How CAR T Cells Breathe

Abstract

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

Keywords: CAR; T cells; chimeric antigen receptor; metabolism; tonic signaling.

Figure 1

Overview of cell metabolism and Seahorse drugs and associated targets. See text for detailed information. FA = Fatty acids; GLS = Glutaminase.

Figure 2

T cells and their metabolic profile. Metabolic reliance of T cells based on their differentiation stage: from right, quiescent T cell, to left, terminally differentiated T cells.

Figure 3

Schematic representation of CAR molecules of the second generation and the metabolic consequences of the different co-stimulatory domains. From left to right: 4-1BB (CD137); CD28; and ICOS co-stimulatory domains.

Figure 4

(A) Seahorse XF Cell Mito Stress Kit. The mitochondrial bioenergetics function is assessed through the measurement of the Oxygen Consumption Rate (OCR). First, a basal OCR value is recorded in triplicate, which reflects mitochondrial activity at a steady state. Then, drugs are sequentially used to challenge components of the mitochondrial respiration chain along OCR measurement 1 = Oligo ATP synthase inhibitor, 2 = FCCP uncoupler of mitochondrial oxidative phosphorylation, and 3 = Rotenone/Antimycin A, inhibitors, respectively, of complex I and III of the ETC. The measurements are repeated 3 times. (B) Overview of the ETC and the targets of the drugs used in the XF Cell Mito stress kit. (C) Seahorse XF Glycolysis stress test kit. The glycolytic function is assessed through the measurement of Extracellular Acidification Rate (ECAR). First, cells are incubated in stress test medium without glucose or pyruvate and the ECAR is measured. Then, the first injection is a saturating concentration of glucose; measurements taken during that time indicate glycolysis under basal conditions. The second injection of Oligomycin, an ATP synthase inhibitor, permits, through the measurement of ECAR, assessment of the maximum glycolytic capacity. Lastly, 2-deoxy-glucose (2-DG) is injected. This is a glucose analog that binds competitively to glucose hexokinase, the first enzyme in the glycolytic pathway. The resulting decrease proves that the ECAR previously measured is due to glycolysis.