The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using high-dimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+DC) heterogeneity originates from two distinct pathways of development. The lymphoid-primed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency.

The IRF8-dependent subset of classical dendritic cells (cDCs), termed cDC1, is important for cross-priming cytotoxic T cell responses against pathogens and tumors. Culture of hematopoietic progenitors with DC growth factor FLT3 ligand (FLT3L) yields very few cDC1s (in humans) or only immature "cDC1-like" cells (in the mouse). We report that OP9 stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) optimize FLT3L-driven development of cDC1s from murine immortalized progenitors and primary bone marrow cells. Co-culture with OP9-DL1 induced IRF8-dependent cDC1s with a phenotype (CD103+ Dec205+ CD8α+) and expression profile resembling primary splenic cDC1s. OP9-DL1-induced cDC1s showed preferential migration toward CCR7 ligands in vitro and superior T cell cross-priming and antitumor vaccination in vivo. Co-culture with OP9-DL1 also greatly increased the yield of IRF8-dependent CD141+ cDC1s from human bone marrow progenitors cultured with FLT3L. Thus, Notch signaling optimizes cDC generation in vitro and yields authentic cDC1s for functional studies and translational applications.

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

The homozygous K108E mutation of interferon regulatory factor 8 (IRF8) is reported to cause dendritic cell (DC) and monocyte deficiency. However, more widespread immune dysfunction is predicted from the multiple roles ascribed to IRF8 in immune cell development and function. We sought to describe the effect on hematopoiesis and immunity of the compound heterozygous R83C/R291Q mutation of IRF8, which is present in a patient with recurrent viral infection, granuloproliferation, and intracerebral calcification. Variant IRF8 alleles were identified by means of exome sequencing, and their function was tested by using reporter assays. The cellular phenotype was studied in detail by using flow cytometry, functional immunologic assay transcriptional profiling, and antigen receptor profiling. Both mutations affected conserved residues, and R291Q is orthologous to R294, which is mutated in the BXH2 IRF8-deficient mouse. R83C showed reduced nuclear translocation, and neither mutant was able to regulate the Ets/IRF composite element or interferon-stimulated response element, whereas R291Q retained BATF/JUN interactions. DC deficiency and monocytopenia were observed in blood, dermis, and lung lavage fluid. Granulocytes were consistently increased, dysplastic, and hypofunctional. Natural killer cell development and maturation were arrested. TH1, TH17, and CD8+ memory T-cell differentiation was significantly reduced, and T cells did not express CXCR3. B-cell development was impaired, with fewer memory cells, reduced class-switching, and lower frequency and complexity of somatic hypermutation. Cell-specific gene expression was widely disturbed in interferon- and IRF8-regulated transcripts. This analysis defines the clinical features of human biallelic IRF8 deficiency, revealing a complex immunodeficiency syndrome caused by DC and monocyte deficiency combined with widespread immune dysregulation.

Ikaros family zinc finger 1 (IKZF1) is a haematopoietic transcription factor required for mammalian B-cell development. IKZF1 deficiency also reduces plasmacytoid dendritic cell (pDC) numbers in mice, but its effects on human DC development are unknown. Here we show that heterozygous mutation of IKZF1 in human decreases pDC numbers and expands conventional DC1 (cDC1). Lenalidomide, a drug that induces proteosomal degradation of IKZF1, also decreases pDC numbers in vivo, and reduces the ratio of pDC/cDC1 differentiated from progenitor cells in vitro in a dose-dependent manner. In addition, non-classical monocytes are reduced by IKZF1 deficiency in vivo. DC and monocytes from patients with IKZF1 deficiency or lenalidomide-treated cultures secrete less IFN-α, TNF and IL-12. These results indicate that human DC development and function are regulated by IKZF1, providing further insights into the consequences of IKZF1 mutation on immune function and the mechanism of immunomodulation by lenalidomide.