Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 1 Downregulation of IRF4 expression in CAR T cells. (A) Schematic of CAR constructs. (B) CAR expression in T cells detected by staining with a phycoerythrin (PE)-labeled goat anti-human IgG antibody before (upper panels) and after (lower panels) magnetic cell separation (MACS). One representative donor out of four is shown. (C) Intracellular staining of IRF4 after stimulation with CEA+ BxPC-3 cells at the indicated time points in CD8+ (left panel) and CD4+ CAR T cells (right panel). CAR T cells were generated as described in the materials and methods section by activation of PBMCs followed by retroviral transduction. Untransduced cells were generated by activation of PBMCs and subsequent expansion with IL-2, but without retroviral transduction. Data represent means ± SEM of five donors, p values were calculated by Student´s t test, *indicates p ≤0.05, ***indicates p ≤0.001.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Expressing, Construct, Staining, Labeling, Magnetic Cell Separation, Generated, Activation Assay, Retroviral, Transduction

Journal: Frontiers in immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities.

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: FIGURE 3 Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 105 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA+ BxPC-3 cells (1 × 105 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3+ CAR+) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤0.05. (B-G) Phenotypic analysis of CD8+ CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B). CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C), PD-1 (D), TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO+ CD62L+), EM = effector-memory (CD45RO+ CD62L-), CM = central-memory (CD45RO+ CD62L+), E = effector (CD45RO- CD62L-) (F), and CD27 expression (G). Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤0.05, **indicates p ≤0.01.

Article Snippet: Four hours later, T cells were stained with the viability dye eFluor 780, the goat F(ab’)2 antihuman IgG-PE antibody, a BV421-conjugated anti-CD8 antibody (BD), and an APC-conjugated anti-CD4 antibody (Miltenyi Biotech).

Techniques: Labeling, Cytometry, Staining, Expressing, Cell Differentiation

Journal: eLife

Article Title: ImmunoCluster provides a computational framework for the nonspecialist to profile high-dimensional cytometry data

doi: 10.7554/eLife.62915

Figure Lengend Snippet:

Article Snippet: 3156033D , Fluidigm , 156 , CD4 , 100 , , .

Techniques: Concentration Assay

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 1. Comparison of exhaustion marker expression (top panels) and memory marker expression (bottom panels) from flow cytometry of freshly digested tumor biopsies or patient-matched circulating T cells for CD4 and CD8 subsets. ***p<0.001, **p<0.01. Exhaustion markers assessed in n = 57 samples (n = 29 TIL and n = 28 circulating T cell). Memory markers assessed in n = 52 samples (n = 24 TIL and n = 28 circulating T cell).

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Comparison, Marker, Expressing, Flow Cytometry

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 2. Comparison of exhaustion marker expression (top panels) and memory marker expression (bottom panels) from flow cytometry of expanded TILs or patient- matched circulating T cells at early (day 4–5 post-activation) and late (day 10–11 post-activation) for CD4 and CD8 subsets. ***p<0.0001, +p = 0.038, *p = 0.050. Exhaustion markers assessed in n = 6 samples (n = 3 TIL and n = 3 circulating T cell). Memory markers assessed in n = 4 samples (n = 2 TIL and n = 2 circulating T cell).

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Comparison, Marker, Expressing, Flow Cytometry, Activation Assay

Journal: Translational oncology

Article Title: Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture.

doi: 10.1016/j.tranon.2024.101899

Figure Lengend Snippet: Fig. 4. After TIL and circulating T cell isolation, T cells from P012 were rested overnight and underwent evaluation with IsoLight single-cell proteomics immediately after CD3/CD28 activation. CD8 and CD4 TILs secreted effector proteins to a greater extent than patient-matched CD8 and CD4 circulating T cells. In particular, CD8 TILs demonstrated polyfunctional secretion of both effector and chemoattractive proteins.

Article Snippet: For immunophenotyping of digested HNSCC specimens, the following antibodies were used: VioBlue antihuman CD223 (Miltenyi, clone REA351), VioGreen anti-human CD4 (Miltenyi, clone M-T466), FITC anti-human CD45 (Miltenyi, clone 5B1), PE anti-human TIGIT (Miltenyi, clone REA 1202), PE-Vio770 antihuman CD279 (Miltenyi, clone REA1165), APC anti-human TIM3 (Miltenyi, clone F38–2E2), APC-Vio770 anti-human (Miltenyi, clone REA734), VioBlue anti-human CD45RA (Miltenyi, clone REA562), FITC anti-human CD3 (Miltenyi, clone REA613), PE-Vio770 anti-human CD62L (Miltenyi, clone REA615), APC anti-human CD45RO (Miltenyi, clone REA611).

Techniques: Cell Isolation, Activation Assay

Journal: Cell reports

Article Title: High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status

doi: 10.1016/j.celrep.2021.108974

Figure Lengend Snippet: (A) GSEA using Hallmark and curated gene sets. The p values, normalized enrichment score (NES), and enrichment scores (ES) are listed in the figure. (B) Heatmap of correlation between WGCNA module eigengenes and immune cell log ratios from mass cytometry evaluation of BAL. The p value and Spearman’s rho are indicated in the figure. (C) Barplot of −log10(p values) for gene ontology (GO) term enrichment of WGCNA modules of interest based on correlation to cells from BAL. (D) Plotting pink module absolute gene significance (GS) for correlation to CD206 + CD11b + CCR4 + macrophages versus module membership (MM). (E) Visualization of pink module network hub genes (diamonds) with next closest network members (circles). (F) Plotting magenta module GS for correlation to FcεRI + CD127 + CCR4 − innate cells versus MM. (G) Visualization of magenta module network hub genes (diamonds) with next closest network members (circles). (H) Plotting green module GS for correlation to CD8 or CD4 T cells versus MM. (I) Visualization of green module network hub genes (diamonds) with next closest network members (circles).

Article Snippet: Anti-Human CD4 (RPA-T4)-145Nd , Fluidigm , Cat# 3145001; RRID:AB_2661789.

Techniques: Mass Cytometry

Journal: Cell reports

Article Title: High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status

doi: 10.1016/j.celrep.2021.108974

Figure Lengend Snippet: (A) Correlogram of gene module to cell cluster interactions using the ICLite algorithm. Spearman correlation of module scoring with immune cell log ratio was used construct a correlogram of BAL gene modules (x axis) versus cell lineages (y axis). Only associations with false discovery rate (FDR) corrected p < 0.05 are illustrated. (B) Barplot of −log10(p values) for GO term enrichment of ICLite modules 13 and 15, which correlate with CD206 + CD11b + CCR4 + macrophages. Plotting of patient module score versus immune cell log ratio is adjacent, with Spearman’s rho and p value of comparison as indicated in the figure. Red hashed line indicates linear regression model for data. (C) Barplot of −log10(p values) for GO term enrichment of ICLite modules 10 and 11, which correlate with FcεRI + CD127 + CCR4 − innate cells and CD206 + FcεRI + macrophages, respectively. Plotting of respective patient module score versus immune cell log ratio is adjacent, with Spearman’s rho and p value of comparison as indicated in the figure. Red hashed line indicates linear regression model for data. (D) Barplot of −log10(p values) for GO term enrichment of ICLite modules 1 and 20, which correlate with CD4 and CD8 EMs, CMs, and TRMs. Plotting of respective patient module score versus CD4 TRM log ratio is adjacent, with Spearman’s rho and p value of comparison as indicated in the figure. Red hashed line indicates linear regression model for data.

Article Snippet: Anti-Human CD4 (RPA-T4)-145Nd , Fluidigm , Cat# 3145001; RRID:AB_2661789.

Techniques: Construct, Comparison

Journal: Cell reports

Article Title: High-dimensional profiling clusters asthma severity by lymphoid and non-lymphoid status

doi: 10.1016/j.celrep.2021.108974

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Human CD4 (RPA-T4)-145Nd , Fluidigm , Cat# 3145001; RRID:AB_2661789.

Techniques: Purification, Control, Antibody Labeling, Flow Cytometry, Software, Staining, Blocking Assay

Journal: PLoS ONE

Article Title: Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model

doi: 10.1371/journal.pone.0101764

Figure Lengend Snippet: A, B) Influx of CD4 + and CD8 + tumor infiltrating lymphocytes respectively was higher in brains of animals treated with immunotherapy with 4-1BB activation and CTLA-4 blockade and triple therapy compared to brains from non-tumor bearing mice ( p <0.05). C, D) Cervical lymph nodes from naïve mice showed a higher baseline of CD4 + and CD8 + T cells which resulted in comparable densities of T cells between all groups. Error bars represent standard error of the mean (SEM) and p values were calculated with the student t-test.

Article Snippet: Hamster and rat IgG to serve as control antibodies were purchased from Rockland Immunochemicals Inc. CD4-FITC and CD8-PE antibodies were used for FACS analysis (BD-Pharmingen).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Focal Radiation Therapy Combined with 4-1BB Activation and CTLA-4 Blockade Yields Long-Term Survival and a Protective Antigen-Specific Memory Response in a Murine Glioma Model

doi: 10.1371/journal.pone.0101764

Figure Lengend Snippet: Kaplan Meier survival curves for untreated animals, triple therapy (no depletion), triple therapy with depleted CD4 + T cells and triple therapy with depleted CD8 + T cells ( n = 5–7 mice/group). CD4 + T cell depletion abolishes the survival benefit of triple therapy ( p <0.001). Depletion of CD8 + T cells did not interfere with the efficacy of triple therapy and resulted in long-term survival.

Article Snippet: Hamster and rat IgG to serve as control antibodies were purchased from Rockland Immunochemicals Inc. CD4-FITC and CD8-PE antibodies were used for FACS analysis (BD-Pharmingen).

Techniques: