Whole tumour cell-based vaccines: tuning the instruments to orchestrate an optimal antitumour immune response.

Immunotherapy, particularly those based on immune checkpoint inhibitors (ICIs), has become a useful approach for many neoplastic diseases. Despite the improvements of ICIs in supporting tumour regression and prolonging survival, many patients do not respond or develop resistance to treatment. Thus, therapies that enhance antitumour immunity, such as anticancer vaccines, constitute a feasible and promising therapeutic strategy. Whole tumour cell (WTC) vaccines have been extensively tested in clinical studies as intact or genetically modified cells or tumour lysates, injected directly or loaded on DCs with distinct adjuvants. The essential requirements of WTC vaccines include the optimal delivery of a broad battery of tumour-associated antigens, the presence of tumour cell-derived molecular danger signals, and adequate adjuvants. These factors trigger an early and robust local innate inflammatory response that orchestrates an antigen-specific and proinflammatory adaptive antitumour response capable of controlling tumour growth by several mechanisms. In this review, the strengths and weaknesses of our own and others' experiences in studying WTC vaccines are revised to discuss the essential elements required to increase anticancer vaccine effectiveness.

Hypoxic Melanoma Cells Deliver microRNAs to Dendritic Cells and Cytotoxic T Lymphocytes through Connexin-43 Channels.

Alterations in microRNA (miRNA) profiles, induced by tumor microenvironment stressors, like hypoxia, allow cancer cells to acquire immune-resistance phenotypes. Indeed, hypoxia-induced miRNAs have been implicated in cancer progression through numerous cancer cell non-autonomous mechanisms, including the direct transfer of hypoxia-responsive miRNA from cancer to immune cells via extracellular vesicles. Connexin-43 (Cx43)-constituted gap junctions (GJs) have also been involved in miRNA intercellular mobilization, in other biological processes. In this report, we aimed to evaluate the involvement of Cx43-GJs in the shift of miRNAs induced by hypoxia, from hypoxic melanoma cells to dendritic cells and melanoma-specific cytotoxic T lymphocytes (CTLs). Using qRT-PCR arrays, we identified that miR-192-5p was strongly induced in hypoxic melanoma cells. Immune cells acquired this miRNA after co-culture with hypoxic melanoma cells. The transfer of miR-192-5p was inhibited when hypoxic melanoma cells expressed a dominant negative Cx43 mutant or when Cx43 expression was silenced using specific short-hairpin RNAs. Interestingly, miR-192-5p levels on CTLs after co-culture with hypoxic melanoma cells were inversely correlated with the cytotoxic activity of T cells and with ZEB2 mRNA expression, a validated immune-related target of miR-192-5p, which is also observed in vivo. Altogether, our data suggest that hypoxic melanoma cells may suppress CTLs cytotoxic activity by transferring hypoxia-induced miR-192-5p through a Cx43-GJs driven mechanism, constituting a resistance strategy for immunological tumor escape.

Connexin-Mediated Signaling at the Immunological Synapse.

The immunological synapse (IS) is an intercellular communication platform, organized at the contact site of two adjacent cells, where at least one is an immune cell. Functional IS formation is fundamental for the modulation of the most relevant immune system activities, such as T cell activation by antigen presenting cells and T cell/natural killer (NK) cell-mediated target cell (infected or cancer) killing. Extensive evidence suggests that connexins, in particular connexin-43 (Cx43) hemichannels and/or gap junctions, regulate signaling events in different types of IS. Although the underlying mechanisms are not fully understood, the current evidence suggests that Cx43 channels could act as facilitators for calcium ions, cyclic adenosine monophosphate, and/or adenosine triphosphate uptake and/or release at the interface of interacting cells. These second messengers have relevant roles in the IS signaling during dendritic cell-mediated T and NK cell activation, regulatory T cell-mediated immune suppression, and cytotoxic T lymphocyte or NK cell-mediated target tumor cell killing. Additionally, as the cytoplasmic C-terminus domain of Cx43 interacts with a plethora of proteins, Cx43 may act as scaffolds for integration of various regulatory proteins at the IS, as suggested by the high number of Cx43-interacting proteins that translocate at these cell-cell interface domains. In this review, we provide an updated overview and analysis on the role and possible underlying mechanisms of Cx43 in IS signaling.

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell-tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

Tumor lysate-based vaccines: on the road to immunotherapy for gallbladder cancer.

Immunotherapy based on checkpoint blockers has proven survival benefits in patients with melanoma and other malignancies. Nevertheless, a significant proportion of treated patients remains refractory, suggesting that in combination with active immunizations, such as cancer vaccines, they could be helpful to improve response rates. During the last decade, we have used dendritic cell (DC) based vaccines where DCs loaded with an allogeneic heat-conditioned melanoma cell lysate were tested in a series of clinical trials. In these studies, 60% of stage IV melanoma DC-treated patients showed immunological responses correlating with improved survival. Further studies showed that an essential part of the clinical efficacy was associated with the use of conditioned lysates. Gallbladder cancer (GBC) is a high-incidence malignancy in South America. Here, we evaluated the feasibility of producing effective DCs using heat-conditioned cell lysates derived from gallbladder cancer cell lines (GBCCL). By characterizing nine different GBCCLs and several fresh tumor tissues, we found that they expressed some tumor-associated antigens such as CEA, MUC-1, CA19-9, Erb2, Survivin, and several carcinoembryonic antigens. Moreover, heat-shock treatment of GBCCLs induced calreticulin translocation and release of HMGB1 and ATP, both known to act as danger signals. Monocytes stimulated with combinations of conditioned lysates exhibited a potent increase of DC-maturation markers. Furthermore, conditioned lysate-matured DCs were capable of strongly inducing CD4+ and CD8+ T cell activation, in both allogeneic and autologous cell co-cultures. Finally, in vitro stimulated CD8+ T cells recognize HLA-matched GBCCLs. In summary, GBC cell lysate-loaded DCs may be considered for future immunotherapy approaches.

High CD8+ and absence of Foxp3+ T lymphocytes infiltration in gallbladder tumors correlate with prolonged patients survival.

BACKGROUND: Gallbladder cancer (GBC), although infrequent in industrialized countries, has high incidence rates in certain world regions, being a leading cause of death among elderly Chilean women. Surgery is the only effective treatment, and a five-year survival rate of advanced-stage patients is less than 10%. Hence, exploring immunotherapy is relevant, although GBC immunogenicity is poorly understood. This study examined the relationship between the host immune response and GBC patient survival based on the presence of tumor-infiltrating lymphocytes at different disease stages. METHODS: Tumor tissues from 80 GBC patients were analyzed by immunohistochemistry for the presence of CD3+, CD4+, CD8+, and Foxp3+ T cell populations, and the results were associated with clinical stage and patient survival. RESULTS: The majority of tumor samples showed CD3+ T cell infiltration, which correlated with better prognosis, particularly in advanced disease stages. CD8+, but not CD4+, T cell infiltration correlated with improved survival, particularly in advanced disease stages. Interestingly, a < 1 CD4+/CD8+ T cell ratio was related with increased survival. Additionally, the presence of Foxp3+ T cells correlated with decreased patient survival, whereas a ≤ 1 Foxp3+/CD8+ T cell ratio was associated with improved patient survival. CONCLUSIONS: Depending on the disease stage, the presence of CD8+ and absence of Foxp3+ T cell populations in tumor tissues correlated with improved GBC patient survival, and thus represent potential markers for prognosis and management of advanced disease, and supports testing of immunotherapy.

The Selective Degradation of Synaptic Connexin 43 Protein by Hypoxia-induced Autophagy Impairs Natural Killer Cell-mediated Tumor Cell Killing.

Although natural killer (NK) cells play an important role in the control of melanoma, hypoxic stress in the tumor microenvironment may impair NK-mediated tumor cell killing by mechanisms that are not fully understood. In this study, we investigated the effect of hypoxia on the expression and channel activity of connexin 43 (Cx43) in melanoma cells and its impact on their susceptibility to NK cell-mediated lysis. Our results demonstrated that hypoxic stress increases Cx43 expression in melanoma cells via hypoxia-inducible factor-1α (HIF-1α) transcriptional activity. Hypoxic cells displaying increased Cx43 expression were less susceptible to NK cell-mediated lysis compared with normoxic cells expressing a moderate level of Cx43. Conversely, when overexpressed in normoxic tumor cells, Cx43 improves their susceptibility to N cell-mediated killing. We show that the NK cell immune synapse formed with normoxic melanoma cells is more stable and contains a high level of gap-junctional Cx43 whereas that formed with hypoxic cells is less stable and contains a significant lower level of gap-junctional Cx43. We provide evidence that the activation of autophagy in hypoxic melanoma cells selectively degrades gap-junctional Cx43, leading to the destabilization of the immune synapse and the impairment of NK cell-mediated killing. Inhibition of autophagy by genetic or pharmacological approaches as well as expression of the non-degradable form of Cx43 significantly restore its accumulation at the immune synapse and improves N cell-mediated lysis of hypoxic melanoma cells. This study provides the first evidence that the hypoxic microenvironment negatively affects the immune surveillance of tumors by NK cells through the modulation of Cx43-mediated intercellular communications.

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.

Tumor plasticity interferes with anti-tumor immunity.

Since tumor cell plasticity was first shown to be crucial in tumor promotion and immune surveillance evasion, it has become an issue of intense investigation. Several mechanisms are associated with the acquisition of tumor cell plasticity and immune evasion, including loss of epithelial phenotype through epithelial-to-mesenchymal transition (EMT). We discuss recent evidence revealing that tumor cell plasticity may lead to the emergence of immunoresistant variants and how the tumor microenvironment evolves to shape this plasticity. We argue that targeting carcinoma cell plasticity represents a novel strategy to better control the emergence of resistant variants and to ensure more effective cancer therapies. In this context, the design of innovative integrative immunotherapy approaches is warranted.

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.

Co-Expression of Immunohistochemical Markers MRP2, CXCR4, and PD-L1 in Gallbladder Tumors Is Associated with Prolonged Patient Survival.

Gallbladder cancer (GBC) is a rare pathology in Western countries. However, it constitutes a relevant health problem in Asia and Latin America, with a high mortality in middle-aged Chilean women. The limited therapeutic options for GBC require the identification of targetable proteins with prognostic value for improving clinical management support. We evaluated the expression of targetable proteins, including three epithelial tumor markers, four proteins associated with multidrug and apoptosis resistance, and eleven immunological markers in 241 primary gallbladder adenocarcinomas. We investigated correlations between tumor marker expression, the primary tumor staging, and GBC patients' survival using automated immunohistochemistry, a semi-automatic method for image analysis, univariate and multivariate statistical analyses, and machine learning algorithms. Our data show a significant association between the expression of MRP2 (p = 0.0028), CXCR4 (p = 0.0423), and PD-L1 (p = 0.0264), and a better prognosis for patients with late-stage primary tumors. The expression of the MRP2/CXCR4/PD-L1 cluster of markers discriminates among short-, medium-, and long-term patient survival, with an ROC of significant prognostic value (AUC = 0.85, p = 0.0012). Moreover, a high MRP2/CXCR4/PD-L1 co-expression is associated with increased survival time (30 vs. 6 months, p = 0.0025) in GBC patients, regardless of tumor stage. Hence, our results suggest that the MRP2/CXCR4/PD-L1 cluster could potentially be a prognostic marker for GBC.

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.

Connexin channels modulation in pathophysiology and treatment of immune and inflammatory disorders.

Connexin-mediated intercellular communication mechanisms include bidirectional cell-to-cell coupling by gap junctions and release/influx of molecules by hemichannels. These intercellular communications have relevant roles in numerous immune system activities. Here, we review the current knowledge about the function of connexin channels, mainly those formed by connexin-43, on immunity and inflammation. Focusing on those evidence that support the design and development of therapeutic tools to modulate connexin expression and/or channel activities with treatment potential for infections, wounds, cancer, and other inflammatory conditions.

Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells.

Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells.

Connexins in melanoma: Potential role of Cx46 in its aggressiveness.

Melanoma is the most aggressive skin cancer, and in metastatic advanced states, it is completely refractory to chemotherapy. Therefore, it is relevant to understand the molecular bases that rule their aggressiveness. Connexins (Cxs) are proteins that under normal physiological conditions participate in intercellular communication, via the exchange of signaling molecules between the cytoplasm and extracellular milieu and the exchange of ions/second messengers between the cytoplasm of contacting cells. These proteins have shown important roles in cancer progression, chemo- and radiotherapy resistance, and metastasis. Accordingly, Cx26 and Cx43 seem to play important roles in melanoma progression and metastasis. On the other hand, Cx46 is typically expressed in the eye lens, where it seems to be associated with oxidative stress protection in fiber lens cells. However, in the last decade, Cx46 expression has been associated with breast and brain cancers, due to its role in potentiation of both extracellular vesicle release and cancer stem cell-like properties. In this review, we analyzed a potential role of Cx46 as a new biomarker and therapeutic target in melanoma.

The Evaluation of 17 Gastrointestinal Tumor Markers Reveals Prognosis Value for MUC6, CK17, and CD10 in Gallbladder-Cancer Patients.

Gallbladder cancer (GBC) is an aggressive and highly lethal disease with relatively low global incidence, but one that constitutes a major health problem in Asian and Latin American countries, particularly in Chile. The identification of new tumor-associated markers with potential prognosis value is required for GBC clinical practice. Using immunohistochemistry/tumor tissue microarray, we evaluated the expression of 17 gastrointestinal tumor-associated protein markers (CK7, CK17, CK19, CK20, CKLMW, CKHMW, MUC1, MUC2, MUC5AC, MUC6, CA125, CD10, CEA, vimentin, villin, claudin-4, and CDX2) in primary gallbladder adenocarcinomas from 180 Chilean patients and analyzed potential associations with their pathological and clinical characteristics. Younger female patients with well- to moderately differentiated tumors had a better prognosis than that of older female or male patients with tumors with a similar tumor differentiation grade. Among all analyzed markers, MUC6 expression was associated with better prognosis in patients with well- to moderately differentiated tumors, whereas CK17 or CD10 was associated with worse prognosis in patients with poorly differentiated tumors. In addition, the MUC6+CK17- expression pattern was strongly associated with better prognosis in patients with well- to moderately differentiated tumors, whereas patients with poorly differentiated tumors and with the CK17+CD10+ expression pattern showed worse prognosis. Our results suggest that tumor MUC6, CK17, and CD10 can be considered as potential prognosis markers for GBC.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth.

BACKGROUND: Immune checkpoint blocker (ICB) therapy has shown survival benefits for some patients with cancer. Nevertheless, many individuals remain refractory or acquire resistance to treatment, motivating the exploration of complementary immunotherapies. Accordingly, cancer vaccines offer an attractive alternative. Optimal delivery of multiple tumor-associated antigens combined with potent adjuvants seems to be crucial for vaccine effectiveness. METHODS: Here, a prototype for a generic melanoma vaccine, named TRIMELVax, was tested using B16F10 mouse melanoma model. This vaccine is made of heat shock-treated tumor cell lysates combined with the Concholepas concholepas hemocyanin as adjuvant. RESULTS: While B16F10 lysate provides appropriate melanoma-associated antigens, both a generic human melanoma cell lysate and hemocyanin adjuvant contributes with danger signals promoting conventional dendritic type 1 cells (cDC1), activation, phagocytosis and effective antigen cross-presentation. TRIMELVax inhibited tumor growth and increased mice survival, inducing cellular and humoral immune responses. Furthermore, this vaccine generated an increased frequency of intratumor cDC1s but not conventional type 2 dendritic cells (cDC2s). Augmented infiltration of CD3+, CD4+ and CD8+ T cells was also observed, compared with anti-programmed cell death protein 1 (PD-1) monotherapy, while TRIMELVax/anti-PD-1 combination generated higher tumor infiltration of CD4+ T cells. Moreover, TRIMELVax promoted an augmented proportion of PD-1lo CD8+ T cells in tumors, a phenotype associated with prototypic effector cells required for tumor growth control, preventing dysfunctional T-cell accumulation. CONCLUSIONS: The therapeutic vaccine TRIMELVax efficiently controls the weakly immunogenic and aggressive B16F10 melanoma tumor growth, prolonging tumor-bearing mice survival even in the absence of ICB. The strong immunogenicity shown by TRIMELVax encourages clinical studies in patients with melanoma.

Comparative EST transcript profiling of peach fruits under different post-harvest conditions reveals candidate genes associated with peach fruit quality.

BACKGROUND: Cold storage is used to inhibit peach fruit ripening during shipment to distant markets. However, this cold storage can negatively affect the quality of the fruit when it is ripened, resulting in disorders such as wooliness, browning or leathering. In order to understand the individual and combined biological effects that factors such as cold storage and ripening have on the fruit and fruit quality, we have taken a comparative EST transcript profiling approach to identify genes that are differentially expressed in response to these factors. RESULTS: We sequenced 50,625 Expressed Sequence Tags (ESTs) from peach mesocarp (Prunus persica O'Henry variety) stored at four different postharvest conditions. A total of 10,830 Unigenes (4,169 contigs and 6,661 singletons) were formed by assembling these ESTs. Additionally, a collection of 614 full-length and 1,109 putative full-length cDNA clones within flanking loxP recombination sites was created. Statistically analyzing the EST population, we have identified genes that are differentially expressed during ripening, in response to cold storage or the combined effects of cold storage and ripening. Pair-wise comparisons revealed 197 contigs with at least one significant difference in transcript abundance between at least two conditions. Gene expression profile analyses revealed that the contigs may be classified into 13 different clusters of gene expression patterns. These clusters include groups of contigs that increase or decrease transcript abundance during ripening, in response to cold or ripening plus cold. CONCLUSION: These analyses have enabled us to statistically identify novel genes and gene clusters that are differentially expressed in response to post-harvest factors such as long-term cold storage, ripening or a combination of these two factors. These differentially expressed genes reveal the complex biological processes that are associated with these factors, as well as a large number of putative gene families that may participate differentially in these processes. In particular, these analyzes suggest that woolly fruits lack the increased boost of metabolic processes necessary for ripening. Additionally, these results suggest that the mitochondria and plastids play a major role in these processes. The EST sequences and full-length cDNA clones developed in this work, combined with the large population of differentially expressed genes may serve as useful tools and markers that will enable the scientific community to better define the molecular processes that affect fruit quality in response to post-harvest conditions and the organelles that participate in these processes.