Development of Dendritic Cell-Based Immunotherapy for Autoimmunity

Dendritic cells are professional antigen-presenting cells that maintain immune tolerance to self-antigens by deleting or controlling the pathogenicity of auto-reactive T-cells. Dendritic cell-based immunotherapies show great promise for the restoration of tolerance in autoimmune disease. Dendritic cells can be modified ex vivo to induce stable tolerogenic function and be used as cellular ‘vaccines’ or they can be targeted in vivo with sophisticated antigen delivery systems. Tolerogenic dendritic cells induce antigen-specific T-cell tolerance in vivo and have therapeutic effects in animal models of autoimmunity. The current challenge is to bring tolerogenic dendritic cell therapy to the clinic.     

树突状细胞在多发性骨髓瘤免疫治疗中的应用进展

多发性骨髓瘤(multiple myeloma,MM)患者在常规治疗达完全缓解后,体内仍会存在微小残留病变(minimal residual disease,MRD),而成为复发的根源,此时依靠机体有效的抗肿瘤免疫反应是消除MRD的一个有效的方法。树突状细胞(dendritic cell,DC)是目前功能最强大、唯一能激活初始型T细胞的抗原递呈细胞(antigen presenting cell,APC),能有效的把MM的肿瘤抗原呈递给T细胞,产生特异性CTL而达到杀灭肿瘤细胞的目的。对DC在MM免疫治疗中的基础研究和临床应用进展的研究很有意义。     

Epidermal Dendritic Cell Populations in the Flaky Skin Mutant Mouse

Flaky skin (gene symbol: fsn) is an autosomal recessive mouse mutation that causes pathologic changes in the skin yielding a papulosquamous disease resembling human psoriasis. Preliminary studies of epidermal sheets from foot pads of fsn/fsn mice stained for Ia⁺ Langerhans cells (LC) or Thy-1⁺ dendritic epidermal cells (Thy-1⁺ DEC) indicated a rise in LC numbers at the time of weaning, when the skin lesion becomes clinically evident. To further investigate this observation, epidermal sheets were obtained from the ear, dorsal skin, and foot pads from replicates of 6 female mice (both mutants and normal littermates) on weekly intervals from birth to 8 weeks of age. Dorsal skin epidermal thickness was quantitated by computer assisted image analysis and found to be significantly thickened from one week onward in the mutant mice. Using immunofluorescence microscopy, epidermal dendritic cell numbers were determined following staining with antibodies for the following markers: Ia, NLDC-145, and S-100 (for LC) or Thy 1.2 and asialc-GM1 (for Thy-1⁺ DEC). Use of all 5 markers to evaluate skin from 3 different locations yielded a subtle but significant increase in LC and Thy-1⁺ DEC in flaky skin mice. Of the three sites evaluated, the dorsal skin and ear epidermal sheets were most informative, which corresponded to the degree of pathological involvement. Mice doubly homozygous for fsn and for the severe combined immunodeficiency (scid) mutation developed the psoriasiform dermatitis. Bone marrow grafts from fsn/fsn homozygotes to homozygous scid/scid mice reproduce the skin lesion. These studies suggest that the psoriasiform dermatitis in the flaky skin mouse mutation is associated with abnormalities at the level of hematopoietic progenitor cells.     

Attempts to Optimize Active Specific Immunotherapy for Melanoma

During the past 5 years, we have been conducting clinical trials with a therapeutic melanoma vaccine (melanoma“theraccine”). Mechanical lysates of two melanoma cell lines chosen for their complementary characteristics were combined with the adjuvant DETOX and injected subcutaneously on weeks 1, 2, 3, 4 and 6 for one or two courses, and then monthly in patients with objective clinical responses. Of 109 patients, 22 (20%) have had objective clinical regression of tumor masses, with 5% complete responses. Ten patients have lived more than a year. Eight of the 10 are still alive, five of whom have lived more than 3 years. It was not necessary to achieve complete remissions to cause an increase in survival, and most of the long-surviving patients have one or more (stable) residual nodules. The pace of the disease process has clearly been slowed in those individuals.

A rise in the level of cytotoxic T lymphocyte precursors in the blood (pCTL) has correlated with clinical response. Only one patient without such a rise in pCTL has had a response, and assays in that patient were considered unreliable. Both CD4+ and CD8+ CTL have been cloned from the blood of immunized patients. Both types of CTL killed a number of melanoma cell lines, but not other types of tumor or normal cells (lymphoblasts and melanocytes). CD8+ CTL have not been restricted to killing the autologous melanoma. MHC restriction by the HLA-A2 locus was identified. CD4+ CTL were not restricted only by Class IIHLA antigens. Many CD4+ clones killed HLA Class H-negative melanomas, and we were able to block cytotoxicity of a particular clone with either anti-HLA Class I or anti-Class II MHC monoclonal antibodies, or both.

An association of clinical response to the theraccine with certain HLA phenotypes, notably HLA-C3, -A2 (and the cross-reactive HLA-A28), B12 (and the related alleles (HLA-B44 and -B45) and perhaps DR4, particularly when combinations of those alleles were present, was suggested by our analysis of 70 patients. It is possible that this simply indicates the sharing of MHC antigens between the immunizing melanomas and the patient's melanoma. However, these MHC molecules may be important in their own right in presenting melanoma-associated antigens in CTL in vivo.

Subtractive hybridization of mRNA from lung squamous carcinoma cells from cDNA of the M-l melanoma cell line has yielded several DNA sequences unique to melanoma. Those are now being analyzed for possible immunogenicity, with cytotoxicity by CTL from immunized patients as the major criterion. Our hope is eventually to produce a wholly synthetic melanoma theraccine incorporating a group of critical antigens proved to be immunogenic to humans.     

Adjuvant Peptide Pulsed Dendritic Cell Vaccination in Addition to T Cell Adoptive Immunotherapy for Cytomegalovirus Infection in Allogeneic Hematopoietic Stem Cell Transplantation Recipients

Adoptive cellular immunotherapy has been shown to be effective in the management of cytomegalovirus (CMV) reactivation and disease. Whether adjuvant dendritic cell (DC) vaccination will provide additional benefit in prophylaxis or treatment of CMV in hematoietic cell transplantation (HSCT) recipients is unknown. In this study, we administered prophylactic CMV-peptide specific T cell infusions, followed by 2 doses of intradermal CMV peptide-pulsed DC vaccine, to 4 HSCT recipients. There were no immediate adverse events associated with T cell infusion or DC vaccinations. One of the 4 patients developed grade III acute gut graft-versus-host disease. Immune reconstitution against CMV was detected in all 4 patients. Patients receiving CMV peptide-specific T cells and DC vaccination had peak immune reconstitution at least 10 days after the second DC vaccination. In summary, combining DC vaccine with T cell infusion appears feasible, although further study is required to ascertain its safety and efficacy in augmenting the effects of infusing donor-derived CMV-specific T cells.     

Evaluation of Immunotherapy Efficiency in Mouse CaO-1 Ovarian Carcinoma Treated by Vaccines Based on Dendritic Cells

Injection of dendritic cells, pulsated by tumor lysate or mucin, containing CA 125 antigen, led to a more than 50% inhibition of tumor growth in female CBA mice with transplanted mouse pseudomucinous CaO-1 ovarian carcinoma in comparison with the control. Tumor-associated CA 125 antigen can be used for obtaining dendritic cell vaccines against ovarian malignant tumors. This trend will extend the potentialities of application of antitumor vaccines based on dendritic cells, as clinical use of this technology is limited by the need in patient’s tumor material. Mucin, containing Ca 125 antigen, can be isolated from patient’s serum or obtained by gene engineering technologies as a recombinant peptide. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 2, pp. 187-189, February, 2009     

Development of a Unique T Cell Receptor Gene-Transferred Tax-Redirected T Cell Immunotherapy for Adult T Cell Leukemia

Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by infection with human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains extremely poor. Tax, the most important regulatory protein for HTLV-1, is associated with the aggressive proliferation of host cells and is also a major target antigen for CD8⁺ cytotoxic T cells (CTLs). Based on our previous findings that Tax-specific CTLs with a T cell receptor (TCR) containing a unique amino-acid sequence motif exhibit strong HLA-A*24:02-restricted, Tax_301-309-specific activity against HTLV-1, we aimed to develop a Tax-redirected T cell immunotherapy for ATL. TCR-ɑ/β genes were cloned from a previously established CTL clone and transduced into peripheral blood mononuclear cells (PBMCs) of healthy volunteers using a retroviral siTCR vector. Then the cytotoxic efficacy against HTLV-1-infected T cells or primary ATL cells was assessed both in vitro and in vivo. The redirected CTLs (Tax-siCTLs) produced a large amount of cytokines and showed strong killing activity against ATL/HTLV-1-infected T cells in vitro, although they did not have universal activity against ATL cells. Next, in a xenograft mouse model using an HTLV-1-infected T cell line (MT-2), in all mice treated with Tax-siCTLs, the tumor rapidly diminished and finally disappeared without normal tissue damage, although all mice that were untreated or treated with non-gene-modified PBMCs died because of tumor progression. Our findings confirm that Tax-siCTLs can exert strong anti-ATL/HTLV-1 effects without a significant reaction against normal cells and have the potential to be a novel immunotherapy for ATL patients.     

Dendritic Cell Immunotherapy Combined with Cytokine-Induced Killer Cells Effectively Suppresses Established Hepatocellular Carcinomas in Mice

Background: The response of hepatocellular carcinoma (HCC) to immunotherapy is often disappointing and new strategies are clearly needed. The aim of the present study was to investigate whether cytokine-induced killer (CIK) cells combined with a dendritic cell vaccination enhanced cytotoxicity against hepatocarcinoma tumor cells in an in vivo animal model.

Methods: CIKs and DCs were prepared from C3H/HeJ mice by conventional methods, the dendritic cell (DC) pulsed with a MH134 cell lysate, DC or CIK alone were used as controls. Cell phenotypes were analyzed by flow cytometry, cytokine secretion levels were determined by enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was assessed by means of an in vitro lactate dehydrogenase (LDH) release assay. A mouse hepatocarcinoma cell MH134-bearing mice model was established to test the in vivo anti-tumor efficacy of the system.

Results: CIK cells combined with DC therapy resulted in significant inhibition of tumor growth compared with the control group, whereas the decrease in tumor growth in mice that had been treated with CIK or DC alone did not reach the level of statistical significance. The combination therapy led to a further increase in the population of cytotoxic T cells (CTLs) in vivo, compared to the CIK or DC alone therapy. In addition, the combination therapy significantly enhanced cytotoxic activity against MH134 cells.

Conclusion: Taken together, these results show that a DC + CIK vaccination is more effective than DC or CIK alone therapy for the treatment of hepatocarcinoma cancer.     

Human Dendritic Cell Subsets for Vaccination

TProtective immunity results from the interplay of antigen (Ag)-nonspecific innate immunity and Ag-specific adaptive immunity. The cells and molecules of the innate system employ non-clonal recognition pathways such as lectins and TLRs. B and T lymphocytes of the adaptive immune system employ clonal receptors recognizing Ag or peptides in a highly specific manner. An essential link between innate and adaptive immunity is provided by dendritic cells (DCs). As a component of the innate immunc system, DC organize and transfer information from the outside world to the cells of the adaptive immune system. DC can induce such contrasting states as active immune responsiveness or immunological tolerance. Recent years have brought a wealth of information regarding DC biology and pathophysiology that shows the complexity of this cell system. Thus, presentation of antigen by immature (non-activated) DCs leads to tolerance, whereas mature, antigen-loaded DCs are geared towards the launching of antigen-specific immunity. Furthermore, DCs are composed of multiple subsets with distinct functions at the interface of the innate and adaptive immunity. Our increased understanding of DC pathophysiologywill permit their rational manipulation for therapy such as vaccination to improve immunity.     

Role of Gangliosides in Active Immunotherapy with Melanoma Vaccine

Among various tumor associated cell surface antigens, gangliosides, the glycosphingolipids that contain sialic acids, offer a variety of epitopes, some of which are preferentially expressed on melanoma cells. These surface components of the bilayered lipid membrane of tumor cells are the targets of active immunotherapy with melanoma vaccine. Purified gangliosides in aqueous solution form micelles and, at high density, form lactones. Their antigenic expression (physical conformation and orientation) on the cell surface is governed by the nature of the sphingosine and the fatty acids they contain. Evidence is accruing to show that the nature of the fatty acid moiety of gangliosides differs in normal and neoplastic cells. Gangliosides per se are not immunogenic and require extrinsic adjuvanticity. Preparation of a melanoma cell vaccine for active immunotherapy requires an understanding of the ganglioside profile of melanoma, the ganglioside-associated heterogeneity of melanoma, and the role of shed melanoma gangliosides in the immunosuppression of cell mediated and humoral immunity. In addition, the role of some of the anti-ganglioside antibodies in the elimination of shed gangliosides, the cytotoxic killing of tumor cells, as well as in the down-regulation of lymphocyte functions must be considered in the formulation of vaccine. Different strategies for augmenting the immunogenicity of melanoma associated gangliosides with melanoma vaccine are evaluated.     

Increases in Serum TARC/CCL17 Levels Are Associated with Progression-Free Survival in Advanced Melanoma Patients in Response to Dendritic Cell-Based Immunotherapy

Introduction  Changes in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy. Materials and Methods  Using an array for 42 cytokines, chemokines, or growth factors, sera from normal controls and metastatic melanoma patients at baseline and week 4 were analyzed for qualitative changes. Quantitative determination of the levels of the chemokine thymus and activation-regulated chemokine (TARC/CCL17) was determined by enzyme-linked immunosorbent assay (ELISA). Results  Significant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (P = 0.026) and a significant increase in TARC/CCLl7 (P = 0.008) from week 0 to week 4 which was associated with improved overall survival (P = 0.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (P = 0.005). Receiver–operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.     

Formyl Peptide Receptor Suppresses Melanoma Development and Promotes NK Cell Migration

In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, wild-type mice were inoculated with melanoma cell line B16-F10 (1?×?10⁶/mouse) and treated with the formyl peptide receptor (FPR) agonist WKYMVm or the FPR antagonist WRW⁴. Growth of melanoma cell line B16-F10 was significantly inhibited in WKYMVm-treated mice and markedly promoted in WRW⁴-treated mice compared with control. Decreased number of myeloid-derived suppressor cells (MDSCs) and increased NK cell infiltration in tumor tissues were detected from WKYMVm-treated mice. Next, we showed that depletion of NK cell significantly increased tumor development in B16 tumor-bearing mice compared with the control group, and the suppressed tumor-developing effect of WKYMVm in B16 melanoma was abrogated with NK cell depletion. We also found that WKYMVm stimulates chemotactic migration in NK cells via the FPR family, and this was dependent on extracellular signal-related kinase (ERK) activation. Moreover, in our further experiment, we showed that the increased infiltration of NK cell and promoted NK cell chemotaxis in B16 melanoma induced by WKYMVm were both abolished with ERK inhibitor PD98059 administration. In conclusion, the FPR family promoted NK cell migration through ERK activation and inhibited B16 melanoma growth in a murine model.     

Levels of Dendritic Cell Populations and Regulatory T Cells Vary Significantly Between Two Commonly Used Mouse Strains

Dendritic cells (DC) are a heterogeneous group of professional antigen-presenting cells (APC) involved in both initiating immune responses and maintaining tolerance. Roughly, DC can be divided into plasmacytoid DC (pDC) and conventional DC (cDC). By controlling regulatory T cells (Treg), DC can influence the outcome of both immunity and autoimmunity. Since the use of mice as in vivo models became a practical tool for researchers studying pathological events in all kind of human diseases, we decided to compare levels of cDC, pDC and Treg in both spleen and blood between two inbred mouse strains. Here we show that two commonly used mouse strains, BALB/c and C57BL/10J mice, have significantly different levels of distinct CD11c⁺/CD4^–/CD8a⁺, CD11c⁺/CD4⁺/CD8a^– and CD11c⁺/CD4^–/CD8a^– cDC populations, pDC and Treg. Therefore, we emphasize the importance of considering the proper model when comparing data sets from different mouse strains.     

小鼠脾脏树突状细胞的分离与鉴定

本文根据Steinman方法并稍加改良从小鼠脾脏分离树突状细胞获得成功。经抗小鼠树突状细胞单克隆抗体(33D_1)作间接免疫荧光染色、微量细胞毒试验及EA花环形成试验等方法鉴定,其纯度大于80％。且经透射和扫描电镜观察,其形态结构明显不同于巨噬细胞。     

Human Dendritic Cell Interactions with Whole Recombinant Yeast: Implications for HIV-1 Vaccine Development

Defects in number and function of dendritic cells (DCs) have been observed during HIV-1 infection, so therapeutic HIV-1 vaccine approaches that target or activate DCs may improve vaccine immunogenicity. To determine the potential of recombinant Saccharomyces cerevisiae yeast as an HIV-1 vaccine, we investigated interactions between yeast and human DCs. Yeast induced direct phenotypic maturation of monocyte-derived DCs (MDDCs) and enriched blood myeloid DCs (mDCs), but only indirectly matured blood plasmacytoid DCs (pDCs). Yeast-pulsed MDDCs and blood mDCs produced inflammatory cytokines and stimulated strong allo-reactive T cell proliferation. Both blood DC subsets internalized yeast, and when pulsed with yeast recombinant for HIV-1 Gag protein, both stimulated in vitro expansion of Gag-specific CD8⁺ memory T cells. These results suggest that S. cerevisiae yeast have potent adjuvant effects on human DCs. Furthermore, recombinant yeast-derived antigens are processed by human blood DCs for MHC class-I cross-presentation. These DC-targeting characteristics of yeast suggest that it may be an effective vaccine vector for induction of HIV-1-specific cellular immune responses.     

Dendritic Cell Therapy of High-Grade Gliomas

The prognosis of patients with malignant glioma is poor in spite of multimodal treatment approaches consisting of neurosurgery, radiochemotherapy and maintenance chemotherapy. Among innovative treatment strategies like targeted therapy, antiangiogenesis and gene therapy approaches, immunotherapy emerges as a meaningful and feasible treatment approach for inducing long-term survival in at least a subpopulation of these patients. Setting up immunotherapy for an inherent immunosuppressive tumor located in an immune-privileged environment requires integration of a lot of scientific input and knowledge of both tumor immunology and neuro-oncology. The field of immunotherapy is moving into the direction of active specific immunotherapy using autologous dendritic cells (DCs) as vehicle for immunization. In the translational research program of the authors, the whole cascade from bench to bed to bench of active specific immunotherapy for malignant glioma is covered, including proof of principle experiments to demonstrate immunogenicity of patient-derived mature DCs loaded with autologous tumor lysate, preclinical in vivo experiments in a murine orthotopic glioma model, early phase I/II clinical trials for relapsing patients, a phase II trial for patients with newly diagnosed glioblastoma (GBM) for whom immunotherapy is integrated in the current multimodal treatment, and laboratory analyses of patient samples. The strategies and results of this program are discussed in the light of the internationally available scientific literature in this fast-moving field of basic science and translational clinical research.