Cristián Pereda

Prolonged Survival of Dendritic Cell–Vaccinated Melanoma Patients Correlates With Tumor-Specific Delayed Type IV Hypersensitivity Response and Reduction of Tumor Growth Factor β-Expressing T Cells

PURPOSE The aim of this work was to assess immunologic response, disease progression, and post-treatment survival of melanoma patients vaccinated with autologous dendritic cells (DCs) pulsed with a novel allogeneic cell lysate (TRIMEL) derived from three melanoma cell lines. PATIENTS AND METHODS Forty-three stage IV and seven stage III patients were vaccinated four times with TRIMEL/DC vaccine. Specific delayed type IV hypersensitivity (DTH) reaction, ex vivo cytokine production, and regulatory T-cell populations were determined. Overall survival and disease progression rates were analyzed using Kaplan-Meier curves and compared with historical records. RESULTS The overall survival for stage IV patients was 15 months. More than 60% of patients showed DTH-positive reaction against the TRIMEL. Stage IV/DTH-positive patients displayed a median survival of 33 months compared with 11 months observed for DTH-negative patients (P = .0014). All stage III treated patients were DTH positive and remained alive and tumor free for a median follow-up period of 48 months (range, 33 to 64 months). DTH-positive patients showed a marked reduction in the proportion of CD4+ transforming growth factor (TGF) beta+ regulatory T cells compared to DTH-negative patients (1.54% v 5.78%; P < .0001). CONCLUSION 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.

Functional gap junctions facilitate melanoma antigen transfer and cross-presentation between human dendritic cells.

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.

Functional Gap Junctions Accumulate at the Immunological Synapse and Contribute to T Cell Activation

Gap junction (GJ) mediates 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 Ag 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 dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk 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 dendritic cell–T cell signaling machinery that regulates T cell activation.

The capacity of Salmonella to survive inside dendritic cells and prevent antigen presentation to T cells is host specific

Infection 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 Salmonella‐derived 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.

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells

We have previously reported a novel method for the production of tumour‐antigen‐presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma‐derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell‐based immunotherapy induces T‐cell‐mediated immune responses resulting in improved long‐term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti‐tumour immunization. This mobilization of DCs is mainly driven by the C‐C chemokine receptor type 7 (CCR7), which is up‐regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient‐derived DCs, and also on the monocytic/macrophage cell line THP‐1. Moreover, in vitro assays showed that TRIMEL‐stimulated DCs and THP‐1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC‐based vaccines for treating aggressive melanomas.

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses

IntroductionImmunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH+ with respect to DTH− unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood.Materials and methodsHealthy donors and melanoma patient’s lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release.Results/discussionPeripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4+ TGF-β+) regulatory T lymphocytes compared with healthy donors. Notably, DTH+ patients showed a threefold reduction of Th3 cells compared with DTH− patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH+ with respect to DTH− patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response.ConclusionsOur results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.

Acceptability of drugs for male fertility regulation: A prospectus and some preliminary data

Hormonal substances for male fertility regulation administered orally or by injection are currently undergoing clinical evaluation. These trials, sponsored by the World Health Organization, provide unique opportunities for intensive study of the acceptability of such an approach to fertility regulation, and of these drugs in particular. The research employs repeated interviews over a 15-month period and is conducted by social scientists collaborating with biomedical scientists at each of seven sites (Bangkok, Hong Kong, London, Mexico City, Santiago, Seoul, and Toronto). The focus is upon gauging male users evaluations of hormonal methods (several androgen/gestagen combinations as well as cyproterone acetate) relative to their evaluations of other male methods they know about or have experienced. Of particular importance is to determine whether the hormonal methods modify or interfere with sexual desire, feelings, and behavior. The research is also assessing specific ways in which various perceived properties of fertility regulating methods relate to their acceptability in different socio-cultural settings.

TAPCells, the Chilean dendritic cell vaccine against melanoma and prostate cancer

Here we summarize 10 years of effort in the development of a biomedical innovation with global projections. This innovation consists of a novel method for the production of therapeutic dendritic-like cells called Tumor Antigen Presenting Cells (TAPCells®). TAPCells-based immunotherapy was tested in more than 120 stage III and IV melanoma patients and 20 castration-resistant prostate cancer patients in a series of phase I and I/II clinical trials. TAPCells vaccines induced T cell-mediated memory immune responses that correlated with increased survival in melanoma patients and prolonged prostate-specific antigen doubling time in prostate cancer patients. Importantly, more than 60% of tested patients showed a Delayed Type Hypersensitivity (DTH) reaction against the lysates, indicating the development of anti-tumor immunological memory that correlates with clinical benefits. The in vitro analysis of the lysate mix showed that it contains damage-associated molecular patterns such as HMBG-1 protein which are capable to improve, through Toll-like receptor-4, maturation and antigen cross-presentation of the dendritic cells (DC). In fact, a Toll-like receptor-4 polymorphism correlates with patient clinical outcomes. Moreover, Concholepas concholepas hemocyanin (CCH) used as adjuvant proved to be safe and capable of enhancing the immunological response. Furthermore, we observed that DC vaccination resulted in a three-fold increase of T helper-1 lymphocytes releasing IFN-γ and a two-fold increase of T helper-17 lymphocytes capable of producing IL-17 in DTH+ with respect to DTH- patients. Important steps have been accomplished for TAPCells technology transfer, including patenting, packaging and technology assessment. Altogether, our results indicate that TAPCells vaccines constitute an exceptional Chilean national innovation of international value.

Molecular signatures associated with tumor-specific immune response in melanoma patients treated with dendritic cell-based immunotherapy

Purpose We previously showed that autologous dendritic cells (DCs) loaded with an allogeneic heat shock (HS)-conditioned melanoma cell-derived lysate, called TRIMEL, induce T-cell-mediated immune responses in stage IV melanoma patients. Importantly, a positive delayed-type hypersensitivity (DTH) reaction against TRIMEL after vaccination, correlated with patients prolonged survival. Furthermore, we observed that DTH reaction was associated with a differential response pattern reflected in the presence of distinct cell subpopulations in peripheral blood. Detected variations in patient responses encouraged molecular studies aimed to identify gene expression profiles induced after vaccination in treated patients, allowing the identification of new molecular predictive markers. Methods Gene expression patterns were analyzed by microarrays during vaccination, and some of them confirmed by quantitative real-time reverse transcriptase PCR (qRT-PCR) in the total leukocyte population of a representative group of responder and non-responder patients. New candidates for biomarkers with predictive value were identified using bioinformatics, molecular analysis, and flow cytometry. Results Seventeen genes overexpressed in responder patients after vaccination respect to non-responders were identified after a mathematical analysis, from which ten were linked to immune responses and five related to cell cycle control and signal transduction. In immunological responder patients, increased protein levels of the chemokine receptor CXCR4 and the Fc-receptor CD32 were observed on cell membranes of CD8+ T and B cells and the monocyte population, respectively, confirming gene expression results. Conclusions Our study contributes to finding new molecular markers associated with clinical outcome and better understanding of clinically relevant immunological responses induced by anti-tumor DC-vaccines.

Proteomic Identification of Heat Shock-Induced Danger Signals in a Melanoma Cell Lysate Used in Dendritic Cell-Based Cancer Immunotherapy

Autologous dendritic cells (DCs) loaded with cancer cell-derived lysates have become a promising tool in cancer immunotherapy. During the last decade, we demonstrated that vaccination of advanced melanoma patients with autologous tumor antigen presenting cells (TAPCells) loaded with an allogeneic heat shock- (HS-) conditioned melanoma cell-derived lysate (called TRIMEL) is able to induce an antitumor immune response associated with a prolonged patient survival. TRIMEL provides not only a broad spectrum of potential melanoma-associated antigens but also danger signals that are crucial in the induction of a committed mature DC phenotype. However, potential changes induced by heat conditioning on the proteome of TRIMEL are still unknown. The identification of newly or differentially expressed proteins under defined stress conditions is relevant for understanding the lysate immunogenicity. Here, we characterized the proteomic profile of TRIMEL in response to HS treatment. A quantitative label-free proteome analysis of over 2800 proteins was performed, with 91 proteins that were found to be regulated by HS treatment: 18 proteins were overexpressed and 73 underexpressed. Additionally, 32 proteins were only identified in the HS-treated TRIMEL and 26 in non HS-conditioned samples. One protein from the overexpressed group and two proteins from the HS-exclusive group were previously described as potential damage-associated molecular patterns (DAMPs). Some of the HS-induced proteins, such as haptoglobin, could be also considered as DAMPs and candidates for further immunological analysis in the establishment of new putative danger signals with immunostimulatory functions.