Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

ABSTRACT The efficiencies with which activated B lymphocytes proliferate and develop into antibody (Ab)- secreting plasma cells are critical determinants of adaptive humoral immunity and central to sustaining certain autoimmune diseases. Increasing evidence indicates that specific pathways in intermediary metabolism, or their substrate supply, influence lymphocyte differentiation and function. We now show that although stringent restriction of glutamine supply decreases proliferation and differentiation of B cells into plasma cells, glutaminolysis - a major means of metabolism of this amino acid - was only conditionally crucial in B cells and the Ab responses derived from them. Strikingly, Gls, the gene encoding the main glutaminase of lymphocytes, promoted anti-NP Ab responses at the primary and recall phases only when either glucose uptake into B cells or pyruvate into their mitochondria was also impaired. This synthetic auxotrophy involved support to a progressive expansion of mitochondrial respiration followed by plasma cell differentiation. Surprisingly, impairment of glutaminase and the mitochondrial pyruvate channel not only decreased the coupling of IL-21 stimulation to STAT3 induction, but also interferon stimulation of STAT1 activation. Together, our findings establish not only a powerful collaboration of metabolic pathways in promoting increased respiration and the development of Ab-secreting cells, but also a capacity of metabolism to modulate cytokine receptor signaling. Competing Interest Statement JCR is a Founder (with no stock or stock options) and receives consultant fee as a Scientific Advisory Board member for Sitryx Therapeutics. No other authors report potential conflicts of interest or competing interests. Abbreviations used - 2-DG - 2-deoxyglucose - 4OHT - 4-hydroxytamoxifen - αKG - α-ketoglutarate - Ab - antibody - Ag - antigen - ASC - Ab-secreting cell - BAFF - B-cell activating factor - 2-deoxyglucose - BCR - B cell Ag receptor - BrdU - bromodeoxyuridine - BSA - bovine serum albumin - WT - wildtype - KO - knockout - MZB - marginal zone B cell - PC - plasma cell - MBC - memory B cell - Ig - immunoglobulin - CD - cluster of differentiation - CoA - coenzyme A - CTV - CellTrace Violet - DMK - dimethyl-ketoglutarate - DMSO - dimethylsulfoxide - ECAR - extracellular acidification rate - ELISA - enzyme-linked immunosorbent assay - ELISpot - enzyme-linked immunosorbent spot - ETC - electron transport chain - FAO - fatty acid oxidation - FCCP - carbonyl cyanide 4-phenylhydrazone - GC - germinal center - GLS - glutaminase - GSEA - gene set enrichment analyses - H2DCFDA - dichlorodihydrofluorescein diacetate - HIF - hypoxia-inducible factor - HCQ - hydroxychloroquine - IFN - interferon - IL- - interleukin- - Jak - Janus kinase - MPC - mitochondrial pyruvate channel - NP-OVA - 3-Nitrophenylacetyl (NP)-ovalbumin (OVA) - OCR - oxygen consumption rate - PDH - pyruvate dehydrogenase - PSA - porcine serum albumin - ROS - reactive oxygen species - mtROS - mitochondrial ROS - SDS-PAGE - sodium dodecyl-sulfate polyacrylamide gel electrophoresis - SEM - standard error of means - SRBC - sheep red blood corpuscule - STAT - signal transducer and activator of transcription - TCA cycle - tricarboxylic acid cycle - TLR - Toll-like receptor - TMRE - tetramethylrhodamine ester - Tmx - tamoxifen