Immunity induced by a broad class of inorganic crystalline materials is directly controlled by their chemistry

There is currently no paradigm in immunology that enables an accurate prediction of how the immune system will respond to any given agent. Here we show that the immunological responses induced by members of a broad class of inorganic crystalline materials are controlled purely by their physicochemical properties in a highly predictable manner. We show that structurally and chemically homogeneous layered double hydroxides (LDHs) can elicit diverse human dendritic cell responses in vitro. Using a systems vaccinology approach, we find that every measured response can be modeled using a subset of just three physical and chemical properties for all compounds tested. This correlation can be reduced to a simple linear equation that enables the immunological responses stimulated by newly synthesized LDHs to be predicted in advance from these three parameters alone. We also show that mouse antigen-specific antibody responses in vivo and human macrophage responses in vitro are controlled by the same properties, suggesting they may control diverse responses at both individual component and global levels of immunity. This study demonstrates that immunity can be determined purely by chemistry and opens the possibility of rational manipulation

Survival of metastatic melanoma patients after dendritic cell vaccination correlates with expression of leukocyte phosphatidylethanolamine-binding protein 1/Raf kinase inhibitory protein

Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 (_PEBP1_)/ Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low _PEBP1_ expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of _PEBP1_ after, but not prior to, DC vaccination. Moreover, the change in _PEBP1_ expression upon vaccination correlated well with survival. Further analyses revealed that _PEBP1_ expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including _STAT3, NOTCH1,_ and _MAPK1_. Concordantly, _PEBP1_ inversely correlated with the myeloid/ lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease

Intranodal vaccination with mRNA-optimized dendritic cells in metastatic melanoma patients

Contains fulltext : 153217.pdf (Publisher’s version ) (Open Access)Autologous dendritic cell (DC) therapy is an experimental cellular immunotherapy that is safe and immunogenic in patients with advanced melanoma. In an attempt to further improve the therapeutic responses, we treated 15 patients with melanoma, with autologous monocyte-derived immature DC electroporated with mRNA encoding CD40 ligand (CD40L), CD70 and a constitutively active TLR4 (caTLR4) together with mRNA encoding a tumor-associated antigen (TAA; respectively gp100 or tyrosinase). In addition, DC were pulsed with keyhole limpet hemocyanin (KLH) that served as a control antigen. Production of this DC vaccine with high cellular viability, high expression of co-stimulatory molecules and MHC class I and II and production of IL-12p70, was feasible in all patients. A vaccination cycle consisting of three vaccinations with up to 15x106 DC per vaccination at a biweekly interval, was repeated after 6 and 12 months in the absence of disease progression. mRNA-optimized DC were injected intranodally, because of low CCR7 expression on the DC, and induced de novo immune responses against control antigen. T cell responses against tyrosinase were detected in the skin-test infiltrating lymphocytes (SKIL) of two patients. One mixed tumor response and two durable tumor stabilizations were observed among 8 patients with evaluable disease at baseline. In conclusion, autologous mRNA-optimized DC can be safely administered intranodally to patients with metastatic melanoma but showed limited immunological responses against tyrosinase and gp100

Dendritic Cells as Arbiters of Peritoneal Immune Responses

During the past few years, there has been a substantial increase in the understanding of innate immunity. Dendritic cells are emerging as key players in the orchestration of this early phase of immune responses, with a role that will translate into the subsequent type of adaptive immune response against infection. Here we provide an overview of dendritic cell differentiation and function, with particular emphasis on those features unique to the immune defense of the peritoneal cavity and in the context of peritoneal dialysis-associated immune responses. The reader is referred to the primary references included in the accompanying list for specific details in this fascinating field

Interleukin-3 and granulocyte–macrophage colony-stimulating factor enhance the generation and function of dendritic cells

Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte–macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo-stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I- and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross-priming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL)

Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205−; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1− DEC-205+ F4/80−), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy

Analysis of mouse dendritic cell migration in vivo upon subcutaneous and intravenous injection

Dendritic cells (DC) have an increasingly important role in vaccination therapy; therefore, this study sought to determine the migratory capacity and immunogenic function of murine bone-marrow (BM)-derived DC following subcutaneous (s.c.) and intravenous (i.v.) injection in vivo. DC were enriched from BM cultures using metrizamide. Following centrifugation, the low-buoyant density cells, referred to throughout as DC, were CD11chigh, Iab high, B7-1high and B7-2high and potently activated alloreactive T cells in mixed lymphocyte reactions (MLR). In contrast, the high-density cells expressed low levels of the above markers, comprised mostly of granulocytes based on GR1 expression, and were poor stimulators in MLR. Following s.c. injection of fluorescently labelled cells into syngeneic recipient mice, DC but not granulocytes migrated to the T-dependent areas of draining lymph nodes (LN). DC numbers in LN were quantified by flow-cytometric analysis, on 1, 2, 3, 5 and 7 days following DC transfer. Peak numbers of around 90 DC per draining LN were found at 2 days. There was very little migration of DC to non-draining LN, thymus or spleen at any of the time-points studied. In contrast, following i.v. injection, DC accumulated mainly in the spleen, liver and lungs of recipient mice but were largely absent from peripheral LN and thymus. The ability of DC to induce T-cell-mediated immune responses was examined using trinitrobenzenesulphate (TNBS)-derivatized DC (TNBS-DC) to sensitize for contact hypersensitivity responses (CHS) in naive syngeneic recipients. Following s.c. injection, as few as 105 TNBS-DC, but not TNBS-granulocytes, sensitized for CHS responses. However, the same number of TNBS-DC failed to induce CHS following i.v. injection. In summary, this study provides new and quantitative data on the organ specific migration of murine BM-derived DC following s.c. and i.v. injection. The demonstration that the route of DC administration determines the potency of CHS induction, strongly suggests that the route of immunization should be considered in the design of vaccine protocols using DC