Antigen-specific Cytotoxic T Lymphocytes Responses Research Articles

In Vivo Depletion of CD4+CD25+ Regulatory T Cells Enhances the Antigen-Specific Primary and Memory CTL Response Elicited by Mature mRNA-Electroporated Dendritic Cells

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-α secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo. Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8+ CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4+CD25+ regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.

Interleukin-12 Increases Proliferation and Interferon-γ Production but Not Cytolytic Activity of Human Antigen-Specific Effector Memory Cytotoxic T Lymphocytes: Power of the Effect Depends on the Functional Avidity of the T Cell and the Antigen Concentration

Cytotoxic T lymphocytes (CTLs) play an important role in the defense against viral infections and malignant diseases. Interleukin (IL)-12 plays a crucial role in induction of antigen-specific primary CTL responses and enhances proliferation, interferon-γ (IFN-γ) production, and cytolytic activity of mitogen-stimulated T cells. However, the effects of IL-12 on proliferation and effector functions of previously in vitro or in vivo primed antigen-specific CTLs are less clear. Our results show that IL-12 induces an increase in proliferation of and IFN-γ production by influenza peptide-specific CTLs, but no increase in cytolytic activity on a per cell basis was observed in bulk cultures. Stimulation of a CTL clone confirmed these results; IL-12 supported an increase in IFN-γ production, but did not increase cytolytic activity. The extent of the effect of IL-12 on IFN-γ production differs per CTL clone and depends on the avidity of the clone and the peptide concentration on its target. Our data suggest that IL-12 is a good adjuvant for boosting CTL responses, in terms of proliferation and IFN-γ production, the latter particularly for CTLs with low to intermediate avidity, such as tumor-associated self-antigen-specific CTLs.

Transcutaneous immunization in mice: Induction of T-helper and cytotoxic T lymphocyte responses and protection against human papillomavirus-induced tumors

Previous reports have shown that transcutaneous immunization (TCI) with proteins or peptides in combination with adjuvants efficiently induces specific cellular and humoral immune responses. However, depending on the kind of skin pretreatment, induction of cellular immune responses was restricted to generation of either specific cytotoxic T lymphocytes (CTLs) or T-helper (Th) cells. In this study, we induced antigen-specific CTL responses together with the appropriate Th responses by TCI of C57BL/6 mice. We applied ovalbumin protein or an ovalbumin-derived fusion peptide containing a CTL and Th epitope together with a combination of cholera toxin (CT) and CpG oligodeoxynucleotide (CpG) onto cold wax-depilated and hydrated bare skin. TCI with the ovalbumin fusion peptide induced more robust CTL and Th responses than that with ovalbumin protein. The fusion peptide in combination with the nontoxic CT derivative CTA1-D2D1 and CpG induced an antigen-specific CTL response, albeit less efficiently than in combination with complete CT. Further, we compared the potency of HPV-16 E7 oncoprotein-derived peptides containing single (CTL) or multiple (CTL + Th + B cell) epitopes to induce effective CTL responses. Strong E7-specific CTL responses were detected only after TCI with the E7 multiepitope peptide. This peptide was also shown to protect mice against tumor growth after challenge with HPV-16 E7-positive tumor cells. TCI with E7 protein and CT/CpG led to formation of an E7-specific humoral immune response.

Cotransfection of dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes

Ribonucleic acid (RNA) transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. We analyzed whether electroporation of DCs with RNA coding for a tumor-associated antigen (TAA) would elicit antigen-specific effector cytotoxic T lymphocyte (CTL) responses and whether these responses could be modulated by cotransfection with a second specific synthetic RNA. Therefore in vitro generated human monocyte-derived DCs were electroporated with in vitro transcribed RNA (in vitro transcript, IVT) encoding the TAA HER-2/neu. Additionally, these cells were cotransfected with IVT coding for human 4-1BBL. Transfection of DCs with 4-1BBL-IVT did not alter their typical phenotype. However, it increased the expression of the costimulatory molecules CD80 and CD40. Coadministration of HER-2/neu- and 4-1BBL-IVT resulted in an increased specific lysis of target cells by the in vitro induced CTL lines, indicating that 4-1BBL enhances their ability to elicit primary CTL responses. Interestingly, transfection of DCs with 4-1BBL-IVT did not augment their capacity to stimulate allogeneic lymphocyte responses. The here established approach of cotransfection of DCs with tumor-RNA and a second specific IVT could improve and optimize the in vitro manipulation of DCs for the induction of antigen-specific CTL responses.

316. Dendritic Cell-Based Genetic Immunotherapy for Prostate Cancer

Top of pageAbstract Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but heretofore the lengthy ex vivo manipulation of DCs has limited the utility of this approach. Our goal was to improve upon DC vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenoviral (Ad) vector expressing PSMA directly to DCs in situ as an efficient means for activation and antigen presentation to T-cells. We initially developed a mouse model of prostate cancer by generating several clonal derivatives of the mouse RM-1 prostate cancer cell line that express high levels of human PSMA (RM-1-PSMA). We then tested the efficacy of the CD40-targeted Ad-PSMA vector to stimulate a cytotoxic T-cell (CTL) response in vivo. In order to maximize antigen presentation in target cells, we induced both MHC class I and Tap protein expression in RM-1 cells by treatment with interferon-gamma (IFN-g). Using RM-1-PSMA cells pretreated with IFN-g as target cells, we demonstrated that DCs infected ex vivo with Ad-PSMA, as well as those infected by direct intraperitoneal injection of the CD40-targeted Ad-PSMA resulted in high levels of tumor-specific CTL responses, compared to responses with a control adenovirus (Ad-luc1). Thus, we have demonstrated that direct immunization of DCs in situ with a CD40-targeted Ad together with IFN-g treatment of target cells is effective in inducing an antigen-specific CTL response to PSMA. We have initiated animal studies to test the efficacy of the CD40-targeted PSMA vector versus a non-targeted vector with and without stimulation by IFN-g in protecting against a lethal RM-1-PSMA tumor challenge. At day 14 post-tumor inoculation, only those animals treated with the CD40-targeted adenovirus and IFN-g were resistant to challenge with RM-1-PSMA tumor cells. Our next aim is to determine the lowest effective dosage of targeted vector and the optimal combination of cytokine/chemokine stimulation to protect against tumor challenge as well as test the efficacy of the targeted vector strategy in a minimal residual disease model. The in vitro studies coupled with the syngeneic tumor survival model will allow us to systematically analyze this approach and generate data directly applicable for a human Phase I clinical trial.

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor-associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte-derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA-derived CEA and MUC1 antigens and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4(+) and CD8(+) T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN-gamma. More importantly, coculture of fusion cells with patient-derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen-specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1-positive and HLA partially matched target cells. Antigen-specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA- and MUC1-specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen-specific CTL responses. Our report demonstrates the simultaneous induction of CRCA-specific CTL responses restricted by HLA-A2 and -A24.

Depletion of CD25+ cells from human T-cell enriched fraction eliminates immunodominance during priming with dendritic cells genetically modified to express a secreted protein.

The ability of dendritic cells (DCs), genetically modified with one of two types of plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses, is compared. The first type, also called "secreted" vaccine (sVac), encodes for the full length of the human prostate-specific antigen (PSA) with a signal peptide sequence so that the expressed product is glycosylated and directed to the secretory pathway. The second type, truncated vaccines (tVacs), encodes for either hPSA or human prostate acidic phosphatase (hPAP), both of which lack signal peptide sequences and are retained in the cytosol and degraded by the proteasomes following expression. Monocyte-derived dendritic cells are transiently transfected with either sVac or one of two tVacs. The DCs are then used to activate CD25+-depleted or nondepleted autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs, peptide-pulsed DCs or monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tVacDCs and sVacDCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted towards one of the three antigen-derived epitopes when priming and boosting is performed with sVacDCs. In contrast, tVac-transfected DCs prime T cells towards all antigen-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance. While CD25+ cell depletion prior to priming with sVacDCs alleviates immunodominance, cotransfection of dendritic cells with GITR-L does so in some but not all cases.

Comparison of PSA-specific CD8+ CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins.

The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8+ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.

Generation of tumor-specific cytotoxic T lymphocyte and prolongation of the survival of tumor-bearing mice using interleukin-18-secreting fibroblasts loaded with an epitope peptide

There is currently much interest in generating cytotoxic T lymphocyte (CTL) responses against tumor antigens as a therapy for cancer. In this study mouse fibroblasts (H-2 b) were genetically modified to express a costimulatory B7.1 and a mature interleukin (IL)-18, and then loaded with an ovalbumin (OVA) epitope (SIINFEKL, H-2K b restricted) as a model antigen, and tested for the induction of OVA-specific CTLs in C57BL/6 mice (H-2 b). The genetically modified fibroblasts lacking either IL-18 or B7.1 were also constructed. Immunization with the IL-18/B7.1-transfected fibroblasts induced strong cytotoxic activities against OVA-expressing EL4 (EG7) tumor cells, but not against other H-2 b tumor cells such as EL4, C1498, and B16F1 cells. The magnitude of the cytotoxic response in mice with the IL-18/B7.1-transfected fibroblasts was significantly higher than the response in mice immunized with any other cell constructs. CD8 + T cells with OVA-specific cytotoxic activities were predominant in mice immunized with the IL-18/B7.1-transfected fibroblasts. Furthermore, treatment with the IL-18/B7.1-transfected fibroblasts significantly prolonged the survival period of EG7 tumor-bearing mice. Anti-tumor CTL immunity by the IL-18/B7.1-transfected fibroblasts could be induced without the help of host antigen-presenting cells (APCs) and NK1.1 + cells, whereas partially decreased by the depletion of CD4 + T cells at the inductive stage. These results support the ability of IL-18/B7.1 gene transfer to enhance the antigen-presenting capacity of fibroblasts for inducing antigen-specific CTL response.

Human dendritic cells genetically engineered to express cytosolically retained fragment of prostate-specific membrane antigen prime cytotoxic T-cell responses to multiple epitopes.

The ability of two plasmid DNA vaccines to stimulate lymphocytes from normal human donors and to generate antigen-specific responses is demonstrated. The first vaccine (truncated; tPSMA) encodes for only the extracellular domain of prostate-specific membrane antigen (PSMA). The product, expressed following transfection with this vector, is retained in the cytosol and degraded by the proteasomes. For the "secreted" (sPMSA) vaccine, a signal peptide sequence is added to the expression cassette and the expressed protein is glycosylated and directed to the secretory pathway. Monocyte-derived dendritic cells (DCs) are transiently transfected with either sPSMA or tPSMA plasmids. The DCs are then used to activate autologous lymphocytes in an in vitro model of DNA vaccination. Lymphocytes are boosted following priming with transfected DCs or with peptide-pulsed monocytes. Their reactivity is tested against tumor cells or peptide-pulsed T2 target cells. Both tPSMA DCs and sPSMA DCs generate antigen-specific cytotoxic T-cell responses. The immune response is restricted toward one of the four PSMA-derived epitopes when priming and boosting is performed with sPSMA. In contrast, tPSMA-transfected DCs prime T cells toward several PSMA-derived epitopes. Subsequent repeated boosting with transfected DCs, however, restricts the immune response to a single epitope due to immunodominance.

MART-1 adenovirus-transduced dendritic cell immunization in a murine model of metastatic central nervous system tumor.

Dendritic cells (DCs) are potent antigen-presenting cells that have been shown to play a critical role in the initiation of host immune responses against tumor antigens. In this study, a recombinant adenovirus vector encoding the melanoma-associated antigen, MART-1, was used to transduce murine DCs, which were then tested for their ability to activate cytotoxic T lymphocytes (CTLs) and induce protective immunity against B16 melanoma tumor cells implanted intracranially. Genetic modifications of murine bone marrow-derived DCs to express MART-1 was achieved through the use of an E1-deficient, recombinant adenovirus vector. Sixty-two C57BL/6 mice were immunized subcutaneously with AdVMART-1-transduced DCs (n = 23), untransduced DCs (n = 17), or sterile saline (n = 22). Using the B16 murine melanoma, which naturally expresses the MART-1 antigen, all the mice were then challenged intracranially with viable, unmodified syngeneic B16 tumor cells 7 days later. Splenocytes from representative animals in each group were harvested for standard cytotoxicity (CTL) and enzyme-linked immunospot (ELISPOT) assays. The remaining mice were followed for survival. Immunization of C57BL/6 mice with DCs transduced with an adenoviral vector encoding the MART-1 antigen elicited the development of antigen-specific CTL responses. As evidenced by a prolonged survival curve when compared to control-immunized mice with intracranial B16 tumors, AdMART-1-DC vaccination was able to elicit partial protection against central nervous system tumor challenge in vivo.

Mimovirus: a novel form of vaccine that induces hepatitis B virus-specific cytotoxic T-lymphocyte responses in vivo.

CD8(+) cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine-mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg(28-39), with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg(28-39)-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

CD40L in autoimmunity and mucosally induced tolerance.

The administration of soluble antigens to the oral or nasal mucosa can lead to systemic unresponsiveness to a subsequent challenge with the same antigens (1). Such mucosally induced tolerance probably protects the body from hypersensitivity reactions to food proteins, pollen, and commensal bacterial antigens present in the normal mucosa and thus helps maintain an appropriate immunological balance between the host and its normal flora. Numerous molecular and cellular inhibitory mechanisms are involved in this unique and important immunological phenomenon. Several lines of evidence show that mucosally induced tolerance does not occur passively, by means of anergy, clonal deletion, or the simple absence of autoreactive immune cells, but also involves ignorance, receptor downregulation, or active cellular suppression (ref. 2; Figure ​Figure11a). Figure 1 Models for the effects on mucosally induced tolerance of perturbing the CD40L-CD40 interaction. (a) In the untreated wild-type mouse, CD40 on dendritic cells (DCs) and other APCs interacts with CD40L expressed on T cells of several subsets, including ... The fact that mucosally induced tolerance can suppress antigen-specific T cell proliferation, delayed-type hypersensitivity responses, and antibody production suggests attractive ap-proaches for the prevention and control of autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, encephalomyelitis, and type 1 diabetes (3). However, although mucosal administration of different autoantigens has been tested in human trials, this strategy has not yet yielded a successful therapy (4, 5). Some previous studies have suggested that this failure of human trials was due to concomitant induction of harmful cytotoxic T lymphocytes (CTLs) by mucosally administered antigen (6, 7). In an attempt to overcome this obstacle, as reported in this issue of the JCI, Hanninen et al. (8) have induced a temporary blockade of the interaction between the costimulatory protein CD40 and its ligand CD40L. This interaction has been suggested to foster mucosally induced tolerance by several mechanisms, as shown in Figure ​Figure1a.1a. In their present report, the authors show that blocking antibodies to CD40L can prevent the development of antigen-specific CTL responses without affecting the development of oral tolerance. Hanninen and colleagues (8) tested this approach in rat insulin promotor-OVA (RIP-OVA) mice, a diabetic transgenic strain in which the expression of the model antigen chicken ovalbumin (OVA) in the pancreatic β-islet cells provokes an autoimmune response to these cells. The consequent destruction of these islet cells thus provides a model for the events seen in type I diabetes. Oral administration of a high dose of OVA can promote mucosally induced tolerance, but this treatment also elicits CTL specific for OVA. However, Hanninen et al. observed that transient blockade of CD40L in this system prevented CTL responses without affecting mucosally induced tolerance toward OVA, either in wild-type or in RIP-OVA transgenic mice (Figure ​(Figure1b).1b). Furthermore, the severity of diabetes, as measured, for example, by blood glucose, was significantly improved after transient blockade of CD40L, suggesting that modulation of CD40L-CD40 interaction could be used clinically to uncouple the desired systemic, autoantigen-specific unresponsiveness from the oral induction of CTLs.

Functional detection of epithelial cell adhesion molecule specific cytotoxic T lymphocytes in patients with lung cancer, colorectal cancer and in healthy donors

Epithelial cell adhesion molecule (Ep-CAM) derived antigenic peptides have been identified that can be recognized by cytotoxic T lymphocytes (CTL) in a major histocompatibility complex (MHC) class I restricted fashion. Thus, altered expression of Ep-CAM in a variety of human tumors might render a potential target for T cell mediated therapy. We have examined, whether the novel HLA-A*0201 restricted peptide ILYENNVIT (184–192) corresponding to Ep-CAM and one heteroclitic modified variant peptide previously demonstrated to be immunogenic in the human system can elicit antigen specific CTL responses in HLA-A2 positive patients with history of Ep-CAM expressing cancer of lung and colon. Specific CTL recognition of T2 target cells pulsed with the native peptide as well as of the lung cancer cell line A549 indicates that an appropriate T cell repertoire can be expanded from peripheral blood from patients in clinical remission and with advanced cancer. Despite an overall low frequency, peptide specific precursor CTLs could be readily expanded from peripheral blood from 6/8 patients that were diagnosed previously with Ep-CAM expressing lung cancer and 4/8 control individuals (2/5 healthy donors and 2/3 colon cancer patients). CTLs from three of five lung cancer patients tested also lyzed the HLA-A2 + and Ep-CAM expressing lung cancer cell line A549. We did not detect an increased frequency of pCTLs after peripheral blood monocytes (PBMCs) were stimulated with the heteroclitic compound peptide. The results of our study indicate that Ep-CAM specific precursor CTL can be expanded in vitro and a specific T cell response against this epitope can be elicited in patients at various stages of lung cancer.

INDUCTION OF A CYTOTOXIC T LYMPHOCYTE (CTL) RESPONSE TO PLASMID DNA DELIVERED VIA LIPODINE™ LIPOSOMES*

ABSTRACTWe have previously shown that liposome-mediated plasmid DNA immunisation may be a preferred alternative to the use of naked DNA. Lipodine™ DNA formulations consist of liposomes containing entrapped DNA plasmid by the dehydration–rehydration (DRV) method. Such liposome formulations are distinct from liposomes with externally complexed DNA in that the majority of the DNA is “internal” to the liposome structure and hence protected from DNAase degradation. Previous studies on the immune response induced by DNA vaccines entrapped in Lipodine™ have focused on the humoural response. In the present study, we have expanded the analysis profile in order to include the cytotoxic T lymphocyte (CTL) component of the immune response. We have analysed the immune response induced by DNA entrapped in Lipodine™ compared to that induced by DNA alone when delivered subcutaneously, a route of administration not normally inducing significant plasmid DNA mediated immune activation. Our results indicate that delivery of a small dose of plasmid DNA in Lipodine™ results in an improved antibody response to the plasmid encoded antigen and a strong antigen specific CTL response compared to that induced by DNA delivered alone.

Development of Th1-mediated CD8+ effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes

There have been many studies for tumor therapy mediated by cytotoxic T lymphocytes (CTL) that recognize tumor-associated antigen. It is generally accepted that CTL responses are induced when antigen is delivered into the cytosol. The pH-sensitive liposomes as vehicles are well known for their capacity to deliver the antigen into the cytosol. In this work, immunization of mice with CTL epitope peptides from Hantaan nucleocapsid protein (M6) or human papilloma virus E7 encapsulated in pH-sensitive liposomes induced effective antigen-specific CTL responses. The CTL responses induced by M6 peptide encapsulated in pH-sensitive liposomes blocked the formation of tumor mass from Hantaan NP transfected B16 melanoma cells in C57BL/6 mice and delayed the growth of preinoculated melanoma cells. During the blockade of the tumor growth, the CTL response was maintained for at least approximately 6 weeks, and the mice secreted Th1 type cytokines such as IL-2 and IFN-γ. These results suggested that the pH-sensitive liposomes might provide an effective peptide delivery system for CTL-mediated tumor therapy.

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens.

Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 × 106OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency. © 2001 Cancer Research Campaign http://www.bjcancer.com

Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T-cell activation, binds to CD47 with high affinity, and is expressed on immature CD34+CD38−hematopoietic cells

AbstractSignal-regulatory proteins (SIRPs) represent a new family of inhibitory/activating receptor pairs. They consist of 3 highly homologous immunoglobulin (Ig)–like domains in their extracellular regions, but differ in their cytoplasmic regions by the presence (SIRPα) or absence (SIRPβ) of immunoreceptor tyrosine-based inhibitory motifs (ITIMs). To analyze the differential expression on hematopoietic cells, function and ligand binding capacity of SIRPα and SIRPβ molecules, soluble fusion proteins consisting of the extracellular domains of SIRPα1, SIRPα2, and SIRPβ1, as well as SIRPα/β-specific and SIRPβ-specific monoclonal antibodies (MoAbs) were generated. In contrast to SIRPα1 and SIRPα2, no adhesion of SIRPβ1 to CD47 could be detected by cell attachment assays and flow cytometry. Using deletion constructs of SIRPα1, the epitope responsible for SIRPα1 binding to CD47 could be confined to the N-terminal Ig-like loop. Flow cytometry analysis with SIRPα/β- and SIRPβ-specific MoAbs revealed that SIRPα but not SIRPβ is expressed on CD34+CD38− hematopoietic cells. In addition, a strong SIRPα expression was also observed on primary myeloid dendritic cells (DCs) from peripheral blood as well as on in vitro generated DCs. Analysis of the T-cell stimulatory capacity of in vitro generated DCs in the presence of soluble SIRPα1 fusion proteins as well as SIRPα/β-specific and CD47-specific MoAbs revealed a significant reduction of T-cell proliferation in mixed lymphocyte reaction and inhibition of induction of primary T-cell responses under these conditions. In contrast, soluble SIRPα or SIRPβ-specific antibodies had no effect. The data suggest that the interaction of SIRPα with CD47 plays an important role during T-cell activation and induction of antigen-specific cytotoxic T-lymphocyte responses by DCs.

Mucosal immune network in the gut for the control of infectious diseases.

The common mucosal immune system (CMIS) consists of an integrated cross-communication pathway of lymphoid tissues made up of inductive and effector sites for host protection against pathogenic microorganisms. Major effector molecules of the CMIS include IgA antibodies and cytokines, chemokines and their corresponding receptors. Secretory IgA (S-IgA), the major immunoglobulin, is induced by gut-associated lymphoreticular tissue (GALT)-derived B cells with the help of Th1- and Th2-type CD4(+) T lymphocytes. Cytotoxic T lymphocytes (CTLs) in the mucosal epithelium, a subpopulation of intraepithelial lymphocytes (IELs), also help maintain the mucosal barrier. The CMIS is unique in that it can provide both positive and negative signals for the induction and regulation of immune responses in both the mucosal and systemic compartments after oral or nasal antigen exposure. Prevention of infection through mucosal surfaces can be achieved by the CMIS through connections between inductive (e.g. GALT) and effector tissues. When vaccine antigens are enterically administered together with mucosal adjuvants [e.g. cholera toxin (CT), heat-labile toxin produced by Escherichia coli (LT) and IL-12], antigen-specific Th1/Th2 and IgA B cell responses are induced simultaneously in the mucosal effector compartment. Since these antigen-specific immune responses are not generated by oral vaccine without mucosal adjuvant, safe and effective adjuvants for the induction of antigen-specific S-IgA and CTL responses are essential for the development of mucosal vaccines for protection against infectious diseases. Finally, recent findings suggest the presence of a CMIS-independent IgA induction pathway, which also must be considered in the development of mucosal vaccines.

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals.

Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo.