Introduction:
T cell metabolic programming shapes cell fate and function. Efficient metabolism is crucial for activation, proliferation, effector activity, and memory formation. However, metabolic regulation of CD4+ T cells and their subsets in allergic disease remains incompletely understood.
Methods:
In this study, we performed in vitro and ex vivo experiments evaluating primary human CD4+ T and Th2 cells. UHPLC-QTOF- MS based metabolomics was employed to assess circulating memory CD4+ T cells and sera from allergic patients and healthy controls. Seahorse Extracellular flux and Single Cell ENergetIc metabolism by profilIng Translation inhibition (SCENITH) assays were used to evaluate cellular energy metabolism. siRNA-based knockdown approaches were used to confirm Phe mediated induction of Interleukin 4 induced gene 1 (IL4I1) in Helper T cell subsets. qRT-PCR, Western blotting, and flow cytometry were used to study transcription factor phosphorylation, type 2 cytokine transcription, and translation and immunophenotyping. Finally, RNA-sequencing data generated as part of this study and published transcriptomic datasets were bioinformatically analysed by standard established pipelines.
Results:
To address the mentioned knowledge-gap, we first performed metabolomic profiling of circulating memory CD4+ T effector (Teff) and regulatory (Treg) cells from healthy individuals, revealing enrichment of amino acid pathways, particularly L-phenylalanine (Phe) metabolism. We therefore examined effect of Phe on CD4+ T cell energy regulation and observed that Phe increased glycolysis in memory CD4+ T cells while limiting OXPHOS. It also inhibited their proliferation through an IL4I1-dependent mechanism, confirmed using siRNA-based knockdown techniques. Using SCENITH, we found Phe selectively enhanced the Th2 cell glycolytic capacity while reducing their mitochondrial dependence among helper T cell subsets. In vitro–differentiated Th2 cells exposed to Phe displayed reduced proliferation, STAT6 phosphorylation, reduced mTOR phosphorylation and increased AMPK phosphorylation. Furthermore, expression of key type 2 transcription factors and cytokines, including mTOR, BACH2, BATF, IL-4, IL-5, and IL-13 were significantly reduced. Phe also reduced expression of CD161, a documented marker of pathogenic Th2a cells in allergy. Metabolomic and ex vivo analyses of allergic patients showed reduced intracellular Phe in circulating memory CD4+ Teff cells in a subset of severe cases and elevated serum Phe levels. Levels of pathogenic Th2a and CRTH2+ Treg cells were significantly elevated in severe allergic patients. Finally, analysis of allergic clinical cohorts revealed impaired Phe metabolism and expression of LAT1, an important Phe transporter, negatively correlated with serum Phe only in patients with allergy.
Conclusion:
Altogether, these findings identify Phe as a regulator of Th2 metabolism and this mechanism appears impaired in severe allergic disease.