Alterations in lipid metabolism accompanied by changes in protein and carotenoid content as spectroscopic markers of human T cell activation

This work aims to understand better the mechanism of cellular processes accompanying the activation of human T cells and to develop a novel, fast, label-free approach to identify molecular biomarkers for this process. The standard methodology for confirming the activation state of T cells is based on flow cytometry and using antibodies recognizing activation markers. The method provide high specificity detection but may be susceptible to background staining or non-specific secondary antibody reactions. Here, we evaluated the potential of Raman-based molecular imaging in distinguishing non-activated and activated human T cells. Confocal Raman microscopy was performed on T cells followed by chemometrics to obtain comprehensive molecular information, while Stimulated Raman Scattering imaging was used to quickly provide high-resolution images of selected cellular components of activated and non-activated cells. For the first time, carotenoids, lipids, and proteins were shown to be important biomarkers of T-cell activation. We found that T-cell activation was accompanied by lipid accumulation and loss of carotenoid content. Our findings on the biochemical, morphological, and structural changes associated with activated mature T cells provide insights into the molecular changes that occur during therapeutic manipulation of the immune response. The methodology for identifying activated T cells is based on a novel imaging method and supervised and unsupervised chemometrics. It unambiguously identifies specific and unique molecular changes without the need for staining, fixation, or any other sample preparation.