay

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Activation and exhaustion genes differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of T cell activation genes ( CD69 , CD62L , CD44 , CD107 , and IFN-γ ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of T cell exhaustion genes ( PD-1 , LAG-3 , CTLA4 , ICOS , and BLIMP1 ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of specific genes representing memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets from TG of SYMP vs. ASYMP HLA Tg rabbits (left panel). Average frequencies of CD8 + T RM cells expressing various exhaustion genes in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (top right panel). Average frequencies of functional CD107 a/b+ IFN- γ + CD8 + T RM cells in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (lower right panel). ( c ) Bulk RNA sequencing showing expression of T-cell activation (left panel) and T-cell exhaustion genes (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Frequencies of memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets detected by FACS in HSV-1 infected TG of SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of CD8 + T cells in HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits. TG sections were co-stained using DAPI and mAb specific to rabbit CD8 + T cells (magnification, × 20). Blue, DAPI: DNA, green: CD8 + T cells.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Activation Assay, Infection, Expressing, RNA Sequencing, Functional Assay, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Genes of cytokines/chemokines and receptors differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of genes of chemokines and chemokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of genes of cytokines and cytokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of CD8 + T RM cells expressing genes of cytokines/chemokines and receptors in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( c ) Bulk RNA sequencing showing expression of genes of chemokines/chemokine receptors (left panel) and genes of cytokines/cytokine receptors (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Representative (left panels) and average (right panels) frequencies of CCR3 + CD8 + T RM cells in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits by CD8 + T cells expressing T cell-attracting chemokines and receptors (i.e., CXCR3, CXCL9, CXCL10, and CXCL11) (magnification, × 20). Blue: DAPI (DNA stain); red: CXCR3 cells.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Infection, Expressing, RNA Sequencing, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: TG-resident HSV-specific memory CD8 + T RM cells downregulate the T cell exhaustion associated pathway and confer protection from ocular herpes in HSV-1 infected asymptomatic humans and HLA transgenic rabbits. ( 1 ) Upon exposure to stressors, the HSV-1 enters into the cornea and travels through neurons to Trigeminal ganglia. ( 2 ) Following primary HSV-1 infection, the vast majority (up to 95%) of antiviral effector CD8 + T cells die, leaving behind only about 5% of CD8 + T cells destined to differentiate into a heterogeneous pool of memory CD8 + T cells. ( 3 ) The effector memory (T EM ) and tissue-resident memory (T RM ) CD8 + T-cell subsets are found mainly in the HSV-infected but "naturally protected" asymptomatic subjects, whereas the lymphoid organ-resident central memory (T CM ) CD8 + T cell subsets are mainly present in non-protected Symptomatic subjects. ( 4 ) Reduced viral reactivation was observed among asymptomatic subjects possessing a higher frequency of CD8 + T RM cells resulting in a less severe herpes disease. ( 5 ) The findings study suggests that by blocking immune checkpoints, there is a reduced expression of T cell exhaustion molecules ( PD-1 , LAG-3 , PSGL-1 , CTLA-4 , TIM3 , and TIGIT ) and T cell exhaustion associated Cell Adhesion Molecule pathway and increased retention of CD8 + T RM cell population in asymptomatic subjects. This memory CD8 + T cell population mediates recall responses and halts attempts of virus reactivation in the infected TG, thus accelerating viral clearance. More-so, reduced expression of T cell exhaustion pathway also gives rise to higher expression of genes associated with T cell function ( CD107 , IFN-γ ), T cell homing ( CXCR3 , CCR7 ), and T-cell keeping ( IL7R , IL15R ). This helps in reducing the ocular herpes infection and recurrent herpetic disease.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Infection, Transgenic Assay, Blocking Assay, Expressing, Virus, Cell Function Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 1. OX40 expression on pDCs in the TME of HNSCC. (A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on differ- ent immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8+ T cells (n = 16), CD4+ T cells (n = 17), CD4+ Th1 T cells (n = 12), and CD4+ Treg cells (n = 14). T cell subsets were gated from live CD45+CD3+ cells. Th1 cells were defined as CD4+Tbet+ T cells and Treg cells were defined as CD4+Foxp3+ cells. (B) Gating strategy for FACS analysis and sorting of OX40+ and OX40lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DRhiLineage– cells, followed by CD11c–CD123+ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN– (n = 50) or dLN+ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (A, D, and E).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 3. OX40+ pDCs promote antigen-specific CD8+ T cell responses. (A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40+/OX40lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8+ T cells for 5 to 6 days, at which point antigen-specific CD8+ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8+ T cells cocultured with OX40+ or OX40lo/– pDCs sorted from their tumors. CD8+ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8+ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40+ or OX40lo/– pDCs from the TME versus non-TME (dLN–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40+ or OX40lo/– pDCs in the top chamber were separated from autologous CD8+ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB+ CD8+ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8+ T cells cocultured with E7-loaded mDCs and OX40+ or OX40lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40+ and OX40lo/– pDCs with mDCs, based on cytolytic CD8+ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8+ T cells in the presence or absence of OX40+/OX40lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimen- tal repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (C–E and G).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: In Vitro, Control, Co-culture Assay, Immunopeptidomics, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 4. The OX40-OX40L axis is utilized by pDCs in the TME. (A) OX40L expression measured by flow cytometry on OX40+ and OX40lo/– pDCs (n = 7). Correla- tion (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123+ (green) cells, including one with OX40 expression (orange), next to a CD8+ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L+CD68+ macrophages with phenotyped OX40+ pDCs. (E and F) Percentage of phenotyped OX40+/ OX40lo/– pDCs touching other OX40L+ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8+ T cell counts within 30 μm of pDC subsets to total counts of CD123+ pDC subsets (OX40+/OX40lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 6. OX40+ pDCs correlate to survival in cancer patients and suppress tumor growth. (A) Prospective recurrence-free survival (log-rank, Mantel-Cox test) of HNSCC cohort (n = 80), stratified by median (45%) intratumoral pDC OX40 expression, as measured by flow cytometry. (B) Overall survival (log-rank, Mantel-Cox test) of HNSCC patients (n = 500) from the GDC data portal, stratified first by median pDC gene signature Z scores followed by stratification of mean TNFRSF4 (encodes OX40) mRNA levels. (C) Correlation (Pearson, with line of best fit) of TNFRSF4 log2 mRNA levels (among cases with pDChi gene signatures) with CD8+ T effector scores in HNSCC (n = 172). (D) OX40 expression on intratumoral pDCs from different murine tumor mod- els. n =4; 4 experimental replicates. (E) gp100-specific Pmel-1 CD8+ T cell IFN-γ production by proliferating (eFluor 450–low) CD8+ T cells, measured in the presence or absence of pDCs from the dLNs of B16-F10– and B16CCR7-bearing mice. n = 2; 2 experimental repeats. (F) gp100-specific proliferating (eFluor 450–low) Pmel-1 CD8+ T cells in the presence or absence of B16CCR7 pDCs prestimulated with Resiquimod and OX86. n = 2; 2 experimental repeats. (G) Effect of pDC depletion (anti-PDCA1) in B16-F10– and B16CCR7-bearing mice compared with controls (anti-polyclonal IgG). Data are pooled from at least 2 independent experiments with 3 to 5 mice per group. (H) Quantification (by flow cytometry) of conventional cDCs (CD11c+CD11b–) and CD8a+ cDCs from B16CCR7-bearing mice treated with anti-PDCA1 or anti-polyclonal IgG. Data are pooled from individual experiments and normalized to 5 × 105 live cells. One-way ANOVA followed by Tukey’s post hoc test (D), 2-way ANOVA with Sidak’s test for multiple comparisons (H), and unpaired Student’s t test (G). **P < 0.01; ***P < 0.001. Tumor burden data and bar graph data are mean ± SEM.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry

Journal: Acta Neuropathologica

Article Title: The structural differences between patient-derived α-synuclein strains dictate characteristics of Parkinson’s disease, multiple system atrophy and dementia with Lewy bodies

doi: 10.1007/s00401-020-02157-3

Figure Lengend Snippet: Assessment of immune response after inoculation of different patient-derived homogenates and PMCA-amplified αSYN strains in association with rAAV2/7-mediated αSYN expression. a Representative photomicrographs of the immune response stained for Iba1 (microglia), MHCII (reactive microglia), CD4 (T helper cells) and CD8 (cytotoxic T cells) in the SN 5 months after co-injection of different patient-derived homogenates (left) or PMCA-amplified αSYN assemblies (right) with rAAV2/7-αSYN overexpression. Cells immunoreactive for CD4 and CD8 are marked with black arrows. Scale bar Iba1 = 50 µm, MHCII – CD4 = 100 µm. b Table with scoring system for the different immune markers Iba1, MHCII, CD4 and CD8 in the rat SN 5 months after inoculation with patient-derived homogenates (left) or PMCA-amplified αSYN assemblies (right) combined with human αSYN expression. The scoring system ranges from not present (−) to abundantly present (++++). A total of three animals per group was included. The data were presented as a table with scoring system to demonstrate the presence of an inflammatory response in the rat SN

Article Snippet: IHC was performed on free-floating sections using antibodies against tyrosine hydroxylase (TH, rabbit polyclonal, Ab152, 1:5000, Merck Millipore, Massachusetts, US), phosphorylated αSYN (P-S129 αSYN, mouse 11A5, 1:5000, provided by Elan Pharmaceuticals, Inc., Dublin, Ireland and rabbit, Ab51253, 1:5000, Abcam, Cambridge, UK), Iba1 (goat polyclonal, Ab107159, 1:1000, Abcam), MHC Class II (mouse, MCA46G, 1:250, Serotec), CD4 (mouse, MCA55GA, 1:500, Serotec) and CD8 (mouse, MCA48GA, 1:500, Serotec).

Techniques: Derivative Assay, Amplification, Expressing, Staining, Injection, Over Expression