The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.

A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.

Langerhans cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation.

Dendritic cell (DC) reconstitution experiments and phenotypic analysis of DC subpopulations have allowed the definition in the mouse of two main DC categories: CD8+ lymphoid DCs and CD8- myeloid DCs. With regard to Langerhans cells (LCs), which represent immature DCs differentiating into mature DCs on migration to the lymph nodes after an antigenic stimulation, although classically considered as myeloid DCs, there is no experimental evidence of their origin. It has been recently shown that mouse LCs, negative for CD8 and LFA-1, undergo CD8/LFA-1 up-regulation on migration, suggesting that LCs belong to the CD8+ lymphoid DC lineage. To further reinforce this hypothesis, we have analyzed the modulation of CD8 expression by LCs on culture with molecules known to induce LC maturation. Our results show that LC acquired a CD8+ lymphoid phenotype on CD40 ligation.

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha.

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.

Mouse thymic dendritic cell subpopulations.

Mouse thymic dendritic cells (DC) have been isolated after collagenase digestion, selection of the low-density cell fraction, then depletion of T-lineage cells and other non-DC by treatment with specific monoclonal antibodies (mAb) and removal with anti-Ig-coated magnetic beads. The resulting DC preparation represented 0.1-0.2% of total thymic cells and contained 70-80% DC. Flow cytometry analysis of MHC class II (MHC II) expression by DC showed that 40% of DC expressed intermediate levels of MHC II, and 60% expressed high levels of this marker. Moreover, immunofluorescent 2-colour staining allowed the characterization of two clearly distinguishable DC subpopulations: MHC IIinter DC were CD45hi, CD44hi, HSAhi, whereas MHC IIhi DC were CD45lo, CD44lo, HSAlo. These results are discussed with regard to the functional significance of MHC IIinter and MHC IIhi DC subpopulations in the mouse thymus.

Rat thymic dendritic cells: cell surface marker variations in culture.

Rat thymic dendritic cells (DC) have been analyzed by flow cytometry in order to study the variations on the cell surface marker expression upon culture at 37 degrees C. Our results demonstrate that whereas expression of major histocompatibility complex (MHC) molecules, CD45, Mac-1, LFA-1, B-cell markers, macrophage markers and some T-cell markers (as CD2, CD4 and CD8) did not undergo changes in culture, the level of expression of the adhesion molecules VLA-4 and ICAM-1, and the T-cell markers CD5, CD25 and Thy-1 increased after 14 h incubation at 37 degrees C. VLA-4, ICAM-1 and Thy-1 expression was up-regulated from intermediate to high levels, the percentage of CD5+ cells increased from 20% to 50%, and the interleukin-2 (IL-2) receptor alpha chain (CD25) was induced in 50% of DC after the culture period. These results are discussed with regard to the functional significance of DC phenotypic variations, and their implications concerning the development of in vitro systems designed for T-cell differentiation studies involving purified DC.

Ultrastructure and changes during metamorphosis of the lympho-hemopoietic tissue of the larval anadromous sea lamprey Petromyzon marinus.

The ultrastructure of the lympho-hemopoietic tissue of the typhlosole and opisthonephros of larval Petromyzon marinus was studied throughout the life cycle. In both locations, lympho-hemopoietic tissue houses a connective tissue stroma and is irrigated by a system of enlarged blood sinuses lined by a continuous endothelium. The typhlosolar and opisthonephric lympho-hemopoietic tissue consists of mature and developing blood cells belonging to erythroid, lymphoid, monocytic and granulocytic lineages. During metamorphosis there is a total degeneration of the larval opisthonephros and a full transformation of the digestive apparatus. As a consequence of these modifications the typhlosole and the opisthonephros lose their hemopoietic capacity. These changes are discussed in view of the role played in them by the so-called "hemopoietic inductive microenvironments", principally the stroma supporting apparatus and vascularization pattern.

Isolation of mouse thymic dendritic cells.

The method described in this chapter for the isolation of mouse thymic dendritic cells (DC) is an optimization of our previously published methods (1,2) and involves the following major steps: 1. Enzymatic digestion of thymic fragments with collagenase and DNase. 2. Separation of a very-low-density cell fraction (VLDF). 3. Magnetic depletion of T-lineage cells, B cells, macrophages, and granulocytes. 4. Positive selection of DCs by magnetic cell sorting (MACS).

Cell surface marker analysis of mouse thymic dendritic cells.

Cell surface markers of mouse thymic dendritic cells have been studied by flow cytometry after isolation by collagenase digestion, separation of the low-density cell fraction and differential adherence. The dendritic cell preparation had a purity of greater than 90%, the contaminating population being essentially composed of thymocytes, macrophages constituting less than 1%. Dendritic cells displayed high forward and low-intermediate side angle scatter, and expressed high levels of major histocompatibility complex (MHC) class I and class II molecules, the heat-stable antigen (HSA), the adhesion molecules Pgp-1 (CD44), LFA-1, ICAM-1 and low levels of Mac-1 and the leukocyte common antigen CD45. Thymic dendritic cells are negative for the stem cell antigen-2 (Sca-2), the B cell-specific form of CD45 (B220), the mouse macrophage markers Fc receptor and F4/80, and the granulocyte marker Gr-1. However, although they do not express the T cell markers Thy-1, CD2, CD3, CD4 and CD5, 20%-30% of dendritic cells are positive for the interleukin 2 receptor alpha chain (CD25), and about 30% express intermediate levels of CD8. These results are discussed with regard to the functional significance of the expression of CD8 by thymic dendritic cells, and the existence of different dendritic cell subpopulations in the murine thymus.

The pharyngeal lymphoid tissue of lampreys. A morpho-functional equivalent of the vertebrate thymus?

PE, pharyngeal epithelium; S, blood sinus; T, connective tissue trabecula; End, endothelial cell; Er, erythrocyte; L, lymphocyte; Lb, lymphoblast; Lm, lymphoblast in mitosis; Ma, macrophage; Ret, reticular cell; col, collagen fibres; db, dense body; g, Golgi apparatus; gr, granules; n, nucleus; nu, nucleolus; m, mitochondria; rer, rough endoplasmic reticulum.

Surface markers of axolotl lymphocytes as defined by monoclonal antibodies.

In an attempt to identify urodele amphibian lymphocyte subpopulations by their surface markers, we prepared hybridomas from BALB/c mice spleen immunized with axolotl (Ambystoma mexicanum) blood and splenic leucocytes and purified immunoglobulins. Sixty-five hybridomas were selected and subsequently subcloned. Among numerous monoclonal antibodies (mAbs) thus obtained, four mAbs were extensively characterized by immunoblotting, single and double fluorescence and immunohistology. MAb 34.38.6 recognizes polypeptides between 65,000 and 72,000 MW and labels in immunofluorescence nearly all thymocytes, 60-63% splenic lymphocytes of normal animals but only 9% splenic lymphocytes in thymectomized animals. MAb 19.14.2 reacts with a 98,000 MW protein and labels a restricted lymphocyte population in thymus (52-77%) and spleen (20-25%). The immunohistological study demonstrates that 34.38.6 and 19.14.2 label most thymocytes and a large proportion of spleen leucocytes including lymphocytes, granulocytes and macrophages. In addition, 19.14.2 labels some large interdigitating cells in thymic epithelial areas and splenic cords. MAbs 33.45.1 and 33.101.2, respectively, recognize heavy (72,000-88,000 MW) and light (20,000-27,000 MW) axolotl immunoglobulin chains. They do not react with thymocytes but label a splenic lymphocyte population not labelled by mAb 34.38.6. The proportion of surface immunoglobulin-positive (sIg+) lymphocytes in spleen is not altered by thymectomy. MAb 33.101.2 labels 40-48% of splenic lymphocytes, 33.45.1 stains only 14% of these same cells. This suggests some interesting heavy-chain isotypic differences in axolotl. For the first time in urodele amphibians, mAbs differentiate T-like and B-like lymphocyte populations by their membrane markers. This will allow further analysis of the axolotl immune system.

In vitro negative selection of viral superantigen-reactive thymocytes by thymic dendritic cells.

Intrathymic expression of endogenous mouse mammary tumor virus (MMTV)-encoded superantigens (SAg) induces the clonal deletion of T cells bearing SAg-reactive T-cell receptor (TCR) Vbeta elements. However, the identity of the thymic antigen-presenting cells (APC) involved in the induction of SAg tolerance remains to be defined. We have analyzed the potential of dendritic cells (DC) to mediate the clonal deletion of Mtv-7-reactive TCR alphabeta P14 transgenic thymocytes in an in vitro assay. Our results show that both thymic and splenic DC induced the deletion of TCR transgenic double positive (DP) thymocytes. DC appear to be more efficient than splenic B cells as negatively selecting APC in this experimental system. Interestingly, thymic and splenic DC display a differential ability to induce CD4+ SP thymocyte proliferation. These observations suggest that thymic DC may have an important role in the induction of SAg tolerance in vivo.

Lamellar body-subsurface cistern complexes in the paraventricular nuclei of the hamster.

Structures identified as lamellar bodies and subsurface cisterns have been observed in the paraventricular nuclei of the hamster hypothalamus. These neuronal structures can be found in the cytoplasm as single organelles or forming lamellar body-subsurface cistern complexes, which are connected with the nuclear envelope. The morphological features of the complexes are described and their possible functional significance with regard to the ageing process are discussed.

Ultrastructure of gut-associated lymphoid tissue (GALT) in the amphibian urodele, Pleurodeles waltlii.

The ultrastructure of gut-associated lymphoid tissue (GALT) has been studied in the salamander, Pleurodeles waltlii. Lymphoid accumulations appear as true infiltrates scattered throughout the lamina propria cell elements. The most important components of these infiltrates are small and medium sized lymphocytes, and, in lesser amounts, developing and mature plasma cells, macrophages and granulocytes. Migrating lymphoid cells massively invade the intestinal epithelium inducing noticeable modifications, such as the disappearance of the basement membrane and decreased numbers of mucous cells. Thus, in its organization and cell composition, the GALT of P. waltlii appears to represent a primitive phylogenetic precursor of the mammalian "intestinal-immunologic" barrier.

Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population.

Dendritic cells, a minor cell population in lymphoid tissues, are specialized for presentation of antigenic peptides to T lymphocytes. Thymic dendritic cells are involved in the deletion of self-reactive T lymphocytes. Although all dendritic cells are ultimately of bone-marrow origin, it has not been clear whether thymic dendritic cells are produced in the adult thymus from a precursor cell or whether they migrate there preformed from the periphery. Recently we isolated from adult mouse thymus a population of early T precursors that could still form B lymphocytes, but not erythroid or myeloid cells, when transferred intravenously. Here we show that these thymic lymphoid precursor cells, as well as bone-marrow haematopoietic stem cells, are able to form both dendritic cells and T-cell progeny when transferred into an irradiated thymus. Such linked development may ensure that developing T cells are negatively selected predominantly by self antigens presented on newly formed thymic dendritic cells.

In vitro characterization of rat thymic macrophages.

Thymic macrophages have been isolated from Wistar rats and maintained in long-term culture. Day 1-isolated thymic macrophages expressed MHC class I and II antigens, Thy-1, CR3, CD4, ED1, ED2, as well as acid phosphatase and non-specific esterase activities. Whereas cultured macrophages were positive for the activation antigen recognized by the mAb OX48, which was not expressed after isolation, MHC class II and Thy-1 expression were down-regulated during the culture, but could be induced with human rIL-2 or with Con A-SCM, which are also inducers of IL-2R, a cell-surface marker not expressed on Day 1 or on cultured macrophages.

Rat thymic cultures: morphological and phenotypical characterization.

Rat thymic cultures have been established in order to analyse the morpho-functional characteristics of thymic epithelial and non-epithelial cells in vitro. Stromal cultures, originating from implanted thymic fragments, consist of fibroblasts occupying most of the culture surface, epithelial cells forming discrete colonies, thymocytes and bone marrow-derived cells. Epithelial cells show a low class II MHC antigen expression, which is highly increased in semi-adherent cells, and do not interact with thymocytes. Thymocytes proliferate extensively at the beginning of the culture, but almost disappear at the end of the first week; however, restarting of thymocyte proliferation occurs during the second week of culture. Bone marrow-derived cells include ED- Ia+ CR3- IL-2R- dendritic cells (DC), ED+ Ia+ CR3+ IL-2R+ non-adherent thymic phagocytic cells (PTR) and ED+ Ia- CR3- IL-2R- adherent type 1 and 2 macrophages, derived from PTR. Both PTR and DC establish lympho-stromatic complexes with thymocytes present in the cultures. These results suggest that PTR and DC present in rat thymic cultures belong to different cell lineages, and that they are, respectively, the in vitro equivalents of intrathymic macrophages and interdigitating cells.

Cell-surface marker analysis of rat thymic dendritic cells.

Rat thymic dendritic cells have been isolated by collagenase digestion, separation of the low-density cell fraction by centrifugation on metrizamide, and differential adherence. The resulting dendritic cell preparation had a purity of > 90%, and has been analysed by flow cytometry (FCM) using a large panel of monoclonal antibodies (mAb). Dendritic cells expressed major histocompatibility (MHC) class I and class II molecules, the leucocyte common antigen CD45, the rat leucocyte antigen OX44, the rat macrophage marker ED1, and the adhesion molecules Mac-1, LFA-1 and ICAM-1. They were negative for the T- and B-cell-specific forms of CD45, CD45R and B220, and the B-cell marker OX12. Concerning T-cell marker expression, they were negative for T-cell receptor (TcR) and OX40, but they expressed CD2, CD4 and CD8, and interestingly, 50% of DC were CD5+, 50% expressed the alpha-chain of interleukin-2 receptor (IL-2R), and 80% were positive for the T-cell activation antigen recognized by the mAb OX48. Moreover, 60% of DC expressed high levels of Thy-1, whereas 40% displayed intermediate levels of this T-cell marker.