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

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

Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8α+ dendritic cells

In mouse, a subset of dendritic cells (DCs) known as CD8α+ DCs has emerged as an important player in the regulation of T cell responses and a promising target in vaccination strategies. However, translation into clinical protocols has been hampered by the failure to identify CD8α+ DCs in humans. Here, we characterize a population of human DCs that expresses DNGR-1 (CLEC9A) and high levels of BDCA3 and resembles mouse CD8α+ DCs in phenotype and function. We describe the presence of such cells in the spleens of humans and humanized mice and report on a protocol to generate them in vitro. Like mouse CD8α+ DCs, human DNGR-1+ BDCA3hi DCs express Necl2, CD207, BATF3, IRF8, and TLR3, but not CD11b, IRF4, TLR7, or (unlike CD8α+ DCs) TLR9. DNGR-1+ BDCA3hi DCs respond to poly I:C and agonists of TLR8, but not of TLR7, and produce interleukin (IL)-12 when given innate and T cell–derived signals. Notably, DNGR-1+ BDCA3+ DCs from in vitro cultures efficiently internalize material from dead cells and can cross-present exogenous antigens to CD8+ T cells upon treatment with poly I:C. The characterization of human DNGR-1+ BDCA3hi DCs and the ability to grow them in vitro opens the door for exploiting this subset in immunotherapy

Type I Interferon Drives Dendritic Cell Apoptosis via Multiple BH3-Only Proteins following Activation by PolyIC In Vivo

BACKGROUND: DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs

Hierarchy of Susceptibility of Dendritic Cell Subsets to Infection by Leishmania major: Inverse Relationship to Interleukin-12 Production

Dendritic cells (DCs) are professional antigen-presenting cells which initiate and regulate T-cell immune responses. Here we show that murine splenic DCs can be ranked on the basis of their ability to phagocytose and harbor the obligately intracellular parasite Leishmania major. CD4(+) CD8(−) DCs are the most permissive host cells for L. major amastigotes, followed by CD4(−) CD8(−) DCs; CD4(−) CD8(+) cells are the least permissive. However, the least susceptible CD4(−) CD8(+) DC subset was the best interleukin-12 producer in response to infection. Infection did not induce in any DC subset production of the proinflammatory cytokine gamma interferon and nitric oxide associated with the induction of Th1 responses. The number of parasites phagocytosed by DCs was low, no more than 3 organisms per cell, compared to more than 10 organisms per macrophage. In infected DCs, the parasites are located in a parasitophorous vacuole containing both major histocompatibility complex (MHC) class II and lysosome-associated membrane protein 1 molecules, similar to their location in the infected macrophage. The parasite-driven redistribution of MHC class II to this compartment indicates that infected DCs should be able to present parasite antigen