Promoting Type-1 CD4+ T Cell Immune Responses Against Tumor-Associated Antigen MAGE-A6

One of the main challenges facing tumor immunologists is to develop strategies that would effectively stimulate Type-1 anti-tumor T cell responses, which have been correlated with better clinical outcome and prolonged survival of cancer patients. As CD4+ T cells were shown to play a critical role in mediating these responses, it was of interest to examine novel ways of effectively stimulating and enhancing Type-1 CD4+ T cell responses. For these studies I used MAGE-A6, a tumor associated antigen (TAA) expressed by a broad range of human cancer types. Two novel MAGE-A6 T-helper epitopes were identified and were shown to be recognized by CD4+ T cells isolated from the majority of normal donors or patients with melanoma, regardless of their HLA genotype (i.e. poly-DR presented epitopes). Furthermore, peptide-specific T cells also recognized autologous monocytes pulsed with recombinant MAGE-A6 protein, supporting the natural processing and MHC presentation of these epitopes. Interestingly, one of the novel MAGE-A6 epitopes possesses a high-degree of homology with a microbial peptide. CD4+ T cells stimulated in vitro with this microbial peptide cross-reacted against the MAGE-A6 homologue peptide, and could recognize naturally-processed MAGE-A6 epitopes more effectively than T cells stimulated with MAGE-A6 peptides. This study showed that it is possible to stimulate, and even enhance tumor-specific T cell responses using microbial epitopes that are homologous to TAA-derived peptides. In the final study, human dendritic cells (DC) were engineered to secrete high levels of IFN-ƒ×-inducing cytokines IL-12p70 and IL-18 via recombinant adenoviral infection to generate an in vitro stimulus capable of promoting previously deficient patient Th1-type responses. DC engineered to secrete both of these cytokines simultaneously (DC.IL-12/18) were highly effective at stimulating MAGE-A6-specific Th1-type CD4+ T cell responses from patients with melanoma, particularly when loaded with MAGE-A6 protein. Poly-DR presented epitopes and MAGE-A6 protein defined in this thesis, if loaded onto DC.IL-12/18, could prove clinically useful as a vaccine modality capable of promoting the recovery and/or enhancement of tumor antigen-specific, Th1-type CD4+ T cell responses in the majority of patients harboring MAGE-A6+ cancers

Alpha fetoprotein directly induces a unique pro-inflammatory, IL-2 hyperresponsive phenotype in human natural killer cells

Alpha fetoprotein (AFP) is an oncofetal antigen commonly produced by hepatocellular carcinomas (HCC). Previous studies have shown that tumor-derived AFP (tAFP) has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cell frequency and function have partially been attributed to tAFP-mediated immunosuppression of DC function. However, a direct tAFP effect on NK cells remains unclear. Here we examine the ability of cord blood-derived AFP (nAFP) and tAFP to modulate human NK cell activity in vitro. We show that exposure to tAFP or, especially, nAFP proteins induces a unique pro-inflammatory NK cell activation profile as measured by CD69 upregulation, IL-1β and IL-6 secretion, and enhanced tumor cell killing. Interestingly, AFP-treated plus interleukin-2 (IL-2) stimulated cultures promote a degree of NK cell activation that is higher than that of NK cells activated with IL-2 alone by both phenotypic and functional measures, including elevated IFN-γ and GM-CSF secretion. To confirm that the observed effects are directly mediated by AFP protein, we confirmed that NK cells can readily bind to and take up nAFP and tAFP. The observed synergism between AFP and IL-2 may be mediated by the ability of AFP to modulate IL-2 receptor signaling, as shown by the ability of AFP to upregulate CD25, and downregulate CD122 and CD132 on NK cells. Overall, these data show that nAFP and tAFP induce a unique pro-inflammatory, IL-2 hyperresponive phenotype in NK cells. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients, and for the development of an effective HCC-targeting immunotherapy

Tumor-derived alpha fetoprotein directly impacts human natural killer cell activity and viability

Alpha-fetoprotein (AFP) is an oncofetal antigen produced by hepatocellular carcinomas (HCC). Previous studies demonstrated that tumor-derived AFP (tAFP) is a glycoprotein that has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cells have been attributed to tAFP-mediated immunosuppression of DC. However, a direct tAFP effect on NK cells remains unexplored. Here we compared the ability of cord blood-derived AFP (nAFP) to that of tAFP to modulate human NK cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique pro-inflammatory, IL-2 hyperresponsive phenotype in healthy donor NK cells as measured by CD69 upregulation, IL-1β, IL-6 and TNF secretion, and enhanced tumor cell killing. In contrast, extended co-culture with tAFP, but not nAFP, inhibited NK cell proliferation and viability. NK cell activation was directly mediated by the AFP protein itself, while their viability was affected by the low molecular mass cargo that co-purified with tAFP. Overall, these data show that nAFP and tAFP induce similar yet distinct changes in NK cell function and viability, respectively. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients

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.National Institutes of Health (U.S.) (R01 CA96504

Editorial: Cancer Immunotherapies: From Efficacy to Resistance Mechanisms

International audienceNo abstract availabl