Hypothesis

TET2-mediated deposition of 5-hydroxymethylcytosine (5hmC) at promoters and enhancers of autophagy genes increases their transcription, thereby elevating autophagic flux during chronic antigen exposure. This heightened autophagy acts as a rationing mechanism that degrades mitochondria and key metabolic enzymes, limiting bioenergetic capacity and pushing CD8+ T cells from a stem‑like progenitor state toward terminal exhaustion. Conversely, loss of TET2 reduces 5hmC at these loci, dampens autophagy, preserves mitochondrial fitness, and sustains TCF1+ stem‑like exhausted cells.

Mechanistic Rationale

1. Epigenetic priming – TET2 oxidizes 5mC to 5hmC at CpG islands of autophagy regulators (e.g., ATG5, ATG7, LC3B, TFEB). 5hmC is known to recruit transcription factors that favor open chromatin, leading to higher mRNA expression.
2. Metabolic consequence – Increased autophagy accelerates turnover of mitochondrial mass (mitophagy) and consumes amino acids via lysosomal degradation, reducing ATP production and mTORC1 signaling. Low mTORC1 activity favors a catabolic state associated with exhaustion.
3. Feedback to exhaustion program – Diminished mitochondrial output lowers acetyl‑CoA levels, decreasing histone acetylation at stemness loci (TCF7, LEF1) while permitting sustained expression of inhibitory receptors (PD‑1, TIM3, LAG3) driven by TET2‑dependent enhancers.
4. Reversibility – Pharmacological inhibition of autophagy (chloroquine) or genetic ablation of essential autophagy genes (ATG5) should rescue the exhausted phenotype in TET2‑sufficient cells, whereas rapamycin‑induced autophagy should exacerbate exhaustion in TET2‑deficient cells.

Experimental Plan

• 5hmC mapping – Perform hMeDIP‑seq or oxidative bisulfite sequencing on CD8+ T cells isolated from chronic LCMV infection at day 8 (peak effector) and day 30 (exhausted). Compare 5hmC levels at autophagy gene promoters between WT and TET2‑KO cells.
• Transcriptional readout – Quantify mRNA and protein of ATG5, LC3B, TFEB by qPCR and flow cytometry in the same samples.
• Flux assay – Measure autophagic flux using LC3‑II turnover with bafilomycin A1 or mitochondrial mass with MitoTracker‑Red after lysosomal inhibition.
• Functional rescue – Treat WT exhausted T cells with chloroquine (50 µM) or transduce with ATG5 shRNA; assess PD‑1, TIM3, LAG3 expression and proliferative capacity after secondary antigen challenge.
• Epigenetic editing – Use dCas9‑TET1 catalytic domain to specifically increase 5hmC at the ATG5 promoter in naïve T cells; evaluate whether this alone drives autophagy up‑regulation and accelerates exhaustion markers.
• In vivo validation – Transfer WT or TET2‑KO P14 CD8+ T cells into LCMV‑Clone13 mice; administer autophagy modulators and track viral control, cytokine production, and memory formation.

Predictions & Falsifiability

• If TET2 directly augments autophagy via 5hmC, then loss of TET2 will produce a measurable decrease in 5hmC at autophagy loci, reduced LC3‑II flux, higher mitochondrial mass, and a sustained TCF1+ phenotype despite chronic antigen.
• If autophagy modulation does not alter exhaustion markers in TET2‑deficient cells, the hypothesis is falsified.
• Conversely, forced increase of 5hmC at autophagy genes should phenocopy TET2‑sufficient exhaustion even when TET2 is genetically absent.

This framework links TET2‑driven epigenetic remodeling to a metabolic rationing system, positioning autophagy not as a generic cleanup process but as a decisive gatekeeper of T‑cell fate during persistent infection.