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Metabolic reprogramming of T cells to optimize adoptive T cell therapyWaller, Alexandra 18 June 2019 (has links)
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.
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Localized Heat Therapy Improves Mitochondrial Function in Human Skeletal MuscleMarchant, Erik D. 15 April 2022 (has links)
Physical activity results in various types of stress in skeletal muscle including energetic, oxidative, and heat stress. Acute exposure to stress impairs skeletal muscle mitochondrial function. In contrast, chronic intermittent exposure to mild stress through exercise training results in increased mitochondrial content and respiratory capacity. While oxidative and energetic stress have received much attention regarding their long-term effect on skeletal muscle mitochondria, heat stress is not well understood. The purpose of this work was to investigate the effects of localized heat therapy on human skeletal muscle mitochondria, and to compare these effects to those of high-intensity interval exercise training. To accomplish this purpose, 35 subjects were assigned to receive 6 weeks of sham therapy, heat therapy, or exercise training; all localized to the quadriceps muscles of the right leg. Two-hour sessions of short-wave diathermy were used for the heat therapy, and identical sessions were used for sham therapy, but the diathermy units were not activated. Forty-minute sessions of single-leg extension, high-intensity interval training were used for the exercise intervention. All interventions took place three times per week. Muscle biopsies were performed at baseline, and after three and six weeks of intervention. Muscle fiber bundles were isolated and permeabilized for measurement of oxygen consumption via high-resolution respirometry. The primary finding of this work was that heat therapy improves mitochondrial respiratory capacity by 24.8 ± 6.2% compared to a 27.9 ± 8.7% improvement following exercise training. Both heat and exercise significantly increased mitochondrial respiration compared to baseline measures (p<0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p<0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. Neither heat nor exercise training increased mitochondrial respiratory protein content. Overall these results suggest that heat therapy significantly improves mitochondrial function, but not to the same degree as exercise training.
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