Journal: Oncoimmunology
Article Title: Combined targeting of soluble latent TGF-ß and a solid tumor-associated antigen with adapter CAR T cells
doi: 10.1080/2162402X.2022.2140534
Figure Lengend Snippet: A soluble antigen is sufficient to activate AdCAR T cells by forming a tertiary complex with antigen and adapter. (A) Concept of triggering AdCAR T cells with soluble antigens within the TME of solid tumors. (B) AdCAR1 T cells co-expressing the transduction marker LNGFR (LNGFR + ) were incubated for 24 h with 10 ng/mL aLAP with or without 125 ng/mL hLAP. CD69 and CD25 expression was analyzed by flow cytometry. (C) Analysis of cytokine secretion by cytokine multiplex assay. Data plotted with mean ± 1 standard deviation (SD). (D) LNGFR + AdCAR1 T cells were incubated for 90 h with 10 ng/mL aLAP with or without 125 ng/mL hLAP. CAR signaling was analyzed by GFP expression. Integrated GFP intensity was normalized to start values. (E) LNGFR + AdCAR1 T cells were incubated for 24 h with 250 ng/mL of soluble hLAP and indicated concentrations of aLAP. Histograms of CD69 and CD25 expression shown for one exemplary donor. (F) LNGFR + AdCAR1 T cells were co-incubated with indicated amounts of hLAP and 100 ng/mL aLAP for 24 h. MFI values were normalized to T cell activation in absence of soluble hLAP and aLAP. Means are plotted as dashed lines. Data shown were obtained from (n=3) independent donors measured in duplicates if not indicated differently. Statistical analysis was performed with Two-way ANOVA (B) or One-way ANOVA (D). Correction for multiple comparisons was done with Holm-Sidak (D). Illustrations created with BioRender.com.
Article Snippet: After 24 h supernatants were removed for cytokine multiplex analysis (Miltenyi Biotec, catalog no.: 130-099-169), and the activation levels of CAR T cells were quantified by flow cytometry using the respective antibody conjugates.
Techniques: Expressing, Transduction, Marker, Incubation, Flow Cytometry, Multiplex Assay, Standard Deviation, Activation Assay
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