Tumor cell p38 inhibition to overcome immunotherapy resistance.

Patients with tumors that do not respond to immune-checkpoint inhibition often harbor a non-T cell-inflamed tumor microenvironment, characterized by the absence of IFN-γ-associated CD8+ T cell and dendritic cell activation. Understanding the molecular mechanisms underlying immune exclusion in non-responding patients may enable the development of novel combination therapies. p38 MAPK is a known regulator of dendritic and myeloid cells however a tumor-intrinsic immunomodulatory role has not been previously described. Here we identify tumor cell p38 signaling as a therapeutic target to potentiate anti-tumor immunity and overcome resistance to immune-checkpoint inhibitors (ICI). Molecular analysis of tumor tissues from patients with human papillomavirus-negative head and neck squamous carcinoma reveals a p38-centered network enriched in non-T cell-inflamed tumors. Pan-cancer single-cell RNA analysis suggests that p38 activation may be an immune-exclusion mechanism across multiple tumor types. P38 knockdown in cancer cell lines increases T cell migration, and p38 inhibition plus ICI in preclinical models shows greater efficacy compared to monotherapies. In a clinical trial of patients refractory to PD1/L1 therapy, pexmetinib, a p38 inhibitor, plus nivolumab demonstrated deep and durable clinical responses. Targeting of p38 with anti-PD1 has the potential to induce the T cell-inflamed phenotype and overcome immunotherapy resistance.

PD-L1 Tumor Expression as a Predictive Biomarker of Immune Checkpoint Inhibitors' Response and Survival in Advanced Melanoma Patients in Brazil.

Immune checkpoint blockade (ICB) agents are prominent immunotherapies for the treatment of advanced melanoma. However, they fail to promote any durable clinical benefit in a large cohort of patients. This study assessed clinical and molecular predictors of ICB response and survival in advanced melanoma. A retrospective analysis was performed on 210 patients treated with PD-1 or CTLA-4 inhibitors at Barretos Cancer Hospital, Brazil. PD-L1 expression was assessed by immunohistochemistry using formalin-fixed paraffin-embedded tumor tissues collected prior to ICB therapy. Patients were divided into responders (complete and partial response and stable disease for more than 6 months) and non-responders (stable disease for less than 6 months and progressive disease). Among them, about 82% underwent anti-PD-1 immunotherapy, and 60.5% progressed after the ICB treatment. Patients that received ICB as first-line therapy showed higher response rates than previously treated patients. Higher response rates were further associated with superficial spreading melanomas and positive PD-L1 expression (>1%). Likewise, PD-L1 positive expression and BRAF V600 mutations were associated with a higher overall survival after ICB therapy. Since ICBs are expensive therapies, evaluation of PD-L1 tumor expression in melanoma patients should be routinely assessed to select patients that are most likely to respond.

Improving head and neck cancer therapies by immunomodulation of the tumour microenvironment.

Targeted immunotherapy has improved patient survival in head and neck squamous cell carcinoma (HNSCC), but less than 20% of patients produce a durable response to these treatments. Thus, new immunotherapies that consider all key players of the complex HNSCC tumour microenvironment (TME) are necessary to further enhance tumour-specific T cell responses in patients. HNSCC is an ideal tumour type in which to evaluate immune and non-immune cell differences because of two distinct TME aetiologies (human papillomavirus (HPV)-positive and HPV-negative disease), multiple anatomic sites for tumour growth, and clear distinctions between patients with locally advanced disease and those with recurrent and/or metastatic disease. Recent technological and scientific advancements have provided a more complete picture of all cellular constituents within this complex TME and have evaluated the interplay of both immune and non-immune cells within HNSCC. Here, we include a comprehensive analysis of the complete ecosystem of the HNSCC TME, performed utilizing data-rich resources such as The Cancer Genome Atlas, and cutting-edge techniques, such as single-cell RNA sequencing, high-dimensional flow cytometry and spatial multispectral imaging, to generate improved treatment strategies for this diverse disease.

An individualized causal framework for learning intercellular communication networks that define microenvironments of individual tumors.

Cells within a tumor microenvironment (TME) dynamically communicate and influence each other's cellular states through an intercellular communication network (ICN). In cancers, intercellular communications underlie immune evasion mechanisms of individual tumors. We developed an individualized causal analysis framework for discovering tumor specific ICNs. Using head and neck squamous cell carcinoma (HNSCC) tumors as a testbed, we first mined single-cell RNA-sequencing data to discover gene expression modules (GEMs) that reflect the states of transcriptomic processes within tumor and stromal single cells. By deconvoluting bulk transcriptomes of HNSCC tumors profiled by The Cancer Genome Atlas (TCGA), we estimated the activation states of these transcriptomic processes in individual tumors. Finally, we applied individualized causal network learning to discover an ICN within each tumor. Our results show that cellular states of cells in TMEs are coordinated through ICNs that enable multi-way communications among epithelial, fibroblast, endothelial, and immune cells. Further analyses of individual ICNs revealed structural patterns that were shared across subsets of tumors, leading to the discovery of 4 different subtypes of networks that underlie disparate TMEs of HNSCC. Patients with distinct TMEs exhibited significantly different clinical outcomes. Our results show that the capability of estimating individual ICNs reveals heterogeneity of ICNs and sheds light on the importance of intercellular communication in impacting disease development and progression.

Co-expression of TNF receptors 1 and 2 on melanomas facilitates soluble TNF-induced resistance to MAPK pathway inhibitors.

BACKGROUND: The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAFV600E+ melanomas and its role in solTNF-driven resistance reprogramming to MAPKi. METHODS: Flow cytometry was used to test TNFR1, TNFR2 and CD271 expression on, as well as NF-kB phosphorylation in human BRAF-mutant melanoma. The ability of melanoma cell lines to acquire MAPKi resistance in response to recombinant or macrophage-derived TNF was evaluated using the MTT cytotoxicity assay. Gene editing was implemented to knock out or knock in TNF receptors in melanoma cell lines. Knockout and knock-in cell line variants were employed to assess the intrinsic roles of these receptors in TNF-induced resistance to MAPKi. Multicolor immunofluorescence microscopy was utilized to test TNFR2 expression by melanoma in patients receiving MAPKi therapy. RESULTS: TNFR1 and TNFR2 are co-expressed at various levels on 4/7 BRAFV600E+ melanoma cell lines evaluated in this study. In vitro treatments with solTNF induce MAPKi resistance solely in TNFR2-expressing BRAFV600E+ melanoma cell lines. TNFR1 and TNFR2 knockout and knock-in studies indicate that solTNF-mediated MAPKi resistance in BRAFV600E+ melanomas is predicated on TNFR1 and TNFR2 co-expression, where TNFR1 is the central mediator of NF-kB signaling, while TNFR2 plays an auxiliary role. solTNF-mediated effects are transient and can be abrogated with biologics. Evaluation of patient specimens indicates that TNFR2 is expressed on 50% of primary BRAFV600E+ melanoma cells and that MAPKi therapy may lead to the enrichment of TNFR2-expressing tumor cells. CONCLUSIONS: Our data suggest that TNFR2 is essential to solTNF-induced MAPKi resistance and a possible biomarker to identify melanoma patients that can benefit from solTNF-targeting therapies.

Human B Cells Mediate Innate Anti-Cancer Cytotoxicity Through Concurrent Engagement of Multiple TNF Superfamily Ligands.

The essential innate immunity effector cells, natural killer and dendritic cells, express multiple plasma membrane-associated tumor necrosis factor (TNF) superfamily (TNFSF) ligands that, through simultaneous and synergistic engagement, mediate anti-cancer cytotoxicity. Here, we report that circulating B cells, mediators of adaptive humoral immunity, also mediate this innate anti-cancer immune mechanism. We show that resting human B cells isolated from peripheral blood induce apoptosis of, and efficiently kill a large variety of leukemia and solid tumor cell types. Single-cell RNA sequencing analyses indicate, and flow cytometry data confirm that B cells from circulation express transmembrane TNF, Fas ligand (FasL), lymphotoxin (LT) α1ß2 and TNF-related apoptosis-inducing ligand (TRAIL). The cytotoxic activity can be inhibited by individual and, especially, combined blockade of the four transmembrane TNFSF ligands. B cells from tumor-bearing head and neck squamous cell carcinoma patients express lower levels of TNFSF ligands and are less cytotoxic than those isolated from healthy individuals. In conclusion, we demonstrate that B cells have the innate capacity to mediate anti-cancer cytotoxicity through concurrent activity of multiple plasma membrane-associated TNFSF ligands, that this mechanism is deficient in cancer patients and that it may be part of a general cancer immunosurveillance mechanism.

Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing.

Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV- HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.

Expression of Tim-3 drives phenotypic and functional changes in Treg cells in secondary lymphoid organs and the tumor microenvironment.

Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3+ Treg cells may be a relevant therapeutic target cell type for the treatment of cancer.

Neoadjuvant nivolumab for patients with resectable HPV-positive and HPV-negative squamous cell carcinomas of the head and neck in the CheckMate 358 trial.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are common malignancies caused by carcinogens, including tobacco and alcohol, or infection with human papillomavirus (HPV). Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1) pathway are effective against unresectable recurrent/metastatic HNSCC. Here, we explored the safety and efficacy of anti-PD-1 therapy in at-risk resectable HPV-positive and HPV-negative HNSCC in the neoadjuvant setting. METHODS: The phase I/II CheckMate 358 trial in virus-associated cancers assessed neoadjuvant nivolumab in patients with previously untreated, resectable HPV-positive or HPV-negative HNSCC. Patients received nivolumab 240 mg intravenously on days 1 and 15, with surgery planned by day 29. Safety/tolerability (primary endpoint) was assessed by monitoring adverse events (AEs) and surgical delays. Radiographic response was measured before surgery using RECIST v1.1, adapted for a single post-nivolumab evaluation. Pathologic specimens were examined for treatment response using immune-based criteria. RESULTS: From November 2015 to December 2017, 52 patients with AJCC (seventh edition) stage III-IV resectable HNSCC received neoadjuvant nivolumab (26 HPV-positive, 26 HPV-negative). Any-grade treatment-related AEs (TRAEs) occurred in 19 patients (73.1%) and 14 patients (53.8%) in the HPV-positive and HPV-negative cohorts, respectively; grade 3-4 TRAEs occurred in five (19.2%) and three patients (11.5%), respectively. No patient had a protocol-defined TRAE-related surgical delay (>4 weeks). Thirty-eight patients were reported as undergoing complete surgical resection, 10 had a planned post-nivolumab biopsy instead of definitive surgery due to a protocol misinterpretation, and four did not undergo surgery or biopsy, including two with tumor progression. Radiographic response rates in 49 evaluable patients were 12.0% and 8.3% in the HPV-positive and HPV-negative cohorts, respectively. There were no complete pathologic responses by site or central review in operated patients. Among 17 centrally evaluable HPV-positive tumors, one (5.9%) achieved major pathological response and three (17.6%) achieved partial pathologic response (pPR); among 17 centrally evaluable HPV-negative tumors, one (5.9%) achieved pPR. CONCLUSIONS: Neoadjuvant nivolumab was generally safe and induced pathologic regressions in HPV-positive (23.5%) and HPV-negative (5.9%) tumors. Combinatorial neoadjuvant treatment regimens, and continued postoperative therapy for high-risk tumors, are warranted in future trials to enhance the efficacy of this approach. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02488759; https://clinicaltrials.gov/ct2/show/NCT02488759.

Impact of checkpoint blockade on cancer vaccine-activated CD8+ T cell responses.

Immune and molecular profiling of CD8 T cells of patients receiving DC vaccines expressing three full-length melanoma antigens (MAs) was performed. Antigen expression levels in DCs had no significant impact on T cell or clinical responses. Patients who received checkpoint blockade before DC vaccination had higher baseline MA-specific CD8 T cell responses but no evidence for improved functional responses to the vaccine. Patients who showed the best clinical responses had low PD-1 expression on MA-specific T cells before and after DC vaccination; however, blockade of PD-1 during antigen presentation by DC had minimal functional impact on PD-1high MA-specific T cells. Gene and protein expression analyses in lymphocytes and tumor samples identified critical immunoregulatory pathways, including CTLA-4 and PD-1. High immune checkpoint gene expression networks correlated with inferior clinical outcomes. Soluble serum PD-L2 showed suggestive positive association with improved outcome. These findings show that checkpoint molecular pathways are critical for vaccine outcomes and suggest specific sequencing of vaccine combinations.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells.

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes. Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells. Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Multiple antigen-engineered DC vaccines with or without IFNα to promote antitumor immunity in melanoma.

BACKGROUND: Cancer vaccines are designed to promote systemic antitumor immunity and tumor eradication. Cancer vaccination may be more efficacious in combination with additional interventions that may build on or amplify their effects. METHODS: Based on our previous clinical and in vitro studies, we designed an antigen-engineered DC vaccine trial to promote a polyclonal CD8+ and CD4+ T cell response against three shared melanoma antigens. The 35 vaccine recipients were then randomized to receive one month of high-dose IFNα or observation. RESULTS: The resulting clinical outcomes were 2 partial responses, 8 stable disease and 14 progressive disease among patients with measurable disease using RECIST 1.1, and, of 11 surgically treated patients with no evidence of disease (NED), 4 remain NED at a median follow-up of 3 years. The majority of vaccinated patients showed an increase in vaccine antigen-specific CD8+ and CD4+ T cell responses. The addition of IFNα did not appear to improve immune or clinical responses in this trial. Examination of the DC vaccine profiles showed that IL-12p70 secretion did not correlate with immune or clinical responses. In depth immune biomarker studies support the importance of circulating Treg and MDSC for development of antigen-specific T cell responses, and of circulating CD8+ and CD4+ T cell subsets in clinical responses. CONCLUSIONS: DC vaccines are a safe and reliable platform for promoting antitumor immunity. This combination with one month of high dose IFNα did not improve outcomes. Immune biomarker analysis in the blood identified several predictive and prognostic biomarkers for further analysis, including MDSC. TRIAL REGISTRATION: NCT01622933 .

Melanoma vaccines: clinical status and immune endpoints.

It has been known for decades that the immune system can be spontaneously activated against melanoma. The presence of tumor infiltrating lymphocytes in tumor deposits is a positive prognostic factor. Cancer vaccination includes approaches to generate, amplify, or skew antitumor immunity. To accomplish this goal, tested approaches involve administration of tumor antigens, antigen presenting cells or other immune modulators, or direct modulation of the tumor. Because the success of checkpoint blockade can depend in part on an existing antitumor response, cancer vaccination may play an important role in future combination therapies. In this review, we discuss a variety of melanoma vaccine approaches and methods to determine the biological impact of vaccination.

CD56dim CD16- Natural Killer Cell Profiling in Melanoma Patients Receiving a Cancer Vaccine and Interferon-α.

Natural killer (NK) cells are innate cytotoxic and immunoregulatory lymphocytes that have a central role in anti-tumor immunity and play a critical role in mediating cellular immunity in advanced cancer immunotherapies, such as dendritic cell (DC) vaccines. Our group recently tested a novel recombinant adenovirus-transduced autologous DC-based vaccine that simultaneously induces T cell responses against three melanoma-associated antigens for advanced melanoma patients. Here, we examine the impact of this vaccine as well as the subsequent systemic delivery of high-dose interferon-α2b (HDI) on the circulatory NK cell profile in melanoma patients. At baseline, patient NK cells, particularly those isolated from high-risk patients with no measurable disease, showed altered distribution of CD56dim CD16+ and CD56dim CD16- NK cell subsets, as well as elevated serum levels of immune suppressive MICA, TN5E/CD73 and tactile/CD96, and perforin. Surprisingly, patient NK cells displayed a higher level of activation than those from healthy donors as measured by elevated CD69, NKp44 and CCR7 levels, and enhanced K562 killing. Elevated cytolytic ability strongly correlated with increased representation of CD56dim CD16+ NK cells and amplified CD69 expression on CD56dim CD16+ NK cells. While intradermal DC immunizations did not significantly impact circulatory NK cell activation and distribution profiles, subsequent HDI injections enhanced CD56bright CD16- NK cell numbers when compared to patients that did not receive HDI. Phenotypic analysis of tumor-infiltrating NK cells showed that CD56dim CD16- NK cells are the dominant subset in melanoma tumors. NanoString transcriptomic analysis of melanomas resected at baseline indicated that there was a trend of increased CD56dim NK cell gene signature expression in patients with better clinical response. These data indicate that melanoma patient blood NK cells display elevated activation levels, that intra-dermal DC immunizations did not effectively promote systemic NK cell responses, that systemic HDI administration can modulate NK cell subset distributions and suggest that CD56dim CD16- NK cells are a unique non-cytolytic subset in melanoma patients that may associate with better patient outcome.

ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker.

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed.

Tumor-Derived α-Fetoprotein Directly Drives Human Natural Killer-Cell Activation and Subsequent Cell Death.

Hepatocellular carcinoma (HCC) patients with reduced natural killer (NK)-cell numbers and function have been shown to have a poor disease outcome. Mechanisms underlying NK-cell deficiency and dysfunction in HCC patients remain largely unresolved. α-Fetoprotein (AFP) is an oncofetal antigen produced by HCC. Previous studies demonstrated that tumor-derived AFP (tAFP) can indirectly impair NK-cell activity by suppressing dendritic cell function. However, a direct tAFP effect on NK cells remains unexplored. The purpose of this study was to examine the ability of cord blood-derived AFP (nAFP) and that of tAFP to directly modulate human NK-cell activity and longevity in vitro Short-term exposure to tAFP and, especially, nAFP proteins induced a unique proinflammatory, IL2-hyperresponsive phenotype in NK cells as measured by IL1ß, IL6, and TNF secretion, CD69 upregulation, and enhanced tumor cell killing. In contrast, extended coculture with tAFP, but not nAFP, negatively affected long-term NK-cell viability. NK-cell activation was directly mediated by the AFP protein itself, whereas their viability was affected by hydrophilic components within the low molecular mass cargo that copurified with tAFP. Identification of the distinct impact of circulating tAFP on NK-cell function and viability may be crucial to developing a strategy to ameliorate HCC patient NK-cell functional deficits. Cancer Immunol Res; 5(6); 493-502. ©2017 AACR.

Inhibition of Soluble Tumor Necrosis Factor Prevents Chemically Induced Carcinogenesis in Mice.

TNF is a potent promoter of carcinogenesis and potentially important target for cancer prevention. TNF is produced as functionally distinct transmembrane and soluble molecules (tmTNF and sTNF, respectively), but their individual roles in carcinogenesis are unexplored. We investigated the participation of tmTNF and sTNF in chemically induced carcinogenesis in mice. We found that injection of XPro1595, a dominant-negative TNF biologic (DN-TNF) and specific antagonist of sTNF, decreased tumor incidence and growth, and prolonged survival of 3-methylcholanthrene (MCA)-injected mice. Similar results were obtained following the exclusion of both TNF forms by either TNF-receptor 2-Fc fusion protein (TNFR2-Fc) treatment or TNF gene deletion. In addition, gene deletion of TNFR1, which is preferentially triggered by sTNF, was temporarily blocked, whereas gene deletion of TNFR2, which is preferentially triggered by tmTNF, enhanced MCA-induced carcinogenesis. Concomitantly with carcinogenesis induction, MCA increased circulating IL1α, accumulation of myeloid-derived suppressor cells (MDSC), STAT3 phosphorylation, and immunosuppression in the spleen. In sharp contrast, DN-TNF treatment dramatically decreased IL1α and increased the essential immunoregulatory cytokines IL1ß, IL12p70, and IL17 in the peripheral blood of MCA-injected mice. In addition, MDSC accumulation, STAT3 phosphorylation, and immunosuppression in MCA-injected mice were prevented by DN-TNF treatment, TNFR2-Fc treatment, and/or gene deletion of TNF or TNFR1, but not deletion of TNFR2. These findings reveal that sTNF is both an essential promoter of carcinogenesis and a pivotal regulator of MDSCs, and indicate that sTNF could be a significant target for cancer prevention and therapy. Cancer Immunol Res; 4(5); 441-51. ©2016 AACR.

Route of antigen delivery impacts the immunostimulatory activity of dendritic cell-based vaccines for hepatocellular carcinoma.

BACKGROUND: Dendritic cells (DC) are uniquely equipped to capture, process, and present antigens from their environment. The context in which an antigen is acquired by DC helps to dictate the subsequent immune response. Cancer vaccination promotes antitumor immunity by directing an immune response to antigens expressed by tumors. We have tested the tumor-associated antigen alpha-fetoprotein (AFP) as an immunotherapy target. The majority of hepatocellular carcinomas (HCC) upregulate and secrete this oncofetal antigen. METHODS: To develop cancer vaccines for HCC capable of promoting potent tumor-specific T cell responses, we tested adenovirally-encoded synthetic AFP, with or without its signal sequence, as well as protein forms of AFP and compared intracellular routing and subsequent antigen-specific CD8+ and CD4+ T cell responses. RESULTS: Surprisingly, the secreted form of antigen was superior for both CD4+ and CD8+ T cell activation. We also examined the mechanism through which AFP protein is endocytosed and trafficked in human DC. We identify the mannose receptor (MR/CD206) as the primary uptake pathway for both normal cord blood-derived AFP (nAFP) and tumor-derived AFP (tAFP) proteins. While in healthy donors, nAFP and tAFP were cross-presented to CD8+ T cells similarly and CD4+ T cell responses were dependent upon MR-mediated uptake. In HCC patient cells, tAFP was more immunogenic, and CD4+ T cell responses were not MR-dependent. CONCLUSIONS: Secreted, cytoplasmically retained, and endocytosed forms of AFP utilize unique uptake and processing pathways, resulting in different immunologic responses from the induced antigen-specific CD4+ and CD8+ T cells and between healthy donors and HCC patients. Collectively, these data elucidate pathways of spontaneous and induced anti-tumor immunity in HCC patients to this secreted antigen.

Molecular mimicry of MAGE-A6 and Mycoplasma penetrans HF-2 epitopes in the induction of antitumor CD8+ T-cell responses.

A promising vaccine strategy for the treatment of cancer involves the use of vaccines incorporating tumor antigen-derived synthetic peptides that can be coordinately recognized by specific CD4+ and CD8+ T-cells. Previously, we reported that a MAGE-A6-derived peptide (MAGE-A6172-187) and its highly-immunogenic and cross-reactive homolog derived from Mycoplasma penetrans HF-2 permease (HF-2216-229) are promiscuously presented by multiple HLA-DR alleles to responder CD4+ T-cells obtained from healthy donors and melanoma patients. Here, we investigated whether these same peptides could concomitantly stimulate cross-reactive MAGE-A6-specific CD8+ T-cell responses in vitro using cells isolated from HLA-A*0201 (HLA-A2)+ healthy individuals and patients with melanoma. We now show that MAGE-A6172-187 and, even more so, HF-2216-229, induce memory CD8+ T cells that recognize HLA-A2+ MAGE-A6+ tumor target cells. The immunogenicity of these peptides was at least partially attributed to their embedded MAGE-A6176-185 and HF-2220-229 "homologous" sequences. The functional avidity of HF-2216-229 peptide-primed CD8+ T cells for the MAGE-A6172-187 peptide was more than 100-fold greater than that of CD8+ T cells primed with the corresponding MAGE-A6 peptide. Additionally, these 2 peptides were recognized in interferon γ (IFNγ) and granzyme B ELISPOT assays by CD8+ T-cell clones displaying variable T-cell receptor (TCR) Vß usage. These data suggest that the immune cross-reactivity of the MAGE-A6172-187 and HF-2216-229 peptides extends to CD8+ T cells, at least in HLA-A2+ donors, and supports the potential translational utility of these epitopes in clinical vaccine formulations and for immunomonitoring of cancer patients.