T-lymphocytes from AIDS Patients Are Unable to Synthesize Ribonucleotides de Novo in Response to Mitogenic Stimulation

Abstract

Sensitive high performance liquid chromatography techniques, which differentiate between purine and pyrimidine ribonucleoside and deoxyribonucleoside triphosphates, were used to quantify pools in phytohemagglutinin-stimulated T-lymphocytes (98% CD4+ and CD8+) from healthy volunteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with 14C-radiolabeled precursors (40 microM), azaserine (20 microM; a glutamine antagonist), and ribavirin (50 microM; an IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocytes meet their metabolic requirements by salvage. Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h. These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.

mTORC1 signaling and the metabolic control of cell growth

“…One might speculate that mTORC1 activates T cells by driving a similar metabolic program to that of tumor cells. It is interesting to note that, like rapamycin, inhibitors of nucleotide synthesis are commonly used immunosuppressants [113–115], as activated lymphocytes exhibit increased de novo nucleotide synthesis on which they are highly dependent [116,117]. As discussed above, rapamycin also attenuates de novo nucleotide synthesis [40–42,102], suggesting an important underlying mechanism that might contribute to its immunosuppressant effects.…”

Similarities and Distinctions of Cancer and Immune Metabolism in Inflammation and Tumors

“…Purine and pyrimidine pools expand several fold in T cell activation (Fairbanks et al, 1995). Indeed, the dependence of lymphocytes on de novo nucleotide synthesis may account for the immunosuppressive action of inosine 5’-monophosphate dehydrogenase (IMPDH) inhibitors, mizoribin and mycophenolate mofetil, the dihydroorotate dehydrogenase inhibitor, leflunomide, and azathioprine, which was initially discovered as an antileukemic drug (Allison, 2000).…”

Ancillary Activity: Beyond Core Metabolism in Immune Cells

“…Ribose-5-phosphate is phosphorylated to phosphoribosyl-pyrophosphate that is used together with amino groups (NH 4 + ) from glutamine, and amino acids to generate nucleotides. This de novo pathway is complex and energy-expensive and used by actively proliferating cells, such as T cells; resting cells instead use the salvage pathway that recycles free bases and nucleosides released from nucleic acid breakdown (Fairbanks et al, 1995). Nucleotide biosynthesis inhibition has obvious effects on cell proliferation and effector functions (Turka et al, 1991), however the underlying mechanisms are often unexpected.…”