Different Subpopulations of Developing Thymocytes are Associated with Adherent (Macrophage) or Nonadherent (Dendritic) Thymic Rosettes

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8+CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROSassociated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex

Characterization of Thymic Nurse-Cell Lymphocytes, Using an Improved Procedure for Nurse-Cell Isolation

Thymic nurse cells (TNC), multicellular complexes consisting of lymphoid cells enclosed within cortical epithelial cells, were isolated from mouse thymus by a modified procedure allowing immunofluorescent labeling and flow cytometric analysis of their lymphoid contents (TNC-L). Collagenase was the only protease used for tissue digestion, to ensure that surface antigen markers remained intact. Zonal unit-gravity elutriation was used to enrich the TNC on the basis of their high sedimentation rate, followed by immunomagnetic bead depletion to remove residual mononuclear cell contaminants and a density separation to remove debris. The TNC-L were then released from inside TNC by a short period of culture. The measured contamination of TNC-L with exogenous thymocytes was around 0.5%. Three-color immunofluorescent labeling revealed that TNC-L included, as well as a maiority of immature CD4+8+3low thymocytes, about 12% of apparently mature CD4+8-3high and CD4-8+3high thymocytes. TNC are located in the cortex, where mature cells are rare; the occurrence of mature phenotype cells within these structures suggests that they represent a microenvironment for the selection and generation of mature T cells

Mouse Plasmacytoid Cells: Long-lived Cells, Heterogeneous in Surface Phenotype and Function, that Differentiate Into CD8+ Dendritic Cells Only after Microbial Stimulus

The CD45RAhiCD11cint plasmacytoid predendritic cells (p-preDCs) of mouse lymphoid organs, when stimulated in culture with CpG or influenza virus, produce large amounts of type I interferons and transform without division into CD8+CD205− DCs. P-preDCs express CIRE, the murine equivalent of DC-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN). P-preDCs are divisible by CD4 expression into two subgroups differing in turnover rate and in response to Staphylococcus aureus. The kinetics of bromodeoxyuridine labeling and the results of transfer to normal recipient mice indicate that CD4− p-preDCs are the immediate precursors of CD4+ p-preDCs. Similar experiments indicate that p-preDCs are normally long lived and are not the precursors of the short-lived steady-state conventional DCs. However, in line with the culture studies on transfer to influenza virus-stimulated mice the p-preDCs transform into CD8+CD205− DCs, distinct from conventional CD8+CD205+ DCs. Hence as well as activating preexistant DCs, microbial infection induces a wave of production of a new DC subtype. The functional implications of this shift in the DC network remain to be determined

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity

Dendritic cell subsets in the intestinal lamina propria: ontogeny and function

The intestinal mucosa is exposed to large amounts of foreign antigen (Ag) derived from commensal bacteria, dietary Ags, and intestinal pathogens. Dendritic cells (DCs) are believed to be involved in the induction of tolerance to harmless Ags and in mounting protective immune responses to pathogens and, as such, to play key roles in regulating intestinal immune homeostasis. The characterization of classical DCs (cDCs) in the intestinal lamina propria has been under intense investigation in recent years but the use of markers (including CD11c, CD11b, MHC class II), which are also expressed by intestinal MΦs, has led to some controversy regarding their definition. Here we review recent studies that help to distinguish cDCs subsets from monocyte-derived cells in the intestinal mucosa. We address the phenotype and ontogeny of these cDC subsets and highlight recent findings indicating that these subsets play distinct roles in the regulation of mucosal immune responses in vivo

Pro-inflammatory Cytokine Expression of Spleen Dendritic Cells in Mouse Toxoplasmosis

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1α, IL-1β, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8α+/CD11c+ splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis

Putative IKDCs are functionally and developmentally similar to natural killer cells, but not to dendritic cells

Interferon-producing killer dendritic cells (IKDCs) have been described as possessing the lytic potential of NK cells and the antigen-presenting capacity of dendritic cells (DCs). In this study, we examine the lytic function and antigen-presenting capacity of mouse spleen IKDCs, including those found in DC preparations. IKDCs efficiently killed NK cell targets, without requiring additional activation stimuli. However, in our hands, when exposed to protein antigen or to MHC class II peptide, IKDCs induced little or no T cell proliferation relative to conventional DCs or plasmacytoid DCs, either before or after activation with CpG, or in several disease models. Certain developmental features indicated that IKDCs resembled NK cells more than DCs. IKDCs, like NK cells, did not express the transcription factor PU.1 and were absent from recombinase activating gene-2–null, common γ-chain–null (Rag2−/−Il2rg−/−) mice. When cultured with IL-15 and -18, IKDCs proliferated extensively, like NK cells. Under these conditions, a proportion of expanded IKDCs and NK cells expressed high levels of surface MHC class II. However, even such MHC class II+ IKDCs and NK cells induced poor T cell proliferative responses compared with DCs. Thus, IKDCs resemble NK cells functionally, and neither cell type could be induced to be effective antigen-presenting cells

Notch1 Deficiency Dissociates the Intrathymic Development of Dendritic Cells and T Cells

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1−/− BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1−/− T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors