CD205 (DEC-205): a recognition receptor for apoptotic and necrotic self.

CD205 is an endocytic receptor that is expressed at high levels by cortical thymic epithelial cells and by dendritic cell (DC) subsets, including the splenic CD8+ DC population that is responsible for cross-presentation of apoptotic cell-derived antigens. Antigen endocytosed via CD205 enters the MHC class I and MHC class II antigen presentation pathways and is subsequently presented to both CD4+ and CD8+ T cells. Despite the known role of CD205 in antigen uptake, the nature of the ligands bound by CD205 has not been determined, and most studies have relied on the use of monoclonal antibodies as surrogate ligands. To go beyond this approach, we created a panel of CD205-IgG fusion proteins spanning the extracellular portion of CD205 and used these to identify the physiological distribution of CD205 ligands. Our data demonstrate that two areas of the CD205 molecule, within C-type lectin-like domains (CTLDs) 3+4 and 9+10, recognise ligands expressed during apoptosis and necrosis of multiple cell types, and are additionally expressed by live cells of the dendritic cell line DC2.4. Thus, CD205 acts as a recognition receptor for dying cells, potentially providing an important pathway for the uptake of self-antigen in intrathymic and peripheral tolerance.

Altered expression and endocytic function of CD205 in human dendritic cells, and detection of a CD205-DCL-1 fusion protein upon dendritic cell maturation.

CD205 (DEC-205) is a member of the macrophage mannose receptor family of C-type lectins. These molecules are known to mediate a wide variety of biological functions including the capture and internalization of ligands for subsequent processing and presentation by dendritic cells. Although its ligands await identification, the endocytic properties of CD205 make it an ideal target for those wishing to design vaccines and targeted immunotherapies. We present a detailed analysis of CD205 expression, distribution and endocytosis in human monocyte-derived dendritic cells undergoing lipopolysaccharide-induced maturation. Unlike other members of the macrophage mannose receptor family, CD205 was up-regulated upon dendritic cell maturation. This increase was a result of de novo synthesis as well as a redistribution of molecules from endocytic compartments to the cell surface. Furthermore, the endocytic capacity of CD205 was abrogated and small amounts of the recently identified CD205-DCL-1 fusion protein were detected in mature DC. Our results suggest that CD205 has two distinct functions -- one as an endocytic receptor on immature dendritic cells and a second as a non-endocytic molecule on mature dendritic cells -- and further highlight its potential as an immuno-modulatory target for vaccine and immunotherapy development.

Creation of tolerogenic human dendritic cells via intracellular CTLA4: a novel strategy with potential in clinical immunosuppression.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complexes (MHCs) and costimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T-cell activation without costimulation can lead to anergy. In this study, we developed a novel strategy to inhibit expression of B7 molecules (CD80/86) by transfecting APCs with a gene construct encoding a modified cytotoxic T lymphocyte antigen 4 (CTLA4) molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). APCs expressing this construct failed to express CD80/86 on their surface, were unable to stimulate allogeneic and peptide-specific T-cell responses, and induced antigen-specific anergy of the responding T cells. Cells expressing CTLA4-KDEL do not up-regulate the indoleamine 2, 3-dioxygenase enzyme, unlike cells treated with soluble CTLA4-immunoglobin (Ig). This gene-based strategy to knock out surface receptors is an attractive alternative to using immature dendritic cells for preventing transplant rejection and treating of autoimmune diseases.

Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination.

Genetic manipulation of dendritic cells (DCs) is important in the context of using either mature DCs to immunize patients or immature DCs to induce tolerance. Here, we describe a novel method of transfecting monocyte-derived human DCs using immunolipoplexes containing anti-CD71 or anti-CD205 monoclonal Abs. This results in up to 20% transfection, which can be increased to 20-30% if the immunolipoplexes are used to transfect CD14+ monocytes prior to differentiation into DCs. Transfected DCs can be substantially enriched using a drug-selection protocol during differentiation. Unlike adenoviral transduction, this nonviral transfection does not alter the expression of costimulatory molecules or the production of proinflammatory cytokines by DCs. In addition, DC function is unaltered, as assessed by mixed lymphocyte reactions. To test the feasibility of the immunolipoplexes and selection protocol for therapeutic intervention, we transfected DCs with the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Allogeneic T cells exposed to IDO-expressing DCs did not proliferate, secreted more IL-10 and less Th1 and Th2 cytokines, and had a higher amount of apoptosis than T cells incubated with control DCs. Furthermore the remaining T cells were rendered anergic to further stimulation by allogeneic DC. These immunolipoplexes, which can be easily and rapidly assembled, have potential for clinical immunization, in particular for tolerance induction protocols.

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

IL-13 production by donor T cells is prognostic of acute graft-versus-host disease following unrelated donor stem cell transplantation.

Despite the success of human leukocyte antigen (HLA) typing in allogeneic stem cell transplantation (SCT) it is rare to find an unrelated donor that is perfectly matched, making identification of "permissive" mismatches of paramount importance. Here, we describe novel associations between donor T-cell cytokine production during donor-antipatient mixed lymphocyte reactions (MLRs) and acute graft-versus-host disease (aGVHD). The data reveal positive correlations between both Th1-type and Th2-type cytokine production and GVHD and the assay established could potentially represent a useful tool for identification of permissible unrelated SCT donors. Associations between interleukin 13 (IL-13) levels and aGVHD were by far the strongest predictor of a GVHD (P =.0002). All patients suffering severe (grade III) aGVHD following SCT had donors who produced very high pretransplantation IL-13 responses, while those developing little or no aGVHD (grades 0-I) produced no IL-13 at all. IL-13 levels were independent of all other cytokines measured as well as cytotoxic T-lymphocyte precursor (CTLp) frequencies. The cytokines IL-5, interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) also predicted development of aGVHD (P <.05 for all 3), appearing to be coproduced in the assay and correlating with estimated CTLp frequencies. The data challenge the notion that aGVHD is purely a Th1-type cytokine-driven response, high-lighting a novel and highly significant link between the Th2-type cytokine IL-13 and aGVHD.

Thymic epithelial cells promote survival of human T-cell acute lymphoblastic leukemia blasts: the role of interleukin-7.

BACKGROUND AND OBJECTIVES: T-cell lymphoblastic leukemia (T-ALL) cells originate within the thymus from the clonal expansion of T cell precursors. Among thymic stromal elements, epithelial cells (TEC) are known to exert a dominant inductive role in survival and maturation of normal, immature T-cells. In this study we explored the possible effect of TEC on T-ALL cell survival and analyzed the role of interleukin-7 (IL-7) within the microenvironment generated by T-ALL-TEC interactions. DESIGN AND METHODS: T-ALL blasts derived from 10 adult patients were cultured with TEC obtained from human normal thymuses. The level of blast apoptosis was measured by annexin V-propidium iodide co-staining and flow cytometry. The proliferative response of leukemic cells to interaction with TEC was evaluated by thymidine incorporation at various time intervals of culture. To assess the role of IL-7, lympho-epithelial co-cultures were carried out in the presence of anti-IL-7 or anti IL-7R blocking antibodies and the level of apoptosis of T-ALL blasts was analyzed. RESULTS: When T-ALL cells were cultured in the presence of TEC monolayers, the percentage of viable cells increased significantly and this survival was sustained with time in culture. In addition, the interaction with TEC induced a considerable proliferative response in T-ALL cells (15-fold greater than that of the control cells after 7 days of culture). The presence of IL-7 or IL-7R blocking antibodies in lympho-epithelial co-cultures consistently reduced the TEC-mediated apoptosis inhibition in T-ALL blasts (70% decrease). INTERPRETATION AND CONCLUSIONS: These results point to the role of thymic epithelium in the regulation of T blast survival. In addition, they show that interaction between IL-7 and its receptor has the major role in modulating T-ALL survival within the microenvironment generated by the T-ALL/TEC interaction.

One for all and all for one: thymic epithelial stem cells and regeneration.

It has long been believed that the thymic epithelial microenvironment originates from both the endodermal and ectodermal germ cell layers. However, a growing body of evidence indicates that such a dual origin is not the case, and that the diverse thymic epithelial populations all develop from a common epithelial stem cell. This article explores these data, investigates the identity of such cells and the signals that might control their expansion and differentiation, and considers the possibility of stem cell transplantation for thymic regeneration.

Somatostatin is expressed in the murine thymus and enhances thymocyte development.

A functional interaction between the immune and the nervous system has been suggested, with neuropeptides acting as immunomodulators. Somatostatin (SOM) is a neuropeptide, mainly produced in the brain, that binds to five different receptors (SSTR). It is believed that SOM along with one of its receptors, SSTR2, is expressed in the murine thymus, although their exact localization is unresolved. We found that SOM is highly expressed in both cortical and medullary epithelial cells whereas its receptor SSTR2 is expressed on thymocytes. In order to elucidate its role in thymopoiesis, SOM was added in fetal thymic organ culture (FTOC) and found to increase thymocyte numbers and enhance maturation. SOM increased the cellular proliferation of total splenocytes but inhibited proliferation of thymocytes and purified splenic T cells. Furthermore, SOM was able to induce the migration of thymocytes. We also investigated the effect of four other neuropeptides in FTOC and found that, vasoactive intestinal peptide had a marginal effect, whereas substance P increased thymic cellularity, at intermediate but not at low or high concentrations. In contrast, both neuropeptide Y and calcitonin gene-related peptide reduced thymocyte numbers. This study supports the hypothesis for a role of neuropeptides, particularly somatostatin, in immune regulation and development.

A new human antitumor immunoreagent specific for ErbB2.

PURPOSE: Our aim was to isolate a novel human mini-antibody(scFv) that specifically targets ErbB2-positive cancer cells. ErbB2, a tyrosinekinase receptor, is overexpressed in clinically significant tumors, such as breast, ovary, and lung carcinomas. In normal tissues, it is expressed only in certain epithelial cell types. EXPERIMENTAL DESIGN: A large phagemid library (Griffin.1 library) of human scFv was used for the isolation of the ErbB2-specific scFv. A very effective strategy was developed for the isolation, consisting in a double subtractive selection, the use of two different combinations of "positive," i.e., ErbB2-bearing, and "negative" cell lines. RESULTS: Here we report the isolation of the first human anti-ErbB2 mini-antibody endowed with antitumor action. Both in its soluble and phage format, it binds specifically to ErbB2, inhibits its autophosphorylation, is internalized by target cells, and exerts a strong and specific antiproliferative action on ErbB2-positive target cells. A correlation was found between the extent of this antiproliferative effect and the expression levels of ErbB2 on target cells, with a strong cytotoxicity for hyper-expressing cells, such as SKBR3, in which apoptosis was evidenced. CONCLUSIONS: This scFv is a potentially effective immunoreagent for diagnostics and therapeutics of certain cancers, both as a readily diffused molecule in solid tumors and as an essential asset for the construction of fully human anticancer drugs.

Optimal analysis of composite cytokine responses during alloreactivity.

The one-way mixed lymphocyte reaction (MLR) is a useful model of the graft-vs.-host (GvH) response that occurs following bone-marrow transplantation (BMT). Previous studies of the MLR have shown high levels of type-1 cytokine production, such as IL-1, IL-6, IFN-gamma and TNF-alpha, but low or undetectable levels of type-2 cytokines, such as IL-4 and IL-10. Here, through establishing optimal conditions for the examination of levels and kinetics of a more definitive panel of type-1/type-2 cytokines (IL-4, IL-5, IL-10 and IL-13, IFN-gamma, TNF-alpha and the soluble IL-4 receptor) we show that, contrary to previously published data, the human alloresponse is truly heterogeneous, resulting in abundant type-2 as well as type-1 cytokine secretion. The kinetics of cytokine levels in the MLR show surprising complexity, suggesting a well-defined regulation as the alloresponse develops over time. Furthermore, each MLR responder:stimulator combination tested produces a composite cytokine profile that is intrinsic to that particular pairing. These combination-specific cytokine responses are reproducible when tested on multiple occasions over time. These data reveal a potential clinical application for the cytokine MLR in selecting donors for BMT with the least inflammatory cytokine profile. Additional analysis of this system reveals that the bulk of cytokine measured is both allospecific and T-cell-derived, with comparatively low levels produced through an autologous mechanism. Interestingly, although most of the cytokine detected is produced by CD45RO+ 'mature/activated' T cells, CD45RA+ 'naive' T cells are responsible for transient early production of IL-4. This novel finding suggests that naive T cells themselves could regulate type-1/type-2 developmental fate through an autocrine IL-4 mechanism.