Nrf2 regulates activation driven-expansion of CD4+T-cells by differentially modulating glucose and glutamine metabolism [ATAC-Seq]

Upon antigenic stimulation, CD4+T-cells undergo clonal expansion elevating their bioenergetic demands and utilization of nutrients like glucose and glutamine The nuclear factor erythroid 2-related factor2 (Nrf2) is a well-known regulator of oxidative stress, but its involvement in modulating the metabolism of CD4+T-cells remain unexplored. We report that Nrf2 protein levels are temporally regulated in activated CD4+T-cells, with elevated expression during early activation followed by a decline. T-cell-specific constitutive activation of Nrf2 by deletion of its regulator Keap1, enhances early activation and IL-2 secretion, upregulates TCR-signaling, and increases activation-driven expansion of CD4+T-cells. Metabolically, high Nr2 alters glucose metabolism and promotes glutamine metabolism via glutaminolysis to support increased CD4+T-cell proliferation. Mechanistically, elevated Nrf2 activity in activated CD4+T-cells leads to increased chromatin accessibility and proliferation-associated gene expression. In summary, we elucidate the role of Nrf2 beyond traditional antioxidation in modulating the activation-driven expansion of CD4+T-cells by influencing their nutrient metabolism. Overall design: Stimulated CD4+T-cells from WT and Keap1-KO mice were harvested after 36 h. Cells were transferred to a 15mL or 50mL conical tube and centrifuged at 500 x g for 5 minutes. The media was aspirated, and cells were resuspended in 10mL of pre-warmed growth media, followed by the addition of 1:100 volume of 100x DNase buffer and DNase solution (250mM MgCl2 and 50mM CaCl2 in dH2O). Incubated the cells at 37°C for 30 minutes and centrifuged again at 500 x g for 5 minutes at 4°C, resuspended in 5mL ice-cold PBS (or 500µL for Eppendorf tubes), and 300,000 cells were quantified using a hemocytometer for cryopreservation. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37oC water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al. 2013), with some modifications based on (Corces et al. 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the ATAC-Seq Kit (Active Motif cat# 53150). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the iSeq (Illumina) and sequenced with PE42 sequencing on the NovaSeq 6000 sequencer (Illumina). Reads were aligned using the BWA algorithm (mem mode; default settings). Duplicate reads were removed, only reads mapping as matched pairs and only uniquely mapped reads (mapping quality >= 1) were used for further analysis. Alignments were extended in silico at their 3'-ends to a length of 200 bp and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peaks were identified using the MACS 2.1.0 algorithm at a cutoff of p-value 1e-7, without control file, and with the –nomodel option. Signal maps and peak locations were used as input data to Active Motif's proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations, and gene annotations.