Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable.

BACKGROUND: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. METHODS: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. RESULTS: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. CONCLUSION: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Induction of memory-like CD8+ T cells and CD4+ T cells from human naive T cells in culture.

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here, we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits, both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia, if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats.

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development.

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56-CD14- NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.