The clinical efficacy of adoptive T cell therapies including CAR T therapy are limited by poor in vivo persistence and moderate anti-tumor efficacy. According to the literature, metabolism plays a critical role in the phenotypic state and fate of T cells during antigen-driven expansion. During different stages of a T cell life cycle, the predominant pathway used for metabolism changes. Naïve T rely on oxidative phosphorylation, but as the T cells becomes activated, their metabolic profile switches to become more reliant on glycolysis. Most T cells become terminally differentiated and become senescent once they have performed their cytotoxic function. A minority of the activated T cells gradually start to rely on oxidative phosphorylation once again and become memory T cells. Memory T cells can become either effector memory or central memory T cells. These memory T cells, specifically central memory T cells, are the key to T cells persistence during both ex vivo and in vivo expansion and following disappearance of the antigenic stimulus. Since the metabolic profile of the T cells plays a critical role in its differentiation state, we tested the hypothesis that inhibitors of intermediary metabolism could promote a metabolic profile that is more desirable for the optimal phenotype consistent with the memory phenotype that would favor persistence in spite of strong activation signals. The four inhibitors screened were: a PFKFB3 inhibitor, an inhibitor of a key step in glycolysis; ibrutinib, an inhibitor of Bruton’s tyrosine kinase; idelalisib, an inhibitor of PI3K subunit; and duvelisib, an inhibitor of PI3K and PI3K gamma subunits. To test this hypothesis, T cells were cultured with or without each compound and then the analysis included: phenotypic analysis by flow cytometry, quantitative analysis by counting cells with ethidium bromide acridine orange, and metabolic profiling by the Seahorse assay.
This study was conducted using T cells from a human healthy volunteer that were collected by apheresis. T cells were cultured in a G-Rex plate for 15 days with complete media supplemented with recombinant human IL-2 (30 U/mL). Cells were activated on day 1 and day 8 by the addition of anti-CD3/CD28 beads and test metabolic inhibitor compounds were added every 4 days.
T cells cultured with idelalisib, duvelisib, and ibrutinib had increased expansion (approximately50-fold: idelalisib/ duvelisib and 21-fold ibrutinib) when compared to control (cells with beads alone) with only 6-fold expansion. Phenotypic analysis performed using flow cytometry showed an increased percentage of CD27+ CD28+ in the CD8+ and CD4+ T cell cell populations in the idelalisib treated group and decreased number of senescent T cells that are double negative for CD27 and CD28. Consistent with our hypothesis, metabolic analysis showed that cells treated with idelalisib and duvelisib were more reliant on oxidative phosphorylation, rather than glycolysis as compared to the control cultures. Cells treated with duvelisib also showed an increased spare respiratory capacity (SRC), which is associated with more efficacious memory T cells.
The results of these studies show that metabolism plays a critical role in the long-term survival of T cells. We demonstrate that inhibiting intermediary metabolism, specifically inhibiting PI3K, favorably alters the metabolic state of the T cells leading to increased cell numbers and T cells with a phenotype consistent with enhanced ex vivo and in vivo proliferation and persistence.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/36720 |
| Date | 18 June 2019 |
| Creators | Waller, Alexandra |
| Contributors | Gerstenfeld, Louis C., Waller, Edmund K. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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