Journal: Journal of Translational Medicine

Article Title: Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading

doi: 10.1186/s12967-023-04064-z

Figure Lengend Snippet: C215Fab-SEA treatment induced a rapid T-cell influx into TDLNs and enhanced T-cell infiltration into the TME. A Violin plots showing the percentage of Vb3 CD4 (left) or CD8 (right) T cells of total CD45 + cells in the TDLNs as determined by FC. B Violin plots showing the cell density (cells/mg tumor tissue) of Vβ3 CD4 + (left) or CD8 + (right) T cells in tumors as determined by FC. C Violin plots showing the percentage of non-Vβ3 CD4 (left) or CD8 (right) T cells among total CD45 + cells in the TDLNs as determined by FC. D Non-Vβ3 CD8 + /CD4 + T-cell ratios in TDLNs. E Violin plots showing the percentage of CCR7 + cells among non-Vβ3 CD8 T cells in the TDLNs. F Violin plots showing the cell density of non-Vβ3 CD4 (left) or CD8 (right) T cells in the TME. G Violin plots showing the CD8/CD4 T-cell ratios of non-Vβ3 cells found in the TME. A – G FC analysis; n = 3–4/group/cycle; timepoints are referred to as C1-C4 (cycles 1–4, respectively). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control; for C4 Combo versus C215Fab-SEA H Graphs displaying the mean (± SEM) log2-fold-change of immune populations according to their gene signatures (Additional file : Table S1) at each treatment cycle compared to a matched control group in tumors. n = 3–4/group/cycle. Statistical significance was determined by nSolver software I. Representative immunohistochemical (IHC) CD3 staining analysis of frozen sections (n = 2–3/group/cycle) of MC38-hEpCAM tumors from the control and treatment groups 24 h after the completion of cycle 3. Scale bar, 100 µm; IHC score (upper right): 1 + = few positive cells, 1–2 + = few to moderate numbers, 2 + = moderate numbers, 2–3 + moderate to high numbers, 3 + high numbers. J Heatmap displaying the relative (mean log2-fold-change) expression of selected chemokine and adhesion genes associated with T-cell infiltration and chemotaxis compared to a matched control group in the TME (left). n = 3–4/group/cycle. The color gradient indicates the fold-change over the matched control. The right heatmap shows the p values of the significant differences in the left heatmap. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test

Article Snippet: Primary antibodies and dilutions were as follows: CD3 (KT3, 1:200; Serotec Bio-Rad), CD45 (13/2.3, 1:150; BioDesign, Carmel Hamlet, New York, United States), Granzyme B-FITC (NGZB, 1:500; Invitrogen), CD8-Viobright FITC (REA601, 1:15; Milteneyi Biotech), FoxP3-Vio667 (REA788, 1:15; Milteneyi Biotech), CD4-FITC (RM4-5, 1:100; eBioscience, San Diego, California, United States), Gr-1-FITC (REA810, 1:15; Milteneyi Biotech), and CD11b-FITC (M1/70, 1:500; Thermo Scientific).

Techniques: Control, Software, Immunohistochemical staining, Staining, Expressing, Chemotaxis Assay, Quantitative Proteomics

Journal: Journal of Translational Medicine

Article Title: Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading

doi: 10.1186/s12967-023-04064-z

Figure Lengend Snippet: C215Fab-SEA promoted CD8 T-cell cytotoxic function and reduced the Treg cell number in the TME. A Heatmap displaying the mean log2-fold-change values of the expression of selected genes associated with T cells in tumors following different treatments (left). n = 3–4/group/cycle. The color gradient indicates the fold increase relative to the designated control. The right heatmap shows the p values of the significant differences in the left heatmap. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test. B Representative immunohistochemical (IHC) double staining analysis (n = 2–3/group/cycle) of CD8 (red) and granzyme B (green). Frozen sections of MC38-hEpCAM tumors from the control and treatment groups were taken 24 h after the completion of cycle 3 for IHC analysis. Scale bar, 100 µm. C Violin plots show the percentage of CD25 + Foxp3 + cells among the total CD4 + T cells in the tumors as determined by FC. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. n = 3–4/group/cycle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control; for C4 Combo versus C215Fab-SEA. D Pseudocolor plots showing Treg gating strategy and representative FC scatter plots (n = 3–4/group/cycle) demonstrating the reduction in Tregs in the TME following C215Fab-SEA. E Graph displaying CD8/Treg ratios in the TME according to NanoString gene expression analysis (left) and FC (right) of MC38-hEpCAM tumors after the completion of each treatment cycle. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. n = 3–4/group/cycle. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control, for C4 Combo versus C215Fab-SEA. In all figures, timepoints are referred to as C1-C4 (cycles 1-4, respectively).

Article Snippet: Primary antibodies and dilutions were as follows: CD3 (KT3, 1:200; Serotec Bio-Rad), CD45 (13/2.3, 1:150; BioDesign, Carmel Hamlet, New York, United States), Granzyme B-FITC (NGZB, 1:500; Invitrogen), CD8-Viobright FITC (REA601, 1:15; Milteneyi Biotech), FoxP3-Vio667 (REA788, 1:15; Milteneyi Biotech), CD4-FITC (RM4-5, 1:100; eBioscience, San Diego, California, United States), Gr-1-FITC (REA810, 1:15; Milteneyi Biotech), and CD11b-FITC (M1/70, 1:500; Thermo Scientific).

Techniques: Expressing, Control, Quantitative Proteomics, Immunohistochemical staining, Double Staining Immunohistochemistry, Gene Expression

Journal: Journal of Translational Medicine

Article Title: Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading

doi: 10.1186/s12967-023-04064-z

Figure Lengend Snippet: TTS treatments increased the number of M1 macrophages and CD8 T-cell abundance and activity. A Graph displaying CD8/TAM ratios in the TME of MC38-hEpCAM tumors after the completion of each treatment cycle, according to FC analysis (left) and NanoString gene expression analysis (right). The TAM (CD11B + CD11C − F4/80 + ) FC gating strategy can be found in Additional file : Fig. S1B. B Representative pseudocolor plots showing MHC II and CD206 expression in CD11b + F4/80 + TAMs on treatment cycle 3 as determined by FC for the detection of TAM M1 and M2 subsets as described in the upper left legend. C Violin plots show the tumor density (cells/mg tumor) of CD206 low MHCII high M1-TAMs (left) and CD206 high MHCII low M2-TAMs (right) over the treatment cycles as determined by FC. D Violin plots show the tumor M1 to M2 TAM ratios over the study period. A – D n = 3–4/group/cycle; timepoints are referred to as C1-C4 (cycles 1–4, respectively). Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control, for C4 Combo versus C215Fab-SEA. E Heatmap displaying the mean log2-fold expression values of changed genes (left) associated with M1 macrophage activity in tumors upon different treatments over the treatment cycles. n = 3–4/group/cycle. The color gradient indicates the fold increase relative to the matched control. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test

Article Snippet: Primary antibodies and dilutions were as follows: CD3 (KT3, 1:200; Serotec Bio-Rad), CD45 (13/2.3, 1:150; BioDesign, Carmel Hamlet, New York, United States), Granzyme B-FITC (NGZB, 1:500; Invitrogen), CD8-Viobright FITC (REA601, 1:15; Milteneyi Biotech), FoxP3-Vio667 (REA788, 1:15; Milteneyi Biotech), CD4-FITC (RM4-5, 1:100; eBioscience, San Diego, California, United States), Gr-1-FITC (REA810, 1:15; Milteneyi Biotech), and CD11b-FITC (M1/70, 1:500; Thermo Scientific).

Techniques: Activity Assay, Gene Expression, Expressing, Control, Quantitative Proteomics

Journal: Journal of Translational Medicine

Article Title: Tumor-targeted superantigens produce curative tumor immunity with induction of memory and demonstrated antigen spreading

doi: 10.1186/s12967-023-04064-z

Figure Lengend Snippet: C215Fab-SEA induced increased frequencies of Ag-specific memory T cells. A , B Heatmaps displaying the mean log 2 -fold expression values of selected genes of T-cell costimulatory and coinhibitory receptors (A-up) and genes associated with T-cell exhaustion and memory development (B-left) in tumors upon different treatments over the treatment cycles (C1–C3). The color gradient indicates the fold increase relative to the matched control. The right (A) or bottom B heatmaps show the p values of the significant differences in the up ( A ) or left ( B ) heatmaps, respectively. White represents a nonsignificant expression change compared to the matched control. Differential expression analysis was performed with Welch’s t test. n = 3–4/group/timepoint. C Violin plots showing the percentage of CD127-expressing cells among total non-Vβ3 CD8 + T cells in the TDLNs (left graphs) and tumors (right graphs). D Violin plots showing the percentage of CD39-expressing cells among the total non-Vβ3 CD8 + T cells in the TDLNs (left graphs) and tumors (right graphs). The timepoints in all figures are indicated as C1-C4 (cycles 1–4, respectively). n = 3–4/group/timepoint. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus the timepoint-matched control, for C4 Combo versus C215Fab-SEA

Article Snippet: Primary antibodies and dilutions were as follows: CD3 (KT3, 1:200; Serotec Bio-Rad), CD45 (13/2.3, 1:150; BioDesign, Carmel Hamlet, New York, United States), Granzyme B-FITC (NGZB, 1:500; Invitrogen), CD8-Viobright FITC (REA601, 1:15; Milteneyi Biotech), FoxP3-Vio667 (REA788, 1:15; Milteneyi Biotech), CD4-FITC (RM4-5, 1:100; eBioscience, San Diego, California, United States), Gr-1-FITC (REA810, 1:15; Milteneyi Biotech), and CD11b-FITC (M1/70, 1:500; Thermo Scientific).

Techniques: Expressing, Control, Quantitative Proteomics

Journal: Cancer Science

Article Title: Downregulated mitochondrial transcription factor A enhances mycoplasma infection to promote the metastasis of hepatocellular carcinoma

doi: 10.1111/cas.15715

Figure Lengend Snippet: Primary Abs used for western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF)

Article Snippet: Custom‐made rabbit polyclonal anti‐P37 , ABclonal , IHC 1:1000, WB 1:1000.

Techniques: Western Blot, Immunohistochemistry, Immunofluorescence, Concentration Assay