Integrated Metabolomic and Transcriptomic Profiling Reveals Novel Activation Induced Metabolic networks in Human T cells

The targeting of metabolic pathways is emerging as an exciting new approach for modulating immune cell function and polarization states. In this study, carbon tracing and systems biology approaches integrating metabolomic and transcriptomic profiling data were used to identify adaptations in human T cell metabolism important for fueling pro-inflammatory T cell function. Results of this study demonstrate that T cell receptor (TCR) stimulation leads to a significant increase in glucose and amino acid metabolism that trigger downstream biosynthetic processes. Specifically, increased expression of several enzymes such as CTPS1, IL4I1, and ASL results in the reprogramming of amino acid metabolism. Additionally, the strength of TCR signaling resulted in different metabolic enzymes utilized by T cells to facilitate similar biochemical endpoints. Furthermore, this study shows that cyclosporine represses the pathways involved in amino acid and glucose metabolism, providing novel insights on the immunosuppressive mechanisms of this drug. To explore the implications of the findings of this study in clinical settings, conventional immunosuppressants were tested in combination with drugs that target metabolic pathways. Results showed that such combinations increased efficacy of conventional immunosuppressants. Overall, the results of this study provide a comprehensive resource for identifying metabolic targets for novel combinatorial regimens in the treatment of intractable immune diseases. Primary human CD4+ T cell isolation, culture, and activation Blood was collected from healthy human patients in accordance with IRB standards. CD4+ T cells were purified using RosetteSep isolation kit (StemCell) as per manufacturer’s protocol. Cells were cultured in RPMI supplemented with 10% heat inactivated FBS, HEPES, penicillin/streptomycin, non-essential amino acids, glutamax, and sodium pyruvate. Cells were initially activated for 72 hours on plate-bound CD3 (200 ng/mL; OKT3; BioXCell) and CD28 (200 ng/mL; 9.3; BioXCell) on goat-anti-mouse (10 µg/mL; Jackson Immuno Research Labs) pre-coated high- binding plates (Corning Costar). After 72 hours, cells were removed from plate activation and were expanded until Day 8-12, maintained at ~1X106 cells/mL by supplementing with fresh media as required. Cells were then washed with PBS and either rested or re-activated at 0.5-1x106 cells/mL in fresh media with plate-bound CD3/CD28 (100 ng/mL each) or with soluble CD3/CD28 Immunocult (1:100 dilution; StemCell). Cells were treated with Rapamycin (100nM) and/or Cyclosporin A (2µM), or equivalent volume of DMSO during re-activation. Lentiviral transduction and shRNA knockdown Lenti-X 293T (Clontech) cells were plated on poly-D-lysine (0.1 mg/mL; Sigma) coated plates at a density of 1.67x105 cells/mL. After 72 hours, cells were transfected with psPAX2 (1.5µg), pCMV-VSV-G (0.5µg), and pZIP-mCMV-ZsGreen-shRNA NT Control or mTOR plasmids (2 µg; Transomic) using the TransIT-LT1 transfection reagent (Mirus) as per manufacturer’s protocol. Virus was collected after an additional 72 hours and CD4+ T cells were spin infected after 48 hours of activation using undiluted virus (~1x108 IFU yielding a MOI of ~70) supplemented with polybrene (10 µg/mL; AmericanBio Inc). After an additional 24 hours of activation, the transduced CD4+ T cells were removed from activation and expanded in the presence of puromycin (1 µg/mL) until Day 8-12, maintained at ~1x106 cells/mL by supplementing with fresh media as required. Cells were then washed and activated as described above. CRISPR/Cas-9 knockout Cells were activated on plate-bound CD3/CD28 as described above. Cells were removed from plate activation after 72 hours and were rested overnight at ~1X106 cells/mL. The following day the conditioned media was saved before washing the cells and resuspending in buffer T (Neon Transfection System; ThermoFisher) at 25x106 cells/mL. Guides targeting human mTOR and ZAP70 were designed using the Broad Institute’s online sgRNA designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)22 (see Sup Table 1). 5 guides per gene were synthesized by IDT and duplexed in a 1:1 ratio with Alt-R-CRISPR-Cas9-tracrRNA (IDT) as per manufacturer’s protocol. Duplexed guides were incubated with Alt-R S.p. Cas9 Nuclease (IDT) before mixing with 2.5x106 cells and electroporating using the Neon Transfection System (ThermoFisher; 100uL tip at 1550V, 10 milliseconds, 3 pulses). Cells were allowed to recover for 1 hour in fresh media before adding back conditioned media in a 1:1 ratio conditioned to fresh media. Cells were expanded in the presence of IL-2 (2 ng/mL; R&D) until Day 8-12, maintained at ~1x106 cells/mL by supplementing with fresh media as required. Cells were then washed and activated as described above.