The dependence of CD4+ T?cells on CD46 costimulation for normal mTORC1 function was further underscored by the inability of T?cells from patient CD46-3 to induce either mTOR or p70S6K phosphorylation at substantial levels under any activation condition tested (Figures 4D and S4), and by a significant reduction in mTOR and p70S6K phosphorylation in T?cells from HDs treated with CD46-specific siRNA (not shown). mutations of patient CD46-2 were heterozygous variants c.175C>T (p.R59X) and c.G104G>A (p.C35Y), and patient CD46-3 had a homozygous variant in c.286+1G>C (RefSeq “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_172359″,”term_id”:”1677500949″,”term_text”:”NM_172359″NM_172359) in exon 2. These mutations were confirmed by Sanger sequencing (not shown). NOTE: Positions with normalized SIFT probabilities less than 0.05 are predicted to be deleterious, those greater than or equal to 0.05 are predicted to be tolerated. Also, columns EVS and 1KG indicate the minor allele frequencies in two large population genetics databases, and all positions observed are not rare and are thus not suggestive of causing a rare immune defect. The seemingly rare mutation in TARP 38299727 occurs very frequently in our inhouse database of normal donors and is therefore also not suggestive of being causal. mmc2.xlsx (20K) GUID:?CEB67271-A017-4C62-8F06-6A16D730FB92 Table S2. Related to Figure?3 Shown is a list of genes in which mutations have been known to cause monogenetic immune defects and which have been screened for mutations in the CD46-deficient patients. mmc3.xlsx (11K) GUID:?C8F924E7-9F48-45A7-998A-4E7B05046532 Table S3. Related to Figure?4 Gene list of the 403 genes differently expressed between CD3+CD46-activated T?cells (2?hr) isolated from Patient CD46 3 and an age- and sex-matched healthy donor. Arrays were performed as technical triplicates. mmc4.xlsx (43K) GUID:?B21B4276-CCF6-498F-834D-64E006E72B5D Table S4. Related to Figure?3 Genes differently expressed in T?cells from Patient CD46-3 and a healthy MAT1 donor identified by GMO that specifically functioning in metabolic processes of the cells. mmc5.xlsx (45K) GUID:?98A4428C-8B21-4B09-8D20-B6864DE6B20F Document S2. Article plus Supplemental Information mmc6.pdf (15M) GUID:?49129815-D909-4892-8110-C99293ABA2DC Summary Expansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. 3b-Hydroxy-5-cholenoic acid Here we found that in activated human T?cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T?cells from CD46-deficient patients, characterized 3b-Hydroxy-5-cholenoic acid by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN- production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T?cell effector function. Graphical Abstract Open in a separate window Introduction Naive T?cells are metabolically quiescent, primarily depending on oxidative phosphorylation (OXPHOS) for homeostatic adenosine triphosphate (ATP) generation (Gubser et?al., 2013; Pearce et?al., 2013; Rathmell, 2012; van der Windt et?al., 2012, 2013). Ligation of the T?cell receptor (TCR) and costimulatory molecules initiates significant changes in nutrient uptake and usage of metabolic pathways, jointly supporting bioenergetic and non-bioenergetic requirements of activated T?cells (Gerriets and Rathmell, 2012; Jacobs et?al., 2008; Pearce et?al., 2013; Wang et?al., 2011). Enhanced cellular uptake of amino acids (AA) is mediated by increased expression of several system L amino-acid transportersparticularly SLC7A5 (which together with SLC3A2 forms the neutral AA transporter LAT1). but did not identify additional mutations in candidate genes mediating T?cell function or genes known to cause monogenic immune defects (Table S1 and S2). While expression of CD3 and CD28 on T?cells from all three patients was within normal range (Figure?S1B), their CD4+ T?cells demonstrated impaired acquisition of Th1 cell effector function in response to TCR ligation and costimulation via either CD46 or CD28 (Cardone et?al., 2010; Le Friec et?al., 2012) (Figure?1Bi). The phenotype of T?cells from HDs treated with CD46-specific siRNA (Figure?S1Ci) was comparable, with a specific reduction in IFN- and IL-10, but normal IL-5 production (Figure?1Bii), and reduced upregulation of CD25 (Ni Choileain et?al., 2011), but unaltered expression of CD69 (Figure?S1Cii) (Le Friec et?al., 2012). Only T?cells from patient CD46-3, which lacked CD46 expression entirely (Figure?S1B, legend), were unable to produce IL-17. Open in a separate window Figure?1 Autocrine CD46-CYT-1 Activation Drives Glycolysis and Oxidative Phosphorylation in CD4+ T Cells (A) TCR and CD28-induced Th1 3b-Hydroxy-5-cholenoic acid cell cytokine production correlates with CD46 ligand C3b generation as assessed 1?hr post activation. (B) Cytokines produced by (Bi) CD4+ T?cells from age- and sex-matched healthy donors (HD1 to HD6) and patients CD46-1 (open circle), CD46-2 (open square), and CD46-3 (open triangle) or by (Bii) T?cells from HDs treated with CD46 siRNA (n?= 3 with duplicate samples [mean]). (C) Basal glycolysis (ECAR) and oxidative phosphorylation (OXPHOS, OCR) rates in resting and activated CD4+ T?cells (Ci) from CD46-deficient patients (n = 3) and HDs (n = 6) or from.