Date of Award

Spring 5-3-2022

Degree Type

Thesis

Degree Name

Master of Science in Mechanical Engineering

Department

Mechanical Engineering

First Advisor

Sikder, Prabaha

Second Advisor

Borkar, Tushar

Third Advisor

Farahani, Saeed

Subject Headings

Biology, Biomedical Research, Molecular Biology

Abstract

Gliomas express mutant isocitrate dehydrogenases producing excessive amounts of D 2-hydroxyglutarate (D2HG) and releasing some of it into the environment. The immune surveillance is reduced as a result, however, the mechanisms behind lymphocyte suppression by the D2HG stereoisomer remain unknown. I incubated Jurkat T cells with D2HG at concentrations present within and surrounding gliomas, or its obverse L2HG stereoisomer, and quantified 2HG isomers within washed cells by TSPC derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was found in quiescent cells in double the amount of L2HG. External D2HG or L2HG increased the level of the provided stereoisomer in a transient, concentration-dependent process. IL-2 expression, even when maximally elicited by A23187 and PMA, was inhibited and ultimately abolished by exogenous D2HG in a concentration-dependent manner. Notably, a significant reduction occurred at just twice its basal intracellular level. In contrast, L2HG was only moderately inhibitory. IL-2 expression is regulated by increased intracellular Ca++ that stimulates Calcineurin to dephosphorylate cytoplasmic phospho-NFAT to enable its nuclear translocation. Besides, to induce IL-2, AP-1 complex which is downstream of ERK needs to ligate. D2HG inhibited p-ERK in Jurkat T cells, impairing the AP-1 complex. D2HG abolished expression of a stably-integrated NFAT-driven luciferase reporter, and this concentration-dependent inhibition precisely paralleled inhibition of IL-2. D2HG did not affect intracellular Ca++. Rather, surface Plasmon resonance showed D2HG, but not L2HG, bound Calcineurin. D2HG, but not L2HG, inhibited Ca++-dependent Calcineurin phosphatase activity. Thus, D2HG is a stereoselective Calcineurin phosphatase inhibitor that prevents NFAT dephosphorylation, and so abolishes IL-2 transcription in stimulated Jurkat cells. This occurs at D2HG concentrations found within and adjacent to gliomas independent of delayed epigenetic modulation of transcription.

Download
COinS