Journal:

Article Title: Inducing Efficient Cross-priming Using Antigen-coated Yeast Particles

doi: 10.1097/CJI.0b013e318181c87f

Figure Lengend Snippet: DCs fed MS74NEY2-coated yeast and yeast hulls stimulated the expansion of cells recognizing an NY-ESO-1 peptide/MHC complex from a naive CD8+ pool. CD8+ CD45RO- cells from 3 HLA-A2 donors were added to immature DCs fed either yeast hulls alone, whole yeast coated with 1 layer of MS74NEY2, or yeast hulls with 3 coats (each at a dose equivalent to 10 yeast/DC). The T cells before coculture and after 10 days (IL-2 and IL-7 were added from day 3 onwards) were labeled with α-CD8-FITC and PE-conjugated SLLMWITQV/HLA-A*0201 pentamer. Cells (2 × 106) were labeled per test and 2 × 105 CD8+ events are shown in each dot plot. The proportions of events (out of CD8+ events) falling in the top right quadrant (in bold italics) and in the rectangular gate are shown. DC indicates dendritic cell; FITC, fluorescein isothiocyanate; MHC, major histocompatibility complex; PE, phycoerythrin.

Article Snippet: In addition, 2 × 10 6 naive T cells from each donor were labeled with 20 μL α-CD8-FITC and 10 μL SLLMWITQV/HLA-A*0201 peptide-MHC pentamer (ProImmune) in a total volume of 100 μL for 30 minutes on ice before analysis by flow cytometry (day 0 results).

Techniques: Labeling

Journal:

Article Title: Inducing Efficient Cross-priming Using Antigen-coated Yeast Particles

doi: 10.1097/CJI.0b013e318181c87f

Figure Lengend Snippet: Peptide/MHC pentamer-positive T cells primed by yeast particles continue to multiply after restimulation with peptide-pulsed autologous PBMCs. On day 10 of the experiment described in Figure 4, PBMCs from each donor were thawed, gamma-irradiated, and pulsed with 40 μM SLLMWITQV peptide for 3 hours. Day 10 T cells from each test condition (5 × 106/well) were cocultured with 107 donor-matched PBMCs in 4 mL CTL medium (with 10 μM peptide) in a 12-well plate. The cells were fed and split as necessary. Flow cytometry analysis after labeling with α-CD8-FITC and PE-conjugated SLLMWITQV/HLA-A*0201 pentamer was performed on day 20. FITC indicates fluorescein isothiocyanate; MHC, major histocompatibility complex; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin.

Article Snippet: In addition, 2 × 10 6 naive T cells from each donor were labeled with 20 μL α-CD8-FITC and 10 μL SLLMWITQV/HLA-A*0201 peptide-MHC pentamer (ProImmune) in a total volume of 100 μL for 30 minutes on ice before analysis by flow cytometry (day 0 results).

Techniques: Irradiation, Flow Cytometry, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: On CD28/CD40 Ligand Costimulation, Common γ -Chain Signals, and the Alloimmune Response 1

doi:

Figure Lengend Snippet: Acquisition of primed phenotype by dividing CD4+ and CD8+ T cells in vivo. A, CFSE-labeled B6AF1 cells were recovered from irradiated DBA/2 hosts 3 days after adoptive transfer. Cells were stained with CyChrome anti-mouse CD4 and anti-CD8 as well as PE-conjugated mAbs against CD25, CD44, and CD62L. Expression of the activation markers was analyzed by gating onto CFSE+CD4+ or CFSE+CD8+ cells. Cells stained with isotype control Abs were used as a control to set up the quadrants for analysis. B, Expression of CD25, CD44, and CD62L in each cell division was analyzed, and the mean fluorescence intensity (MFI) was calculated and plotted against the number of cell divisions. Representative data of five experiments shown.

Article Snippet: The following staining Abs were obtained from BD PharMingen (San Diego, CA): CyChrome anti-mouse CD4 (GK1.5), CyChrome anti-mouse CD8 α (clone 53-6.7), PE anti-mouse CD44 (clone IM7), PE anti-mouse CD62L (L-selectin, clone MEL-14), PE anti-mouse CD25 (clone PC61), PE anti-mouse IL-2 (clone JES6-5H4), PE anti-mouse TNF- α (clone MP6-XT22), PE anti-mouse IFN- γ (clone XMG 1.2), and PE isotype control Abs.

Techniques: In Vivo, Labeling, Irradiation, Adoptive Transfer Assay, Staining, Expressing, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc and treated with VV and/or α-PD-L1 as described. Single cells were made from primary tumours collected from tumour-bearing mice at day 5 post first treatment, blocked with α-CD16/32 Ab and then stained with antibodies against CD45, CD8, CD4, PD-1, ICOS, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and Foxp3 to determine the quantities of CD8 + T cells ( a ), CD8 + T-cell activation ( b – d ), CD8 + T-cell exhaustion ( e – h ), Treg cells ( i ), CD8 + /CD4 + Foxp3 + T cells ( j ) and CD4 + Foxp3 − T cells ( k , l ) in the TME. Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent phenotypic analysis. Data were analysed using Student's t- test (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Staining, Activation Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: ( a ) B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Splenic CD8 + T cells (4 × 10 5 ) were isolated from naive and MC38-luc-bearing mice that received different treatments 18 days post tumour cell injection and restimulated with mitomycin C-treated MC38-luc or B16 cancer cells (4 × 10 4 cells each) in the presence of 4000-rad-irradiated CD8-depleted naive B6 splenocytes (2 × 10 6 ) in 200 μl RPMI-1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 for 2 days. The concentration of IFN-γ in the culture supernatants was tested by ELISA. The statistical analyses were performed with t -test. ( b ) Naive or MC38-luc-bearing B6 mice with dual treatments, which survived for more than 60 days, were s.c. rechallenged with 1 × 10 6 MC38-luc cancer cells. The primary tumour size was measured and presented here. ( c ) In a separate experiment, B6 mice were inoculated with 5 × 10 5 MC38-luc cells i.p. and treated with VV plus α-PD-L1 or PBS at day 5 post tumour inoculation, α-PD-L1 Ab was injected every 2 days for a total of four times. α-CD8 Ab (250 μg per injection), α-CD4 Ab (150 μg per injection) or α-IFN-γ Ab (200 μg per injection) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells or neutralize circulating IFN-γ as scheduled in c , and the overall survival was monitored by Kaplan–Meier analysis and analysed using log rank test ( d ).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Isolation, Injection, Irradiation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: PD-L1/PD-1 crosstalk in colorectal cancer: are we targeting the right cells?

doi: 10.1186/s12885-018-4853-0

Figure Lengend Snippet: Characterisation of infiltrated T-lymphocytes in tumour and peritumour areas in colorectal cancer samples. Percentage of CD3 + cells within tumour (T, grey boxes) and peritumour (PT, white boxes) areas from patients with CRC ( n = 15), as analysed by FACS ( a ). Percentage of CD8 + cells ( b ) and CD4 + cells ( c ) on CD3 + gated populations in A. Expression of PD-1 on CD4 + gated populations in C ( d ). * p < .05, ** p < .01 using a Wilcoxon test

Article Snippet: The following antibodies were used for the FACS analysis: α-CD14, α-CD4, α-CD8, α-CD3 (Immunostep); α-PanK, α-EpCAM, α-PD-1, α-PD-L1, α-CD163, α-CD133, α-CD64, α-EphBR2, α-vimentin (MiltenyiBiotec); α-CD34 (BD Pharmingen); α-CD90 and α-CD45 (Labclinics eBioscience).

Techniques: Expressing

Journal: BMC Cancer

Article Title: PD-L1/PD-1 crosstalk in colorectal cancer: are we targeting the right cells?

doi: 10.1186/s12885-018-4853-0

Figure Lengend Snippet: Proliferative properties of T-lymphocytes cocultured with monocytes/macrophages and colorectal cancer stem cells. Expression of PD-1 by CD4 + ( a ) and CD8 + ( b ) populations within naïve lymphocytes (φ, white boxes) vs. lymphocytes cocultured (T, grey boxes) for 5 days with isolated tumour cells from colorectal tumour samples and monocytes/macrophages. Representative gating strategy to analyse the proliferation of CD4 + T-lymphocytes ( c ) and their proliferation in the presence (+) or not (−) of an α-PD-1 antibody, as measured by CFSE dimming (d). * p < .05, ** p < .01 using a Wilcoxon test

Article Snippet: The following antibodies were used for the FACS analysis: α-CD14, α-CD4, α-CD8, α-CD3 (Immunostep); α-PanK, α-EpCAM, α-PD-1, α-PD-L1, α-CD163, α-CD133, α-CD64, α-EphBR2, α-vimentin (MiltenyiBiotec); α-CD34 (BD Pharmingen); α-CD90 and α-CD45 (Labclinics eBioscience).

Techniques: Expressing, Isolation