Exploring cell death mechanisms in spheroid cultures using a novel application of the RIP3-caspase3-assay.

This study explores the application of the RIP3-caspase3-assay in heterogeneous spheroid cultures to analyze cell death pathways, emphasizing the nuanced roles of apoptosis and necroptosis. By employing directly conjugated monoclonal antibodies, we provide detailed insights into the complex mechanisms of cell death. Our findings demonstrate the assay's capability to differentiate between RIP1-independent apoptosis, necroptosis, and RIP1-dependent apoptosis, marking a significant advancement in organoid research. Additionally, we investigate the effects of TNFα on isolated intestinal epithelial cells, revealing a concentration-dependent response and an adaptive or threshold reaction to TNFα-induced stress. The results indicate a preference for RIP1-independent cell death pathways upon TNFα stimulation, with a notable increase in apoptosis and a secondary role of necroptosis. Our research underscores the importance of the RIP3-caspase3-assay in understanding cell death mechanisms in organoid cultures, offering valuable insights for disease modeling and the development of targeted therapies. The assay's adaptability and robustness in spheroid cultures enhances its potential as a tool in personalized medicine and translational research.

Intestinal CD8+ T cell responses are abundantly induced early in human development but show impaired cytotoxic effector capacities.

Gastrointestinal viral infections are a major global cause of disease and mortality in infants. Cytotoxic CD8+ T cells are critical to achieve viral control. However, studies investigating the development of CD8+ T cell immunity in human tissues early in life are lacking. Here, we investigated the maturation of the CD8+ T cell compartment in human fetal, infant and adult intestinal tissues. CD8+ T cells exhibiting a memory phenotype were already detected in fetal intestines and increased after birth. Infant intestines preferentially harbored effector CCR7-CD45RA-CD127-KLRG1+/- CD8+ T cells compared to tissue-resident memory CD69+CD103+CD8+ T cells detected in adults. Functional cytotoxic capacity, including cytokine and granzyme B production of infant intestinal effector CD8+ T cells was, however, markedly reduced compared to adult intestinal CD8+ T cells. This was in line with the high expression of the inhibitory molecule PD-1 by infant intestinal effector CD8+ T cells. Taken together, we demonstrate that intestinal CD8+ T cell responses are induced early in human development, however exhibit a reduced functionality. The impaired CD8+ T cell functionality early in life contributes to tolerance during foreign antigen exposure after birth, however functions as an immune correlate for the increased susceptibility to gastrointestinal viral infections in infancy.

Quantitative comparison of human intestinal mononuclear leukocyte isolation techniques for flow cytometric analyses.

Studies on immune cells derived from the human intestine are needed to understand the pathogenesis of gastrointestinal diseases and to develop novel treatment strategies. Isolation techniques to extract these immune cells from intestinal tissue are largely based on murine studies and comparative data on isolation from human intestine is scarce. In this study we evaluated cell yield, viability, and surface-molecule expression on mononuclear leukocytes, comparing three techniques to obtain a single immune cell suspension from human intestine; low concentrations of either the enzymes Collagenase D or Liberase TL, and enzyme-free mechanical dissociation with the Medimachine. Both enzymatic isolation techniques provided a higher cell yield than mechanical dissociation. Expression of surface molecules remained intact after Collagenase D treatment, while Liberase TL digestion resulted in a strong decrease in the expression of the CD4 receptor. Taken together, Collagenase D digestion provides the highest yield of mononuclear cells while keeping surface molecule expression intact.