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Background Various proinflammatory cytokines can be detected within the melanoma tumor microenvironment. Interleukin 32 (IL32) is produced by T cells, NK cells and monocytes/macrophages, but also by a subset of melanoma cells. We sought to better understand the biology of IL32 in human melanoma. Methods We analyzed RNA sequencing data from 53 in-house established human melanoma cell lines and 479 melanoma tumors from The Cancer Genome Atlas dataset. We evaluated global gene expression patterns associated with IL32 expression. We also evaluated the impact of proinflammatory molecules TNFα and IFNγ on IL32 expression and dedifferentiation in melanoma cell lines in vitro. In order to study the transcriptional regulation of IL32 in these cell lines, we cloned up to 10.5 kb of the 5′ upstream region of the human IL32 gene into a luciferase reporter vector. Results A significant proportion of established human melanoma cell lines express IL32, with its expression being highly correlated with a dedifferentiation genetic signature (high AXL/low MITF). Non IL32-expressing differentiated melanoma cell lines exposed to TNFα or IFNγ can be induced to express the three predominant isoforms (α, β, γ) of IL32. Cis -acting elements within this 5′ upstream region of the human IL32 gene appear to govern both induced and constitutive gene expression. In the tumor microenvironment, IL32 expression is highly correlated with genes related to T cell infiltration, and also positively correlates with high AXL/low MITF dedifferentiated gene signature. Conclusions Expression of IL32 in human melanoma can be induced by TNFα or IFNγ and correlates with a treatment-resistant dedifferentiated genetic signature. Constitutive and induced expression are regulated, in part, by cis -acting sequences within the 5′ upstream region.

Tumor antigen-reactive T cells must enter into an immunosuppressive tumor microenvironment, continue to produce cytokine and deliver apoptotic death signals to affect tumor regression. Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation and cytotoxicity. In a murine model of adoptive cell therapy, we demonstrate that transgenic Pmel-1 CD8 T cells, rendered insensitive to TGFβ by transduction with a TGFβ dominant negative receptor II (DN), were more effective in mediating regression of established B16 melanoma. Smaller numbers of DN Pmel-1 T cells effectively mediated tumor regression and retained the ability to produce interferon-γ in the tumor microenvironment. These results support efforts to incorporate this DN receptor in clinical trials of adoptive cell therapy for cancer.

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34+ HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8+ and CD4+ T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4–5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8+ and CD4+ T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34+ HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.

Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3 EGFP mice. OT-II Foxp3 EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3 EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor β (TGFβ). B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNgamma-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80–100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A*0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.

Background Alpha fetoprotein (AFP) is an oncofetal antigen over-expressed by many hepatocellular cancers (HCC). We previously demonstrated that HLA-A2-restricted epitopes derived from AFP are immunogenic in vitro and in vivo despite high circulating levels of this oncofetal antigen. In order to test a more broadly applicable, HLA-unrestricted, inexpensive, cell-free vaccine platform capable of activating tumor antigen-specific CD8 + and CD4 + T cells, we tested full length AFP in a plasmid DNA construct in combination with an AFP-expressing replication-deficient adenovirus (AdV) in a prime-boost vaccine strategy. Methods HCC patients who had an AFP + tumor and previous treatment for HCC were screened and two patients received vaccination with three plasmid DNA injections followed by a single AdV injection, all delivered intramuscularly (i.m.). Results The vaccine was well tolerated and safe. Both patients showed immunologic evidence of immunization. The first patient had a weak AFP-specific T cell response, a strong AdV-specific cellular response and recurred with an AFP-expressing HCC at nine months. The second patient developed a strong AFP-specific CD8 + and CD4 + cellular response and an AdV neutralizing antibody response, and recurred at 18 months without an increase in serum AFP. Conclusions The AFP DNA prime-AdV boost vaccine was safe and immunogenic. Circulating anti-AdV neutralizing antibodies at baseline did not prohibit the development of AFP-specific cellular immunity. The patient who developed CD8 + and CD4 + AFP-specific T cell immunity had more favorable progression-free survival. The observations with these two patients support development of this vaccine strategy in a larger clinical trial. Trial registration ClinicalTrials.gov: NCT00093548

Tumor antigen gene-modified dendritic cells (DC) generates robust antigen-specific protective antitumor responses. Though the role of CD4 positive and CD8 positive cells in the immunological response to gene-modified DC has been well-characterized, the role of NK cells in this response has been somewhat less clear. Owing to the significant contribution of innate immunity in other model systems, we postulated that NK cells would hold a critical position in the generation of an immune response following immunization with tumor antigen-engineered DC. Immunization with MART-1 melanoma antigen-engineered DC in C57BL/6 mice resulted in the generation of antigen-specific cytotoxic T lymphocytes and in vivo protective responses to the murine B16 melanoma. These responses were dependent on the presence of functional NK cells, although NK cells alone were not sufficient in generating protective responses. Adoptive transfer of NK cells into an NK-deficient but T-cell-competent environment restored the protective response to gene-modified DC immunization. In conclusion, protective immunity after tumor antigen gene-modified DC immunization requires collaboration between CD4+ and CD8+ T cells and NK cells.

Patients with malignant gliomas continue to have very poor prognosis even after surgical resection, radiation and chemotherapy. Because these tumors often have alterations in the p53 tumor suppressor gene, which plays a key role in the cellular response to DNA damaging agents, we investigated the role of p53 gene therapy in conjunction with ionizing radiation in a rat brain tumor model. Exposure of cultured rat 9L gliosarcoma cells, which contain a mutant p53 gene, to a recombinant adenovirus-vector bearing the wild-type p53 gene (Adp53), induced apoptosis within 24 hours. Although ionizing radiation had no additional effect on apoptosis within this time frame, it caused G1 arrest in non-apoptotic cells after Adp53 therapy. In contrast, wild-type 9L cells demonstrated little G1 arrest after X-irradiation. When animals bearing brain tumors were irradiated after intratumoral Adp53 injections, more than 85% reduction in tumor size was noted. Moreover, the group of rats receiving both radiation and Adp53 therapy had a significant increase in survival as compared to animals receiving either therapy alone. These results support the use of p53 gene therapy as an adjunct to radiation in treatment of malignant brain tumors.