Cell metabolism: Functional and phenotypic single cell approaches.

Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.

Light-inducible T cell engagers trigger, tune, and shape the activation of primary T cells.

To mount appropriate responses, T cells integrate complex sequences of receptor stimuli perceived during transient interactions with antigen-presenting cells. Although it has been hypothesized that the dynamics of these interactions influence the outcome of T cell activation, methodological limitations have hindered its formal demonstration. Here, we have engineered the Light-inducible T cell engager (LiTE) system, a recombinant optogenetics-based molecular tool targeting the T cell receptor (TCR). The LiTE system constitutes a reversible molecular switch displaying exquisite reactivity. As proof of concept, we dissect how specific temporal patterns of TCR stimulation shape T cell activation. We established that CD4+ T cells respond to intermittent TCR stimulation more efficiently than their CD8+ T cells counterparts and provide evidence that distinct sequences of TCR stimulation encode different cytokine programs. Finally, we show that the LiTE system could be exploited to create light-activated bispecific T cell engagers and manipulate tumor cell killing. Overall, the LiTE system provides opportunities to understand how T cells integrate TCR stimulations and to trigger T cell cytotoxicity with high spatiotemporal control.

Plasmatic MMP9 released from tumor-infiltrating neutrophils is predictive for bevacizumab efficacy in glioblastoma patients: an AVAglio ancillary study.

We previously identified matrix metalloproteinase 2 (MMP2) and MMP9 plasma levels as candidate biomarkers of bevacizumab activity in patients with recurrent glioblastoma. The aim of this study was to assess the predictive value of MMP2 and MMP9 in a randomized phase III trial in patients with newly diagnosed glioblastoma and to explore their tumor source. In this post hoc analysis of the AVAglio trial (AVAGlio/NCT00943826), plasma samples from 577 patients (bevacizumab, n = 283; placebo, n = 294) were analyzed for plasma MMP9 and MMP2 levels by enzyme-linked immunosorbent assay. A prospective local cohort of 38 patients with newly diagnosed glioblastoma was developed for analysis of tumor characteristics by magnetic resonance imaging and measurement of plasma and tumor levels of MMP9 and MMP2. In this AVAglio study, MMP9, but not MMP2, was correlated with bevacizumab efficacy. Patients with low MMP9 derived a significant 5.2-month overall survival (OS) benefit with bevacizumab (HR 0.51, 95% CI 0.34-0.76, p = 0.0009; median 13.6 vs. 18.8 months). In multivariate analysis, a significant interaction was seen between treatment and MMP9 (p = 0.03) for OS. In the local cohort, we showed that preoperative MMP9 plasma levels decreased after tumor resection and were correlated with tumor levels of MMP9 mRNA (p = 0.03). However, plasma MMP9 was not correlated with tumor size, invasive pattern, or angiogenesis. Using immunohistochemistry, we showed that MMP9 was expressed by inflammatory cells but not by tumor cells. After cell sorting, we showed that MMP9 was expressed by CD45+ immune cells. Finally, using flow cytometry, we showed that MMP9 was expressed by tumor-infiltrating neutrophils. In conclusion, circulating MMP9 is predictive of bevacizumab efficacy and is released by tumor-infiltrating neutrophils.

Proteostasis in dendritic cells is controlled by the PERK signaling axis independently of ATF4.

In stressed cells, phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation and gene expression known as the integrated stress response. We show here that DCs are characterized by high eIF2α phosphorylation, mostly caused by the activation of the ER kinase PERK (EIF2AK3). Despite high p-eIF2α levels, DCs display active protein synthesis and no signs of a chronic integrated stress response. This biochemical specificity prevents translation arrest and expression of the transcription factor ATF4 during ER-stress induction by the subtilase cytotoxin (SubAB). PERK inactivation, increases globally protein synthesis levels and regulates IFN-ß expression, while impairing LPS-stimulated DC migration. Although the loss of PERK activity does not impact DC development, the cross talk existing between actin cytoskeleton dynamics; PERK and eIF2α phosphorylation is likely important to adapt DC homeostasis to the variations imposed by the immune contexts.

SCENITH: A Flow Cytometry-Based Method to Functionally Profile Energy Metabolism with Single-Cell Resolution.

Energetic metabolism reprogramming is critical for cancer and immune responses. Current methods to functionally profile the global metabolic capacities and dependencies of cells are performed in bulk. We designed a simple method for complex metabolic profiling called SCENITH, for single-cell energetic metabolism by profiling translation inhibition. SCENITH allows for the study of metabolic responses in multiple cell types in parallel by flow cytometry. SCENITH is designed to perform metabolic studies ex vivo, particularly for rare cells in whole blood samples, avoiding metabolic biases introduced by culture media. We analyzed myeloid cells in solid tumors from patients and identified variable metabolic profiles, in ways that are not linked to their lineage or their activation phenotype. SCENITH's ability to reveal global metabolic functions and determine complex and linked immune-phenotypes in rare cell subpopulations will contribute to the information needed for evaluating therapeutic responses or patient stratification.