Neem leaf glycoprotein overcomes indoleamine 2,3 dioxygenase mediated tolerance in dendritic cells by attenuating hyperactive regulatory T cells in cervical cancer stage IIIB patients

Tolerogenic dendritic cells (DCs) are a subset of DCs characterized by abundant indoleamine 2,3 dioxygenase (IDO) expressions. IDO may be co-operatively induced in DCs by regulatory T (Tregs) cells and various DC maturation agents. Tregs are markedly amplified in the physiological system of cancer patients, inducing over tolerance in DCs that leads to the hyper accumulation of immunosuppressive IDO in tumor microenvironment, thereby, hampering anti-tumor immunity. Consequently, a major focus of current immunotherapeutic strategies in cancer is to minimize IDO, which is possible by reducing Tregs and using various IDO inhibitors. Neem leaf glycoprotein (NLGP), a natural and nontoxic immunomodulator, demonstrated several unique immunoregulatory activities. Noteworthy activities of NLGP are to mature DCs and to inhibit Tregs. As Tregs are inducer of IDO in DCs and hyperactive Tregs is a hallmark of cancer, we anticipated that NLGP might abrogate IDO induction in DCs by inhibiting Tregs. Evidences are presented here that in a co-culture of DCs and Tregs isolated from cervical cancer stage IIIB (CaCx-IIIB) patients, NLGP does inhibit IDO induction in DCs by curtailing the over expression of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) on Tregs and concomitantly induces optimal DC maturation. In contrast, in the presence of LPS as maturation agent the DCs displays a tolerogenic profile. This finding suggests the reduction of tolerogenecity of DCs in CaCx-IIIB patients by reducing the IDO pool using NLGP. Accordingly, this study sheds more light on the diverse immunomodulatory repertoire of NLGP.     

Fusion of CpG-ODN-stimulating dendritic cells with Lewis lung cancer cells can enhance anti-tumor immune responses

Immunogenicity of tumor cells is generally weak. Therefore, dendritic cells (DCs) have been used to boost anti-tumor responses of DC-based vaccines. DC function is highly dependent on its subsets and the level of its maturation. Nowadays, DC/tumor cell fusion vaccines are already used in clinical trials, and there are numerous studies discussing the effects of cytidine-phosphate-guanosine-containing oligonucleotides (CpG-ODN) on various cell types including DC. CpG-ODN a powerful immuno-stimulant can drive DCs fully mature, thus improve the efficacy of vaccine therapy. There are two simple ways to help load tumor antigens onto DCs by direct contact with cells themselves: fusion or co-culture of DCs with whole tumor cells. In this study, we combined these two approaches to improve the efficacy of DC/tumor cell-based vaccine. Mature DCs are adept at presenting processed Ag to T cells with loss of its capacity to capture Ag, while immature DCs are on the contrary. Our results emphasize the necessity of considering the stage of DC maturation and corresponding choice of tumor antigen delivery when designing approaches for prophylaxis or therapy of tumors using DC-based immunization protocols. We used CpG-ODN-1826-stimulated mature DCs and non-CpG-ODN-stimulating DCs as sources of tumor antigen carriers to investigate the appropriate Ag-loading ways between fusion and co-culture. Our results displayed that DC/tumor vaccine using CpG-ODN-stimulating mature DCs fused, not co-cultured, with tumor cells can generate a consistent and highly effective anti-tumor immune responses in vivo.     

CD1-dependent dendritic cell instruction

Both microbial products and T cell factors influence dendritic cell (DC) maturation. However, it is not known which T cells are capable of interacting with DCs at the initiation of adaptive immunity, when foreign antigen-specific T cells are rare. We show here that self-reactive CD1-restricted T cells can promote DC maturation by recognizing CD1 in the absence of foreign antigens. T cell recognition of all four CD1 isoforms can trigger DC maturation, but their distinct mechanisms of costimulation lead to profound differences in concomitant interleukin 12 p70 production. Distinct CD1-reactive T cells may thus differentially direct DC development early in the immune response, thereby controlling subsequent polarization of acquired immunity.     

Generation of monocyte-derived dendritic cells in patients with hereditary hemochromatosis

Hereditary hemochromatosis (HH) is a common genetic disease with autosomal recessive transmission and is characterized by a dysregulation of iron metabolism, leading to serum iron overload and its progressive accumulation in most body tissues. The effects of HH on the immune system include altered lymphocytosis and functions of monocytes. Moreover, monocytes can differentiate into dendritic cells (DCs), which play crucial roles in the immune response (capture, processing, and presentation of antigen to effector T cells) and this process was shown to be impaired in several pathologies. The aim of this study was to determine whether the monocytes from HH patients still displayed the ability to differentiate into DCs. To that purpose, purified monocytes from healthy donors and HH patients were cultured in the appropriate medium. The results showed no phenotypic and functional differences, at both the immature and the mature stages. Furthermore, our work reports altered lymphocytosis with expanded CD8+CD28- T cell subset. These monocyte-derived DCs could therefore be a solid vector for DC-based immunotherapy and a powerful tool for investigating the immune regulatory loops and especially the biological relevance of the expanded CD8+CD28- T cells since this population has also been described as suppressor T cells.     

Restoration of dysregulated CC chemokine signaling for monocyte/macrophage chemotaxis in head and neck squamous cell carcinoma patients by neem leaf glycoprotein maximizes tumor cell cytotoxicity

Previous studies have shown that the CC chemokine receptor CCR5 is downregulated on monocyte/macrophage (MO/Mφ) surfaces in head and neck squamous cell carcinoma (HNSCC) patients (stage IIIB). Ligands (RANTES, MIP-1α and MIP-1β) of this chemokine receptor were also secreted in lesser quantity from MO/Mφ of HNSCC patients in comparison with healthy individuals. In an aim to restore this dysregulated receptor–ligand signaling, we have used neem leaf glycoprotein (NLGP), a novel immunomodulator reported from our laboratory. NLGP upregulated CCR5 expression, as evidenced from studies on MO/Mφ of peripheral blood from HNSCC patients as well as healthy individuals. Expression of RANTES, MIP-1α and MIP-1β was also upregulated following NLGP treatment of these cells in vitro. Interestingly, NLGP has little effect on the expression of CCR5 and the ligand RANTES in oral cancer cells. This restored CCR5 receptor–ligand signaling seen in MO/Mφ was reflected in improved CCR5-dependent, p38 mitogen-activated protein kinase (MAPK)-mediated migration of MO/Mφ after NLGP treatment to a standard chemoattractant. NLGP also induces better antigen presentation and simultaneous costimulation to effector T cells by MO/Mφ by upregulating human leucocyte antigen (HLA)-ABC, CD80 and CD86. In addition, NLGP-treated MO/Mφ-primed T cells can effectively lyse tumor cells in vitro. The effects of NLGP on monocyte migration and T cell-mediated oral tumor cell killing were further demonstrated in transwell assays with or without CCR5 neutralization. These results suggest a new approach in cancer immunotherapy by modulating dysregulated CCR5 signals from MO/Mφ.     

Production of functionally deficient dendritic cells from HTLV-I-infected monocytes: implications for the dendritic cell defect in adult T cell leukemia

Adult T cell leukemia (ATL) is induced by an infection with human T lymphotropic virus type I (HTLV-I) and is accompanied by immunodeficiency. Monocyte-derived immature dendritic cells (DCs) donated by 11 ATL patients were suppressed in the ability to take up fluorescein isothiocyanate (FITC)-dextran and were down-regulated in the expression of CD1a and CD86 antigens (Ags). Monocytes from the patients showed impaired expression of CD14 and HLA-DR Ags. These results suggest intrinsic abnormalities of monocytes and a defect of DC maturation in ATL patients. Therefore, we examined the influence of HTLV-I infection of monocytes on their differentiation to DCs. Monocytes obtained from healthy donors were susceptible to HTLV-I infection in vitro. HTLV-I-infected monocytes were down-regulated in the expression of CD14 Ags, and immature DCs obtained from them expressed CD1a poorly and were impaired in the ability to take up FITC-dextran. Mature DCs differentiated from these cells could not stimulate autologous CD4(+) T cell or CD8(+) T cell proliferation, even after being secondarily pulsed with HTLV-I at an immature DC stage. These results suggest that HTLV-I-infected monocytes cannot properly differentiate to DCs and that this might be one of the important mechanisms producing dysfunctional DCs in ATL patients.     

Potent Dendritic Cell Vaccine Loaded with Latent Membrane Protein 2A （LMP2A）

Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.     

Dendritic cell as therapeutic vaccines against tumors and its role in therapy for hepatocellular carcinoma

Dendritic cells （DCs） are the most potent professional antigen-presenting cells, and capable of stimulating naive T cells and driving primary immune responses. DCs are poised to capture antigen, migrate to draining lymphoid organs, and after a process of maturation, select antigen-specific lymphocytes to which they present the processed antigen, thereby inducing immune responses. The development of protocols for the ex vivo generation of DCs may provide a rationale for designing and developing DC-based vaccination for the treatment of tumors. There are now several strategies being applied to upload antigens to DCs and manipulate DC vaccines. DC vaccines are able to induce therapeutic and protective antitumor immunity. Numerous studies indicated that hepatocellular carcinoma （HCC） immunotherapies utilizing DC-presenting tumor-associated antigens could stimulate an antitumour T cell response leading to clinical benefit without any significant toxicity. DC-based tumor vaccines have become a novel immunoadjuvant therapy for HCC. Cellular ＆ Molecular Immunology. 2006;3（3）：197-203.     

Artificially generated dendritic cells misdirect antiviral immune responses

Dendritic cells (DCs) are critical to the outcome of many viral infections. Questions still remain as to the relevance of artificially generated DCs in models of in vivo immune responses. We compared different DC generation pathways, in terms of phenotypic expression, cytokine production, apoptosis, and T cell proliferation, following viral infection. Direct viral infection of monocytes or monocytes cultured with supernatants from virally infected lung epithelial cells (A549 DCs) induce distinct DC subsets compared with viral infection of artificially generated IL-4 DCs and IFN-DCs. These virally infected DC subsets stimulated different cytokine secretion profiles and displayed contrasting sensitivities to viral-induced apoptosis. It is most interesting that we observed marked differences in the proliferation of purified CD3+ T cells from the virally infected DC subsets. In conclusion, artificially generated DCs skew immune responses to viral infections, and direct viral infection of monocytes and DCs, generated from monocytes cultured with supernatants from infected epithelial cells, appears to be a more relevant pathway of producing DCs, which mimic those generated in vivo.     

Generation and characterization of an immunogenic dendritic cell population

Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.     

Interferon γ Stimulates Cellular Maturation of Dendritic Cell Line DC2.4 Leading to Induction of Efficient Cytotoxic T Cell Responses and Antitumor Immunity

Dendritic cells （DCs） are the most potent antigen-presenting cells （APCs） for the initiation of antigen （Ag）-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-α for 7-10 days and stimulated with lipopolysaccharide （LPS）. However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class Ⅱ and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli （IL-1β, IFN-γ, TNF-α CpG and LPS） in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-γ was able to stimulate the maturation of DC2.4 cells. The IFN-γ-activated ovalbumin （OVA）-pulsed DC2.4 cells have capacity to upregulate MHC class Ⅱ, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8^＋ T cell responses and antitumor immunity. Therefore, IFN-γ-activated immortal DC2.4 ceils may prove to be useful in the study of DC biology and antitumor immunity.     

Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell crosspriming in vivo

The nature of dendritic cell(s) (DC[s]) that conditions efficient in vivo priming of CD8+ CTL after immunization via epithelial tissues remains largely unknown. Here, we show that myeloid DCs rapidly recruited by adjuvants into the buccal mucosa or skin are essential for CD8+ T cell crosspriming. Recruitment of circulating DC precursors, including Gr1+ monocytes, precedes the sequential accumulation of CD11c+ MHC class II+ DCs in dermis and epithelium via a CCR6/CCL20-dependent mechanism. Remarkably, a defect in CCR6, local neutralization of CCL20, or depletion of monocytes prevents in vivo priming of CD8+ CTL against an innocuous protein antigen administered with adjuvant. In addition, transfer of CCR6-sufficient Gr1+ monocytes restores CD8+ T cell priming in CCR6( degrees / degrees ) mice via a direct Ag presentation mechanism. Thus, newly recruited DCs likely derived from circulating monocytes are responsible for efficient crosspriming of CD8+ CTL after mucosal or skin immunization.     

Differential levels of dendritic cell maturation on different biomaterials used in combination products

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with films of biomaterials commonly used in combination products (e.g., tissue engineered constructs or vaccines) to assess the resultant dendritic cell (DC) maturation compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The following biomaterials were tested: alginate, agarose, chitosan, hyaluronic acid, 75:25 poly(lactic-co-glycolic acid) (PLGA). The effect of DC culture on these films was undertaken to identify biomaterials which support DC maturation and those biomaterials that did not. Dendritic cells treated with chitosan or PLGA (agarose to a lesser extent) films increased expression levels of CD86, CD40, and HLA-DQ, compared to control iDCs, similar to LPS-matured DCs, whereas DCs treated with alginate or hyaluronic acid films decreased their expression levels of these same molecules. In summary, a differential effect of the biomaterial on which iDCs were cultured was observed as far as the extent of induced DC maturation. The effect of biomaterials on DC maturation, and the associated adjuvant effect, is a novel biocompatibility selection and design criteria for biomaterials to be used in combination products in which immune consequences are potential complications or outcomes.     

The effect of human placenta cytotrophoblast cells on the maturation and T cell stimulating ability of dendritic cells in vitro

The success of pregnancy depends upon regulatory mechanisms that allow the fetus to survive and develop to term in the uterus, despite maternal immune cells' awareness of paternal alloantigens. At least some of these specific mechanisms are mediated by the effect of fetal trophoblast cells. In the present study we examine the effect of human placental cytotrophoblast cells (CTCs) on the maturation of dendritic cells (DCs) in vitro. For that purpose, CTCs were isolated from samples of placentae at 5–11 weeks of gestation and co‐cultured with peripheral blood monocytes under conditions inducing DC maturation. CTC were shown to alter the morphology, phenotype and functional properties of DCs. As a result, a significant portion of cells acquire fibroblast‐like morphology and some of the cells retain the expression of CD14. DCs matured in the presence of CTCs do not differ from usual DCs in terms of CD80, CD83 and CD86 expression, as well as the ability to induce allogenic lymphocytes proliferation. However, CTCs reduce significantly the ability of DCs to stimulate interferon‐γ production and the loss of CD62L by T cells. The results obtained indicate that DCs may be involved in pregnancy‐associated changes of cytokine production and T cell migration.     

IFN-alpha promotes definitive maturation of dendritic cells generated by short-term culture of monocytes with GM-CSF and IL-4

Dendritic cells (DC) generated in vitro have to be viable and phenotypically mature to be capable of inducing T cell-mediated immunity after in vivo administration. To facilitate optimization of DC-based vaccination protocols, we investigated whether the cytokine environment and the mode of activation affect maturation and survival of DC derived from monocytes by a short-term protocol. Monocytes cultured for 24 h with granulocyte macrophage-colony stimulating factor and interleukin-4 were stimulated with proinflammatory mediators for another 36 h to generate mature DC. Additional activation with CD40 ligand and interferon (IFN)-gamma increased viability of DC and promoted definitive maturation as defined by maintenance of a mature phenotype after withdrawal of cytokines. Addition of IFN-alpha to DC cultures prior to stimulation further enhanced definitive maturation: IFN-alpha-primed DC expressed high levels of costimulatory molecules and CC chemokine receptor 7 (CCR7) up to 5 days after cytokine withdrawal. Compared with unprimed DC, IFN-alpha-primed DC displayed equal capacity to migrate upon CCR7 ligation and to prime antigen-specific T helper cell as well as cytolytic T cell responses. In conclusion, we show that optimal maturation and survival of monocyte-derived DC require multiple activation signals. Furthermore, we identified a novel role for IFN-alpha in DC development: IFN-alpha priming of monocytes promotes definitive maturation of DC upon activation.     

Enhanced in vitro stimulation of rhesus macaque dendritic cells for activation of SIV-specific T cell responses

The macaque-simian immunodeficiency virus (SIV) system is one of the best animal models available to study the role of dendritic cells (DCs) in transmission and pathogenesis of HIV, as well as to test DC-based vaccine and therapeutic strategies. To better define and optimize this system, the responsiveness of macaque monocyte-derived DCs to a variety of maturation stimuli was examined. Characteristic immunophenotypic and functional DC maturation induced by standard monocyte conditioned medium (MCM) was compared to the activation induced by a panel of stimuli including soluble CD40L, LPS, Poly I:C, PGE₂/TNF, and a cocktail mixture of PGE₂/TNF/IL-1β/IL-6. Immunophenotypic analysis confirmed that all stimuli induced stable up-regulation of CD25, CD40, CD80, CD83, CD86, HLA-DR, DC-LAMP (CD208), and DEC-205 (CD205). In general, macaque DCs exhibited weaker responses to LPS and Poly I:C than human DCs, and soluble CD40L stimulation induced variable expression of CD25. Interestingly, while the endocytic capacity of CD40L-matured cells was down-modulated comparably to DCs matured with MCM or the cocktail, the T cell stimulatory activity was not enhanced to the same extent. The particularly reproducible and potent T cell stimulatory capacity of cocktail-treated DCs correlated with a more homogenous mature DC phenotype, consistently high levels of IL-12 production, and better viability upon reculture compared to DCs activated by other stimuli. Furthermore, cocktail-matured DCs efficiently captured and presented inactivated SIV to SIV-primed T cells in vitro. Thus, the cocktail represents a particularly potent and useful stimulus for the generation of efficacious immunostimulatory macaque DCs.     

Tumour necrosis factor-alpha (TNF-alpha) transgene-expressing dendritic cells (DCs) undergo augmented cellular maturation and induce more robust T-cell activation and anti-tumour immunity than DCs generated in recombinant TNF-alpha

Tumour antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of anti-tumour immune responses. It has been previously reported that tumour necrosis factor-alpha (TNF-alpha) is required for DC activation and subsequent induction of optimal immune responses, and thus DCs for anti-tumour vaccination are often generated by culture in exogenous TNF-alpha. In the present study, we investigated the effect on anti-tumour immunity of vaccination with Mut1 tumour peptide-pulsed DCs engineered to express a TNF-alpha transgene. Our data shows that transfection of DCs with recombinant adenovirus AdV-TNF-alpha resulted in greater maturation of the DCs than occurred with control DCs cultured in exogenous TNF-alpha, as determined by up-regulated expression of pro-inflammatory cytokines (e.g. interleukins 1beta and 18), chemokines [e.g. interferon-gamma-inducible protein-10 and macrophage inflammatory protein-1beta (MIP-1beta)], the CC chemokine receptor CCR7, and immunologically important cell surface molecules (CD40, CD86 and intercellular adhesion molecule-1). These transgenic DCs stimulated stronger allogeneic T-cell responses in vitro and T-cell activation in vivo; displayed 2.4-fold enhanced chemotactic responses to the MIP-3betain vitro (P<0.05); and, perhaps most importantly, trafficked into the draining lymph nodes dramatically (seven-fold, P<0.01) more efficiently than the control DCs. Our data also demonstrate that vaccination of mice with Mut1 peptide-pulsed, AdV-TNF-alpha-transfected DCs stimulated more efficient in vitro Mut1-specific CD8+ cytotoxic T-cell responses and solid tumour immunity in vivo, when compared to the in vitro TNF-alpha-cultivated DCs. Thus, DCs engineered to secrete TNF-alpha may offer a new strategy in DC cancer vaccines.