Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 4. FD and IL15 fusion protein (FDþ15) further enhance T-cell activity. T cells were retrovirally transduced to coexpress CAR PSCA and FDþ15 and then used in experiments to assess their function. A, Schematic diagram illustrating the FD and IL15 fusion (FDþ15) transgene. B and C, T-cell transduction to assess the expression of CAR PSCA, CAR PSCA, and FD, or CAR PSCA and FDþ15 was measured by flow cytometry 5 days after transduction. Flow cytometry density plots for a representative donor (B) and summary data indicating expression of the CAR PSCA, CAR.FD, and CAR.FDþ15 transgenes (mean SEM, n ¼ 8; C). D, Secretion of FDþ15 molecule by activated T cells was measured using Fas and IL15 ELISAs to separately measure the two components of the fusion molecule. Bar graphs demonstrate the concentration of Fas and IL15 in supernatant obtained from CAR.FDþ15 T cells cultured in the absence or presence of recombinant PSCA (mean SEM, n ¼ 4). Significance was calculated by unpaired t tests. E, Expansion of CAR PSCA, CAR.FD, and CAR.FDþ15 T cells stimulated with irradiated K562-PSCA tumor cells was monitored for 6 days using a flow cytometer (mean SEM, n ¼ 4). Statistical significance was calculated using one-way ANOVA. F and G, Antitumor activity of CAR PSCA, CAR.FD, and CAR.FDþ15 T cells was measured against CAPAN-1-PSCA tumor cells in a coculture assay (E:T of 1:10). Tumor as well as T-cell numbers were quantified by flow cytometry. Starting cell numbers (day 0) were used as a reference to calculate fold change in cell numbers for all time points. Graphs illustrate fold change in tumor cell numbers (F) and T-cell numbers (G) over time (mean SEM, n ¼ 4). Statistical significance was calculated using one-way ANOVA. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: Briefly, supernatant samples harvested from T cells activated in 24-well non-TC plates coated with recombinant PSCA antigen (CUSABIO; catalog no. CSB-YP018840HU) at indicated concentrations or irradiated K562-PSCA cells at indicated E:T. To measure the production of FDþ15 fusionmolecule, samples were used in soluble Fas and IL15 ELISAs (R&D Systems, catalog no. DY247) to measure the two components separately.

Techniques: Activity Assay, Transduction, Expressing, Cytometry, Flow Cytometry, Concentration Assay, Cell Culture, Recombinant, Irradiation, Co-culture Assay