Figure 2.

mTOR and JAK but not Src inhibitors prevent CD19-TCB-induced cytokine release while retaining tumor cell killing and T cell activation. (A) PBMCs were stimulated on CTV-labeled SU-DHL-8 tumors cell by CD19-TCB in the presence of Src inhibitor (dasatinib), mTOR inhibitors (sirolimus, temsirolimus, everolimus) and JAK inhibitors (ruxolitinib, tofacitinib, baricitinib). (B) Effect of JAK, mTOR and Src inhibitors on CD19-TCB-induced killing of SU-DHL-8 tumor cells. At 24 hours, technical replicates were pooled and stained with Live/Dead NIR, allowing measurement of dead SU-DHL-8 cells by flow cytometry by gating on dead NIR positive cells. The EC50 values of each individual killing curve for n=3 donors are summarized in the bar plot (except for dasatinib, where EC50 values could not be calculated). (C) The levels of CD69 and CD25 on CD4⁺ and CD8⁺ T cells induced by CD19-TCB were measured by flow cytometry (24 hours). (B–C) Mean of n=3 donors+SD with *p≤0.05, **p≤0.01 by one-way ANOVA (Friedman test). (D) The culture supernatants were pooled and the levels of TNF-α, IFN-γ, IL-2, IL-6 and GM-CSF were measured by Luminex (24 hours), mean of n=3 donors±SD. The statistical difference to CD19-TCB treatment are summarized in the table with *p≤0.05, **p≤0.01, ***p≤0.001 by one-way ANOVA (Friedman test). ANOVA, analysis of variance; CTV, Cell Trace Violet; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; PBMCs, peripheral blood mononuclear cells; TCB, T cell bispecific antibody; TNF, tumor necrosis factor.