Hypothesis

Simultaneous inhibition of the NLRP3 inflammasome and pharmacological activation of AMPK during the first two weeks after antigen exposure will preserve the naïve CD8+ T‑cell pool, delay expression of exhaustion markers (PD‑1, TIM‑3, KLRG1), and enhance vaccine‑induced immunity in aged organisms.

Rationale

NLRP3‑driven IL‑1β production fuels systemic low‑grade inflammation that accelerates immunosenescence [[https://doi.org/10.1016/j.cmet.2013.09.010]]. However, broad NF‑κB blockade worsens T‑cell dysfunction because NF‑κB signaling is required for tumor control and survival [[https://pmc.ncbi.nlm.nih.gov/articles/PMC4308877/]]. Recent work shows that NIK maintains T‑cell metabolic fitness via an NF‑κB‑independent mechanism by stabilizing hexokinase 2 [[https://pmc.ncbi.nlm.nih.gov/articles/PMC7855506/]]. We propose that NLRP3‑derived IL‑1β also suppresses AMPK activity through ROS‑mediated oxidation of upstream kinases, thereby limiting HIF‑1α stabilization and glycolytic flux independent of NIK. Inhibiting NLRP3 would relieve this suppression, allowing AMPK activation to promote HIF‑1α‑dependent glycolysis and sustain T‑cell effector function without needing NIK‑mediated NF‑κB signaling.

AMPK activation (e.g., with metformin or AICAR) directly phosphorylates and stabilizes HIF‑1α, enhancing glycolysis and mitochondrial biogenesis [[https://pmc.ncbi.nlm.nih.gov/articles/PMC12711513/]]—a metabolic state known to resist exhaustion. Because exhaustion becomes functionally irreversible after ~14 days of persistent antigen exposure [[https://pmc.ncbi.nlm.nih.gov/articles/PMC10135681/]], intervening within this window is critical. Early NLRP3 inhibition reduces inflammasome output, while AMPK activation provides a parallel metabolic rescue that does not rely on NIK, thereby addressing the NF‑κB paradox from two angles.

Predictions

1. Aged mice receiving MCC950 (NLRP3 inhibitor) plus an AMPK activator will show higher frequencies of naïve CD44lo CD62Hi CD8+ T cells compared with either monotherapy or vehicle.
2. Expression of PD‑1, TIM‑3, and KLRG1 on tumor‑infiltrating CD8+ T cells will be significantly lower in the combination group at day 7 and day 14 post‑tumor challenge.
3. Ex vivo stimulation of splenocytes from combination‑treated mice will yield increased IFN‑γ and granzyme B production upon antigenic restimulation.
4. Vaccination with a model antigen (e.g., OVA) in aged mice will produce superior antibody titers and faster viral clearance when the adjuvant regimen includes both MCC950 and an AMPK activator.

Experimental Design

• Model: 18‑month‑old C57BL/6 mice challenged with subcutaneous B16‑F10 melanoma or immunized with OVA‑alum.
• Groups: (1) Vehicle, (2) MCC950 alone (50 mg/kg i.p. q48h), (3) AMPK activator alone (metformin 250 mg/kg diet or AICAR 500 mg/kg i.p. q24h), (4) Combination (MCC950 + AMPK activator). Treatment begins at tumor inoculation or immunization and continues for 14 days.
• Readouts: Flow cytometry for naïve/memory CD8+ subsets, exhaustion markers, intracellular HK2 and p‑AMPK; Seahorse glycolysis assay; ELISA for serum IL‑1β, IL‑6, TNF‑α; viral titer or tumor growth kinetics; antibody titers post‑vaccination.

Falsifiability

If the combination fails to increase naïve CD8+ T‑cell frequencies or does not reduce exhaustion marker expression relative to monotherapies, or if vaccine responses are not enhanced, the hypothesis would be falsified. Conversely, a significant improvement only when both agents are present would support the proposed synergistic mechanism linking inflammasome suppression to AMPK‑driven metabolic rescue.