Our findings strongly suggest that TRIMEL-pulsed DCs provide a standardized and widely applicable source of melanoma antigens, very effective in evoking antimelanoma immune response. To our knowledge, this is the first report describing a correlation between vaccine-induced reduction of CD4+TGFbeta+ regulatory T cells and in vivo antimelanoma immune response associated to improved patient survival and disease stability.

Our results indicate that lysates derived from heat-shocked tumor cells are an optimal source of tumor-associated Ags, which are crucial for the generation of DCs with improved Ag cross-presentation capacity and clinically effective immunogenicity.

Previously, we found that human dendritic cells (hDCs) pulsed with a melanoma cell lysate (MCL) and stimulated with TNF-α (MCL/TNF) acquire a mature phenotype in vitro and are able to trigger tumor-specific immune responses when they are used in melanoma immunotherapy in patients. In this study, we describe that MCL/TNF induces gap junction (GJ)-mediated intercellular communications and promotes melanoma Ag transfer between ex vivo produced hDCs from melanoma patients. hDCs also exhibit increased expression of the GJ-related protein connexin 43, which contributes to GJ plaque formation after MCL/TNF stimulation. The addition of GJ inhibitors suppresses intercellular tumor Ag transfer between hDCs, thus reducing melanoma-specific T cell activation. In summary, we demonstrate that MCL/TNF-stimulated hDCs can establish functional GJ channels that participate in melanoma Ag transfer, facilitating Ag cross-presentation and an effective dendritic cell-mediated melanoma-specific T cell response. These results suggest that GJs formed between hDCs used in cancer vaccination protocols could be essentials for the establishment of a more efficient antitumor response.

Gap junction (GJ) mediate intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was antigen-specific and time-dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and DCs is bidirectional and specifically mediated by Cx43. Moreover, this intercellular crosstalk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca2+ responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the DC-T cell signaling machinery that regulates T cell activation.

SummaryInfection with Salmonella enterica serovar Typhimurium (S. Typhimurium) causes a severe and lethal systemic disease in mice, characterized by poor activation of the adaptive immune response against Salmonelladerived antigens. Recently, we and others have reported that this feature relies on the ability of S. Typhimurium to survive within murine dendritic cells (DCs) and avoid the presentation of bacteria-derived antigens to T cells. In contrast, here we show that infection of murine DCs with either S. Typhi or S. Enteritidis, two serovars adapted to different hosts, leads to an efficient T-cell activation both in vitro and in vivo. Accordingly, S. Typhi and S. Enteritidis failed to replicate within murine DCs and were quickly degraded, allowing T-cell activation. In contrast, human DCs were found to be permissive for survival and proliferation of S. Typhi, but not for S. Typhimurium or S. Enteritidis. Our data suggest that Salmonella host restriction is characterized by the ability of these bacteria to survive within DCs and avoid activation of the adaptive immune response in their specific hosts.

Background:Recently, we produced a tumour antigen-presenting cells (TAPCells) vaccine using a melanoma cell lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer cell lysates (PCCL) as an antigen source, and TRIMEL as a dendritic cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients.Methods:The lysate mix capacity, for inducing T-cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8+ memory T cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients.Results:The lysate mix induced functional mature DCs that were capable of activating PCCL-specific T cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH+ patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8+ Tm were detected.Conclusion:Treatment of CRPC patients with lysate-loaded TAPCells and CCH as an adjuvant is safe: generating biochemical and memory immune responses. However, the limited number of cases requires confirmation in a phase II clinical trial.

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR-RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8(+) T cells determined by ELISpot (p < 0.01). Moreover, CD8(+) T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p < 0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p < 0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.

In 2008, 563,294,000 people were living in Latin America (LA), of which 6.6% were older than 65. The region is going through a fast demographic and epidemiologic transition process, in the context of an improvement in socio-economic indices. The Latin American Dialysis and Renal Transplant Registry has collected data since 1991, through an annual survey completed by 20 affiliated National Societies. Renal replacement treatment (RRT) prevalence and incidence showed an increase year by year. The prevalence rate (in all modalities) correlated with the World Bank country classification by income and the epidemiologic transition stage the countries were experiencing. RRT prevalence and kidney transplantation rates correlated significantly with gross national income (GNI), health expenditure in constant dollars (HeExp), % older than 65, life expectancy at birth, and % of the population living in urban settings. Kidney transplantation increased also, year by year, with more than 50% of transplants performed using kidneys from deceased donors. Double transplants were performed in six countries. RRT prevalence and incidence increased in LA, and are associated with indexes reflecting higher and more evenly distributed national wealth (GNI and HeExp), and the stage of demographic and epidemiological transition.