Figure 3.
Figure 3. Ligand-induced c-MPL activation supports sequential killing activity and T-cell expansion in TCR-transgenic T cells. (A) Serial coculture with BV173 leukemia cells, E:T 1:5. Residual BV173 cells (left) and T cells (right) were quantified by FACS every 3 to 4 days from a total of 8 replicate wells per donor, and BV173 cells were added-back to untouched wells (+) at each point. Cultures in noCK (gray circles), IL-2 25 U/mL (purple squares), TPO 5 ng/mL (red squares), TPO 50 ng/mL (blue triangles), EP 0.1 μg/mL (green triangles), plate-bound anti-CD28 (orange triangles, dotted line), IL-2 (25 U/mL) + plate-bound anti-CD28 (purple diamond, dotted line); n = 3 for IL-2, anti-CD28, IL-2+anti-CD28, TPO5, EP; n = 6 for noCK, TPO50. Lines of individual donors are shown for tumor cell counts; mean ±SD for T-cell counts. (Left) Serial killing activity was analyzed by Kaplan-Meier analysis: overall, P < .0001; noCK vs TPO5, P = .007; noCK vs TPO50, P < .0001; noCK vs EP, P = .003; noCK vs IL-2, P < .0001; noCK vs aCD28, P = .038; noCK vs aCD28+IL-2, P < .0001. TPO50 vs IL-2, P = NS; TPO50 vs aCD28, P = .003; TPO50 vs aCD28+IL-2, P = NS. (Right) T-cell expansion in noCK vs TPO5, P = NS; noCK vs TPO50, P = .003; noCK vs EP, P = NS; noCK vs IL-2, P = .001; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .001. TPO5 vs TPO50, P = .02; TPO5 vs EP, P = NS; TPO50 vs EP, P = .03; TPO50 vs IL-2, P = NS; TPO50 vs aCD28+IL-2, P = NS. Student t test on log area under the curve. (B) Cytokine levels in coculture supernatants 24 hours after tumor cell challenge for the first, third, fifth, and seventh tumor challenge on days 1, 8, 15, and 22 of coculture, respectively; n = 3; mean ± SD, analyzed in duplicates. Student t test on log transformed data (days 1, 8), 1 sample Student t test compares with null hypothesis of 0 on log transformed data (days 15, 22). *P < .05, **P < .01. (C-D) T-cell phenotype of CD3+CD8+TCR+c-MPL+ T cells recovered from cocultures at each time-point; n = 3-6 (as in A); mean ± SD. (C) Percentages of CD45RA+CD45RO+ cells. No CK vs TPO5, P = .002; noCK vs TPO50, P < .0001; noCK vs EP, P = .002; noCK vs IL-2, P = .002; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .004. TPO50 vs IL-2, P = .05; TPO50 vs aCD28, P < .0001; TPO50 vs aCD28+IL-2, P = .005. Student t test on log area under the curve. (D) Naive, central memory (CM), effector memory (EM), and effector T cells. Naïve: P = NS, except noCK vs TPO50, P = .05; noCK vs aCD28+IL-2, P = .05; CM: noCK vs TPO5, P = .003; noCK vs TPO50, P < .0001; noCK vs EP, P = .002; noCK vs IL-2, P = .005; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .009. TPO50 vs IL-2, P = NS; TPO50 vs aCD28, P = .001, TPO50 vs aCD28+IL-2, P = .03. EP vs aCD28+IL-2, P = .04; Student t test on log area under the curve day 14.

Ligand-induced c-MPL activation supports sequential killing activity and T-cell expansion in TCR-transgenic T cells. (A) Serial coculture with BV173 leukemia cells, E:T 1:5. Residual BV173 cells (left) and T cells (right) were quantified by FACS every 3 to 4 days from a total of 8 replicate wells per donor, and BV173 cells were added-back to untouched wells (+) at each point. Cultures in noCK (gray circles), IL-2 25 U/mL (purple squares), TPO 5 ng/mL (red squares), TPO 50 ng/mL (blue triangles), EP 0.1 μg/mL (green triangles), plate-bound anti-CD28 (orange triangles, dotted line), IL-2 (25 U/mL) + plate-bound anti-CD28 (purple diamond, dotted line); n = 3 for IL-2, anti-CD28, IL-2+anti-CD28, TPO5, EP; n = 6 for noCK, TPO50. Lines of individual donors are shown for tumor cell counts; mean ±SD for T-cell counts. (Left) Serial killing activity was analyzed by Kaplan-Meier analysis: overall, P < .0001; noCK vs TPO5, P = .007; noCK vs TPO50, P < .0001; noCK vs EP, P = .003; noCK vs IL-2, P < .0001; noCK vs aCD28, P = .038; noCK vs aCD28+IL-2, P < .0001. TPO50 vs IL-2, P = NS; TPO50 vs aCD28, P = .003; TPO50 vs aCD28+IL-2, P = NS. (Right) T-cell expansion in noCK vs TPO5, P = NS; noCK vs TPO50, P = .003; noCK vs EP, P = NS; noCK vs IL-2, P = .001; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .001. TPO5 vs TPO50, P = .02; TPO5 vs EP, P = NS; TPO50 vs EP, P = .03; TPO50 vs IL-2, P = NS; TPO50 vs aCD28+IL-2, P = NS. Student t test on log area under the curve. (B) Cytokine levels in coculture supernatants 24 hours after tumor cell challenge for the first, third, fifth, and seventh tumor challenge on days 1, 8, 15, and 22 of coculture, respectively; n = 3; mean ± SD, analyzed in duplicates. Student t test on log transformed data (days 1, 8), 1 sample Student t test compares with null hypothesis of 0 on log transformed data (days 15, 22). *P < .05, **P < .01. (C-D) T-cell phenotype of CD3+CD8+TCR+c-MPL+ T cells recovered from cocultures at each time-point; n = 3-6 (as in A); mean ± SD. (C) Percentages of CD45RA+CD45RO+ cells. No CK vs TPO5, P = .002; noCK vs TPO50, P < .0001; noCK vs EP, P = .002; noCK vs IL-2, P = .002; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .004. TPO50 vs IL-2, P = .05; TPO50 vs aCD28, P < .0001; TPO50 vs aCD28+IL-2, P = .005. Student t test on log area under the curve. (D) Naive, central memory (CM), effector memory (EM), and effector T cells. Naïve: P = NS, except noCK vs TPO50, P = .05; noCK vs aCD28+IL-2, P = .05; CM: noCK vs TPO5, P = .003; noCK vs TPO50, P < .0001; noCK vs EP, P = .002; noCK vs IL-2, P = .005; noCK vs aCD28, P = NS; noCK vs aCD28+IL-2, P = .009. TPO50 vs IL-2, P = NS; TPO50 vs aCD28, P = .001, TPO50 vs aCD28+IL-2, P = .03. EP vs aCD28+IL-2, P = .04; Student t test on log area under the curve day 14.

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