The MET-FLAM Faculty

Martin Helmut STRADNER

Scientific Interests:

Signs of immune cell aging occur early in life in patients suffering from autoimmune diseases. Even naïïve T cells, which have not encountered antigen yet show signs of premature aging in young patients (Fig. 1). Senescent naïve T cells are characterized by telomerase insufficiency, increase of senescence-associated β-galactosidase and drastically reduced levels of T-cell receptor excision circles in autoimmune diseases [1]. We found a similar phenotype in regulatory T cells (Treg) of patients with autoimmune disease [2]. These Tregs have an impaired suppressive function and might contribute to the loss of tolerance in autoimmunity. Furthermore, premature senescence is linked to dysfunctional homeostatic proliferation of T cells leading to peripheral lymphopenia in patients with autoimmune diseases such as Sjögren’s syndrome or systemic lupus erythematosus (SLE). In our lab we aim to find out how premature aging of T cells links to the occurrence of autoimmunity. Therefore, we analyze T cells of patients with early Sjögren’s syndrome and those with latent autoimmunity prone to develop the disease [3]. Furthermore, we investigate the link between premature senescence and cellular metabolism in Sjögren’s syndrome and SLE (Fig. 2). Thereby, we aim to better understand and potentially reverse the effects of premature aging on T-cell function.

Proposed Dissertation Topic:

Background: Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease associated with considerable morbidity and mortality. T cells play a critical role in the pathogenesis of SLE. We have previously shown that naïve CD4⁺ T cells of young SLE patients show features of premature senescence and extensive post-thymic cell division [1]. These hallmarks of immuno­senescence have been closely linked to metabolic alterations including mitochondrial dysfunction, increase in reactive oxygen species (ROS), defective autophagy, and deregulated nutrient sensing [4]. Intriguingly, mitochondrial hyperpolarization towards increased glucose-derived oxidative phosphorylation resulting in increased production of ROS and active mTOR signalling are hallmarks of SLE T cells [5]. Furthermore, our preliminary data indicate increased glycolysis in senescent naïve CD4⁺ T cells — a feature known to be deregulated in T effector cells in SLE [5].

Hypothesis and objectives: We hypothesize that premature immunosenescence of naive T cells drives metabolic changes in T cells associated with SLE pathogenesis. The PhD candidate will (i) define the metabolic characteristics of senescent naïve T cells from SLE patients; (ii) analyse the impact of immunosenescence on T cell receptor (TCR) signalling and T effector cell polarization; (iii) unveil the molecular and mechanistic basis of these findings by in vivo and in vitro experiments.

Methods and approaches: To achieve the objectives, the PhD candidate will analyse mitochondrial biology (mitochondrial mass, membrane potential, mitochondrial reactive oxygen species), autophagic activity, lipid metabolism and accumulation of lipid droplets, overall cellular stress via determination of reactive oxygen species, energetic metabolism using SCENITH and seahorse technology in naïve T cells of SLE patients and healthy controls. These findings will be associated with analyses of cellular senescence and post-thymic cell division such as telomere length, levels of senescence-associated β-galactosidase and TCR excision circles (1st year). In the second year, TCR activation thresholds and in vitro T effector cell polarization (using cytokine combinations for the various T helper subsets) of senescent SLE naïve T cells will be compared to naïve T cells of healthy individuals. Untargeted metabolomics using NMR metabolic profiling of cells and supernatants will identify metabolic traits of these cells. Chemical inhibitors of the metabolic pathways identified will indicate their functional relevance in these in vitro assays (2nd and 3rd year). Starting in the 3rd year, the PhD candidate will use suitable mouse models to investigate the role of these metabolic pathways in in nephrotoxic serum nephritis in vivo.

Pitfalls and alternative approaches: In case no suitable mouse model is available to address the metabolic pathways identified, the PhD candidate will extend the in vitro analyses in the 3rd year. Using chemical modifiers of the metabolic pathways identified the student will examine its influence on T-cell differentiation of human naïve T cells to T_FH cells and B cell–T cell interaction.

Involved Faculty members: Martin H. Stradner (PI), Simon Sedej (autophagy), Dagmar Kratky (lipid metabolism), Stefano Angiari (glucose metabolism) and Kathrin Eller (mouse models).

International Collaborations: Divi Cornec (Brest, France) and Ananda Goldrath (San Diego, USA).

Facilities: The Stradner lab is a team of two post docs, a lab manager, two PhD candidates and several Master students. The team has access to samples of more than two thousand patients with autoimmune diseases. It is equipped with facilities for cell culture, immuno­fluorescence, western blot, real-time qPCR, multiplex cytokine analysis, automated cell sorting, and two high end flow cytometers. Furthermore, we have established single cell energetic metabolism by profiling translation inhibition (SCENITH). The lab has full access to the animal facilities, Seahorse XFe96 Analyzer, Cytek Aurora (Spectral Flow Cytometry) and single cell RNAseq pipeline using the Chromium Controller (10X Genomics). NMR metabolomics is performed in a long-standing cooperation with Tobias Madl at Molecular Biology and Biochemistry of our University.

Preparatory Findings:

Figure 1. Naïve T cells of patients with Sjögren’s syndrome and SLE have shortened telomeres and reduced levels of T cell receptor excision circles indicative of extensive replicative history and premature senescence.
T cell receptor excision circles were analysed by pRT-PCR in naïve T cells of healthy controls (HC) and patients suffering from spondyloarthritis (SpA), rheumatoid arthritis (RA), SLE, and primary Sjögren’s syndrome (pSS). Published in [1].

Figure 2. Decreased concentrations of glucose and lactate in the supernatant of cultured senescent naïve T cells indicating increased glucose utilisation and oxidative phosphorylation.
Supernatants of naïve T cells were cultured 3 days in the presence of IL-7 and were analysed by NMR metabolomics. Comparison of healthy non-senescent T cells to senescent T cells from SLE patients.

1. Fessler J, Fasching P, Raicht A, Hammerl S, Weber J, Lackner A, Hermann J, Dejaco C, Graninger WB, Schwinger W, Stradner MH: Lymphopenia in primary Sjögren’s syndrome is associated with premature aging of naïve CD4+ T cells. Rheumatology (Oxford), 2021; 60(2):​588–597.
   
2. Fessler J, Raicht A, Husic R, Ficjan A, Schwarz C, Duftner C, Schwinger W, Graninger WB, Stradner MH, Dejaco C: Novel Senescent Regulatory T-Cell Subset with Impaired Suppressive Function in Rheumatoid Arthritis. Front Immunol, 2017; 8:300.
   
3. Zenz S, Erlacher L, Windisch E, Dreo B, Javorova P, Lackner A, D’Orazio M, Thiel J, Cornec D, Stradner M: Identifying individuals at risk for Sjögren’s syndrome – The pre-Sjögren syndrome targeted immunology evaluation (PRESTIGE) study. Ann Rheum Dis, 2022; 81(Suppl. 1):​680 (POS0786).
   
4. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G: The hallmarks of aging. Cell, 2013; 153(6):​1194–1217.
   
5. Sharabi A, Tsokos GC: T cell metabolism: new insights in systemic lupus erythematosus pathogenesis and therapy. Nat Rev Rheumatol, 2020; 16(2):​100–112.