Journal: PLoS ONE

Article Title: The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells

doi: 10.1371/journal.pone.0231896

Figure Lengend Snippet: A) Effects of PSL and TO-207 on the cytotoxicity of CAR T cells. K562/CD19/fLucEGFP cells (3 × 10 3 ) were co-cultured with CD4 + or CD8 + CAR T cells (1.5 × 10 4 ), and treated with different concentrations of PSL and TO-207. Viable target cells were quantified using the luciferase assay after 72 h of co-culture. Values were normalized to viable K562/CD19/fLucEGFP cells cultured alone. The error bars represent SDs from three independent experiments. B) Effects of PSL and TO-207 on CAR T cell degranulation. K562/CD19 cells (5 × 10 5 ) and CAR T cells (5 × 10 5 ) were co-cultured in 500 μl T-cell expansion medium in the absence or presence of PSL or TO-207. Following 68 h of co-culture, monensin (2 μM) and APC–anti-human CD107a antibody were added. Cells were incubated for an additional 4 h, and membrane expression of CD107a was determined by flow cytometry. Representative data of four independent experiments are shown. C) Mean fluorescence intensity (MFI) of samples in Fig 5B. The error bars represent SEs from four independent experiments. D) Sustained cytotoxicity of CAR T cells following TO-207 treatment. K562/CD19/fLucEGFP cells (1 × 10 4 ), CAR T cells (5 × 10 4 ), and CD14 + cells (5 × 10 4 ) were co-cultured in the absence or presence of PSL or TO-207 for 72 h, and viable target cells were quantified by the luciferase assay. Values were normalized to the well containing K562/CD19/fLucEGFP cells alone. The error bars represent SDs from three independent experiments. The linear dose-response relationship was assessed using log-transformed dose values (to the base 10) in a mixed model, in which the zero dose was replaced by the log (minimal dose) - 1. P < 0.05 was considered statistically significant. n.s.: not significant.

Article Snippet: Flow cytometry was performed using allophycocyanin (APC)-conjugated IgG 1 (BioLegend), APC–anti-human CD19 antibody (BioLegend), APC–anti human CD107a (BioLegend), APC–Cy7–anti-human CD3 antibody (BioLegend), V500–anti-human CD4 antibody (BD Biosciences, San Jose, CA, USA), and PerCP–Cy5.5–anti-human CD8 antibody (TONBO Biosciences, San Diego, CA, USA).

Techniques: Cell Culture, Luciferase, Co-Culture Assay, Incubation, Membrane, Expressing, Flow Cytometry, Fluorescence, Transformation Assay

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: (A) IFN-γ spots, each representing a CD8 T cell responding with IFN-γ production, formed upon sensitization of NLV peptide-specific murine (mCD8-NLV; left panel) and human (hCD8-NLV; right panel) cytolytic CD8 T-cell lines (CTLL) by NLV-peptide-pulsed NSG/HHD MEF at an effector-to-stimulator cell ratio of 0.1:1. MEF were pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and were exogenously loaded with synthetic NLV- or non-cognate gp100 280-288 -peptide at the indicated concentrations. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range. (B) Structural avidity of antigen binding to mCD8-NLV and hCD8-NLV CTLL was quantified by dose-dependent HLA-A2.1/NLV tetramer binding in flow cytometry. The respective dissociation constant (K D ) was calculated from half-maximal tetramer binding obtained by Scatchard plot analysis. Dotted curves border the 95% confidence regions of the log-linear regression lines. MFI, mean fluorescence intensity. (C) HLA-A2.1 restricted presentation of NLV epitope by NSG/HHD MEF pretreated with murine IFN-γ for 48h (filled bars) or left untreated (open bars) and infected at an MOI of 4 with the indicated viruses for a total time of 22h until the end of the assay. Peptide presentation on the infected MEF during that period was detected in an IFN-γ ELISpot assay with mCD8-NLV (left panel) and hCD8-NLV (right panel) CTLL at an effector-to-stimulator cell ratio of 0.1:1. Data are shown as mean of duplicates from one experiment representative of two performed. Error bars represent the range.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Infection, Enzyme-linked Immunospot

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: (A) Experimental strategy and schedule of adoptive transfer of NLV-specific CD8 T cells into infected NSG/HHD recipients that were preconditioned by total-body γ-irradiation with a dose of 2 Gy. Intraplantar infection was performed throughout with 1x10 5 PFU of chimeric mCMV. Recipients (n = 4–5 per group) were infected with mCMV-NLV (filled symbols) or mCMV-NLV Ala (open symbols), followed by i.v. transfer of graded numbers of (B) murine or (C) human NLV-specific CD8 T cells (mCD8-NLV or hCD8-NLV CTLL, respectively). Infectivity was quantified at d11 post-transfer in spleen and lungs by standard plaque (plaque-forming unit, PFU, assay). In the livers of the same mice, infected cells (filled and open circles for mCMV-NLV and mCMV-NLV Ala , respectively) and tissue-infiltrating CD8 T cells (filled and open squares, correspondingly) were stained by 2C-IHC and counted in representative 10-mm 2 areas of tissue sections. Symbols represent individual mice and horizontal bars mark median values. Statistical analysis for group differences of most interest (bracketed) was performed after log-transformation using Student’s t-test (unpaired, two-sided; p<0.05 considered significant) with Welch’s correction.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Adoptive Transfer Assay, Infection, Irradiation, Staining, Transformation Assay

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: Corresponding to , 2C-IHC of liver tissue sections taken on d11 after transfer of (A) mCD8-NLV CTLL or (B) hCD8-NLV CTLL show mCMV-NLV or mCMV-NLV Ala infected hepatocytes (iHc, red staining of intranuclear IE1 protein) with typical intranuclear inclusion bodies, and infiltrating CD8 T cells (CD8-T, black cytoplasmic and membrane staining of CD3ε), forming nodular inflammatory foci (NIF) or foci of infection in absence of CD8 T cells (IF), respectively. Upper row images give overviews (A,B; a1, b1), higher-magnification lower row images (A, B; a2, b2) reveal details. Bar markers represent 50 μm throughout.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Infection, Staining

Journal: PLoS Pathogens

Article Title: Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice

doi: 10.1371/journal.ppat.1005049

Figure Lengend Snippet: Groups of 10 γ-irradiated (2 Gy) NSG/HHD mice were infected (A) with 1x10 5 PFU or (B) with 1x10 3 PFU of mCMV-NLV, and in (A; B,b) they received the 1:4 mixtures (see the legend to ) of TCR NLV -transduced (solid graphs) or mock-transduced (dashed graphs) human CD4 and CD8 T cells on the day of infection as a pre-emptive therapy. (B,a) As a reference for comparison, recipients received 1x10 7 cells of CTLL hCD8-NLV (solid graph) or were left with no T cell transfer (dashed graph, w/o T cells). Survival rates over time are displayed as Kaplan Meier survival plots. Statistical significance of differences in survival was calculated using the log-rank test and the Gehan-Wilcoxon test. In the most efficient therapy with the mixture of TCR NLV -transduced CD4 and CD8 T cells (B,b), the median survival time was 26d (range: 21-27d) compared to 21d (range 16-22d) in the mock-transfected control group.

Article Snippet: Fluorochrome-labeled monoclonal Antibodies (mAb) were anti-mouse H-2K b (clone AF6-88.5), CD62L-PE-Cy7 (clone MEL-14), CD8-FITC (clone 53–6.7), anti-human CD8-PerCP (clone SK1), CD8-APC (clone RPA-T8), CD4-APC (clone RPA-T4), CD28-FITC (clone CD28.2), CD95-PE (clone DX2), CD45RA-PE (clone HI100), CD45RO-PE (clone UCHL1), CD62L-FITC (clone DREG-56), HLA-A2.1-PE (clone BB7.2) (all BD Biosciences), anti-mouse CD44-PB (clone IM7) (BioLegend, San Diego, CA, USA), and anti-human CCR7-FITC (clone 150503) (R&D Systems, Minneapolis, MN, USA).

Techniques: Irradiation, Infection, Transfection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Myeloma-specific multiple peptides able to generate cytotoxic T lymphocytes: A potential therapeutic application in multiple myeloma and other plasma cell disorders

doi: 10.1158/1078-0432.CCR-11-2776

Figure Lengend Snippet: MP-CTL generated with a cocktail of XBP1 US, XBP1 SP, CD138, and CS1 peptides were evaluated for their respective peptide-specific responses. Specific activities of MP-CTL were measured in response to the individual peptide pulsed APC by detecting the increase of CD107a+ or IFN-γ+ cells.

Article Snippet: Reagents Mouse anti-human CD3, CD4, CD8, CCR7, CD45RO, CD69, CD107a, IFN-γ, and HLA-A2 mAbs conjugated with FITC, PE, PerCP, PerCP-Cy5.5, APC, Pacific Blue, APC-H7 or PE-Cy7 were purchased from Becton Dickinson (BD)/Pharmingen or BD/Biosciences (San Diego, CA).

Techniques: Generated

Journal: Scientific Reports

Article Title: CD4 T cell autophagy is integral to memory maintenance

doi: 10.1038/s41598-018-23993-0

Figure Lengend Snippet: ( A ) Thymocytes or peripheral CD4 + T cells were isolated from spleens of littermate or Atg5 f/f dLck-cre mice. Cells were stimulated when indicated by 50 ng/mL PMA and 1 µg/mL Ionomycin for 18 hours. During the last 4 hours of stimulation, cells were treated when indicated with pepstatin A and E64d. Cell lysates were then processed by SDS-PAGE and blotted against ATG5 and LC3. Representative experiment of at least three replicates. Complete images of different blotting of the same membrane, including data with wild type C57BL/6 (B6) mice, are shown in Figure . ( B ) Left: Representative dot plots of thymocytes stained by anti-CD4 and anti-CD8 Abs (left) from B6, littermate (LM), Atg5 f/f dLck-cre mice. Right: proportions among thymocytes of double negative (DN), CD4 single positive (CD4 SP), CD8 single positive (CD8 SP) or double positive (DP) cells (n = 5 LM and n = 4 Atg5 f/f dLck-cre mice) ( C ). Representative dot plots of spleen cell staining by anti-CD4 and anti-CD8 Abs, after gating on TCRβ + B220 − cells from B6, littermate LM, Atg5 f/f dLck cre mice. ( D ) Percentages of CD4 + , CD8 + T cells, and B cells among spleen cells (n = 13 B6, n = 17 LM and n = 17 Atg5 f/f dLck-cre mice). ( E ) Absolute numbers of naive, effector memory (EM, CD44 hi CD62 lo ) and central memory (CM, CD44 hi CD62L hi ) CD4 + and CD8 + T cells in spleens (n = 12 for each genotype) among TCR-β + -gated cells. Each point represents the value obtained with one mouse. LM and Atg5 f/f dLck-cre mice are compared by Mann Whitney U Test. *p < 0.05, ***p < 0.001.

Article Snippet: For surface stainings: allophycocyanin (APC)-cyanine 7-labelled anti-mouse TCR-β (clone H57-597, BD Biosciences 553139), phycoerythrin (PE)-labelled anti-mouse CD62L (clone MEL-14, BD Biosciences 553151), fluorescein isothiocyanate (FITC), APC or peridinin chlorophyll (PerCP) cyanine 5.5-labelled anti-mouse B220 (clone RA3-6B2, BD Biosciences, 553087, 553092, and 552771), FITC-labelled anti-mouse CD3ε (clone 145-2C11, BD Biosciences 553061), APC cyanine7, PE or PerCP cyanine 5.5, APC-labelled anti-mouse CD4 (clone GK1.5,552051, eBioscience 12-0041 or clone RM4-5, BD Biosciences, 550954, 553051), PerCP cyanine 5.5-labelled anti-mouse CD8 (clone 53-6.7, BD Biosciences, 551162), APC cyanine 7 or FITC-labelled anti-mouse CD44 (clone IM7, BD Biosciences 559250, 103027, 553133), PE-labelled CD127 (IL-7R, clone A7R43 eBioscience 12-1271-82).

Techniques: Isolation, SDS Page, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: CD4 T cell autophagy is integral to memory maintenance

doi: 10.1038/s41598-018-23993-0

Figure Lengend Snippet: ( A ) Percentages of dead cells measured by Annexin V staining ex vivo from the spleen for the following populations: effector memory (EM, CD44 hi CD62 lo ) and central memory (CM, CD44 hi CD62L hi ) after gating on TCRβ + CD4 + cells or TCRβ + CD8 + cells, from littermate (LM) and Atg5 f/f dLck-cre mice (n = 8 for each gentotype). ( B ) Percentages of viable cells defined as Annexin V − /propidium iodide (PI) − , defined after stimulation of purified CD4 + (left) or CD8 + T cells (right) under the indicated conditions (non-stimulated, stimulated by anti-CD3, by anti-CD3 and anti-CD28, or by 50 ng/mL PMA and 1 µM ionomycin, n = 4 for each genotype). ( C ) Staining of CD4 + or CD8 + T cells purified from LM or Atg5 f/f dLck-cre mice by mitotracker deep red and mitotracker green. Gates could be defined for cells with either high load of mitochondria (mitotracker deep red hi , mitotracker green hi ), low mitochondrial content (mitotracker deep red low , mitotracker green low ), or with damaged mitochondria (mitotracker green hi , mitotracker deep red low ). On the right, histograms show the results of independent experiments (n = 4 for each genotype). ( D ) Ex vivo staining of spleen cells from LM or Atg5 f/f dLck-cre mice, by Mitosox, after gating on TCRβ + CD4 + cells or TCRβ + CD8 + . (Top) representative histograms are shown and (bottom) a summary of different experiments is shown. LM and Atg5 f/f dLck-cre mice are represented and compared by Mann Whitney U Test. **p < 0.01, *p < 0.05.

Article Snippet: For surface stainings: allophycocyanin (APC)-cyanine 7-labelled anti-mouse TCR-β (clone H57-597, BD Biosciences 553139), phycoerythrin (PE)-labelled anti-mouse CD62L (clone MEL-14, BD Biosciences 553151), fluorescein isothiocyanate (FITC), APC or peridinin chlorophyll (PerCP) cyanine 5.5-labelled anti-mouse B220 (clone RA3-6B2, BD Biosciences, 553087, 553092, and 552771), FITC-labelled anti-mouse CD3ε (clone 145-2C11, BD Biosciences 553061), APC cyanine7, PE or PerCP cyanine 5.5, APC-labelled anti-mouse CD4 (clone GK1.5,552051, eBioscience 12-0041 or clone RM4-5, BD Biosciences, 550954, 553051), PerCP cyanine 5.5-labelled anti-mouse CD8 (clone 53-6.7, BD Biosciences, 551162), APC cyanine 7 or FITC-labelled anti-mouse CD44 (clone IM7, BD Biosciences 559250, 103027, 553133), PE-labelled CD127 (IL-7R, clone A7R43 eBioscience 12-1271-82).

Techniques: Staining, Ex Vivo, Purification, MANN-WHITNEY

Journal: Cell reports

Article Title: Pro-resolving lipid mediator lipoxin A 4 attenuates neuro-inflammation by modulating T cell responses and modifies the spinal cord lipidome

doi: 10.1016/j.celrep.2021.109201

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse CD8 PerCP-eFluor 710 , BD Biosciences , Cat# 53–6.7; RRID: AB_1603266.

Techniques: Recombinant, Activation Assay, Software

Journal: Experimental & Translational Stroke Medicine

Article Title: Coadministration of FTY720 and rt-PA in an experimental model of large hemispheric stroke–no influence on functional outcome and blood–brain barrier disruption

doi: 10.1186/2040-7378-5-11

Figure Lengend Snippet: FTY720 leads to a rapid decrease of blood lymphocyte counts. We sampled blood 2 h after i.p. injection of FTY720 (1 mg/kg). FACS analysis showed a significant reduction of both CD4+ and to a lesser extent CD8+ T cells (n = 3/group). Statistical significance between groups was tested with two-tailed Student’s t -test for unpaired values. * p < 0.05.

Article Snippet: After washing with RPMI, samples were incubated with FACS buffer (1% FCS in PBS/0,01% NaN3) and the respective antibodies (Rat anti-mouse CD4 [APC-coupled, Southern Biotech, 1540–11], rat anti-mouse CD8 [PerCP-CTM5.5-coupled, BD Pharmingen, 551162]) for 30 minutes before overnight fixation in 4% PFA in PBS at 4°C.

Techniques: Injection, Two Tailed Test