Loss of the Nuclear Receptor Pparγ Primes T Effector (T_EFF) Differentiation of Antigen-Specific CD8⁺ T Cells

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to the nuclear hormone superfamily, with three distinct types identified: PPARalpha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus dictate the differentiation and fate of immune cells. PPARγ is a lipid-activated transcription factor mainly known to mediate anti-inflammatory effects on both innate and adaptive immune system. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. In the present study we investigated how PPARγ affects the differentiation and function of antigen-specific effector (T_EFF) and memory (T_M) CD8⁺ T cells using OTI TCR transgenic mice and OTI mice with T cell-specific PPARγ deletion (OTI/PPARγ^f/f-CD4-cre; thereafter named OTI/PPARγ-KO). Antigen-specific T_EFF were generated in vitro by stimulation of OTI and OTI/PPARγ-KO T cells with Ova_257-264 followed by culture with IL-2, whereas T_M were generated by stimulation with Ova_257-264 followed by culture with IL-15. Under both T_EFF and T_M conditions in vitro, antigen-specific stimulation of OTI/PPARγ-KO cells resulted in significantly greater proliferation and expansion relative to OTI control. Additionally, OTI/PPARγ-KO T_M cells cultured with IL-15 co-expressed significantly higher CD44⁺/CD62L⁺ fractions indicating a greater expansion of central memory cells. Consistent with these, metabolomics analysis showed that under both culture conditions, PPARγ-deficient OTI cells had increased levels of glycolysis intermediates but also TCA metabolites consistent with a program of metabolically active cells with high levels of glycolysis and OXPHOS. RNA-sequencing studies showed that upon T_EFF differentiation in vitro, OTI/PPARγ-KO T cells displayed a differential gene expression program compared to OTI controls. Pathway enrichment analysis revealed that PPARγ-deficient OTI cells were significantly enriched for genes related to T cell activation and proliferation, integrin activation, cytokine production, ribosomal function and biogenesis, RNA processing and protein translation. During antigen-specific stimulation in vivo performed after adoptive transfer of OTI/PPARγ-KO and OTI cells into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA), PPARγ deletion resulted in a rapid and enhanced T_EFF cell activation and expansion as determined by the higher mitotic index, higher enrichment of Dextramer-positive antigen-specific T cells, and the higher expression of activation markers including KLRG1, CD44 and CD69, but also CTLA-4, PD-1, Lag-3 and Tim-3. These cells also developed higher numbers of TNF-α and IFN-γ-producing T cells, as well as ROR-γt expressing T cells consistent with Tc17 differentiation. In vivo stimulated antigen-specific PPARγ-deficient and control OTI T cells showed no numerical differences in T_M differentiation or expansion. However, upon rechallenge in vitro, PPARγ-deficient T_M cells exhibited improved capacity for T_EFF differentiation with higher release of inflammatory cytokines and expression of CD69, PD-1 and CTLA-4. Our findings uncover a new role of PPARγ ablation in programming CD8⁺ T responses by regulating metabolism-mediated cues, which imprint T cell differentiation and long-term effector capacity.

Vlachos:Botanisol Analytics: Research Funding; NextRNA: Consultancy; Singular Genomics: Research Funding; Takeda Pharmaceutical: Current Employment; National Institute of Health: Research Funding; Department of Defense: Research Funding; Guidepoint Global: Consultancy; Mosaic: Consultancy.