Delivery route, MyD88 signaling and cross-priming events determine the anti-tumor efficacy of an adenovirus based melanoma vaccine.

Adenovirus (Ad)-based vaccines are considered for cancer immunotherapy, yet, detailed knowledge on their mechanism of action and optimal delivery route for anti-tumor efficacy is lacking. Here, we compared the anti-tumor efficacy of an Ad-based melanoma vaccine after intradermal, intravenous, intranasal or intraperitoneal delivery in the B16F10 melanoma model. The intradermal route induced superior systemic anti-melanoma immunity which was MyD88 signaling-dependent. Predominant transduction of non-professional antigen-presenting cells at the dermal vaccination sites and draining lymph nodes, suggested a role for cross-presentation, which was confirmed in vitro. We conclude that the dermis provides an optimal route of entry for Ad-based vaccines for high-efficacy systemic anti-tumor immunization and that this immunization likely involves cross-priming events in the draining lymph nodes

The R620W polymorphism in PTPN22 confers general susceptibility for the development of alopecia areata

The functional R620W (c.1858C > T) variant of the protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) has been associated with a variety of autoimmune disorders. A recent study has suggested that R620W also contributes to the severe form of alopecia areata (AA). We sought to replicate the finding of an association between PTPN22 and severe AA. In addition, we wanted to study the effect of PTPN22 on the general risk to develop AA and on other subtypes of AA (mild AA, early/late age at onset, positive/negative family history). The R620W variant was genotyped in a large case-control sample of Belgian-German origin with 435 patients and 628 controls. Significant results were obtained for the overall collective of patients with AA (P = 0.007). Subdividing the sample according to severity of AA, family history and age at onset, we detected lowest P-values for patients with the severe form of AA (P-corr = 0.036), with a positive family history (P-corr = 0.042) and with an age at onset <= 20 years (P-corr = 0.048). Our results suggest the R620W variant of PTPN22 as a general risk factor in AA with the strongest effect observed among patients with a severe type of AA, a positive family history or an early onset of disease

Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8+ DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8+ DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8+ T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity

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

Efficient genetic modification of murine dendritic cells by electroporation with mRNA

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti-tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow-derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70-80% with a mean fluorescence intensity of 100-200. A maximal expression level was reached 3 hours after electroporation. A clear dose-response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches