The regulatory T cell master transcription factor, Forkhead box P3 (Foxp3), has been detected in cancer cells; however, its role in breast tumor pathogenesis remains controversial. Here we assessed Foxp3 tumor intrinsic effects in experimental breast cancer using a Foxp3 binder peptide (P60) that impairs Foxp3 nuclear translocation. Cisplatin upregulated Foxp3 expression in HER2+ and triple-negative breast cancer (TNBC) cells. Foxp3 inhibition with P60 enhanced chemosensitivity and reduced cell survival and migration in human and murine breast tumor cells. We also developed an adenoviral vector encoding P60 (Ad.P60) that efficiently transduced breast tumor cells, reduced cell viability and migration, and improved the cytotoxic response to cisplatin. Conditioned medium from transduced breast tumor cells contained lower levels of IL-10 and improved the activation of splenic lymphocytes. Intratumoral administration of Ad.P60 in breast-tumor-bearing mice significantly reduced tumor infiltration of Tregs, delayed tumor growth, and inhibited the development of spontaneous lung metastases. Our results suggest that Foxp3 exerts protumoral intrinsic effects in breast cancer cells and that gene-therapy-mediated blockade of Foxp3 could constitute a therapeutic strategy to improve the response of these tumors to standard treatment.

The Sin3 transcriptional regulator homolog A (Sin3A) is the core member of a multiprotein chromatin-modifying complex. Its inactivation at the CD4/CD8 double-negative stage halts further thymocyte development. Among various functions, Sin3A regulates STAT3 transcriptional activity, central to the differentiation of Th17 cells active in inflammatory disorders and opportunistic infections. To further investigate the consequences of conditional Sin3A inactivation in more mature precursors and post-thymic T cell, we have generated CD4-Cre and CD4-CreERT2 Sin3AF/F mice. Sin3A inactivation in vivo hinders both thymocyte development and peripheral T-cell survival. In vitro, in Th17 skewing conditions, Sin3A-deficient cells proliferate and acquire memory markers and yet fail to properly upregulate Il17a, Il23r, and Il22. Instead, IL-2+ and FOXP3+ are mostly enriched for, and their inhibition partially rescues IL-17A+ T cells. Notably, Sin3A deletion also causes an enrichment of genes implicated in the mTORC1 signaling pathway, overt STAT3 activation, and aberrant cytoplasmic ROR¿t accumulation. Thus, together our data unveil a previously unappreciated role for Sin3A in shaping critical signaling events central to the acquisition of immunoregulatory T-cell phenotypes.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Currently, among the possible treatments for hepatocel-lular carcinoma there is group of minimally invasive abla-tion techniques with wide clinical acceptance due to their greater efficacy and safety in comparison to traditional therapies, low cost, and no need of being admitted to hospi-tal (outpatient treatment program). Irreversible electroporation is a non-thermal abla tion technique in which electrical fields are used to create nan-opores in the cell membrane that induce tumor cell death.Irreversible electroporation has shown promising results in numerous clinical trials; however, its control on long-term tumor growth and recurrence is inferior in comparison to that of radiofrequency. Combining irreversible electropora-tion with immunological agents may increase its efficacy in the treatment of focal lesions and metastases. In this work, we present an update on IRE including procedure, mecha-nism of action, application as a treatment for HCC, and the improvements that have been made in the past few years.

Vaccination using optimized strategies may increase response rates to immune checkpoint inhibitors (ICI) in some tumors. To enhance vaccine potency and improve thus responses to ICI, we analyzed the gene expression profile of an immunosuppressive dendritic cell (DC) population induced during vaccination, with the goal of identifying druggable inhibitory mechanisms. RNAseq studies revealed targetable genes, but their inhibition did not result in improved vaccines. However, we proved that immunosuppressive DC had a monocytic origin. Thus, monocyte depletion by gemcitabine administration reduced the generation of these DC and increased vaccine-induced immunity, which rejected about 20% of LLC-OVA and B16-OVA tumors, which are non-responders to anti-PD-1. This improved efficacy was associated with higher tumor T-cell infiltration and overexpression of PD-1/PD-L1. Therefore, the combination of vaccine + gemcitabine with anti-PD-1 was superior to anti-PD-1 monotherapy in both models. B16-OVA tumors benefited from a synergistic effect, reaching 75% of tumor rejection, but higher levels of exhausted T-cells in LLC-OVA tumors co-expressing PD-1, LAG3 and TIM3 precluded similar levels of efficacy. Our results indicate that gemcitabine is a suitable combination therapy with vaccines aimed at enhancing PD-1 therapies by targeting vaccine-induced immunosuppressive DC.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, making them an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We evaluated this possibility by invivo electroporation of Semliki Forest virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. Optimization of saRNA electroporation conditions in tumors was performed using an SFV vector coding for luciferase. Then we evaluated the therapeutic potential of this approach using an SFV saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine with potent antitumor effects. Delivery of SFV-IL-12 saRNA by electroporation led to improvement in tumor control and higher survival compared with mice treated with electroporation or with SFV-IL-12 saRNA alone. The antitumor efficacy of SFV-IL-12 saRNA electroporation increased by combination with systemic PD-1 blockade. This therapy, which was also validated in a hepatocellular carcinoma tumor model, suggests that local delivery of saRNA by electroporation could be an attractive strategy for cancer immunotherapy. This approach could have easy translation to the clinical practice, especially for percutaneously accessible tumors.

The high metabolic activity and insufficient perfusion of tumors leads to the acidification of the tumor microenvironment (TME) that may inhibit the antitumor T cell activity. We found that pharmacological inhibition of the acid loader chloride/bicarbonate anion exchanger 2 (Ae2), with 4,4'-diisothiocyanatostilbene-2,2'-disulfonicacid (DIDS) enhancedCD4(+) andCD8(+) T cell function upon TCR activation in vitro, especially under low pH conditions. In vivo, DIDS administration delayed B16OVA tumor growth in immunocompetent mice as monotherapy or when combined with adoptive T cell transfer of OVA-specificT cells. Notably, genetic Ae2 silencing in OVA-specificT cells improvedCD4(+)/CD8(+) T cell function in vitro as well as their antitumor activity in vivo. Similarly, genetic modification of OVA-specificT cells to overexpress Hvcn1, a selectiveH(+) outward current mediator that prevents cell acidification, significantly improved T cell function in vitro, even at low pH conditions. The adoptive transfer of OVA-specificT cells overexpressing Hvcn1 exerted a better antitumor activity in B16OVA tumor-bearingmice. Hvcn1 overexpression also improved the antitumor activity of CAR T cells specific for Glypican 3 (GPC3) in mice bearing PM299L-GPC3tumors. Our results suggest that preventing intracellular acidification by regulating the expression of acidifier ion channels such as Ae2 or alkalinizer channels like Hvcn1 in tumor-specificlymphocytes enhances their antitumor response by making them more resistant to the acidic TME.

Adoptive cell transfer therapy using CD8+ T lymphocytes showed promising results eradicating metastatic malignancies. However, several regulatory mechanisms limit its efficacy. We studied the role of the expression of the transcription factor FOXP3 on CD8+ T cell function and anti-tumor immunity. Here we show that suboptimal T cell receptor stimulation of CD8+ T cells upregulates FOXP3 in vitro. Similarly, CD8 T cells transferred into tumor-bearing mice upregulate FOXP3 in vivo. Cell-intrinsic loss of FOXP3 by CD8+ T cells resulted in improved functionality after TCR stimulation and better antitumor responses in vivo. Inhibition of the FOXP3/NFAT interaction likewise improved CD8+ T cell functionality. Transcriptomic analysis of cells after TCR stimulation revealed an enrichment of genes implicated in the response to IFN-gamma, IFN-alpha, inflammatory response, IL-6/JAK/STAT, G2M checkpoint and IL-2/STAT signaling in FOXP3-deficient CD8+ T cells with respect to FOXP3-wt CD8+ T cells. Our results suggest that transient expression of FOXP3 by CD8+ T cells in the tumor microenvironment restrains their anti-tumor activity, with clear implications for improving T cell responses during immunotherapy.

Background One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. Methods EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. Results EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3 zeta endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. Conclusions These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.

Background Target antigen (Ag) loss has emerged as a major cause of relapse after chimeric antigen receptor T (CART)-cell therapy. We reasoned that the combination of CART cells, with the consequent tumor debulking and release of Ags, together with an immunomodulatory agent, such as the stimulator of interferon gene ligand (STING-L) 2 ' 3 '-cyclic GMP-AMP (2 ' 3 '-cGAMP), may facilitate the activation of an endogenous response to secondary tumor Ags able to counteract this tumor escape mechanism. Methods Mice bearing B16-derived tumors expressing prostate-specific membrane Ag or gp75 were treated systemically with cognate CART cells followed by intratumoral injections of 2 ' 3 '-cGAMP. We studied the target Ag inmunoediting by CART cells and the effect of the CART/STING-L combination on the control of STING-L-treated and STING-L-non-treated tumors and on the endogenous antitumor T-cell response. The role of Batf3-dependent dendritic cells (DCs), stimulator of interferon gene (STING) signaling and perforin (Perf)-mediated killing in the efficacy of the combination were analyzed. Results Using an immune-competent solid tumor model, we showed that CART cells led to the emergence of tumor cells that lose the target Ag, recreating the cancer immunoediting effect of CART-cell therapy.

Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10(+) DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8(+) T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.

Background/Aim. Irreversible electroporation (IRE) showed promising results for small-size tumors and very early cancers. However, further development is needed to evolve this procedure into a more efficient ablation technique for long-term control of tumor growth. In this work, we show that it is possible to increase the antitumor efficiency of IRE by simmultaneously injecting c-di-GMP, a STING agonist, intratumorally. Materials and Methods. Intratumoral administration of c-di-GMP simultaneously to IRE was evaluated in murine models of melanona (B16.OVA) and hepatocellular carcinoma (PM299L). Results. The combined therapy increased the number of tumor-infiltrating IFN-gamma/TNF-alpha-producing CD4 and CD8 T cells and delayed tumor growth, as compared to the effect observed in groups treated with c-di-GMP or IRE alone. Conclusion. These results can lead to the development of a new therapeutic strategy for the treatment of cancer patients refractory to other therapies.

The aim of this article is to present the research protocol for a prospective cohort study that will assess the olfactory function and the effect of an intervention based on olfactory training in healthy very old adults (>= 75 years old). A convenience sample of 180 older people (50% female) will be recruited in three different environments: hospitalized control group (CH) with stable acute illness (n = 60); ambulatory control group (CA) of community-based living (n = 60); and an experimental odor training group (EOT) from nursing homes (n = 60). The odor training (OT) intervention will last 12 weeks. All the volunteers will be assessed at baseline; CA and EOT groups will also be assessed after 12 weeks. The primary end point will be change in olfactory capacity from baseline to 12 weeks period of intervention or control. The intervention effects will be assessed with the overall score achieved in Sniffin Sticks Test (SST) - Threshold, Discrimination, and Identification (TDI) extended version. Secondary end points will be changes in cognitive tasks, quality of life, mood, immune status, and functional capacity. All these measurements will be complemented with an immune fitness characterization and a deep proteome profiling of the olfactory epithelium (OE) cultured ex vivo. The current study will provide additional evidence to support the implementation of olfactory precision medicine and the development of immunomodulatory nasal therapies based on non-invasive procedures. The proposed intervention will also intend to increase the knowledge about the olfactory function in very elderly people, improve function and quality of life, and promote the recovery of the health.

Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 x 10(4)-3 x 10(5), depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern.

Introduction The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. Methods A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. Results Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. Conclusions YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.

(1) Background: The ability of cancer cells to evade the immune system is due in part to their capacity to induce and recruit T regulatory cells (Tregs) to the tumor microenvironment. Strategies proposed to improve antitumor immunity by depleting Tregs generally lack specificity and raise the possibility of autoimmunity. Therefore, we propose to control Tregs by their functional inactivation rather than depletion. Tregs are characterized by the expression of the Forkhead box protein 3 (FOXP3) transcription factor, which is considered their "master regulator". Its interaction with DNA is assisted primarily by its interaction with other proteins in the so-called "Foxp3 interactome", which elicits much of the characteristic Treg cell transcriptional signature. We speculated that the disruption of such a protein complex by using synthetic peptides able to bind Foxp3 might have an impact on the functionality of Treg cells and thus have a therapeutic potential in cancer treatment. (2) Methods: By using a phage-displayed peptide library, or short synthetic peptides encompassing Foxp3 fragments, or by studying the crystal structure of the Foxp3:NFAT complex, we have identified a series of peptides that are able to bind Foxp3 and inhibit Treg activity. (3) Results: We identified some peptides encompassing fragments of the leuzin zipper or the C terminal domain of Foxp3 with the capacity to inhibit Treg activity in vitro. The acetylation/amidation of linear peptides, head-to-tail cyclization, the incorporation of non-natural aminoacids, or the incorporation of cell-penetrating peptide motifs increased in some cases the Foxp3 binding capacity and Treg inhibitory activity of the identified peptides. Some of them have shown antitumoral activity in vivo. (4) Conclusions: Synthetic peptides constitute an alternative to inhibit Foxp3 protein-protein interactions intracellularly and impair Treg immunosuppressive activity. These peptides might be considered as potential hit compounds on the design of new immunotherapeutic approaches against cancer.

Transforming Growth Factor-beta (TGF-ß) superfamily members are essential for tissue homeostasis and consequently, dysregulation of their signaling pathways contributes to the development of human diseases. In the liver, TGF-ß signaling participates in all the stages of disease progression from initial liver injury to hepatocellular carcinoma (HCC). During liver carcinogenesis, TGF-ß plays a dual role on the malignant cell, behaving as a suppressor factor at early stages, but contributing to later tumor progression once cells escape from its cytostatic effects. Moreover, TGF-ß can modulate the response of the cells forming the tumor microenvironment that may also contribute to HCC progression, and drive immune evasion of cancer cells. Thus, targeting the TGF-ß pathway may constitute an effective therapeutic option for HCC treatment. However, it is crucial to identify biomarkers that allow to predict the response of the tumors and appropriately select the patients that could benefit from TGF-ß inhibitory therapies. Here we review the functions of TGF-ß on HCC malignant and tumor microenvironment cells, and the current strategies targeting TGF-ß signaling for cancer therapy. We also summarize the clinical impact of TGF-ß inhibitors in HCC patients and provide a perspective on its future use alone or in combinatorial strategies for HCC treatment.

Background Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation. Methods PD-1(-) and PD-1(+) CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation. Results Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1(+) fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137(+) cells within the PD-1(+)CD8(+) TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products. Conclusion We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1(+) TILs.

Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified IncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNF alpha- and NF-kappa B-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.

Background In vivo targeting of human papillomavirus (HPV) derived antigens to dendritic cells might constitute an efficient immunotherapeutic strategy against cervical cancer. In previous works, we have shown that the extra domain A from murine fibronectin (mEDA) can be used to target antigens to toll-like receptor 4 (TLR4) expressing dendritic cells and induce strong antigen-specific immune responses. In the present study, we have produced a bivalent therapeutic vaccine candidate consisting of the human EDA (hEDA) fused to E7 proteins from HPV16 and HPV18 (hEDA-HPVE7-16/18) and evaluate its potential as a therapeutic vaccine against cervical cancer. Materials and methods Recombinant fusion proteins containing HPV E7 proteins from HPV16 and HPV18 virus subtypes fused to hEDA were produced and tested in vitro on their capacity to bind TLR4 and induce the production of tumor necrosis factor-alpha or interleukin (IL)-12 by human monocytes and dendritic cells. The immunogenicity and potential therapeutic activity of the vaccine in combination with cisplatin or with the TLR3 agonist molecules polyinosinic-polycytidylic acid (Poly IC) or Poly ICLC was evaluated in mice bearing subcutaneous or genital orthotopic HPV16 TC-1 tumors. Results hEDA-HPVE7-16/18 prototype vaccine binds human TLR4 and stimulate TLR4-dependent signaling pathways and IL-12 production by human monocyte-derived dendritic cell. Vaccination with hEDA-HPVE7-16/18 induced strong HPVE7-specific Cytotoxic T lymphocy

Current immunotherapies based on blockade of immunosuppressive elements provide limited results in liver cancer patients. Here we tested whether combination of this therapy with a vaccine based on the Cold-Inducible RNA Binding Protein (CIRP) would improve its efficacy. Combination of immunotherapy with a CIRP-based vaccine increased vaccine immunogenicity and, when tested in several mouse models of liver cancer, resulted in better therapeutic effects. Despite good immune responses observed in peripheral organs, lymphocytes infiltrating the tumor appeared exhausted, with a weak functional capacity. Finally, by using the same strategy, we prepared a new CIRP-based vaccine containing glypican-3, human antigen commonly found in patients with liver cancer. An equivalent combination enclosing this new vaccine was also highly immunogenic. This suggests that CIRP-based vaccines may enhance the beneficial effects provided by current immunotherapies. However, they should also consider incorporating new elements to overcome limitations observed in tumor lymphocytes. Therapies based on immune checkpoint inhibitors (ICPI) have yielded promising albeit limited results in patients with hepatocellular carcinoma (HCC). Vaccines have been proposed as combination partners to enhance response rates to ICPI. Thus, we analyzed the combined effect of a vaccine based on the TLR4 ligand cold-inducible RNA binding protein (CIRP) plus ICPI. Mice were immunized with vaccines containing ovalbumin linke

Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1¿programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.

Background Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. Objectives Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). Methods We used three PV-IgG fractions from different patients containing high or low levels of anti-Dsg1 and anti-Dsg3 antibodies, and the presence or not of anti-desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. Results Although all of the PV-IgG fractions produced suprabasal acantholysis, only those containing anti-Dsg1/3, but not anti-Dsc2/3 antibodies, induced ADAM10 activation in a Src-dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti-Dsc2/3 antibodies, in addition to anti-Dsg1/3, triggered earlier and ADAM10-independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. Conclusions All PV-IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti-Dsg antibodies or the presence of non-Dsg antibodies, such as anti-Dsc, more severe cell-cell epidermal detachment will occur at different times, and in an ADAM10-dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non-Dsg proteins, and therefore more specific therapeutic approaches in PV should be used. What's already known about this topic? Suprabasal acantholysis in pemphigus vulgaris (PV) may be triggered by both desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient is associated with a distinct intracellular signalling pattern. What does this study add? In patients with PV with anti-Dsg3 and anti-Dsg1, but not anti-desmocollin (Dsc)3 antibodies, ADAM10 activation is induced in an Src-dependent way, together with an increase in the epidermal growth factor receptor (EGFR) ligands EGF and betacellulin. The presence of anti-Dsc3 antibodies triggers an earlier and ADAM10-independent acantholysis, without increasing EGFR ligands, and is associated with more severe epidermal detachment. Lower levels of anti-Dsc3 antibodies are associated with less severe acantholysis. What is the translational message? In some patients with PV, the severity and the timing for cell-cell detachment seem to depend on the level of anti-Dsg1/3 antibodies, although other as yet uncharacterized antibodies may also participate. These patients with PV would exhibit inhibition of acantholysis by Src, ADAM10, EGF and EGFR inhibitors. In other patients, the presence of non-Dsg antibodies, such as anti-Dsc2/3, would produce an earlier and more severe ADAM10-independent suprabasal acantholysis.

Immune checkpoint inhibitors are currently tested in different combinations in patients with advanced hepatocellular carcinoma (HCC). Nivolumab, an anti-PD-1 agent, has gained approval in the second-line setting in the USA. Epigenetic drugs have immune-mediated antitumor effects that may improve the activity of immunotherapy agents. Our aim was to study the therapeutic efficacy of checkpoint inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) in combination with the histone deacetylase inhibitor (HDACi) Belinostat. In a subcutaneous Hepa129 murine HCC model, we demonstrated that Belinostat improves the antitumor activity of anti-CTLA-4 but not of anti-PD-1 therapy. This effect correlated with enhanced IFN-gamma production by antitumor T-cells and a decrease in regulatory T-cells. Moreover, the combination induced early upregulation of PD-L1 on tumor antigen-presenting cells and late expression of PD-1 on tumor-infiltrating effector T-cells, suggesting the suitability of PD-1 blockade. Indeed, Belinostat combined with the simultaneous blockade of CTLA-4 and PD-1 led to complete tumor rejection. These results provide a rationale for testing Belinostat in combination with checkpoint inhibitors to enhance their therapeutic activity in patients with HCC.

Background/Aim: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. Materials and Methods: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon gamma (IFN gamma) ELISA. Results: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFN gamma secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. Conclusion: P60 may potentiate CIK cell cytotoxicity against tumor cells.

Adoptive immunotherapy with ex vivo-expanded tumor-infiltrating lymphocytes (TILs) has achieved objective clinical responses in a significant number of patients with cancer. The failure of many patients to develop long-term tumor control may be, in part, due to exhaustion of transferred T cells in the presence of a hostile tumor microenvironment. In several tumor types, growth and survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. We speculated that if transferred T cells could benefit from EGFR ligands produced by the tumor, they might proliferate better and exert their anti-tumor activities more efficiently. We found that CD8(+) T cells transduced with a retrovirus to express EGFR responded to EGFR ligands activating the EGFR signaling pathway. These EGFR-expressing effector T cells proliferated better and produced more IFN-gamma and TNF-alpha in the presence of EGFR ligands produced by tumor cells in vitro. EGFR-expressing CD8 T cells from OT-1 mice were more efficient killing B16-OVA cells than control OT-1 CD8 T cells. Importantly, EGFR-expressing OT-1 T cells injected into B16-OVA tumor bearing mice were recruited into the tumor, expressed lower levels of the exhaustion markers PD1, TIGIT, and LAG3, and were more efficient in delaying tumor growth. Our results suggest that genetic modification of CD8(+) T cells to express EGFR might be considered in immunotherapeutic strategies based on adoptive transfer of anti-tumor T cells against cancers expressing EGFR ligands.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances(1,2). Recent molecular characterization has defined new (epi) genetic drivers and potential targets for bladder cancer(3,4). The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients(5-8). Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (Pten(loxP/loxP); Trp53(loxP/loxP); Rb1(loxP/loxP); Rbl1(-/-)) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-kappa B activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-kappa B and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-kappa B-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.

Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and s

Purpose: To evaluate the therapeutic efficacy of irreversible electroporation (IRE) combined with the intratumoral injection of the immunogenic adjuvant poly-ICLC (polyinosinic-polycytidylic acid and poly-L-lysine, a dsRNA analog mimicking viral RNA) inmediately before IRE. Materials and Methods: Mice and rabbits bearing hepatocellular carcinoma tumors (Hepa.129 and VX2 tumor models, respectively) were treated with IRE (2 pulses of 2500V), with poly-ICLC, or with IRE + poly-ICLC combination therapy. Tumor growth in mice was monitored using a digital caliper and by computed tomography in rabbits. Results: Intratumoral administration of poly-ICLC immediately before IRE elicited shrinkage of Hepa.129 cell-derived tumors in 70% of mice, compared to 30% and 26% by poly-ICLC or IRE alone, respectively (P = .0004). This combined therapy induced the shrinkage of VX-2-based hepatocellular carcinoma tumors in 40% of rabbits, whereas no response was achieved by either individual treatment (P = .045). The combined therapy activated a systemic antitumor response able to inhibit the growth of other untreated tumors. Conclusions: IRE treatment, immediately preceded by the intratumoral administration of an immunogenic adjuvant such as poly-ICLC, might enhance the antitumor effect of the IRE procedure. This combination might facilitate the induction of a long-term systemic response to prevent tumor relapses and the appearance of metastases.

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-beta for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-alpha 1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-alpha or AAV-IFN-alpha fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-alpha resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-alpha-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-gamma) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-alpha for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.

A complex network of interactions exists between the immune, the olfactory, and the central nervous system (CNS). Inhalation of different fragrances can affect immunological reactions in response to an antigen but also may have effects on the CNS and cognitive activity. We performed an exploratory study of the immunomodulatory ability of a series of compounds representing each of the 10 odor categories or clusters described previously. We evaluated the impact of each particular odor on the immune response after immunization with the model antigen ovalbumin in combination with the TLR3 agonist poly I:C. We found that some odors behave as immunostimulatory agents, whereas others might be considered as potential immunosuppressant odors. Interestingly, the immunomodulatory capacity was, in some cases, strain-specific. In particular, one of the fragrances, carvone, was found to be immunostimulatory in BALB/c mice and immunosuppressive in C57BL/6J mice, facilitating or impairing viral clearance, respectively, in a model of a viral infection with a recombinant adenovirus. Importantly, inhalation of the odor improved the memory capacity in BALB/c mice in a fear-conditioning test, while it impaired this same capacity in C57BL/6J mice. The improvement in memory capacity in BALB/c was associated with higher CD3+ T cell infiltration into the hippocampus and increased local expression of mRNA coding for IL-1ß, TNF-¿, and IL-6 cytokines. In contrast, the memory impairment in C57BL/6 was associated with a reduction in CD3 numbers and an increase in IFN-¿. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals. These results highlight the potential of studying odors as therapeutic agents for CNS-related diseases.

Tumor infiltrating lymphocytes have been associated with a better prognostic and with higher response rates in patients treated with checkpoint inhibiting antibodies, suggesting that strategies promoting tumor inflammation may enhance the efficacy of these currently available therapies. Our aim was thus to develop a new vaccination platform based on cold-inducible RNA binding protein (CIRP), an endogenous TLR4 ligand generated during inflammatory processes, and characterize whether it was amenable to combination with checkpoint inhibitors. In vitro, CIRP induced dendritic cell activation, migration and enhanced presentation of CIRP-bound antigens to T-cells. Accordingly, antigen conjugation to CIRP conferred immunogenicity, dependent on immunostimulatory and antigen-targeting capacities of CIRP. When applied in a therapeutic setting, vaccination led to CD8-dependent tumor rejection in several tumor models. Moreover, immunogenicity of this vaccination platform was enhanced not only by combination with additional adjuvants, but also with antibodies blocking PD-1/PD-L1, CTLA-4 and IL-10, immunosuppressive molecules usually present in the tumor environment and also induced by the vaccine. Therefore, priming with a CIRP-based vaccine combined with immune checkpoint-inhibiting antibodies rejected established B16-OVA tumors. Finally, equivalent activation and T-cell stimulatory effects were observed when using CIRP in vitro with human cells, suggesting that CIRP-based vaccination strategies could be a valuable clinical tool to include in combinatorial immunotherapeutic strategies in cancer patients.

Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer. SIGNIFICANCE: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. (C) 2017 AACR.

Although T regulatory cells (Treg) are essential for the prevention of autoimmune diseases, their immunoregulatory function restrains the induction of immune responses against cancer. Thus, development of inhibitors of FOXP3, a key transcription factor for the immunosuppressive activity of Treg, might give new therapeutic opportunities. In a previous work we identified a peptide (named P60) able to enter into the cells, bind to FOXP3, and impair Treg activity in vitro and in vivo. Here we show that P60 binds to the intermediate region of FOXP3 and inhibits its homodimerization as well as its interaction with the transcription factor AML1. Alanine-scanning of P60 revealed the relevance of each position on FOXP3 binding, homodimerization, association with AML1 and inhibition of Treg activity. Introduction of alanine at positions 2, 5 and 11 improved the activity of the original P60, whereas alanine mutations at positions 1, 7, 8, 9, 10 and 12 were detrimental. Multiple mutation experiments allowed us to identify peptides with higher FOXP3 binding affinity and stronger biological activity than the original P60. Head to tail macrocyclization of peptide P60-D2A-S5A improved Treg inhibition and enhanced anti-tumor activity of anti-PD1 antibodies in a model of hepatocellular carcinoma. Introduction of a D-aminoacid at position 2 augmented significantly microsomal stability while maintained FOXP3 binding capacity and Treg inhibition in vitro. In vivo, when combined with the cytotoxic T-cell epitope AH1, it induced protection against CT26 tumor implantation. This study provides important structure¿function relationships essential for further drug design to inhibit Treg cells in cancer.

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

Recent studies have found that tumor-infiltrating lymphocytes (TIL) expressing PD-1 can recognize autologous tumor cells, suggesting that cells derived from PD-1(+) TILs can be used in adoptive T-cell therapy (ACT). However, no study thus far has evaluated the antitumor activity of PD-1-selected TILs in vivo. In two mouse models of solid tumors, we show that PD-1 allows identification and isolationof tumor-specific TILs without previous knowledge of their antigen specificities. Importantly, despite the high proportion of tumor-reactive T cells present in bulk CD8 TILs before expansion, only T-cell products derived fromsorted PD-1(+), but not from PD-1(-) or bulk CD8 TILs, specifically recognized tumor cells. The fold expansion of PD-1(+) CD8 TILs was 10 times lower than that of PD-1(-) cells, suggesting that outgrowth of PD-1(-) cells was the limiting factor in the tumor specificity of cells derived from bulk CD8 TILs. The highly differentiated state of PD-1(+) cells was likely the main cause hampering ex vivo expansion of this subset. Moreover, PD-1 precisely identified marrow-infiltrating, myeloma-specific T cells in a mouse model of multiple myeloma. In vivo, only cells expanded from PD-1(+) CD8 TILs contained tumor progression, and their efficacy was enhanced by PDL-1 blockade. Overall, our data provide a rationale for the use of PD-1-selected TILs in ACT. (C) 2017 AACR.

Vaccination induces immunostimulatory signals that are often accompanied by regulatory mechanisms such as IL-10, which control T-cell activation and inhibit vaccine-dependent antitumor therapeutic effect. Here we characterized IL10-producing cells in different tumor models treated with therapeutic vaccines. Although several cell subsets produced IL-10 irrespective of treatment, an early vaccine-dependent induction of IL-10 was detected in dendritic cells (DC). IL-10 production defined a DC population characterized by a poorly mature phenotype, lower expression of T-cell stimulating molecules and upregulation of PD-L1. These IL-10(+) DC showed impaired in vitro T-cell stimulatory capacity, which was rescued by incubation with IL-10R and PD-L1-inhibiting antibodies. In vivo IL-10 blockade during vaccination decreased the proportion of IL-10(+) DC and improved their maturation, without modifying PD-L1 expression. Similarly, PD-L1 blockade did not affect IL-10 expression. Interestingly, vaccination combined with simultaneous blockade of IL-10 and PD-L1 induced stronger immune responses, resulting in a higher therapeutic efficacy in tumor-bearing mice. These results show that vaccine-induced immunoregulatory IL-10(+) DC impair priming of antitumor immunity, suggesting that therapeutic vaccination protocols may benefit from combined targeting of inhibitory molecules expressed by this DC subset.

LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

The CD6 lymphocyte receptor has been involved in the pathophysiology of different autoimmune disorders and is now considered a feasible target for their treatment. In vitro data show the relevance of CD6 in the stabilization of adhesive contacts between T-cell and antigen-presenting cells, and the modulation of T-cell receptor signals. However, the in vivo consequences of such a function are yet undisclosed due to the lack of suitable genetically modified animal models. Here, the in vitro and in vivo challenge of CD6-deficient (CD6(-/-)) cells with allogeneic cells was used as an approach to explore the role of CD6 in immune responses under relative physiological stimulatory conditions. Mixed lymphocyte reaction (MLR) assays showed lower proliferative responses of splenocytes from CD6(-/-)mice together with higher induction of regulatory T cells (T-reg, CD4(+)CD25(+)FoxP3(+)) with low suppressive activity on T and B-cell proliferation. In line with these results, CD6(-/-)mice undergoing a lupus-like disorder induced by chronic graft-versus-host disease (cGvHD) showed higher serum titers of anti-double-stranded DNA and nucleosome autoantibodies. This occurred together with reduced splenomegaly, which was associated with lower in vivo bromodesoxyuridine incorporation of spleen cells and with increased percentages of spleen follicular B cells (B2, CD21(+)CD23(hi)) and T-reg cells. Interestingly, functional analysis of in vivo-generated CD6(-/-)T(reg) cells exhibited defective suppressive activity. In conclusion, the data from MLR and cGvHD-induced lupus-like models in CD6(-/-)mice illustrate the relevance of CD6 in T (and B) cell proliferative responses and, even more importantly, Treg induction and suppressive function in the in vivo maintenance of peripheral tolerance.

The introduction of beta-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis. Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L lactis-based therapy revealed elevated frequencies of CD4(+)Foxp3(+) T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-beta partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3(+) Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4(+)Foxp3(+) T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients.

Regulatory T (Treg) cells can weaken antitumor immune responses, and inhibition of their function appears as a promising immunotherapeuticimmunotherapy therapeutic approach in cancer patients. Mice with targeted deletion of the gene encoding the Cl-HCO3-anion exchanger AE2 (also termed SLC4A2), a membrane-bound carrier involved in intracellular pH regulation, showed a progressive decrease in the number of Treg cells. We therefore challenged AE2 as a potential target for tumor immune therapy, and generated linear peptides designed to bind the third extracellular loop of AE2, which is crucial for its exchange activity. Peptide p17AE2 exhibited optimal interaction ability and indeed promoted apoptosis in mouse and human Treg cells, while activating effector T-cell function. Interestingly, this linear peptide also induced apoptosis in different types of human B-cell leukemia, lymphoma and multiple myeloma cell lines and primary malignant samples, while it showed only moderate effects on normal B lymphocytes. Finally, a macrocyclic peptide exhibiting increased stability in vivo was effective in mice xenografted with B-cell lymphoma. These data suggest that targeting the anion exchanger AE2 with specific peptides may represent an effective therapeutic approach in B-cell malignancies

Regulatory T cells (Tregs) impair the clinical benefit of cancer immunotherapy. To optimize the antitumor efficacy of therapeutic dendritic cell (DC) vaccines, we aimed to inhibit Foxp3, a transcription factor required for Treg function. Mice bearing established syngeneic LM3 and 4T1 breast tumors were treated with antitumor DC vaccines and a synthetic peptide (P60) that has been shown to inhibit Foxp3. Treatment with P60 improved the therapeutic efficacy of DC vaccines in these experimental models. In addition, monotherapy with P60 inhibited tumor growth in immunocompetent as well as in immuno-compromised animals bearing established tumors. We found expression of Foxp3 in human and murine breast tumor cells. P60 inhibited IL-10 secretion in breast cancer cells that expressed Foxp3. Our results suggest that Foxp3 blockade improves the therapeutic efficacy of DC vaccines by inhibition of Tregs and through a direct antitumor effect. This strategy could prove useful to neutralize the immunosuppressive microenvironment and to boost antitumor immunity in breast cancer.

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ¿XIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ¿XIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-¿, TNF-¿, IL-2, IL-17 and IL-10. ¿XIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ¿XIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ¿XIII as a safe and effective live DIVA vaccine.

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.

The recent approval by the FDA of the combination of anti-CTLA4 and anti-PD-1 mAbs for the treatment of BRAF-unmutated unresectable or metastatic melanoma is a landmark for the development of cancer immunotherapy. On October 18 to 22, 2015, a symposium was held in Pamplona (Spain) to present and discuss the basic and clinical discoveries that have brought us to this milestone and to explore other targets and immunotherapy strategies aimed at attaining more efficacious oncology practice in the short term.

In this work we show a clinically feasible strategy to convert in situ the own tumor into an endogenous vaccine by coating the melanoma cancerous cells with CD28 costimulatory ligands. This therapeutic approach is aimed at targeting T-cell costimulation to chemotherapy-resistant tumors which are refractory and been considered as untreatable cancers. These tumors are usually defined by an enrichment of cancer stem cells and characterized by the higher expression of chemotherapy-resistant proteins. In this work we develop the first aptamer that targets chemotherapy-resistant tumors expressing MRP1 through a novel combinatorial peptide-cell SELEX. With the use of the MRP1 aptamer we engineer a MRP1-CD28 bivalent aptamer that is able to bind MRP1-expressing tumors and deliver the CD28 costimulatory signal to tumor-infiltrating lymphocytes. The bi-specific aptamer is able to enhance costimulation in chemotherapy-resistant tumors. Melanoma-bearing mice systemically treated with MRP1-CD28 bivalent aptamer show reduced growth, thus proving an improved mice survival.Besides, we have designed a technically feasible and translational whole-cell vaccine (Aptvax). Disaggregated cells from tumors can be directly decorated with costimulatory ligand aptamers to generate the vaccine Aptvax. CD28Aptvax made of irradiated tumor cells coated with the CD28-agonistic aptamer attached to MRP1 elicits a strong tumor- cell immune response against melanoma tumors reducing tumor growth.

Blocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immuno

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5¿×¿10(6) or 10(7) autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patients' DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10-producing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine

NY-ESO-1 TCR-engineered T cells have shown activity in solid tumors. Recent work supports their use in multiple myeloma by showing that ex vivo antigen-specific expanded T cells traffic to and persist in bone marrow, are well tolerated, and produce promising response rates when infused after stem cell transplantation.

Calcineurin (CN), a serine and threonine protein phosphatase that depends on Ca2+ and calmodulin for its activity, is the target of the immunosuppressant drugs cyclosporin A (CsA) and tacrolimus (FK506). CN dephosphorylates and activates members of the NFATc (nuclear factor of activated T cells) family of transcription factors in T cells by binding to their conserved PxIxIT motif. Upon dephosphorylation, NFATc proteins translocate to the nucleus, where they stimulate the expression of genes encoding cytokines and chemokines that are required for T cell proliferation and the immune response. We performed a pharmacophore-based virtual screening of ~5.5 million commercially available, "drug-like" compounds to identify nonpeptidic compounds that inhibited the CN-dependent activation of NFATc signaling and that could serve as potential drug candidates for immunosuppressive therapy. Of 32 compounds that mimicked the PxIxIT motif, 7 competed with NFATc for binding to CN in vitro without interfering with the phosphatase activity of CN. Furthermore, in activated human CD4+ T cells, four of the seven compounds inhibited the expression of NFATc-dependent genes, cytokine production, and cell proliferation, suggesting that these may have therapeutic potential as immunosuppressive agents.

Several studies have shown that some liver transplant recipients may tolerate immunosuppression withdrawal. Mechanisms and biomarkers of tolerance are not well known. Methods: Twenty-four LT patients with immunosuppression side-effects underwent progressive immunosuppression withdrawal. Peripheral lymphocyte populations and secretion of cytokines were analyzed at baseline and during withdrawal until tolerance (n = 15) or rejection (n = 9), as well as 3. months after tolerance achievement or rejection resolution (as follow-up). Immunological markers were compared among groups. Results: The percentages of CD3 + CD4 + cells progressively decreased in both groups. CD3 + CD8 + cells gradually increased in tolerant patients. B lymphocytes gradually decreased in tolerant and initially in non-tolerant patients, reverting at rejection. Regulatory T cells progressively increased until rejection in non-tolerants, decreasing to basal levels after renewing immunosuppression; no significant changes were found in tolerant patients. The percentages and absolute counts of natural killer cells significantly increased in both groups, being more evident in tolerant patients. The secretion of several cytokines was higher in non-tolerant patients when rejection was diagnosed. Conclusions: The greater increase of natural killer cells in tolerant patients suggests their potential role in the tolerance phenomenon

Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4+ T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-¿, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-ß. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

Background & Aims: Tremelimumab is a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), an inhibitory co-receptor that interferes with T cell activation and proliferation. The purpose of this pilot clinical trial was to test the antitumor and antiviral effect of tremelimumab in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C virus (HCV) infection; and to study the safety of its administration to cirrhotic patients. Methods: Tremelimumab at a dose of 15 mg/kg IV every 90 days was administered until tumor progression or severe toxicity. Twenty patients were assessable for toxicity and viral response and 17 were assessable for tumor response. Most patients were in the advanced stage and 43% had an altered liver function (Child-Pugh class B). Results: A good safety profile was recorded and no patient needed steroids because of severe immune-mediated adverse events. Some patients had a transient albeit intense elevation of transaminases after the first dose, but not following subsequent cycles. Partial response rate was 17.6% and disease control rate was 76.4%. Time to progression was 6.48 months (95% CI 3.95-9.14). A significant drop in viral load was observed while new emerging variants of the hypervariable region 1 of HCV replaced the predominant variants present before therapy, particularly in those patients with a more prominent drop in viral load. This antiviral effect was associated with an enhanced specific anti-HCV immune response. Conclusions: Tremelimumab safety profile and antitumor and antiviral activity, in patients with advanced HCC developed on HCV-induced liver cirrhosis, support further investigation. (C) 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- ¿ß by TLR4-expressing cells, as well as the production of TNF- ¿ by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.

Peptide vaccines derived from CD8(+) T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8(+) T-cell responses, even in the absence of CD4(+) T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8(+) T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8(+) T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8(+) T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8(+) T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8(+) T cells. Taken together, these results suggest that CD40L expression induced by potent CD8(+) T-cell epitopes can activate antitumor CD8(+) T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8(+) T-cell epitopes and bypassing the requirement for CD4(+) helper T cells in vaccination protocols.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4+, CD8+, and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range¿=¿8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P¿=¿0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P¿=¿0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n¿=¿7 for an interval¿>¿10 years and an SI¿<¿20), 100% reached tolerance; in the second group (n¿=¿10 for an interval¿>¿10 years and an SI¿>¿20 or an interval¿<¿10 years and an SI¿<¿20), 60% reached tolerance; and in the third group (n¿=¿7 for an interval¿<¿10 years and an SI¿>¿20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables¿the time from transplantation and the SI¿may help to identify these patients.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study, we investigated the capacity of lung cancer cells to activate complement and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in nonmalignant bronchial epithelial cells. Notably, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL-6, IL-10, LAG3, and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

The complement system and Toll-like receptors (TLR) are key innate defense systems which might interact synergistically on dendritic cells (DC) to reinforce adaptive immunity. In a previous work, we found that the extra domain A from fibronectin EDA (an endogenous ligand for TLR4) can favour antigen delivery to DC and induce their maturation. Given the potential of anaphylatoxins to cause inflammation and activation of myeloid cells, we hypothesized that a fusion protein between EDA, and anaphylatoxins C3a, C4a or C5a together with an antigen might improve the immunogenicity of the antigen. Naked DNA immunization with a construct expressing the fusion protein between C5a, EDA and the cytotoxic T cell epitope SIINFEKL from ovalbumin, induced strong antigen specific T cell responses. The purified recombinant fusion protein EDA¿SIINFEKL¿C5a induced activation of dendritic cells, the production of proinflammatory cytokines/chemokines and stimulated antigen presenting cell migration and NK cell activation. As compared to EDA¿SIINFEKL, the fusion protein EDA¿SIINFEKL¿C5a did not induce the production of the immunosuppressive molecules IL-10, CCL17, CCL1, CXCL12 or XCL1 by DC. Moreover, EDA¿SIINFEKL¿C5a induced strong specific T cell responses in vivo and protected mice against E.G7-OVA tumor growth more efficiently than EDA¿SIINFEKL or SIINFEKL¿C5a recombinant proteins. Our results suggest that fusion proteins containing EDA, the anaphylatoxin C5a and the antigen may serve as a suitable strategy for the development of anti-tumor or anti-viral vaccines.

Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8+ T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 57 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma.

This work shows that class II-linked humoral lack of response to an antigen can be overcome by joint immunization with the antigen and a T-helper cell determinant (TDh) well recognized by class II molecules of a non-responder individual. Thus, SJL/J mice (H-2s), which are non-responders to the S region of hepatitis B virus surface antigen (HBsAg), were rendered responders by joint immunization with a recombinant surface antigen, only composed of the S region, and a short synthetic TDh peptide well recognized by the H-2s restriction. By contrast, when this peptide is not recognized as TDh, as in B10M mice (H-2f restricted and also non-responders to the S region), no humoral response could be induced against the S region. These results have important implications for therapy and vaccination against hepatitis B virus as well as in enhancing the immunogenicity of other antigens.

The development of effective vaccines against HIV-1 infection constitutes one of the major challenges in viral immunology. One of the protein candidates in vaccination against this virus is p24, since it is a conserved HIV antigen that has cytotoxic and helper T cell epitopes as well as B cell epitopes that may jointly confer enhanced protection against infection when used in immunization-challenge approaches. In this context, the adjuvant effect of EDA (used as EDAp24 fusion protein) and poly(I:C), as agonists of TLR4 and TLR3, respectively, was assessed in p24 immunizations using a recombinant Listeria monocytogenes HIV-1 Gag proteins (Lm-Gag, where p24 is the major antigen) for challenge in mice. Immunization with EDAp24 fusion protein together with poly(I:C) adjuvant induced a specific p24 IFN-¿ production (Th1 profile) as well as protection against a Lm-Gag challenge, suggesting an additive or synergistic effect between both adjuvants. The combination of EDA (as a fusion protein with the antigen, which may favor antigen targeting to dendritic cells through TLR4) and poly(I:C) could thus be a good adjuvant candidate to enhance the immune response against HIV-1 proteins and its use may open new ways in vaccine investigations on this virus.

The Extradomain A from fibronectin (EDA) has an immunomodulatory role as fusion protein with viral and tumor antigens, but its effect when administered with bacteria has not been assessed. Here, we investigated the adjuvant effect of EDA in mice immunizations against Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis). Since lipopolysaccharide (LPS) is a major virulence factor and the LPS O-polysaccharide (O-PS) is the immunodominant antigen in serological diagnostic tests, Salmonella mutants lacking O-PS (rough mutants) represent an interesting approach for developing new vaccines and diagnostic tests to differentiate infected and vaccinated animals (DIVA tests). Here, antigenic preparations (hot-saline extracts and formalin-inactivated bacterins) from two Salmonella Enteritidis rough mutants, carrying either intact (SE Delta waaL) or deep-defective (SE Delta gal) LPS-Core, were used in combination with EDA. Biotinylated bacterins, in particular SE Delta waaL bacterin, decorated with EDAvidin (EDA and streptavidin fusion protein) improved the protection conferred by hot-saline or bacterins alone and prevented significantly the virulent infection at least to the levels of live attenuated rough mutants. These findings demonstrate the adjuvant effect of EDAvidin when administered with biotinylated bacterins from Salmonella Enteritidis lacking O-PS and the usefulness of BEDA-SE Delta waaL as non-live vaccine in the mouse model.

By triggering immunogenic cell death, some anticancer compounds, including anthracyclines and oxaliplatin, elicit tumor-specific, interferon-gamma-producing CD8(+) alpha beta T lymphocytes (Tc1 CTLs) that are pivotal for an optimal therapeutic outcome.

Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-¿B and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.

The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN-gamma], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8(+) T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.

We previously showed that recombinant extra domain A from fibronectin (EDA) purified from Escherichia coli was able to bind to toll-like receptor 4 (TLR4) and stimulate production of proinflammatory cytokines by dendritic cells. Because EDA could be used as an adjuvant for vaccine development, we aimed to express it from the tobacco plastome, a promising strategy in molecular farming. To optimize the amount of recombinant EDA (rEDA) in tobacco leaves, different downstream sequences were evaluated as potential fusion tags. Plants generated by tobacco plastid transformation accumulated rEDA at levels up to 2% of the total cellular protein (equivalent to approximately 0.3 mg/g fresh weight) when translationally fused to the first 15 amino acids of green fluorescence protein (GFP). The recombinant adjuvant could be purified from tobacco leaves using a simple procedure, involving ammonium sulfate precipitation and anion exchange chromatography. Purified protein was able to induce production of tumour necrosis factor-alpha (TNF-alpha) either by bone marrow-derived dendritic cells or THP-1 monocytes. The rEDA produced in tobacco leaves was also able to induce upregulation of CD54 and CD86 maturation markers on dendritic cells, suggesting that the rEDA retains the proinflammatory properties of the EDA produced in E. coli and thus could be used as an adjuvant in vaccination against infectious agents and cancer. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of this protein vaccine adjuvant.

Staphylococcus epidermidis releases a complex of at least four peptides, termed phenol-soluble modulins (PSM), which stimulate macrophages to produce proinflammatory cytokines via activation of TLR2 signalling pathway. We demonstrated that covalent linkage of PSM peptides to an antigen facilitate its capture by dendritic cells and, in combination with different TLR ligands, can favour the in vivo induction of strong and persistent antigen-specific immune responses. Treatment of mice grafted with HPV16-E7-expressing tumor cells (TC-1)with poly(l: C) and a peptide containing alpha Mod linked to the H-2D(b)-restricted cytotoxic T-cell epitope E7(49-57) from HPV16-E7 protein allowed complete tumor regression in 100% of the animals. Surprisingly, this immunomodulatory property of modulin-derived peptides was TLR2 independent and partially dependent upon the EGF-receptor signalling pathway. Our results suggest that alpha or gamma modulin peptides may serve as a suitable antigen carrier for the development of anti-tumoral or anti-viral vaccines. (C) 2010 Elsevier Ltd. All rights reserved.