CD4+ T helper (Th) cells are key drivers of adaptative immune responses that can develop into several different subsets, such as Th1, Th2, Th17, and regulatory Th (Treg) cells. Each subset is involved in different immune responses; particularly, Th2 cells are commonly associated with allergic diseases, such as asthma. Tregs, on the other hand, are responsible for managing immune tolerance via suppression and resolution of inflammation processes.
In recent years, cell metabolism has emerged as a key regulator of T cell function. Alterations in cell metabolism, including glucose energetic metabolism and amino acid metabolism, have been described for immune cells in inflammatory diseases.
Amino acids are primarily considered as the building blocks of proteins. Nonetheless, amino acid processing and metabolism in T cells can also control their function. In this regard, enzymes that process amino acids, specifically, aromatic amino acids (namely, tryptophan-Trp-, tyrosine-Tyr-, and phenylalanine-Phe-), such as IDO1 or IL4I1, have been connected with Treg function.
Recent results from our group have shown that Phe alters Th2 energetic metabolism, proliferation, and function; and a similar trend in Tregs. However, the specific pathway through which they exert this function is yet to be described, since T cells do not express phenylalanine hydroxylase (PAH), the most common Phe-metabolizing enzyme.
Thus, we wondered how Phe is processed in T cells. To investigate it, we performed Phe flux analysis through metabolomics. Briefly, Th2 and Treg cells were cultured in media under 3 different experimental conditions: (1) without Phe, (no Phe) (2) with normal-weight Phe (nw-Phe), or (3) with heavy-weight Phe (hw-Phe). Heavy Phe had all its normal weight carbons substituted with carbons with an extra neutron, allowing us to differentiate it from regular Phe. T cells were cultured during 30min, 2h, 6h, 24h, and 48h; and cells were collected for metabolomics and proteomics at each timepoint. Furthermore, cells cultured for 48h were collected to analyse T cell phenotype.
In this proof of concept, we observed that even after only 30 minutes, cells cultured in no Phe showed a significant depletion of intracellular Phe content; while cells in hw-Phe media had all their Phe replaced with the heavier one. Phe accumulation was slightly higher in activated cells. As expected, no Tyr was labelled with the heavier carbons, confirming the absence of PAH.
Furthermore, we observed that incorporation of hw-Phe into Phe-containing proteins increased with time and was slower than Phe take-up from the media, reaching around 25% after 48h. The type of medium in which cells were cultured did not significantly alter their phenotype.
Thus, we have confirmed that cells are able to incorporate and use heavier phenylalanine without significantly altering their phenotype. Future analyses following which compounds take the heavier carbons will allow us to determine how phenylalanine is processed in these cells.