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: 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: 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:

Journal: Journal of Translational Medicine

Article Title: Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

doi: 10.1186/s12967-021-03073-0

Figure Lengend Snippet: The information of antibodies used for western blot, immunohistochemistry, and flow cytometry

Article Snippet: CD4 and isotype , Human , 5 μl of antibody per test , Elabscience, E-AB-F1109I , Flow cytometry.

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: Antibody panels used for cytotoxic-associated CD4+ T cell phenotypic evaluation.

Article Snippet: CD4 , BV510 , BD Biosciences , 562970 , FITC , Diaclone , 954.031.010.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Lidocaine induced the secretion of IL-10, TGF-β, and IL-35 by CD4 + CD25 + and CD14 + PBMCs and inhibited the secretion of IL-10, TGF-β, and IL-35 by CD4 + CD25 + and CD14 + TIICs. The effect of lidocaine on the secretion of IL-10 ( A , B ), TGF-β ( C , D ), and IL-35 ( E , F ) from CD4 + CD25 + and CD14 + PBMCs and tumor-infiltrating immune cells (TIICs) was investigated. CD4 + CD25 + and CD14 + PBMCs or TIICs were cultured in the absence or presence of graded concentrations of lidocaine (from 0.25 mM to 1.5 mM). The levels of IL-10, TGF-β, and IL-35 in the supernatant were determined at 72 h by ELISA. Cell viability was >95%, as assessed by trypan blue exclusion. Data are presented as the mean ± SEM from three independent experiments, each performed in duplicate, using distinct samples (PBMCs) or three different donors (TIICs). * p < 0.05, ** p < 0.01. The values marked as 0 without lidocaine represent the basal levels of IL-10, TGF-β, and IL-35 in CD4 + CD25 + PBMCs and CD14 + PBMCs.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Lidocaine inhibited IL-35, IL-10, and TGF-β production by CD4 + CD25 + Foxp3 + and CD4 + CD25 + CD127 − TIICs. Sorted CD4 + CD25 + TIICs were treated with 1.5 mM lidocaine for 72 h followed by staining with anti-IL-35, anti-IL-10, anti-TGF-β, and anti-Foxp3 antibodies for flow cytometry analysis. Isotype controls were used to distinguish between positive and negative cells for IL-35, IL-10, TGF-β, and Foxp3. Typical flow cytometry dot plot analysis revealed the percentage of ( A , B ) CD35 + Foxp3 + CD4 + CD25 + TIICs, ( C , D ) IL-10 + Foxp3 + CD4 + CD25 + TIICs, and ( E , F ) TGF-β + Foxp3 + CD4 + CD25 + TIICs treated with lidocaine (1.5 mM). ( G , H ) The expression levels of CD127 in lidocaine-treated IL-35 + CD4 + CD25 + , IL-10 + CD4 + CD25 + , and TGF-β + CD4 + CD25 + cells were analyzed using flow cytometry. The expression levels are shown on histograms. Isotype controls were used to distinguish between positive and negative cells for CD127. Cell viability was >95%, as assessed by trypan blue. RMFI: relative mean fluorescence intensity. Data are from distinct samples and presented as the mean± SEM in three different experiments, each performed in duplicate. * p < 0.05, ** p < 0.01.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Staining, Flow Cytometry, Expressing, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Schematic diagram illustrating the lidocaine-mediated anti-tumoral mechanism through immunogenic cell death targeting PGCCs. Lidocaine inhibited the production of IL-35, IL-10, and TGF-β by CD4 + CD25 + Foxp3 + tumor-infiltrating immune cells (TIICs). Additionally, lidocaine enhanced anti-tumor immunity by reducing PD-1 expression and increasing IFN-γ expression on CD8 + TIICs via the NF-κB signaling pathway. The lidocaine-treated CD8 + TIICs subsequently promoted the immunogenic cell death of primary gastric cancer cells (PGCCs). Long→: treated. Short→: linked. ┬: inhibition.

Article Snippet: Reagents and sources were as follows: lidocaine (Xylocaine ® 2% for Intravenous Injection) (Water for injections) (Cenexi, Fontenay-sous-Bois, France); FITC-conjugated IgG1 anti-human CD14 (BD Bioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD8 (eBioscience, San Diego, CA, USA); FITC-conjugated IgG1 anti-human CD25 (Elabscience, San Diego, CA, USA); IgG1-FITC isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA); PE-conjugated IgG1 anti-human CD4 (Elabscience, San Diego, CA, USA); PE-conjugated IgG1 anti-human IFN-γ (Elabscience, San Diego, CA, USA); IgG1-PE isotype control (Caltag Laboratories, Inc., Burlingame, CA, USA).

Techniques: Expressing, Inhibition