Improving Anti-tumor Immunity by Targeting LDH-A Functions: Beyond the Warburg Effect

The purpose of this subcontract is to fulfill the tasks proposed in Specific Aim 1: To investigate how metabolic changes induced by LDH-A targeting impact on the differentiation program of antigen-specific T cells. These tasks include: 1. Perform stable isotope-resolved metabolomic (SIRM) analysis of antigen-specific T cells subjected to 13C6-glucose tracer treatment. Polar metabolites will be analyzed by 1D 1H and 1H{13C} HSQC NMR, GC-MS, and FT-MS for amino acids, organic acids, phosphorylated metabolites (e.g. nucleotides, sugar nucleotides, phosphorylated sugars and polyols), and antioxidants (e.g. glutathiones), while lipidic metabolites will be analyzed by FT-MS as described previously [1-4]. The effect of LDH-A targeting on various metabolic pathways in T cells will be reconstructed based on the 13C labeling patterns (number and position of labels in metabolites or mass isotopologues and positional isotopomers) of relevant metabolites. These pathways include (but not limited to) glycolysis, pentose phosphate pathway (PPP), Krebs cycle, hexosamine phosphate pathway, nucleotide biosynthesis, glutathione biosynthesis, urea cycle and lipid/oxylipin biosynthesis. 2. Perform SIRM analysis antigen-specific T cells subjected to 13C2-1,2-glucose tracer treatment and reconstruct reprogrammed pathways as in Task 1. This tracer experiment enables a clear delineation between glycolysis and PPP. 3. Perform SIRM analysis antigen-specific T cells subjected to 13C5,15N2-glutamine tracer treatment and reconstruct reprogrammed pathways as in Task 1. This tracer experiment enables tracking of glutamine uptake, glutaminolysis and gluconeogenesis pathways. 4. Perform SIRM analysis antigen-specific T cells subjected to 13C8-octanoate tracer treatment and reconstruct reprogrammed pathways as in Task 1. This tracer experiment enables tracking of fatty acid uptake, beta-oxidation, and incorporation of exogenous fatty acid into cellular lipids and oxylipins.