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The signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor–dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

T cell activation requires that the cell meet increased energetic and biosynthetic demands. We showed that exogenous nutrient availability regulated the differentiation of naïve CD4(+) T cells into distinct subsets. Activation of naïve CD4(+) T cells under conditions of glutamine deprivation resulted in their differentiation into Foxp3(+) (forkhead box P3-positive) regulatory T (Treg) cells, which had suppressor function in vivo. Moreover, glutamine-deprived CD4(+) T cells that were activated in the presence of cytokines that normally induce the generation of T helper 1 (TH1) cells instead differentiated into Foxp3(+) Treg cells. We found that α-ketoglutarate (αKG), the glutamine-derived metabolite that enters into the mitochondrial citric acid cycle, acted as a metabolic regulator of CD4(+) T cell differentiation. Activation of glutamine-deprived naïve CD4(+) T cells in the presence of a cell-permeable αKG analog increased the expression of the gene encoding the TH1 cell-associated transcription factor Tbet and resulted in their differentiation into TH1 cells, concomitant with stimulation of mammalian target of rapamycin complex 1 (mTORC1) signaling. Together, these data suggest that a decrease in the intracellular amount of αKG, caused by the limited availability of extracellular glutamine, shifts the balance between the generation of TH1 and Treg cells toward that of a Treg phenotype.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV–coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.

Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)–6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1β, and tumor necrosis factor-α, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking–induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues.

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