SUMMARY Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD+-dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD+, enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD+ axis could increase the efficacy of anti-tumor adoptive T cell therapy.
Graphical AbstractHighlights d S1PR-independent intrinsic S1P signaling activates PPARg to skew the Treg/Th17 balance d S1P-PPARg activation inversely correlates with lipolysis and regulates Tcm phenotype d Inhibiting SphK1/S1P/PPARg signaling improves T cellmediated tumor control Correspondence ogretmen@musc.edu (B.O.), mehrotr@musc.edu (S.M.) In BriefChakraborty et al. define the role for SphK1/S1P signaling via engaging lipid transcription factor PPARg to attenuate lipolysis and spare respiratory capacity in T cells. Genetic ablation or pharmacological inhibition of SphK1 expression limits intrinsic S1P levels and improves T cell-mediated anti-tumor immunotherapeutic control. SUMMARYSphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARg (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.(G) Cells obtained using strategy in (F) with different cytokines post day 3 (IL-2 at 100 IU/mL, and IL-15 at 100 ng/mL, respectively) were analyzed for determining the flow cytometry-based cell surface expression of CD62L and intracellular expression of SphK1. (H) Day 3 TCR-activated WT T cells were transfected with either WT vector or empty vector for SphK1 enzyme. Cells were subsequently cultured for another 3 days, either in presence of IL-2 (100 IU/mL), or IL-15 (100 ng/mL). After 3 days, expression of CD62L and SphK1 was evaluated in CD8 + T cells by flow cytometry. (I) Day 3 TCR-activated WT T cells were further cultured for 3 days, either in presence of IL-2 (100 IU/mL) or in presence of TGF-b (5 ng/mL) and IL-2 (100 IU/mL). Cells were then used to determine the intracellular expression of SphK1. Data were analyzed using FlowJo software. The numerical values within the fluorescence-activated cell sorting (FACS) overlay plots indicate MFI, and the adjacent bar diagrams represent cumulative data from at least three repeat experiments. Error bars represent mean ± SD; *p < 0.05. See also Figure S1.
Using miRNA microarray analysis, we identified 31 miRNAs that were significantly up-regulated or down-regulated in colon cancer tissues. We chose MIR196B, which was specifically up-regulated in colon cancer, for further study. We identified 18 putative MIR196B target genes by comparing between the mRNAs down-regulated in MIR196B-overexpressed cells and the assumed MIR196B target genes predicted by public bioinformatics tools. The association between MIR196B and FAS was verified in this study. FAS expression was constitutively elevated in normal human colorectal tissues. However, its expression was often reduced in human colorectal cancer. The decrease in FAS expression could be responsible for the reduction of apoptosis in colorectal cancer cells. In colorectal cancer tissue, we showed that MIR196B up-regulation was mutually followed by down regulation of FAS expression. We also showed that MIR196B directly repressed FAS expression in colorectal cells. Furthermore, anti-MIR196B up-regulated FAS expression and increased apoptosis in colorectal cancer cell lines. Our results suggest that the up-regulation of MIR196B modulates apoptosis in colorectal cancer cells by partially repressing FAS expression and that anti-MIR196B could be a potential candidate as an anti-cancer drug in colorectal cancer therapy.
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