Shalin H. Naik

Steady-state and inflammatory dendritic-cell development

The developmental pathways that lead to the production of antigen-presenting dendritic cells (DCs) are beginning to be understood. These are the last of the pathways of haematopoiesis to be mapped. The existence of many specialized subtypes of DC has complicated this endeavour, as has the need to distinguish the DCs formed in steady state from those produced during an inflammatory response. Here we review studies that lead to the concept that different types of DC develop through different branches of haematopoietic pathways that involve different immediate precursor cells. Furthermore, these studies show that many individual tissues generate their own DCs locally, from a reservoir of immediate DC precursors, rather than depending on a continuous flux of DCs from the bone marrow.

Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny

The mononuclear phagocyte system (MPS) has historically been categorized into monocytes, dendritic cells and macrophages on the basis of functional and phenotypical characteristics. However, considering that these characteristics are often overlapping, the distinction between and classification of these cell types has been challenging. In this Opinion article, we propose a unified nomenclature for the MPS. We suggest that these cells can be classified primarily by their ontogeny and secondarily by their location, function and phenotype. We believe that this system permits a more robust classification during both steady-state and inflammatory conditions, with the benefit of spanning different tissues and across species.

Cutting Edge: Generation of Splenic CD8+ and CD8− Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8+ and CD8− conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RAhigh plasmacytoid DC, two distinct CD24high and CD11bhigh cDC subsets were present, and these subsets showed equivalent properties to splenic CD8+ and CD8− cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-α; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-α, MIP-1α, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24high subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.

Differential development of murine dendritic cells by GM-CSF versus Flt3 ligand has implications for inflammation and trafficking.

To gain ample numbers of dendritic cells (DCs) for investigation, or for immunotherapy, the culture of DC precursors from bone marrow in either GM-CSF and IL-4 (GM/IL4-DCs) or Flt3L (FL-DCs) has often been used. Despite their common use, the relationship of these culture-derived DCs to those in vivo, and their relative potential for use in immunotherapy, needs further elucidation. In this study we found that in contrast to FL-DCs, highly purified GM/IL4-DCs were larger and more granular, surface Mac-3+, and were comprised of two populations (CD24lowCD11bhigh and CD24highCD11blow). Functionally, although comparable in T cell activation, GM/IL4-DCs produced more inflammatory mediators including TNF-α, IL-10, CCL-2, and NO than FL-DCs upon TLR ligation. However, FL-DCs migrated more efficiently to draining lymph nodes after s.c. injection and produced a different profile of cytokines to GM/IL4-DCs. Developmentally, unlike GM/IL4-DCs, FL-DCs cannot be differentiated from CD11bhighLy6ChighLy6G− monocytes. Collectively, these data suggest that the GM/IL4-DCs are the equivalents of the TNF-α and inducible NO synthase producing DCs in vivo that emerge after inflammation whereas FL-DCs better represent the steady-state resident DCs. The differences between GM/IL4-DCs and FL-DCs have serious implications for DC-based immunotherapeutic strategies.

Heterogeneous Differentiation Patterns of Individual CD8+ T Cells

Dynamic Protection During an immune response, CD8+ T cells are recruited to provide protection. Most cells differentiate into short-lived effectors that help to clear the pathogen, whereas others form long-lived memory cells to protect against reinfection. Gerlach et al. (p. 635, published online 14 March) and Buchholz et al. (p. 630, published online 14 March) used in vivo fate mapping of mouse T cells with a defined specificity during a bacterial infection to show that the dynamics of the single-cell response are not uniform. The response of a particular T cell population is the average of a small number of clones that expand greatly (“large clones”) and many clones that only proliferate at low amounts (“small clones”). The memory pool arises largely from small clones whereas effectors are primarily made up of large clones. The single-cell dynamics as cytotoxic T cells respond to a bacterial infection are analyzed in mice. Upon infection, antigen-specific CD8+ T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8+ T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8+ T cell heterogeneity and demonstrates that reproducibility of CD8+ T cell responses is achieved through population averaging.

Diverse and heritable lineage imprinting of early haematopoietic progenitors

Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of ‘cellular barcoding’ to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a ‘graded commitment’ model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

The Molecular Basis for the Lack of Immunostimulatory Activity of Vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at ∼3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 μg/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Demystifying the development of dendritic cell subtypes, a little.

The broadest definition of dendritic cells (DCs) is white blood cells that can take up antigen, process it and then present antigen‐derived peptides to activate cognate naive T cells. Although this definition is by no means perfect, it is nevertheless now textbook. The source of frustration more recently has focused on other issues, including the distinction of the DC subtypes, their differential roles in the immune system, their lineage relationship to each other (and other leukocytes) and whether the mouse and human DC findings overlap. Here, I condense the classification of DCs in both the steady state versus infection, with primary focus in the mouse. Emphasis is then given to debates surrounding the in vivo pathways of DC differentiation in different conditions, which culture models best represent these processes (fms‐like tyrosine kinase 3 ligand versus granulocyte‐macrophage colony‐stimulating factor), and what the human and mouse DC subtype equivalents might be. In addition, a model termed ‘graded’ commitment is proposed that, as a departure from the classic binary models of hematopoiesis, attempts to explain the recent clonal data where subtype‐specific DC precursors branch from this pathway.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α+ (CD103+) cDC1 lineage, and the CD11b+ cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H−Ly6C− pre-DCs) or cDC2 lineage (Siglec-H−Ly6C+ pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

Normal proportion and expression of maturation markers in migratory dendritic cells in the absence of germs or Toll-like receptor signaling

Dendritic cells (DCs) play major roles in immunosurveillance. In peripheral tissues, ‘immature’ DCs are dedicated to capturing antigens. Detection of pathogens through Toll‐like receptors (TLRs) triggers DC migration to the lymph nodes (LNs), where they acquire a ‘mature’ phenotype specialized at presenting antigens. However, DCs migrate from tissues and mature even in the absence of overt infections. This has been attributed to detection of commensal flora in the skin, the gut or other peripheral tissues in the steady state. To test this assumption, we have analyzed the DCs contained in the lymphoid organs of germ‐free mice and of mice lacking the TLR adapter molecules, MyD88 and TRIF. We show that the proportion and expression of maturation markers in DC immigrants in the LNs of these mice are similar to those in normal mice. These results suggest that DC migration from tissues, followed by their phenotypic maturation, is regulated in the steady state by an inherent program of DC differentiation or by the release of low levels of inflammatory signals from normal tissues.