Alexander Gelbard

IFN-α Enhances Peptide Vaccine-Induced CD8+ T Cell Numbers, Effector Function, and Antitumor Activity

Type I IFNs, including IFN-α, enhance Ag presentation and promote the expansion, survival, and effector function of CD8+ CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-α as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8+ T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp10025–33 peptide in IFA. IFN-α synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-α dramatically increased the accumulation of gp100-specific, IFN-γ-secreting, CD8+ T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8+ T cells. IFN-α treatment also greatly increased the long-term maintenance of pmel-1 CD8+ T cells with an effector memory phenotype, a process that required expression of IFN-α receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-α as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-α therapy for melanoma.

Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model. Experimental Design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N6-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy. Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity. Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors. Clin Cancer Res; 16(6); 1834–44

Combination Chemotherapy and Radiation of Human Squamous Cell Carcinoma of the Head and Neck Augments CTL-Mediated Lysis

Purpose: The combination of systemic multiagent chemotherapy (5-fluorouracil + cisplatin) and tumor irradiation is standard of care for head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been shown that sublethal doses of radiation or chemotherapeutic drugs in diverse cancer types may alter the phenotype or biology of neoplastic cells, making them more susceptible to CTL-mediated cytotoxicity. However, little is known about the potential synergistic effect of drug plus radiation on CTL killing. Here, we examined whether the combination of two chemotherapeutics and ionizing radiation enhanced CTL-mediated destruction of HNSCC more so than either modality separately, as well as the basis for the enhanced tumor cell lysis. Experimental Design: Several HNSCC cell lines with distinct biological features were treated with sublethal doses of cisplatin and 5-fluorouracil for 24 hours and with 10-Gy irradiation. Seventy-two hours postirradiation, tumor cells were exposed to an antigen-specific CD8+ CTL directed against carcinoembryonic antigen or MUC-1. Results: In three of three tumor cell lines tested, enhanced CTL activity was observed when the two modalities (chemotherapy and radiation) were combined as compared with target cells exposed to either modality separately. CTL-mediated lysis was MHC restricted and antigen specific and occurred almost entirely via the perforin pathway. Moreover, the combination treatment regimen led to a 50% reduction in Bcl-2 expression whereas single modality treatment had little bearing on the expression of this antiapoptotic gene. Conclusions: Overall, these results reveal that (a) CTL killing can be enhanced by combining multiagent chemotherapy and radiation and (b) combination treatment enhanced or sensitized HNSCC to the perforin pathway, perhaps by down-regulating Bcl-2 expression. These studies thus form the rational basis for clinical trials of immunotherapy concomitant with the current standard of care of HNSCC.

Nanoparticle formulated alpha-galactosylceramide activates NKT cells without inducing anergy

Activation of innate immunity is critical for vaccine development and immunotherapy, through triggering antigen specific immune responses. Natural killer T (NKT) cells are a unique type of innate immune cells which exert potent anti-viral and anti-metastasis function, through producing interferon-gamma and activating dendritic cells to present tumor antigens to CD8 T cells. alpha-Galactosylceramide, a synthetic antigen for NKT cells, is an adjuvant for protein antigens which can induce protective immunity against cancer and viral diseases, and has been proven to be safe and immune stimulatory in human cancer and hepatitis patients. Current existing problem for alpha-galactosylceramide is its induction of anergy of NKT cells, due to the non-selective presentation of alpha-galactosylceramide antigen by B cells. We hypothesized that nanoparticle formulated alpha-galactosylceramide may be selectively presented by dendritic cells and macrophages, but not B cells, thus avoiding anergy induction in NKT cells. We have prepared poly-lactic acid based nanoparticles conjugated with alpha-galactosylceramide, examined their stimulation of NKT cells in vitro and in vivo in mice, and showed that nanoparticle formulated alpha-galactosylceramide stimulates NKT cells. In contrast to soluble alpha-galactosylceramide, which caused NKT anergy after single stimulation, nanoparticle formulated alpha-galactosylceramide repeatedly stimulates NKT cells without inducing anergy. Mechanistic studies showed that nanoparticle formulated alpha-galactosylceramide is efficiently presented by mouse CD11c+population containing dendritic cells, and CD11b+population containing macrophages, but very poorly by B220+population containing B cells. Hence, nanoparticle formulated alpha-galactosylceramide is an attractive immunomodulator for immunotherapy and vaccine development. Future studies will be focused on its application as adjuvant for protein and/or peptide antigens.

An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells

An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly ‘purified’ cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

Inhibition of p-STAT3 Enhances IFN-α Efficacy against Metastatic Melanoma in a Murine Model

Purpose: Melanoma is a common and deadly tumor that upon metastasis to the central nervous system has a median survival duration of <6 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma malignancy, including immune suppression in melanoma patients. We hypothesized that WP1193, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of IFN-α against metastatic melanoma. Experimental Design: Combinational therapy of STAT3 blockade agents with IFN-α was investigated in a metastatic and an established syngeneic intracerebral murine tumor model of melanoma. The immunologic in vivo mechanisms of efficacy were investigated by T-cell and natural killer (NK) cell cytotoxic assays. Results: IFN-α immunotherapy was synergistic with WP1193 showing marked in vivo efficacy against metastatic and established intracerebral melanoma. At autopsy, it was noted that there was a decreased trend in mice with melanoma developing leptomeningeal disease treated with combinational therapy. The combinational approach enhanced both NK-mediated and T-cell–mediated antitumor cytotoxicity. Conclusions: The immune modulatory effects of STAT3 blockade can enhance the therapeutic efficacy of IFN-α immunotherapy by enhancing both innate and adaptive cytotoxic T-cell activities. This combination therapy has the potential in the treatment of metastatic melanoma that is typically refractory to this type of immune therapeutic approach. Clin Cancer Res; 16(9); 2550–61. ©2010 AACR.

Causes and consequences of adult laryngotracheal stenosis

Laryngotracheal stenosis (LTS) is largely considered a structural entity, defined on anatomic terms (i.e., percent stenosis, distance from vocal folds, overall length). This has significant implications for identifying at‐risk populations, devising systems‐based preventive strategies, and promoting patient‐centered treatment. The present study was undertaken to test the hypothesis that LTS is heterogeneous with regard to etiology, natural history, and clinical outcome.

Use of radiolabeled monoclonal antibody to enhance vaccine-mediated antitumor effects

Radiolabeled monoclonal antibodies (mAb) have demonstrated measurable antitumor effects in hematologic malignancies. This outcome has been more difficult to achieve for solid tumors due, for the most part, to difficulties in delivering sufficient quantities of mAb to the tumor mass. Previous studies have shown that nonlytic levels of external beam radiation can render tumor cells more susceptible to T cell-mediated killing. The goal of these studies was to determine if the selective delivery of a radiolabeled mAb to tumors would modulate tumor cell phenotype so as to enhance vaccine-mediated T-cell killing. Here, mice transgenic for human carcinoembryonic antigen (CEA) were transplanted with a CEA expressing murine carcinoma cell line. Radioimmunotherapy consisted of yttrium-90 (Y-90)-labeled anti-CEA mAb, used either alone or in combination with vaccine therapy. A single dose of Y-90-labeled anti-CEA mAb, in combination with vaccine therapy, resulted in a statistically significant increase in survival in tumor-bearing mice over vaccine or mAb alone; this was shown to be mediated by engagement of the Fas/Fas ligand pathway. Mice receiving the combination therapy also showed a significant increase in the percentage of viable tumor-infiltrating CEA-specific CD8+ T cells compared to vaccine alone. Mice cured of tumors demonstrated an antigen cascade resulting in CD4+ and CD8+ T-cell responses not only for CEA, but for p53 and gp70. These results show that systemic radiotherapy in the form of radiolabeled mAb, in combination with vaccine, promotes effective antitumor response, which may have implications in the design of future clinical trials.

Cell-intrinsic role for IFN-α–STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

Plasmacytoid dendritic cells (pDCs) reside in bone marrrow and lymphoid organs in homeostatic conditions and typically secrete abundant quantities of type I interferons (IFNs) on Toll-like receptor triggering. Recently, a pDC population was identified within Peyer patches (PPs) of the gut that is distinguished by its lack of IFN production; however, the relationship of PP pDCs to pDCs in other organs has been unclear. We report that PP pDCs are derived from common DC progenitors and accumulate in response to Fms-like tyrosine kinase 3 ligand, yet appear divergent in transcription factor profile and surface marker phenotype, including reduced E2-2 and CCR9 expression. Type I IFN signaling via STAT1 has a cell-autonomous role in accrual of PP pDCs in vivo. Moreover, IFN-α enhances pDC generation from DC progenitors by a STAT1-dependent mechanism. pDCs that have been developed in the presence of IFN-α resemble PP pDCs, produce inflammatory cytokines, stimulate Th17 cell generation, and fail to secrete IFN-α on Toll-like receptor engagement. These results indicate that IFN-α influences the development and function of pDCs by inducing emergence of an inflammatory (Th17-inducing) antigen-presenting subset, and simultaneously regulating accumulation of pDCs in the intestinal microenvironment.

Antitumor Activity Mediated by CpG: The Route of Administration is Critical

Unmethylated CpG oligodeoxynucleotides (CpG) are synthetic toll-like receptor 9 agonists that activate innate immune cells and which have been tested as an immune therapy in a number of cancer clinical trials. Although some antitumor immune responses have been reported, so far the majority of studies have failed to show significant clinical responses to CpG. Here we showed that the route of administration is critical to the antitumor activity of CpG. Although intravenous (i.v.) injection of CpG was capable of inducing the activation and expansion of tumor antigen-specific T cells, most of these activated T cells failed to migrate to tumor sites. By contrast, intratumoral (i.t.) injection of CpG led to extensive tumor infiltration of antigen-specific T cells and subsequent tumor suppression. We further showed that very high levels of inflammatory chemokines [regulated upon activation, normal T-cell expressed, and secreted (RANTES), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, monocyte chemotactic protein (MCP5), macrophage inflammatory proteins (MIP1&agr;, and MIP1&bgr;)] were induced in the tumor microenvironment after i.t. CpG injection, compared with administration by the i.v. route. It is interesting to note that, in vivo depletion of plasmacytoid dendritic cells greatly reduced the levels of chemokines induced; also, T-cell accumulation and antitumor effect were impaired. We also showed that i.t. but not i.v. CpG injection induced a broad antigen-specific T-cell response against tumor-derived antigens. Collectively, our data provides evidence that the route of CpG administration is a critical factor in mediating antitumor activity. By inducing localized inflammatory signals at tumor sites, i.t. CpG effectively promotes the migration, activation and function of immune cells, ultimately leading to improved tumor control.