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