1.2.1 Work Package 1. Define the role of mitochondrial dynamics in T cell differentiation
To characterize whether distinct T cells subsets show specific configurations of their mitochondrial membranes, we first analysed the mitochondrial morphology in naive CD4 T cells differentiated in vitro towards distinct T cell subsets (Th1, Th2, Th17 and Treg). As opposed to other CD4 T cell subsets, Th17 cells show fused mitochondrial network, tight cristae organization, and reduced metabolic activity, a similar configuration as found in quiescent, low-metabolically memory T cells. Genetic manipulation of mitochondrial inner membrane by deletion of OPA1 reduced mitochondrial OXPHOS and increased glycolysis across T cell subsets. However, this metabolic rewiring induced by OPA1-deficiency, only restrained Th17 function. Using in vivo models of Th17 function (experimental autoimmune encephalomyelitis, EAE, or CD3 antibody (Ab) monoclonal injection), we found that OPA1 and mitochondrial inner membrane fusion are required for establishing Th17 autoimmune responses.
1.2.2 Work package 2
Elucidate how mitochondria shapes the transcriptional and epigenetic networks of T cell lineages
To investigate whether manipulation of mitochondrial membrane controls chromatin remodeling and gene expression in T cells, I first interrogated the transcriptome of the distinct T cell subsets upon specific deletion of OPA1 during in vitro polarization experiments. OPA1 deletion up- and downregulated a number of different genes involved in T cell differentiation, cellular metabolism and inflammatory disease.
1.2.3 Work Package 3
Identify mitochondria-to-nucleus signaling pathways controlling T cell differentiation
The third objective aimed to identify metabolites regulating the epigenome and T cell transcriptional programs. To this end, I performed extensive characterization of the metabolome of in vitro differentiated T cells with specific deletion of Opa1. Global metabolite analysis revealed an accumulation of TCA metabolites, concomitant with an impairment in NAD/NADH levels, reduced aspartate biosynthesis, and increased lactate and serine biosynthesis. Notably, we detected the accumulation of a byproduct of the TCA metabolism, the oncometabolite 2-hydroxyglutarate (2-HG) upon OPA-1 deletion, whose accumulation in cancer cells inhibits DNA and histone modifying enzymes and triggers epigenetic reprogramming. Proteomic analysis identified upstream regulators of this metabolic rewiring, being LKB1 a major candidate. OPA-1 deletion activates LKB1 signaling and CRISPR-Cas9 knockdown of LKB1 in OPA1-deficient cells, restores IL-17A production in Th17 cells. In all, these results pointed to a crucial role of LKB1 in controlling OPA1 phenotype. In this line, silencing of LKB1 by CRISPR-Cas9 on OPA1-deficient cells restored the metabolic deficiencies quantified in OPA1 cells (i.e. accumulation of TCA metabolites and 2-HG).
1.2.3 Work Package 4
Identify and validate metabolites changing T cell transcriptional networks in vitro and in vivo
Intrigued by the high specificity of the effect of OPA1 deficiency in Th17 cells and the lack of effect on the effector function in other subsets, we speculated that the different metabolism of these cells would determine their response to OPA1 deficiency (mitochondrial dysfunction). We found that reducing metabolic activity of Th1 cells by culturing them with decreasing amounts of glucose, made Th1 cells susceptible to OPA1 deficiency. These results support the notion that cellular metabolic activity and distinct substrate availability are responsible for the vulnerability of T cells to mitochondrial function/dysfunction. To investigate whether mitochondrial membrane remodeling sustain T cell responses in vivo, we focused our analysis in immune models mediated by Th17 cells and we did not perform any of the cancer models originally proposed. To this end, we generated mice in which OPA1 is deleted specifically in IL17-expressing cells by crossing OPA1 flox mice with a Th17 fate reporter mouse (IL17Cre x R26eYFP, Hirota et al. 2009). In these mice, cells that express IL-17A become fluorescently labelled and show targeted OPA1 expression. OPA1 deletion in Th17 cells fully abrogated disease incidence in a mouse model of multiple sclerosis (EAE), a Th17-related pathology. In addition, mice in which OPA1 and LKB1 are deleted specifically in Th17 cells, recover signs of disease upon immunization, supporting a role for LKB1 as a sensor of OPA1-deficiency in vivo.
