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

THE ROLE OF NONLYMPHOID ACCESSORY CELLS IN THE IMMUNE RESPONSE TO DIFFERENT ANTIGENS

Tissue culture techniques were combined with cell separation procedures to investigate the cellular requirements for a response to antigen, leading to the production of antibody-forming cells. Mouse spleen was dissociated, and the cells were separated into various groups on the basis of density, size, and active adherence. The ability of fractions to initiate a response in vivo, on transfer to an irradiated recipient, was compared to the response in vitro; and this ability was correlated with the presence or absence of phagocytic cells. Two different antigens were studied, sheep erythrocytes (SRC) and polymerized bacterial flagellin (POL). Density distribution analysis of spleen showed a wide density range of cells responding to both antigens in vivo. The same fractions responded to POL in vitro as in vivo. By contrast, only the light density regions responded in vitro to SRC. Response occurred in regions of overlap between lymphocytes and phagocytic macrophages. Separation by active adherence on columns of large glass beads gave a preparation containing large, medium, and small lymphocytes but no detectable phagocytic macrophages and very low levels of phagocytic polymorphs. This lymphocyte preparation responded to both antigens in vivo. In vitro it gave a full response to POL, but no response to SRC. Addition of a small quantity of the adherent fraction, enriched for phagocytic cells, restored response to SRC. The use of strain-specific antisera in a mixed culture containing a C57 phagocytic fraction and CBA lymphocytes showed that the lymphocyte fraction contributed the precursors of the final antibody-forming cells. The accessory cells from C57 spleen banded in the light regions of the density gradient where phagocytic macrophages were found. Irradiated spleen cells also activated the lymphocyte preparation, suggesting that the irradiated host provided the accessory cells for the in vivo response to SRC. Small lymphocytes were purified from spleen by the small glass bead size filtration technique. This sample of small lymphocytes responded less well to POL than the total lymphocyte population, but it responded as well in vitro as in vivo. The small lymphocyte preparation responded in vivo to SRC but not in vitro. Addition of a small quantity of the phagocyte-rich fraction from adherence columns restored the in vitro response to SRC. The results indicated that phagocytic cells are not required in the initiation of an immune response to POL. By contrast some accessory cell, possibly a phagocytic macrophage, is required for a response to SRC. The basis for this marked difference is discussed

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

Found in translation: the human equivalent of mouse CD8+ dendritic cells

The murine dendritic cell network comprises multiple subsets with distinct functions, but few of their human counterparts have been described. New data now reveals the likely human equivalent of the mouse DC subset specialized in cross-presentation

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