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SummaryT cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses. Graphical abstract Highlights?Isolation of leukocytes from murine BM, liver, heart and kidneys?Non-enzymatic dissociation of kidney and heart tissue?Protocol for immunomagnetic enrichment of T cells?Flow cytometry analysis of T cell purity and viability Publisher¡¯s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. T cells are the central effectors and regulators of the adaptive immune response. This protocol provides a step-by-step approach for isolating and enriching total T cells by negative selection from complex murine tissues, including bone marrow (BM), liver, heart, and kidneys. We describe steps for tissue harvesting, preparation of single-cell suspensions, and immunomagnetic enrichment. We then outline procedures for flow cytometric assessment of cell purity and viability. This protocol enables efficient recovery of high-quality T cells for reliable downstream analyses.

AbstractINTRODUCTIONRecent studies have identified important species©\dependent differences in the response of microglia to ¦Â©\amyloid (A¦Â) pathology. Yet, whether human microglia also interact differently with the pathognomonic combination of amyloid and tau pathologies that occur in Alzheimer's disease (AD) remains unclear.METHODSWe generated a xenotolerant mouse model of AD that develops both plaque and tangle pathologies, transplanted stem cell©\derived microglial progenitors and examined the interactions between human microglia and AD pathologies with scRNA sequencing, immunohistochemistry, and in vitro modeling.RESULTSThe combined amyloid and tau pathologies induced robust type©\I interferon and proinflammatory cytokine responses, as well as an increased adoption of a distinct ¡°rod¡± morphology in human microglia. The rod morphology could be induced with type©\I interferon treatment in vitro.DISCUSSIONWe provide new insights into human microglial responses to combined AD pathologies and a novel platform to investigate and manipulate human microglia in vivo.Highlights Amyloid pathology promotes the rapid development of neurofibrillary tangles and neuronal loss in a novel chimeric model of AD.Combined Alzheimer's disease pathologies lead to an expansion of disease©\associated microglia (DAM) and exacerbate Interferon©\responsive and cytokine/chemokine©\enriched states in xenotransplanted human microglia.The combination of amyloid and tau promotes the development of a distinctive rod microglial phenotype that closely correlates with tau pathology and neurodegeneration.Rod morphology and transcriptional changes can be modeled in vitro by treatment of induced pluripotent stem cells (iPSC) ©\microglia with type©\I interferons.

Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless, how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8+ T cell metabolism and function, using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However, low iron profoundly stalls proliferation (without influencing cell viability), alters histone methylation status, gene expression, and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling, aspartate is increased in stalled iron deficient CD8+ T cells but is not utilised for nucleotide synthesis, likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8+ T cells. Overall, iron scarcity creates a mitochondrial-located metabolic bottleneck, which is bypassed by supplying 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. Iron has been shown to be necessary for the activation and differentiation of CD8+ T cells. Here the authors investigate changes in CD8+ T cell metabolism in iron limiting conditions and find that aspartate is increased yet downstream nucleotide synthesis is suppressed and addition of exogenous aspartate partially rescues T cell function.