Journal: The Journal of Clinical Investigation

Article Title: Selective SIRP α blockade reverses tumor T cell exclusion and overcomes cancer immunotherapy resistance

doi: 10.1172/JCI135528

Figure Lengend Snippet: (A) Representative IFN-γ response of human HLA-A2/melan-A–specific CD8+ T clones stimulated 6 hours with human HLA-A2+ DCs crosspresenting the long peptide melan-A/MART-1 and preincubated for 24 hours with the peptide and 10 μg/mL of different mAbs targeting the SIRPα/SIRPγ/CD47 pathway. (B) Quantification of IFN-γ response as in A normalized to the irrelevant control mAb condition for each DC donor. (C) IL-2 secretion by mouse chimeric thymoma cell line transfected with human CD8 and the HLA-A2/melan-A TCR cultured for 48 hours with human HLA-A2+ DC loaded with melan-A long peptide and cultured with indicated mAbs, as in A. (D) Proliferation of isolated human T cells from human PBMCs cultured with 10 μg/mL of indicated mAbs for 3 days with anti-CD3/anti-CD28–coated beads or (E) for 5 days with allogeneic LPS-matured human DCs. Proliferation measured by H3-thymidine incorporation was normalized under control conditions. (F) Human PBMCs from allogeneic healthy donors were cultured 5 days with indicated mAbs at 10 μg/mL. T cell proliferation was assessed by H3-thymidine incorporation, and TNF-α and IFN-γ secretion was quantified by ELISA and normalized to controls. (G) Human T cell migration across a monolayer of TNF-α–activated endothelial cells with 10 μg/mL of indicated mAbs and CXCL-12 (50 ng/mL) as chemoattractant. The number of transmigrated T cells was determined by flow cytometry after 4 hours and normalized under control conditions. *P < 0.05; **P < 0.01; ****P < 0.001, between indicated conditions, unpaired Kruskall-Wallis test, Mann-Whitney U test. ####P < 0.001, compared with control Ab conditions.

Article Snippet: Mixed lymphocyte reaction was assessed by incubating fresh PBMCs with irradiated allogeneic PBMCs from different donors for 5 days with Abs at 10 μg/mL, T cell activation was assessed by thymidine incorporation as mentioned before, and TNF-α and IFN-γ secretion were evaluated in the supernatant by ELISA (BD Biosciences).

Techniques: Clone Assay, Transfection, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Migration, Flow Cytometry, MANN-WHITNEY

Journal:

Article Title: CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

doi: 10.1172/JCI35449

Figure Lengend Snippet: (A) Five clones isolated from a patient with type 1 diabetes stained with anti-CD8 and HLA-A2 tetramers loaded with PPI15–24. Tetramer-stained cells were detected in the upper-right quadrant. A CD8+ T cell clone obtained in the same expansion that was negative for PPI15–24–Tmr (3C9) and a clone (2D9) raised against the CMV pp65 epitope NLVPMVATV are shown. (B) Staining of the same clones with HLA-A2 tetramers loaded with the CMV pp65 epitope NLVPMVATV. (C) PPI15–24–specific T cell clones produce TNF-α in a dose-dependent fashion, shown here as the percentage of 1E6 clone cells staining for intracellular cytokine in response to HLA-A2+ PBMCs pulsed with varying concentrations of PPI15–24. (D) 1E6 clone cells proliferate in response to PPI15–24 peptide-pulsed monocyte-derived DCs, as measured by [3H]thymidine incorporation. No response was observed using antigen-presenting cells lacking HLA-A2 (data not shown). (E) 1E6 clone cells also recognize the PPI15–24 epitope when naturally processed by K562-PPI-A2 cells, since no TNF-α was produced after coculture with K562-A2 cells, but in the presence of PPI15–24 peptide-pulsed K562-A2 (1 μg/ml) or K562-PPI-A2 cells, copious amounts of cytokine were produced. Isotype control staining was similar to control-activated clone cells. Proliferation assays were performed in triplicate; bars represent means, error bars are SEMs. Values inside flow cytometry quadrants indicate percentage of positive cells. Data are representative of more than 5 individual experiments.

Article Snippet: Functional analysis of clones was carried out after stimulation with peptide-pulsed DCs, PBMCs, and variously transfected K562 cell lines, using [ 3 H]thymidine incorporation, TNF-α secretion (measured by ELISA from ImmunoTools), or intracellular TNF-α and IFN-γ expression (measured by flow cytometry).

Techniques: Clone Assay, Isolation, Staining, Derivative Assay, Produced, Flow Cytometry

Journal:

Article Title: CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

doi: 10.1172/JCI35449

Figure Lengend Snippet: (A) Agarose gel showing resolution of products of semiquantitative RT-PCR amplification using insulin- and glucagon-specific primers from total RNA extracted from human islet cells cultured for 14 hours in the presence of 5.6, 11, and 20 mM glucose. Results show increasing insulin mRNA synthesis with increasing glucose concentration. (B) Expressed relative to the density of the β-actin gene PCR product, insulin expression is greater than 4-fold higher at 20 mM compared with 5.6 mM glucose. There is an expected reduction (~2-fold) in glucagon expression at 20 mM compared with 5.6 mM glucose due to the known suppression of glucagon secretion by high glucose and insulin concentrations (55–57). (C) Coculture of islet cells at different glucose concentrations with 1E6 PPI15–24–specific T cell clone results in a significant increase (1-way ANOVA, P < 0.01) in TNF-α production by the clone, with a significant linear trend as glucose concentration increases (P < 0.005), indicating an increase in epitope presentation at higher glucose concentration. (D) A similar analysis of islets precultured at different glucose concentrations and prepulsed with PPI15–24 peptide shows that high glucose alone has no effect on T cell clone activation. Symbols indicate different effector/target ratios of 3:1 (circles), 6:1 (diamonds), 12:1 (inverted triangles), and 25:1 (triangles).

Article Snippet: Functional analysis of clones was carried out after stimulation with peptide-pulsed DCs, PBMCs, and variously transfected K562 cell lines, using [ 3 H]thymidine incorporation, TNF-α secretion (measured by ELISA from ImmunoTools), or intracellular TNF-α and IFN-γ expression (measured by flow cytometry).

Techniques: Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Cell Culture, Concentration Assay, Expressing, Activation Assay

Journal:

Article Title: CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

doi: 10.1172/JCI35449

Figure Lengend Snippet: (A) DCs pulsed with soluble PPI and then matured induce TNF-α production by 1E6 PPI15–24–specific T cells. Cytokine production in the presence of a control protein (the fusion partner of recombinant human PPI) and PPI15–24 peptide are shown for comparison. (B) DCs pulsed with freeze-thawed K562 cells expressing PPI (K562-PPI) and then matured induce TNF-α production by 1E6 clone cells. Cytokine production in the presence of control cells (nontransfected K562) and PPI15–24 peptide are shown for comparison. Bars represent means from triplicate experiments, error bars SEMs; data are representative of 3 independent experiments.

Article Snippet: Functional analysis of clones was carried out after stimulation with peptide-pulsed DCs, PBMCs, and variously transfected K562 cell lines, using [ 3 H]thymidine incorporation, TNF-α secretion (measured by ELISA from ImmunoTools), or intracellular TNF-α and IFN-γ expression (measured by flow cytometry).

Techniques: Recombinant, Expressing