Journal: bioRxiv

Article Title: Potentiating the anti-tumor response of tumor infiltrated T cells by NAD + supplementation

doi: 10.1101/2020.03.21.001123

Figure Lengend Snippet: (a) Whole genome sgRNA screens were performed in both Jurkat and primary human T cells to identify genes involved in T cell activation. The top 50 target gene screening hits common to both cell types (green dots). The orange dot represents the NAMPT gene. (b) Metabolic inhibitor screening in Jurkat (JE6) cells. Jurkat cells were pre-treated with a panel of 199 inhibitors (10nM) for 24 hours, followed by anti-TCR stimulation for 16 hours. Cells were then stained with anti-CD69-APC and anti-ICOS-PE-Cy7 for 40min on ice. The surface levels of these markers were measured by flow cytometry. (c-d) Metabolic inhibitor screening in primary human T cells. Isolated human CD8 + T cells (c) and CD4 + T cells (d) were treated with the same panel of inhibitors (100nM) for 24 hours, followed by transfer into well of 96-well plates pre-coated with anti-CD3 and anti-CD28 antibodies and cultured for 16 hours. After stimulation, cells were stained with anti-CD69-APC, anti-CD25-PE, and anti-ICOS-PE-Cy7 for 40min on ice. The surface levels of these markers were measured based on immunofluorescence. (e) Isolated human T cells among PBMCs (both CD8 + and CD4 + ) were pre-treated as indicated (NAD + , FK866 or FK866 + NAD + ) for 24 hours. Cells were then transferred to a plate coated with anti-CD3 and anti-CD28 for stimulation. Cell volumes and cell-surface expression levels of CD25, ICOS, and CD69 were measured using flow cytometry, respectively. (f) Cell surface expression level of CD69 in anti-TCR activated Jurkat cells cultured with the NAD + synthesis inhibitors (FK866 or STF118804) or with NAD + ; Jurkat cells were first treated with inhibitors or NAD + for 24 hours, followed by stimulation with anti-TCR for 16 hours. (g) Monitoring of the ERK phosphorylation level in Jurkat cells; Jurkat cells were treated as indicated (DMSO, FK866, or FK866 + NAD + ) for 24 hours followed by resting in RPMI1640 medium without FBS for 1 hour. Then cells were further stimulated with anti-TCR antibody and collected at the indicated time points and stained with a phospho-ERK specific antibody. (h) Jurkat cells were treated as indicated (DMSO, FK866 or FK866 + NAD + ) for 24 hours, followed by Calcium probe Indo-1 labeling. Cells were pre-incubated with 2× anti-TCR antibody and 2 μM Ionomycin at 37°C for 5 minutes prior to measurement by flow cytometry|. (i) Immunoblot analysis of the levels of phosphotyrosine, phospho-ZAP70, phospho-ERK, phospho-SRC, phospho-LCK, phospho-Zeta in Jurkat cells stimulated with anti-TCR antibody. Jurkat cells were treated as indicated (DMSO, FK866 or FK866 + NAD + ) for 24 hours, followed by resting in RPMI1640 medium without FBS for 1 hour. Then cells were further stimulated with anti-TCR antibody and collected at the indicated time points for immunoblotting. (j to k) NAD + concentration measurement in paired TILs and T cells among PBMCs of ovarian cancer patient samples (j), and paired TILs and T cells in spleen of melanoma mouse allograft tumors (k). For TILs and T in PBMCs of ovarian cancer patient samples, samples were stained with anti-human CD3 antibody and isolated by FACS. For mouse samples, 6-week-old B6 mice were subcutaneously transplanted with 1×10 6 B16F10 mouse melanoma cancer cells. Two weeks later, both tumors and spleens were harvested and analyzed by sorting for, respectively, CD3 + tumor infiltrated (TIL) cells and spleen CD3 + cells. (l) sgRNA screening of NAD + metabolism related genes apparently required for T cell activation. JX003 cells (a single clone selected from Jurkat cells stably expressing Cas9) were first infected with a lentivirus expressing one of 390 sgRNAs targeting each previously annotated NAD + metabolism related gene (94 genes, 4∼5 sgRNA/gene, Table S3). The viral titer used for infection aimed to infect half of the Jurkat cell population (MOI=0.5). After seven days, cells were stimulated with anti-TCR antibody, and changes in the expression level of CD69 were calculated as follows: the CD69 luminescence intensity of infected Jurkat cells divided by the CD69 luminescence intensity of uninfected Jurkat cells.

Article Snippet: In concert with this genetic screen, and aiming to identify the metabolic pathways that are essential for T cell activation, we also undertook chemical screens of both Jurkat T cell and primary human T cells using the 199 compounds of the Metabolism Compound Library (Selleck, L5700) and monitoring T cell activation with multiple markers ( and Extended Data Fig. 1b).

Techniques: Activation Assay, Staining, Flow Cytometry, Isolation, Cell Culture, Immunofluorescence, Expressing, Phospho-proteomics, Labeling, Incubation, Western Blot, Concentration Assay, Stable Transfection, Infection