Haptoglobin Induces a Specific Proteomic Profile and a Mature-Associated Phenotype on Primary Human Monocyte-Derived Dendritic Cells.

Damage-associated molecular patterns (DAMPs) play a critical role in dendritic cells (DCs) ability to trigger a specific and efficient adaptive immune response for different physiological and pathological scenarios. We have previously identified constitutive DAMPs (HMGB1 and Calreticulin) as well as new putative inducible DAMPs such as Haptoglobin (HP), from a therapeutically used heat shock-conditioned melanoma cell lysate (called TRIMEL). Remarkably, HP was shown to be the most abundant protein in the proteomic profile of heat shock-conditioned TRIMEL samples. However, its relative contribution to the observed DCs phenotype has not been fully elucidated. Human DCs were generated from monocytes isolated from PBMC of melanoma patients and healthy donors. DC lineage was induced with rhIL-4 and rhGM-CSF. After additional stimulation with HP, the proteome of these HP-stimulated cells was characterized. In addition, DCs were phenotypically characterized by flow cytometry for canonical maturation markers and cytokine production. Finally, in vitro transmigration capacity was assessed using Transwell plates. Our results showed that the stimulation with HP was associated with the presence of exclusive and higher relative abundance of specific immune-; energy production-; lipid biosynthesis-; and DAMPs-related proteins. Importantly, HP stimulation enhanced the expression of specific DC maturation markers and pro-inflammatory and Th1-associated cytokines, and an in vitro transmigration of primary human DCs. Taken together, these data suggest that HP can be considered as a new inducible DAMP with an important role in in vitro DC activation for cancer immunotherapy.

Gap junction intercellular communications regulate NK cell activation and modulate NK cytotoxic capacity.

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca(2+) responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell-mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43-GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell-mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca(2+) influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

Dendritic cell chimerism in oral mucosa of transplanted patients affected by graft-versus-host disease.

OBJECTIVE: Graft-versus-host disease (GVHD) is one of the main complications after haematopoietic stem cell transplantation. Clinical features of GVHD include either an acute (aGVHD) or a chronic (cGVHD) condition that affects locations such as the oral mucosa. While the involvement of the host's dendritic cells (DCs) has been demonstrated in aGVHD, the origin (donor/host) and mechanisms underlying oral cGVHD have not been completely elucidated. In this study, we intend to determine the origin of DCs present in mucosal tissue biopsies from the oral cavity of transplanted patients affected by cGVHD. METHODS: We purified DCs, from oral biopsies of three patients with cGVHD, through immunobeads and subsequently performed DNA extraction. The origin of the obtained DCs was determined by PCR amplification of 13 informative short tandem repeat (STR) alleles. We also characterised the DCs phenotype and the inflammatory infiltrate from biopsies of two patients by immunohistochemistry. RESULTS: Clinical and histological features of the biopsies were concordant with oral cGVHD. We identified CD11c-, CD207- and CD1a-positive cells in the epithelium and beneath the basal layer. Purification of DCs from the mucosa of patients affected by post-transplantation cGVHD was >95%. PCR-STR data analysis of DCs DNA showed that 100% of analysed cells were of donor origin in all of the evaluated patients. CONCLUSION: Our results demonstrate that resident DCs isolated from the oral tissue of allotransplanted patients affected by cGVHD are originated from the donor. Further research will clarify the role of DCs in the development and/or severity of oral cGVHD.

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.

Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

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

INTRODUCTION: Immunization 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 METHODS: Healthy 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/DISCUSSION: Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4(+) TGF-ß(+)) 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. CONCLUSIONS: Our 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.

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 Ca(2+) 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.

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.

Enteropathy and gut dysbiosis as obstacles to achieve immune recovery in undetectable people with HIV: a clinical view of evidence, successes, and projections.

Immune performance following antiretroviral therapy initiation varies among patients. Despite achieving viral undetectability, a subgroup of patients fails to restore CD4+ T-cell counts during follow-up, which exposes them to non-AIDS defining comorbidities and increased mortality. Unfortunately, its mechanisms are incompletely understood, and no specific treatment is available. In this review, we address some of the pathophysiological aspects of the poor immune response from a translational perspective, with emphasis in the interaction between gut microbiome, intestinal epithelial dysfunction, and immune system, and we also discuss some studies attempting to improve immune performance by intervening in this vicious cycle.

Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients.

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.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Paradoxical effects of cytokines in tumor immune surveillance and tumor immune escape.

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity.

Inverse Relationship Between Alzheimer's Disease and Cancer: How Immune Checkpoints Might Explain the Mechanisms Underlying Age-Related Diseases.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the adult population. There is evidence of an inverse epidemiological relationship between AD and cancer, another prevalent age-related disease. This has led to hypothesize that there could be a common biological mechanism, deregulated in opposite directions that might explain the phenomenon of mutual protection. The immunological system and its regulatory checkpoints are good candidates to explain why having survived a cancer could protect from developing AD. During cancerous growth, the neoplastic cells induce immune tolerance to block the host's immunity system that would prevent tumor growth. This has led to the development of drugs that block distinct immune checkpoints, such as Programmed Death 1 (PD-1) and its major ligand PD-L1, that have shown great promise in treating diverse types of cancer. We propose that in those individuals who survived a cancer, the immune system is left in a state of diminished tolerance or proinflammatory systemic milieu, after its successful attempt to fight the cancer, that protects them from developing AD.

Dendritic Cells Loaded with Heat Shock-Conditioned Ovarian Epithelial Carcinoma Cell Lysates Elicit T Cell-Dependent Antitumor Immune Responses In Vitro.

Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced stage patients in a series of clinical trials. In these studies, 60% of treated patients showed immunological responses which correlated positively with improved survival. Considering the relevance of ovarian cancer and the promising results of our DC-based vaccine, we show here that heat shock-conditioned cell lysates derived from ovarian epithelial carcinoma cell lines have the potential to induce the phenotypic and functional maturation of human DC, which in turn, is able to induce an efficient CD4+ and CD8+ T cell-mediated immune responses against ovarian cancer cell lines in vitro. In summary, OEC heat shock-conditioned cell lysate-loaded DCs may be considered for future combined immunotherapy approaches against ovarian tumors.

Dexamethasone turns tumor antigen-presenting cells into tolerogenic dendritic cells with T cell inhibitory functions.

BACKGROUND: Dendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses. METHODS: The effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined. RESULTS: Dexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1ß and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-ß). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4+ T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumor-infiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity. CONCLUSIONS: These findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.

Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation.

Hypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.

Pure platelet-rich plasma and supernatant of calcium-activated P-PRP induce different phenotypes of human macrophages.

AIM: This study aimed to evaluate the effect of two platelet preparations used in the clinic, pure platelet-rich plasma (P-PRP) and the supernatant of calcium-activated P-PRP (S-PRP), on the phenotype of human macrophages. MATERIALS & METHODS: Surface markers and cytokine production of human monocyte-derived macrophages were analyzed after 24 h stimulation with P-PRP or S-PRP. RESULTS: P-PRP and S-PRP present no difference in the expression of CD206, a M2 tissue-repair macrophage-related marker. However, these same macrophages presented different levels of CD163, CD86 as well as different IL-10 secretion capacities after 24 h incubation. CONCLUSION: These platelet preparations do not have an equivalent biological effect over macrophages, which suggest that they may present different clinical regenerative potentials.

Antitumor activity and carrier properties of novel hemocyanins coupled to a mimotope of GD2 ganglioside.

Conjugation to carrier proteins is a way to improve the immunogenicity of peptides. Such is the case for peptides mimicking carbohydrate tumor-associated antigens in cancer vaccine development. The most used protein for this purpose is the keyhole limpet hemocyanin (KLH) from Megathura crenulata. Its limited bioavailability has prompted interest in finding new candidates; nevertheless, it is not known whether other hemocyanins might be equally efficient as carrier of carbohydrate peptide mimotopes to promotes anti-tumor responses. Here, we evaluated the carrier and antitumor activity of novel hemocyanins with documented immunogenicity obtained from Concholepas concholepas (CCH) and Fissurella latimarginata (FLH), coupled through sulfo-SMCC to P10, a mimetic peptide of GD2, the major ganglioside constituent of neuroectodermal tumors, and incorporating AddaVax as an adjuvant. The humoral immune responses of mice showed that CCH-P10 and FLH-P10 conjugates elicited specific IgM and IgG antibodies against P10 mimotope, similar to those obtained with KLH-P10, which was used as a positive control. The CCH-P10 and FLH-P10 antisera, exhibited cross-reactivity with murine and human melanoma cells, like anti-CCH and anti-FLH sera suggesting a cross-reaction of CCH and FLH glycosylations with carbohydrate epitopes on the tumor cell surfaces, similar to the KLH antisera. When mice were primed with each hemocyanin-P10 and challenged with melanoma cells, better antitumor effects were observed for FLH-P10 than for CCH-P10 and, as for KLH-P10, irrespective of conjugation. These data demonstrate that CCH and FLH are useful carriers of carbohydrate mimotopes; however, the best antitumor activity of FLH preparations, indicate that is a suitable candidate for further cancer vaccines research.

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.

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.