T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.

• MeSH
• dítě MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• interleukin-7 * farmakologie   MeSH
• lidé MeSH
• lymfoblastická leukemie-lymfom z prekurzorových T-buněk * farmakoterapie   metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• recidiva MeSH
• T-lymfocyty metabolismus   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

• MeSH
• aktivace lymfocytů účinky léků   genetika   MeSH
• buněčné kultury metody   MeSH
• buňky PC-3 MeSH
• chimerické antigenní receptory genetika   metabolismus   MeSH
• elektroporace MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• imunoterapie adoptivní metody   MeSH
• interleukin-4 farmakologie   MeSH
• interleukin-7 farmakologie   MeSH
• interleukiny farmakologie   MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• proteinové inženýrství metody   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• receptory antigenů T-buněk genetika   metabolismus   MeSH
• T-lymfocyty * cytologie   účinky léků   imunologie   metabolismus   MeSH
• transdukce genetická metody   MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH