Extracellular antigens were stained for 15 min at 4 C with an antibody staining mix containing Ghost Dye Red 780 viability dye diluted in PBS. effector T cells to promote coherent physiological responses to decreased feeding, allowing organisms to scale their immune responses according to food availability. for experimental setup). Limiting nutrient intake N-Methylcytisine led to lower splenic weights and decreased total leukocyte counts at the peak of adaptive immune responses on D7 (Fig. 1 and and (= 10) are representative of three independent experiments. Data in (= 10) are pooled from two independent experiments. Statistical significance determined by a two-tailed test (and and < 0.05; **< 0.01; ***< 0.001; ****< 0.0001; ns, not significant. We then assessed the N-Methylcytisine effects of transient food deprivation on the T cell compartment. While the counts for naive and central memory T cells were not altered by fasting and refeeding, effector T cell numbers on D7 were decreased compared to ad-libitumCfed controls (Fig. 1and and and and and and and and and (= 8 to 13) are pooled from at least two independent experiments. Data in (= 4 to 5) are representative of three independent experiments. Statistical significance was determined by a two-tailed test (< 0.05; ns, not significant. We conjectured that lymphocyte-intrinsic responsiveness to food intake may be important for metabolic homeostasis, and that FXR expression in effector T cells may allow for a coherent organismal response to reduced feeding during infection. Indeed, we observed that LCMV-infected T?FXR mice had increased weight loss after fasting compared to WT littermates (Fig. 2and and axis) and log2 fold-change (FC) between OT-IWT FS and OT-IWT AL (axis). Significantly altered N-Methylcytisine transcripts (adjusted value <0.05) are highlighted in red, and two-fold change thresholds are indicated by dotted lines. (axis, highlighted in red) or FS (axis, highlighted in orange) state. (= 2.11= 3). To gain insight into the cellular processes affected by food deprivation, we performed gene-set enrichment analysis (GSEA). We identified cell cycle and apoptosis as among the pathways differentially regulated in OT-IWT cells (Fig. 3and and These changes suggest that effector N-Methylcytisine CD8+ T cells may oxidize alternative substrates in fasted animals. Next, we assessed the effects of FXR deficiency on the transcriptome of effector CD8+ T cells. Analyses of OT-I?FXR and OT-IWT cells revealed that most FXR-dependent changes in gene expression were observed regardless of the absorptive state of the host (Fig. 3and and are plotted as the percentage of basal OCR after injection of inhibitor. (= 3 to 11), representative of at least two independent experiments. Statistical significance determined by one- or two-way ANOVA followed by a Dunnet (< 0.05; **< 0.01; ***< 0.001; ****< 0.0001. To minimize potential confounding effects of cell-extrinsic signals present in vivo, we also performed extracellular flux measurements on in-vitroCgenerated effector T cells. In line with our ex vivo IGFBP1 assessment, T?FXR cells showed a modest but highly reproducible increase in their basal OCR compared to WT (and and and axis) and spleen size on D7 (axis). (and (= 10 to 24) are pooled from three experiments. Data in (= 5 to 10) are representative of at least two independent experiments. Statistical significance determined by Pearson correlation analysis (= 0.0018, test (< 0.05; **< 0.01; ***< 0.001; ns, not significant. Next, we measured the glycemia N-Methylcytisine of LCMV-infected WT and T?FXR animals to assess whether potential differences could explain the higher numbers of effector T cells in the latter. Unexpectedly, we found that blood glucose levels were significantly lower in T?FXR compared to WT following food deprivation ((L. mono) expressing OVA as a model antigen induces a long-lasting anorexic response that is present when adaptive immune responses arise on D5 post infection (Fig. 5infection, bacteria were grown overnight in Brain-Heart-Infusion broth (BD) with agitation (250 for 3 min, and serum was transferred into Eppendorf tubes and stored at ?80 C until analysis. Serum chemistry analyses were performed by the Center for Comparative Medicine and Pathology at MSKCC. Continuous Oxygen Measurement with Clark-type Electrode. Tissue oxygen consumption was measured with a Clark-type oxygen polarographic electrode. Immediately after euthanization of animals, tissues were dissected and weighed. After mincing into 0.2- to 0.5-mm diameter fragments with sterile surgical tools, tissue aliquots were transferred into an oxygenated PBS solution at RT. Tissue suspensions were put into a respirometer microcell linked to an oxygen meter (Strathkelvin Instruments MS200A) and continuously stirred during measurements. Oxygen.