Journal: PLoS ONE

Article Title: MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

doi: 10.1371/journal.pone.0164547

Figure Lengend Snippet: (A) NIH3T3 or (B) MPEK stable cells lines expressing either GFP alone (neg. control), OVA or MC.OVA were incubated for 60 hours. MC.OVA +rim had 10 nM rim present in the culture. After 60 hours, cells were harvested and MHC-I expression was analyzed by flow cytometry. (C-E) MPEK stable cells surface expression of CD80, CD86, and CD40. MFI values of live cells are reported relative to the mean MFI of negative control cells, transduced with an empty-vector, in each respective sample. (F) CD3 + CD8 + OT-1 or WT T cells were purified by MACs and labeled with CellTrace violet (CTV) dye. Then 5 x 10 4 CD3 + CD8 + CTV dye-labeled OT-1 or WT T cells were incubated with 5 x 10 4 B6-MPEK cells for 60 hours. By flow cytometry, live CTV + cells were analyzed for CTV dye dilution as a measure of cell proliferation. (G) Representative histograms of live CTV + cells. Percentages represent the percent of cells proliferating. n = 3, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons.

Article Snippet: Cells were then labeled with CellTrace Violet (CTV) cell proliferation dye (ThermoFisher Scientific, Waltham, MA) at either a high (Hi) concentration (5 μM) or low (Lo) concentration (0.5 μM) for 25 minutes at 37°C, per the manufacturer’s recommendations.

Techniques: Expressing, Control, Incubation, Flow Cytometry, Negative Control, Transduction, Plasmid Preparation, Purification, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Anti-PD-1 therapy triggers Tfh cell–dependent IL-4 release to boost CD8 T cell responses in tumor-draining lymph nodes

doi: 10.1084/jem.20232104

Figure Lengend Snippet: Anti-PD-1 mAb promotes tumor-specific CD8 + T cell proliferation in draining lymph nodes. (A–C) C57BL/6 mice were injected s.c. with MC38-OVA tumor cells (0.5 × 10 6 cells). After 10 days, mice were treated or not with anti-PD-1 (250 µg, i.v.) either alone or anti-PD-1 in combination with FTY720 (20 µg for each injection). (A) Experimental setup. (B) Evolution of tumor volume between day 0 and day 10. (C) Mice survival. Statistical analysis was performed using a log-rank test. Compiled from two independent experiments with 10–12 mice per group. (D and E) Representative FACS contour plots and quantification of H2-K b -OVAp tetramers + among CD8 + T cells 5 days after treatment. Tumor-free mice were included as a control. Compiled from five independent experiments with 13–15 mice per group. (F–I) C57BL/6 mice were injected s.c. with MC38-OVA or EG7 tumor cells. After 10 days, mice were adoptively transferred with naïve CTV-labeled OT-I CD8 + T cells and treated, or not, with anti-PD-1 mAb (250 µg, i.v.). (F) Experimental setup. (G–I) OT-I CD8 + T cell proliferation was assessed on day 3 in the draining lymph node. (G) Representative histograms showing CTV dilution in OT-I CD8 + T cells. (H) Quantification of OT-I CD8 + T cell replication index in lymph nodes from mice bearing MC38-OVA. Anti-PD-1 mAb-treated mice were compared with PBS- or isotype-injected mice with similar results. Compiled from five independent experiments with 12–19 mice per group. (I) Absolute numbers of OT-I CD8 + T cells in tumor-draining lymph nodes were assessed 3 days later. Compiled from four independent experiments with a total of 10–12 mice per group. (J and K) Quantification of OT-I CD8 + T cell (J) replication index and (K) absolute numbers in tumor-draining lymph nodes from mice bearing EG7. Compiled from three independent experiments with a total of seven to nine mice per group. Statistical analyses were performed using t tests (E, I, J and K) or two-way ANOVA (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: OT-I CD8 + T cells were isolated from Ubi-GFP-OT-1 TCR-Rag1 −/− transgenic mice, labeled with the Cell Trace Violet (CTV) Proliferation Dye (Thermo Fisher Scientific) according to the manufacturer’s instructions, and injected i.v. (3 × 10 6 cells per mice).

Techniques: Injection, Control, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Anti-PD-1 therapy triggers Tfh cell–dependent IL-4 release to boost CD8 T cell responses in tumor-draining lymph nodes

doi: 10.1084/jem.20232104

Figure Lengend Snippet: Anti-PD-1 mAb activity in the draining lymph node is BCL6 dependent. (A and B) C57BL/6 mice were injected s.c. with MC38-OVA. After 10 days, mice were treated or not with anti-PD-1 mAb (250 µg, i.v.) and received either three doses of the BCL6 inhibitor FX1 or a vehicle as a control. Representative FACS dot plots (A) and quantification (B) of H2-K b -OVAp tetramers + CD8 + T cells 5 days after treatment. Compiled from two independent experiments with five to eight mice per group. (C and D) C57BL/6 mice were injected s.c. with MC38-OVA. After 10 days, mice were adoptively transferred by CTV-labeled OT-I CD8 + T cells and treated or not with anti-PD-1 mAb (250 µg, i.v.) and received either two doses of the BCL6 inhibitor FX1 or a vehicle as a control. OT-I CD8 + T cell proliferation was assessed 3 days later. (C) Representative histograms showing CTV dilution in OT-I CD8 + T cells. (D) Quantification of OT-I CD8 + T cell replication index in lymph nodes. Compiled from two independent experiments with 6–11 mice per group. (E and F) C57BL/6 mice were injected s.c. with MC38-OVA. After 10 days, mice were treated or not with anti-PD-1 mAb (250 µg, i.v.). Some mice were injected every 2 days with FX1 or vehicle until day 10. (E) Evolution of tumor volume between day 0 and day 10. (F) Survival curves for the indicated groups. The dashed line corresponds to 50% survival. Compiled from two independent experiments with a total of 9–10 mice per group. Statistical analyses were performed using two-way ANOVA (B and D) or a log-rank test (F). *, P < 0.05; **, P < 0.01.

Article Snippet: OT-I CD8 + T cells were isolated from Ubi-GFP-OT-1 TCR-Rag1 −/− transgenic mice, labeled with the Cell Trace Violet (CTV) Proliferation Dye (Thermo Fisher Scientific) according to the manufacturer’s instructions, and injected i.v. (3 × 10 6 cells per mice).

Techniques: Activity Assay, Injection, Control, Labeling

Journal: bioRxiv

Article Title: Trypanosoma cruzi amastigotes have a reduced replication rate during chronic stage infections

doi: 10.1101/2020.08.04.236240

Figure Lengend Snippet: CellTrace Violet (CTV) reduces T. cruzi infectivity and inhibits intracellular proliferation. (A) T. cruzi CL-Luc::Scarlet trypomastigotes were incubated with either 5 or 10 µM CTV for 20 minutes and used to infect Vero cell monolayers at a multiplicity of infection of 10:1 (Materials and methods). 18 hours later, infection efficiency was determined by inspecting a total of 2,203 (control), 3,781 (5 µM), and 3,840 (10 µM) Vero cells. At least 300 of these were infected in each case. Each data point corresponds to a randomly acquired image and represents the mean percentage of cells infected. Differences between columns were analysed using a parametric one-way ANOVA with Tukey's post hoc pair wise comparisons. ****p=≤0.0001. (B) Vero cells infected with CTV−ve or CTV+ve trypomastigotes (as above) were incubated for the time periods indicated. The numbers of amastigotes per infected host cell were then determined by analysing >300 infected cells per treatment. Error bars represent the standard deviation from the mean. Data were analysed using a Wilcoxon rank sum test. (C) Images of Vero cells 36 hours after infection with CTV−ve or CTV+ve trypomastigotes. Red, fluorescent T. cruzi amastigotes. Fluorescent parasites containing the CTV tracer dye appear as purple on a red fluorescent background. Size bars=20 µM (D) Images of Vero cells 5 days after infection with trypomastigotes that had been incubated with various concentrations of CTV, as indicated. Blue, intracellular vesicles containing CVT. See also supplementary Video 1. Size bars=20 µM, except where indicated; *=50 µM.

Article Snippet: T. cruzi trypomastigotes were isolated by centrifugation and allowed to recover for 2 hours at 37ºC in high-glucose DMEM medium with 10% FBS, and then labelled with the CellTrace Violet (CTV) fluorescent dye (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Infection, Incubation, Standard Deviation

Journal: Cancer immunology research

Article Title: Phenotypic and Functional Activation of Hyporesponsive KIR neg NKG2A neg Human NK-Cell Precursors Requires IL-12p70 Provided by Poly(I:C)-Matured Monocyte-Derived Dendritic Cells

doi: 10.1158/2326-6066.CIR-14-0054-T

Figure Lengend Snippet: Freshly isolated NK cells were sorted for the absence of KIR and NKG2A receptors (KIRnegNKG2Aneg), then cocultured for six days either alone (media) or with LCcyto, moDCcyto, moDCLPS, or moDCpoly, but without any additional cytokines in culture. A neutralizing antibody to soluble IL-12p70 (+anti IL-12) or its isotype control (+IgG1) was added to moDCpoly cocultures. After six days’ stimulation, these KIRnegNKG2Aneg NK cells were assayed for NKG2A (A-B) or KIR (C-D) expression, with representative flow cytometric contour plots from the same experimental sample shown. Bar graphs depict pooled phenotypic data from all experiments (mean ± SD). Asterisks above each error bar refer to the statistical difference between that condition and the unstimulated d+6 KIRnegNKG2Aneg NK cells (media), whereas statistical notations above the brackets refer to the indicated comparisons between conditions. (E-F) CellTrace™ Violet (CTV)-labeled KIRnegNKG2Aneg cells were cocultured for six days either alone (media) or with moDCpoly to correlate proliferation with NKG2A (E) or KIR (F) induction. Proliferation was measured by the dilution in fluorescent intensity of CTV. One representative experiment out of three is shown. **p=0.001-0.01; ***p<0.001; ****p=<0.0001; ns=not significant.

Article Snippet: Proliferation assay Sorted KIR neg NKG2A neg cells were labeled using the CellTrace™ violet (CTV) cell proliferation kit (Invitrogen) according to the manufacturer’s instructions.

Techniques: Isolation, Expressing, Labeling

Journal: Frontiers in Immunology

Article Title: Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with rituximab

doi: 10.3389/fimmu.2023.878953

Figure Lengend Snippet: AC EBV -mediated long-term proliferation of EBV-specific memory T cells. Frequency of (A) CTV low T cells (CD3, CD8, CD4) examined after seven days of stimulation with APCs loaded with the EBV antigen cocktail (AC EBV , E:T 10:1) and visualized after subtraction from those stimulated with unloaded APCs. Results of four independent experiments are expressed as mean ± SD. (B) Frequency of A02LMP2A + T cells in HLA-A*02:01 + , EBV-seropositive donors (n=4, grey dots) determined by A02LMP2A dextramer staining before and after stimulation (day 0/7). As control, unloaded APCs were co-cultured with autologous CD3 + T cells (Ø). Frequencies measured using a nonsense dextramer (background control) were subtracted. Results of four independent experiments are displayed as contiguous grey dots for individual donors and as mean values in response to unloaded and AC EBV -loaded. Statistical analysis was performed using the non-parametric Wilcoxon test. CTV, CellTrace Violet.

Article Snippet: To determine the proliferative capacity of EBV-specific memory T cells, freshly isolated CD3 + T cells were labeled with CellTrace Violet (CTV) Proliferation Kit (2 mM, Thermo Fisher Scientifics, Waltham MA) according to the manufacturer’s instructions.

Techniques: Staining, Control, Cell Culture