Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies

 The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1⁺OX6⁺, ED1⁺OX6⁻, and ED1⁻OX6⁺. More then 40% of OX6⁺ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6⁺ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1⁺ED2⁻ cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1⁻ED2⁺ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1⁺ED2⁺, ED1⁺ED2⁻, and ED1⁻ED2⁺ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions. Accepted: 3 November 1997     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Density of DC-LAMP+ mature dendritic cells in combination with activated T lymphocytes infiltrating primary cutaneous melanoma is a strong independent prognostic factor

As the most potent antigen presenting cells, dendritic cells (DCs) play key roles in the immune response against tumors. Their density in the tumor tissue has been associated with prognosis in patients with various cancers. However, few studies have been aimed at the presence and maturation state of DCs in cutaneous melanoma, with regard to their potential clinical correlates. In this study, the density of DCs expressing CD1a and the maturation marker DC-LAMP was determined by immunohistochemistry in primary tumor samples from 82 patients with cutaneous malignant melanoma. Intratumoral and peritumoral cell densities were analyzed in relation to tumor thickness and the subsequent development of metastases, as well as to patients’ survival. CD1a⁺ DCs were found both infiltrating melanoma cell nests and in the surrounding stroma, while DC-LAMP⁺ mature DCs were generally confined to the peritumoral areas, associated with lymphocytic infiltrates. DC density values significantly correlated with the number of activated (CD25⁺ or OX40⁺) T lymphocytes (p < 0.001). The degree of infiltration by CD1a⁺ and DC-LAMP⁺ DCs showed strong inverse correlation with the thickness of melanomas (p < 0.001). High peritumoral density of mature DCs was associated with significantly longer survival (p = 0.0195), while density of CD1a⁺ cells had a prognostic impact of borderline significance (p = 0.0610). Moreover, combination of high peritumoral CD1a⁺ or DC-LAMP⁺ cell density with high number of CD25⁺ or OX40⁺ lymphocytes identified patient subgroups with more favorable survival compared to other subgroups. A multivariate survival analysis involving DC and activated T-cell densities alone and in combinations, as well as traditional prognostic factors, identified high DC-LAMP⁺ cell/high OX40⁺ cell density and Breslow index as independent predictors of good prognosis. These results suggest that the presence of CD1a⁺ DCs primarily depends on the thickness of melanomas, without direct relationship with the patients’ survival. On the other hand, the density of mature DCs, especially in association with that of activated T cells, proved of prognostic importance, suggesting that these parameters could be considered as signs of a functional immune response associated with better outcome of the disease.     

In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 × 10⁶ cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR⁺, CD1a⁺, CD4⁺, CD54⁺, CD80⁺, CD86⁺, CD40⁺, CD3⁻ and CD14⁻). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. Received: 8 January 1998 / Accepted: 30 April 1998     

Close anatomical relationships between nerve fibers and MHC class II-expressing dendritic cells in the rat liver and extrahepatic bile duct

 The anatomical relationships between immunocytochemically identified nerve fibers and MHC class II-expressing antigen presenting dendritic cells were investigated in the rat hepatobiliary system using immunocytochemistry, confocal laser scanning, and electron microscopy. Close proximity of nerve fiber varicosities immunostained for PGP 9.5 and MHC class II-expressing dendritic cells was frequently observed in the wall of extrahepatic bile ducts, in Glisson’s area, around central and hepatic veins, and in the liver capsule. Contacts between nerve fibers staining for substance P, calcitonin gene-related peptide, calretinin, and vasoactive intestinal polypeptide and dendritic cells were more often observed around extrahepatic bile ducts than in Glisson’s area. Nerve fibers immunostaining for tyrosine hydroxylase and neuropeptide Y were numerous both in the wall of extrahepatic bile ducts and in Glisson’s area and frequently contacted dendritic cells there. At the ultrastructural level, close membrane contacts between bare axolemmal areas of unmyelinated nerve fibers and processes of MHC class II-expressing cells were observed. These results demonstrate close anatomical relationships of nerve fibers from various sources with antigen presenting dendritic cells in the visceral domain and suggest modulation of antigen presentation by the autonomic nervous system. Accepted: 29 September 1997     

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4⁺ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11–14 amino acids in length, all P1A-encoded) for their ability to initiate CD4⁺ and CD8⁺ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4⁺ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells. Received: 25 February 1999 / Accepted: 14 April 1999     

Therapeutic effect of a T helper cell supported CTL response induced by a survivin peptide vaccine against murine cerebral glioma

Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides with predicted binding to mouse H2-K^b and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-K^b-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were screened by flow cytometry for binding of dimeric H2-K^b:Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN_57–64 and SVN_82–89) generated specific CD8+ T cells. We chose to focus on the SVN_57–64 peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN_53–67), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell help. We tested the SVN_53–67 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a significant prolongation of survival. The SVN_53–67 vaccine was significantly more effective than the SVN_57–64 core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated cytokine support within a single peptide.     

Detection of immature dendritic cells in the enamel organ of rat incisors by using anti-cystatin C and anti-MHC class II immunocytochemistry.

Dendritic cells in the enamel organ of rat incisors were examined with immunocytochemistry using an anti-cystatin C antibody for immature dendritic cells and macrophages, OX6 for MHC Class II, ED1 for macrophages and dendritic cells, and ED2 for macrophages. Single cells positive for anti-cystatin C appeared in the enamel organ in zones at which ameloblasts secrete enamel matrix proteins. They were also present in transition and enamel maturation zones. In addition, ameloblasts, osteocytes, and osteoclasts were labeled by anti-cystatin C. ED1 and ED2 immunocytochemistry revealed that there was no macrophage population in the enamel organ of secretion, transition, or enamel maturation zone. A double labeling study showed that most anti-cystatin C-positive cells in the enamel maturation zone were also positive for OX6, whereas anti-cystatin C-positive and OX6-negative cells were prevalent in the secretion zone. The results suggest that immature dendritic cells penetrate the enamel organ of the secretion zone and begin to mature in the zones of transition and enamel maturation. (J Histochem Cytochem 48:1243-1255, 2000)     

Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4⁺ T cells, but also on MHC-class I molecules to CD8⁺ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4⁺ and CD8⁺ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin⁺ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin⁺ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4⁺ and CD8⁺ T cell responses emphasizes their potential for epicutaneous immunization strategies. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications,” Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Isolation of CD4and CD8T-Cell Clones from Mice Immunized with Synthetic Peptides on Splenic Dendritic Cells

Splenic dendritic cells (DC) express high levels of MHC, co-stimulator, and adhesion molecules and have been shown to be extremely potent antigen-presenting cells for both CD4⁺and CD8⁺T-cell responses. Previous studies have shown that murine DC can be loaded with exogenous antigens and used to prime CD4⁺, Class II-restricted T-cell responsesin vivo.This article describes protocols for immunization using DC loaded with peptides bound to Class I or Class II molecules and for the derivation and characterization of CD4⁺and CD8⁺antigen-specific T-cell lines and clones. The rationale for using DC as antigen-presenting cells is discussed, together with the advantages and disadvantages of these cells compared with more conventional methods of immunization.     

Role of macrophage colony-stimulating factor in the differentiation and expansion of monocytes and dendritic cells from CD34+ progenitor cells

The present study focused on whether it is possible to expand monocytic cells from CD34⁺ progenitor cells by using macrophage colony-stimulating factor (M-CSF) in the absence and presence of mast cell growth factor (MGF) and IL-6. It was demonstrated that CD34⁺ cells differentiate without expansion to functional mature monocytic cells in the presence of M-CSF or combinations of M-CSF plus IL-6 and MGF. A different response pattern was observed for the number of clonogenic cells. The addition of IL-6 or both IL-6 and MGF to M-CSF containing cultures resulted in significant higher numbers of colony-forming unit-macrophage (CFU-M) as tested in clonogenic and³H-thymidine assays. Furthermore, M-CSF plus both IL-6 and MGF appeared to be the most potent combination to preserve the monocytic precursor in cell suspension culture assays. These results indicate that IL-6 and MGF in conjunction with M-CSF affect CD34⁺ cells especially at precursor level without distinct effect on the more mature stages. Secondly we studied whether M-CSF is only critical for the monocytic lineage or also affects dendritic cell (DC) development. Indeed, we were able to culture CD83⁺ DC from CD34⁺ progenitor cells in the presence of M-CSF in conjunction with TNF-α, IL-4, and MGF although their absolute number is almost threefold lower than the number of CD83⁺ cells yielded from GM-CSF plus TNF-α, IL-4, and MGF stimulated CD34⁺ cells.     

Expression of lymphatic endothelium-specific hyaluronan receptor LYVE-1 in the developing mouse kidney

Our knowledge of the embryonic development of the lymphatic vessels within the kidney is limited. The aim of this study was to establish the time of appearance and the distribution of intra-renal lymphatic vessels in the developing mouse kidney by using the lymphatic marker, LYVE-1. Kidneys from embryonic day 12 (E12) to E18, from neonates at post-natal day 1 (P1) to P21, and from adults were studied. In the adult mouse kidney, LYVE-1 was expressed mainly in the lymphatic endothelial cells (LECs) and in a subset of endothelial cells in the glomerular capillaries. However, in the developing mouse kidney, LYVE-1 was also expressed transiently in F4/80⁺/CD11b^– immature macrophages/dendritic cells and in the developing renal vein. LYVE-1⁺ lymphatic vessels connected with extra-renal lymphatics were detected in the kidney at E13. F4/80⁺/CD11b^–/LYVE-1⁺ immature macrophages/dendritic cells appeared prior to the appearance of LYVE-1⁺ renal lymphatic vessels and were closely intermingled or even formed part of the lymphatic vascular wall. Prox1 was expressed only in the LYVE-1⁺ LECs from fetus to adult-hood, but not in LYVE-1⁺ endothelial cells of the developing renal vein and macrophages/dendritic cells. Thus, lymphatic vessels of the kidney might originate by extension of extra-renal lymphatics through an active branching process possibly associated with F4/80⁺/CD11b^–/LYVE-1⁺ macrophages/dendritic cells.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Three Distinct Subsets of Thymic Epithelial Cells in Rats and Mice Defined by Novel Antibodies

Aim

Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells.

Results

Rat TECs were subdivided on the basis of phenotype into three subsets; ED18⁺ED19^+/−keratin 5 (K5)⁺K8⁺CD205⁺ class II MHC (MHCII)⁺ cortical TECs (cTECs), ED18⁺ED21⁻K5⁻K8⁺ Ulex europaeus lectin 1 (UEA-1)⁺CD205⁻ medullary TECs (mTEC1s), and ED18⁺ED21⁺K5⁺K8^dullUEA-1⁻CD205⁻ medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18⁺ED19^+/−K5⁺K8⁺ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII⁺CD103⁺ but negative for TEC markers, including CD205. Those in the medulla were MHCII⁺CD103⁺ and CD205⁺ cells were found only in the TEC-free area.

Conclusion

Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.     

Development of a standardized protocol for reproducible generation of matured monocyte-derived dendritic cells suitable for clinical application

There is increasing interest in the generation of dendritic cells (DC) for cancer immunotherapy. In order to utilize DC in clinical trials it is necessary to have standardized, reproducible and easy to use protocols. We describe here the process development for the generation of DC as the result of investigation of culture conditions as well as consumption rates of medium and cytokines. Our studies demonstrate that highly viable DC (93 ± 2%) can be produced from CD14⁺ enriched monocytes via immunomagnetic beads in a high yield (31 ± 6%) with X-VIVO 15, 400 U ml⁻¹ GM-CSF and 2000 U ml⁻¹ IL-4 without serum and feeding. For the maturation of DC different cocktails (TNF-α, IL-1β, IL-6, PGE₂ and TNF-α, PGE₂) were compared. In both cases cells expressed typical surface molecules of mature DC and induced high proliferative responses in mixed lymphocyte reactions which led to IFN-γ producing T-lymphocytes. The data suggest that the use of this optimized, easy to use protocol results in highly mature DC. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative Analysis of Transduced Primary Human Dendritic Cells Generated by the Use of Three Different Lentiviral Vector Systems

Lentiviral gene transfer vectors are suitable for genetically modifying non-cycling primary human cells. In this study, we analyzed transduced human dendritic cells (DC) generated by the use of three different GFP-encoding lentiviral vectors, HIV-2 ROD A Δenv-GFP (ROD A), SIVsmm PBj ΔE EGFP (PBj), and SIVmac ΔE EGFP (SIVmac). CD14⁺ monocytes were isolated from buffy coat, transduced, and differentiated to immature and mature DC. Cytofluometric analysis of DC revealed high transduction efficiencies at MOI 1 for simian immunodeficiency virus (SIV)-derived vectors PBj and SIVmac ranging between 80–90 and 70–90%, respectively. In contrast, transduction with ROD A resulted only in approximately 30%-positive DC at the same MOI. Of note, none of the analyzed vectors affected expression of maturation and/or activation markers. Moreover, transduction with PBj or SIVmac did not induce significant cytokine responses whereas ROD A transduction stimulated weak interferon-alpha responses. SIVmac transduced DC showed normal phagocytosis of antigen and normal allo T cell stimulatory capacity when compared with untreated DC. Thus, the SIVmac lentiviral transduction vector is suitable for efficient genetic modification of human DC without affecting phenotype or function and thus qualifies this vector as a versatile tool for use in basic research.     

Apoptosis of dental pulp cells and their elimination by macrophages and MHC class II-expressing dendritic cells.

Apoptosis of dental pulp cells of rat incisors was investigated by the TUNEL method and electron microscopy. The results showed that a considerable amount of apoptosis occurred in the pulp, increasing in extent with incisal direction. OX6, ED1, and ED2 antibodies were used to detect macrophages and dendritic cells in combination with immunoelectron microscopy. Apoptotic fragments were eliminated mainly by MHC Class II-expressing cells, including dendritic cells positive for the OX6 antibody, and by MHC Class II-negative macrophages. Macrophages and dendritic cells positive for OX6, ED1, or ED2 increased from the apical to incisal direction of the incisor. These results indicate that apoptosis contributes to normal pulp formation and maintenance.     

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

 We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR⁺ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3^–, CD19^–, CD20^–, CD14^–, CD11b^–, CD16^–, CD56^–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system. Received: 20 June 1997 / Accepted: 14 August 1997     

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4⁺ and/or CD8⁺ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4⁺ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8⁺ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4⁺ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8⁺ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4⁺ T cell-mediated antitumor activity in vivo.