Genetic Ablation of the Mitochondrial Calcium Uniporter (MCU) Does not Impair T Cell-Mediated Immunity In Vivo

Abstract

T cell activation and differentiation is associated with metabolic reprogramming to cope with the increased bioenergetic demand and to provide metabolic intermediates for the biosynthesis of building blocks. Antigen receptor stimulation not only promotes the metabolic switch of lymphocytes but also triggers the uptake of calcium (Ca2+) from the cytosol into the mitochondrial matrix. Whether mitochondrial Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU) controls T cell metabolism and effector function remained, however, enigmatic. Using mice with T cell-specific deletion of MCU, we here show that genetic inactivation of mitochondrial Ca2+ uptake increased cytosolic Ca2+ levels following antigen receptor stimulation and store-operated Ca2+ entry (SOCE). However, ablation of MCU and the elevation of cytosolic Ca2+ did not affect mitochondrial respiration, differentiation and effector function of inflammatory and regulatory T cell subsets in vitro and in animal models of T cell-mediated autoimmunity and viral infection. These data suggest that MCU-mediated mitochondrial Ca2+ uptake is largely dispensable for murine T cell function. Our study has also important technical implications. Previous studies relied mostly on pharmacological inhibition or transient knockdown of mitochondrial Ca2+ uptake, but our results using mice with genetic deletion of MCU did not recapitulate these findings. The discrepancy of our study to previous reports hint at compensatory mechanisms in MCU-deficient mice and/or off-target effects of current MCU inhibitors.

Acute Downregulation but Not Genetic Ablation of Murine MCU Impairs Suppressive Capacity of Regulatory CD4 T Cells

“…First, we transduced murine T cells with adenovirus-encoding Ca 2+ sensor targeted to the mitochondria (mtCEPIA3). Measurements performed using two independent murine T cell lines-EG7 (Figure 2A) and EL-4 (Figure S1A)-show that siMcu resulted in significant reduction of mitochondrial Ca 2+ uptake induced by ATP (Figures 2B and S1A) or by histamine (Figures 2C and S1B) application, in agreement with the efficient downregulation we observed (Figure 1) and with the previous findings using murine T cells with CD4specific deletion of Mcu (Mcu fl/fl Cd4 Cre ) [8].…”

“…This timely study, however, did not entail investigation of functional consequences of MCU downregulation in T cells, which was investigated systematically in a parallel study [8]. Using a mouse model that specifically deletes Mcu in CD4 T cells, Wu and colleagues showed that MCU is largely dispensable for murine T cells function [8]. In agreement with Yoast and colleagues, Mcu knock-out resulted in elevated cytosolic Ca 2+ in response to thapsigargine (Tg)-mediated store depletion.…”

“…Corresponding analysis of the role of MCU in T cells was mainly lacking until 2021, when Yoast and colleagues showed that downregulation of MCU in a T cell line results in an accumulation of cytosolic Ca 2+ and enhanced NFAT translocation [7]. This timely study, however, did not entail investigation of functional consequences of MCU downregulation in T cells, which was investigated systematically in a parallel study [8]. Using a mouse model that specifically deletes Mcu in CD4 T cells, Wu and colleagues showed that MCU is largely dispensable for murine T cells function [8].…”

“…In the previous study by Vaeth group, Mcu was specifically targeted in Tregs by using a Foxp3 Cre Mcu fl/fl mouse model [8]. Although this approach did not result in Treg phenotypic alterations, and mice showed comparable susceptibility to infection, the question remained whether long-term adaptive mechanisms could be masking a functional Treg-specific effect.…”

Arming a killer: mitochondrial regulation of CD8+ T cell cytotoxicity

“…Calcium transfer via MAMs is thought to occur via inositol triphosphate receptors (IP 3 Rs) in the ER with the voltage-dependent anion channel in the outer mitochondrial membrane, allowing mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) in the inner mitochondrial matrix [34]. MCU is known to regulate calcium influx into the mitochondrial matrix and subsequent NFAT translocation into the nucleus [35]; MCU deletion does not inhibit T cell function or differentiation, as shown in vitro and in vivo in both CD4 + and CD8 + T cells [36]. These data point to the possibility that additional or compensatory mechanisms may exist to regulate calcium flux and ensure T cell activation following mitochondrial dysfunction.…”