c-MPL is functional in survivin-specific TCR-transgenic T cells and enhances antitumor function in vitro. (A) Transduction efficiencies of CD8⁺ activated T cells with survivin-TCR alone (murine constant β chain, mCβ) or in combination with c-MPL. Representative FACS plots (left) and summary (right), n = 13; mean ± SD. (B) TCR⁺c-MPL⁺ T cells expand upon stimulation with survivin peptide pulsed artificial antigen-presenting cells in a TPO dose-responsive manner (right). TCR⁺ T cells only expand in IL-2, but not high-dose TPO (left, n = 6), except for noCK condition (n = 3; mean ± SD). TCR⁺ T cells at end S2: noCK vs IL-2, P = .003; noCK vs TPO500, P = NS. TCR⁺c-MPL⁺ T cells at end S2: noCK vs IL-2, P < .001; noCK vs TPO5, P = NS; noCK vs TPO50, P = .02; noCK vs TPO500, P < .001; IL-2 vs TPO500, P = NS; Student t test. (C) c-MPL⁺ T cells expand in eltrombopag in a dose-responsive manner during activation with OKT3 and CD28 antibodies, NT T cells only expand in IL-2 50 U/mL, analyzed on day 7. One representative of 3 donors. (D) c-MPL ligand (TPO or EP) induced phosphorylation of STAT3 and STAT5 at 1 hour (left) and 24 hours (right). (E) Coculture of expanded NT, TCR⁺, or TCR⁺c-MPL⁺ T cells with U266 myeloma cells (HLA-A*0201⁺survivin⁺) in noCK (gray circles), TPO 5 ng/mL (red squares), TPO 50 ng/mL (blue triangles), or IL-2 25 U/mL (purple squares); effector:target ratio, E:T 1:1. Residual U266 cells (left) and T cells (right) were quantified by FACS on day 5; n = 3; mean ± SD. (F) Coculture with BV173 leukemia cells (HLA-A*0201⁺survivin⁺), E:T 1:3. Residual BV173 cells (left) and T cells (right) were quantified by FACS on day 5; n = 7 for noCK, TPO5, and TPO50; n = 3 for IL-2; mean ± SD. (E, F) *P < .05, **P < .01, ***P < .001, Student t test on log transformed data. NS, not significant.