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Dendritic cells (DCs) are professional antigen presenting cells that play an important role in the induction of T cell responses. Different subsets (cDC1s, cDC2s, pDCs, and moDCs) were described based on the expression of different surface markers and functions. In the context of peritoneum, DCs are also a key population cell orchestrating immune responses against pathogens, malignant cells and tissue-damage. Furthermore, they play an important role in the promotion of an anti-inflammatory microenvironment, which is necessary to maintain tolerance and adipocyte homeostasis. The aim of this review is to summarize the current knowledge of the functional and phenotypic features of peritoneal DCs and shed some light on the importance of these cells within this unique cavity and its associated components: the omentum, the mesentery and gut-associated lymphoid tissue (GALT).

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.

BackgroundNeoantigens, new immunogenic sequences arising from tumor mutations, have been associated with response to immunotherapy and are considered potential targets for vaccination. Hepatocellular carcinoma (HCC) is a moderately mutated tumor, where the neoantigen repertoire has not been investigated. Our aim was to analyze whether tumors in HCC patients contain immunogenic neoantigens suitable for future use in therapeutic vaccination.MethodsWhole-exome sequencing and RNAseq were performed in a cohort of fourteen HCC patients submitted to surgery or liver transplant. To identify mutations, single-nucleotide variants (SNV) originating non-synonymous changes that were confirmed at the RNA level were analyzed. Immunogenicity of putative neoAgs predicted by HLA binding algorithms was confirmed by using in vitro HLA binding assays and T-cell stimulation experiments, the latter in vivo, by immunizing HLA-A*02.01/HLA-DRB1*01 (HHD-DR1) transgenic mice, and in in vitro, using human lymphocytes.ResultsSequencing led to the identification of a median of 1217 missense somatic SNV per patient, narrowed to 30 when filtering by using RNAseq data. A median of 13 and 5 peptides per patient were predicted as potential binders to HLA class I and class II molecules, respectively. Considering only HLA-A*02.01- and HLA-DRB1*01-predicted binders, 70% demonstrated HLA-binding capacity and about 50% were immunogenic when tested in HHD-DR1 mice. These peptides induced polyfunctional T cells that specifically recognized the mutated but not the wild-type sequence as well as neoantigen-expressing cells. Moreover, coimmunization experiments combining CD8 and CD4 neoantigen epitopes resulted in stronger CD8 T cell responses. Finally, responses against neoantigens were also induced in vitro using human cells.ConclusionThese results show that mutations in HCC tumors may generate immunogenic neoantigens with potential applicability for future combinatorial therapeutic strategies.

Immune checkpoint inhibitors (ICI) have been used as immunotherapy for hepatocellular carcinoma (HCC) with promising but still limited results. Identification of immune elements in the tumor microenvironment of individual HCC patients may help to understand the correlations of responses, as well as to design personalized therapies for non-responder patients. Immune-enhancing strategies, such as vaccination, would complement ICI in those individuals with poorly infiltrated tumors. The prominent role of responses against mutated tumor antigens (neoAgs) in ICI-based therapies suggests that boosting responses against these epitopes may specifically target tumor cells. In this review we summarize clinical vaccination trials carried out in HCC, the available information on potentially immunogenic neoAgs in HCC patients, and the most recent results of neoAg-based vaccines in other tumors. Despite the low/intermediate mutational burden observed in HCC, data obtained from neoAg-based vaccines in other tumors indicate that vaccines directed against these tumor-specific antigens would complement ICI in a subset of HCC patients.

BackgroundHepatocellular carcinoma (HCC) is a major global health concern, causing over 700,000 deaths annually [1]. For decades, treatment for advanced HCC was restricted to broad-acting tyrosine kinase inhibitors that conferred limited survival benefits [2]. Recently, immunotherapies have revolutionized treatment of advanced HCC with objective response rates of around 30% [3]. However, with 70% of patients remaining insensitive, there is an urgent need to identify biomarkers of resistance and response and to design novel combination therapies that restore sensitivity in the resistant patients.MethodsTo understand mechanisms of HCC immune evasion, we performed transcriptomic analysis on a collection of immune-escaped murine tumors harboring MYC overexpression and loss of p53 (MYC;p53-/-) [4]. The immune-escaped tumors could be categorized into ”immune-inflamed” and ”immune-desert” through single sample gene set enrichment analysis. We further identified an enrichment signature of Notch signaling in immune-inflamed tumors that was associated with a specific deficit of CD8+ T cells – a result validated in HCC patient samples. To test the role of Notch in immune escape, we generated a novel murine model of HCC based on MYC overexpression, activated Notch1 Intracellular Domain (MYC;NICD1), and customized expression of tumor antigens.ResultsExpression of tumor antigens in MYC;p53-/- mice led to enhanced survival and active immune surveillance/tumor clearance by antigen-specific CD8+ T cells compared to control MYC;p53-/- mice without tumor antigens. However, this survival advantage conferred by tumor antigen expression was abrogated in MYC;NICD1 mice, demonstrating that Notch activation drives immune evasion in HCC in the presence of antigen expression. Mechanistically, Notch activation in the liver led to a reduced number of tumor antigen-specific CD8+ T cells within the tumor microenvironment compared to the MYC;p53-/- mice while the presence of dendritic cells was similar. Adoptive transfer experiments have revealed a potential impairment of CD8+ T cell priming and activation. Further, Notch1-driven tumors were resistant to anti-PD1 monotherapy as well as anti-PDL1/anti-VEGFR2 and anti-PDL1/anti-TGFB1 combination therapies, indicating a multifaceted immune resistance mechanism mediated in part by VEGF and TGFB1. Resistance to anti-PDL1/anti-VEGF combination therapy has been confirmed in HCC patients.ConclusionsTogether, Notch activation promotes a multifaceted immune escape program in HCC. Additionally, it promotes resistance to currently approved immunotherapies, establishing the Notch pathway as a putative biomarker for HCC patient stratification.AcknowledgementsThis study was funded by Damon Runyon-Rachleff Innovator Award and NCI-NIH R37 Award. KEL was supported by 5T32AI078892-12.ReferencesBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394–424.Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008 Jul 24;359(4):378–90.Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, Kudo M, Breder V, Merle P, Kaseb AO, Li D, Verret W, Xu DZ, Hernandez S, Liu J, Huang C, Mulla S, Wang Y, Lim HY, Zhu AX, Cheng AL; IMbrave150 Investigators. Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma. N Engl J Med. 2020 May 14;382(20):1894–1905.Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P, Lindblad KE, Maier B, Sia D, Puigvehi M, Miguela V, Casanova-Acebes M, Dhainaut M, Villacorta-Martin C, Singhi AD, Moghe A, von Felden J, Tal Grinspan L, Wang S, Kamphorst AO, Monga SP, Brown BD, Villanueva A, Llovet JM, Merad M, Lujambio A. ß-Catenin Activation Promotes Immune Escape and Resistance to Anti-PD-1 Therapy in Hepatocellular Carcinoma. Cancer Discov. 2019 Aug;9(8):1124–1141.Ethics ApprovalAll mouse experiments were approved by the ISMMS Animal Care and Use Committee (protocol number IACUC-2014–0229).

PURPOSE: BN-CV301 is a poxviral-based vaccine comprised of recombinant (rec.) Modified vaccinia Ankara (MVA-BN-CV301; prime) and rec. fowlpox (FPV-CV301; boost). Like its predecessor PANVAC, BN-CV301 contains transgenes encoding tumor-associated antigens, MUC1 and CEA, as well as costimulatory molecules (B7.1, ICAM-1, LFA-3). PANVAC was re-engineered to make it safer and more antigenic. EXPERIMENTAL DESIGN: This open-label, 3+3 design, dose-escalation trial evaluated three dose levels (DL) of MVA-BN-CV301: one, two or four subcutaneous injections of 4×10(8) infectious units (Inf.U)/0.5 mL on weeks 0 and 4. All patients received FPV-CV301 subcutaneously at 1×10(9)Inf.U/0.5 mL every 2 weeks for 4 doses, then every 4 weeks. Clinical and immune responses were evaluated. RESULTS: There were no dose limiting toxicities. Twelve patients enrolled on trial (DL1=3, DL2=3, DL3=6). Most side effects were seen with the prime doses and lessened with subsequent boosters. All treatment-related adverse events were temporary, self-limiting, grade ½, and included injection site reactions and flu-like symptoms. Antigen-specific T-cells to MUC1 and CEA, as well as to a cascade antigen, brachyury, were generated in most patients. Single agent BN-CV301 produced a confirmed partial response (PR) in one patient and prolonged stable disease (SD) in multiple patients, most notably in KRAS mutant gastrointestinal tumors. Furthermore, two patients with KRAS mutant colorectal cancer had prolonged SD when treated with an anti-PD-L1 antibody following BN-CV301. CONCLUSIONS: The BN-CV301 vaccine can be safely administered to patients with advanced cancer. Further studies of the vaccine in combination with other agents are planned.

Simple Summary 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. Abstract 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 linked to CIRP (OVA-CIRP), with or without ICPI, and antigen-specific responses and therapeutic efficacy were tested in subcutaneous and orthotopic mouse models of liver cancer. OVA-CIRP elicited polyepitopic T-cell responses, which were further enhanced when combined with ICPI (anti-PD-1 and anti-CTLA-4). Combination of OVA-CIRP with ICPI enhanced ICPI-induced therapeutic responses when tested in subcutaneous and intrahepatic B16-OVA tumors, as well as in the orthotopic PM299L HCC model. This effect was associated with higher OVA-specific T-cell responses in the periphery, although many tumor-infiltrating lymphocytes still displayed an exhausted phenotype. Finally, a new vaccine containing human glypican-3 linked to CIRP (GPC3-CIRP) induced clear responses in humanized HLA-A2.01 transgenic mice, which increased upon combination with ICPI. Therefore, CIRP-based vaccines may generate anti-tumor immunity to enhance ICPI efficacy in HCC, although blockade of additional checkpoint molecules and immunosuppressive targets should be also considered.