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Kronsteiner, B.

Publications and source records attributed to Kronsteiner, B..

3 recordsLinked to original sources

Iron deficiency causes aspartate-sensitive metabolic and proliferative dysfunction in CD8+ T cells

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people, causing symptoms including anaemia and impaired immunity. Nevertheless, precisely how iron deprivation impacts immune cell function remains poorly characterised. We therefore interrogated how physiologically low iron availability affected activated CD8+ T cell metabolism and function, using multi-omic and metabolic labelling approaches. Iron limitation profoundly stalled proliferation without influencing cell viability, altered histone methylation status and disrupted mitochondrial membrane potential. Consistently, metabolism of glucose and glutamine in the TCA cycle was limited, indeed TCA cycle activity was partially reversed to a reductive trajectory. Previous studies have shown mitochondria-derived aspartate is crucial for proliferation of transformed cells. Surprisingly, we found aspartate was increased in stalled iron deficient CD8+ T-cells, but was not utilised cytosolically for nucleotide synthesis, likely due to trapping within depolarised mitochondria. Conversely, exogenous aspartate, which directly accesses the cytosol, markedly rescued the clonal expansion of even severely iron-deficient CD8+ T-cells. Overall, iron scarcity creates a mitochondrial-located metabolic bottleneck impairing T-cells, which can be bypassed by resupplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8+ T cell function, providing mechanistic insight into the basis for immune impairment during iron deficiency.

immunology↗

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus

BackgroundAssessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. Methodology/Principle FindingsWe optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-{gamma}, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. Conclusions/SignificanceIn conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level. Author summaryScrub typhus is an acute febrile illness caused by the bite of infected chigger mites transmitting O. tsutsugamushi, a gramnegative obligate intracellular bacteria in the family Rickettsiaceae. The disease progression and clinical manifestations are closely associated with host immune responses. T cell responses are in strong relation with immune protection against scrub typhus. As there is limited knowledge on specific T cell roles against scrub typhus, we optimized a flow-cytometer based protocol to assess O. tsutsugumushi-specific T cell responses in whole blood samples, which is suitable for studies in limited resource settings. This method requires low blood volumes, but enables multiparametric immunophenotyping assessments. The optimized WBA protocol could be a useful tool for comprehensive immunopathological studies in scrub typhus, and other infectious diseases as well as vaccine studies in areas with limited availability of specialized equipment, while reliably capturing the complexity of the immune response.

immunology↗

Identification of regulatory genes through global gene expression analysis of a Helicobacter pylori co-culture system

Helicobacter pylori is a gram-negative bacterium that establishes life-long infections by inducing immunoregulatory responses. We have developed a novel ex vivo H. pylori co-culture system to identify new regulatory genes based on expression kinetics overlapping with that of genes with known regulatory functions. Using this novel experimental platform, in combination with global transcriptomic analysis, we have identified five lead candidates, validated them using mouse models of H. pylori infection and in vitro co-cultures under pro-inflammatory conditions. Plexin domain containing 2 (Plxdc2) was selected as the top lead immunoregulatory target. Gene silencing and ligand-induced activation studies confirmed its predicted regulatory function. Our integrated bioinformatics analyses and experimental validation platform has enabled the discovery of new immunoregulatory genes. This pipeline can be used for the identification of genes with therapeutic applications for treating infectious, inflammatory, and autoimmune diseases.

immunology↗