Hypomethylating agent alters the immune microenvironment in acute myeloid leukaemia (AML) and enhances the immunogenicity of a dendritic cell/AML vaccine.

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.

Expansion of cancer germline antigen-specific cytotoxic T lymphocytes for immunotherapy.

The cancer germline antigens MAGE-A1, MAGE-A3, and NY-ESO-1 can be used to target relapsed or therapy-resistant malignant solid tumors, and previous studies have demonstrated that these antigens can be epigenetically upregulated on the surface of tumor cells following exposure to low-dose demethylating chemotherapy agents, such as decitabine. The extent to which cancer germline antigen cytotoxic T lymphocytes can be reliably expanded from healthy donors has not been well characterized, specifically in terms of whether these T cells consistently kill antigen-bearing targets or simply produce interferon-γ in the presence of the antigen. Cancer germline antigen cytotoxic T lymphocytes were generated using conventional method and high-density lymphocyte culture method. We demonstrate that there is no difference in the extent of antigen-specific killing with or without CD25 depletion when interleukin-21 is added to the cultures. Cancer germline antigen-specific killer cells could be expanded from 8/12 healthy donors using overlapping peptide mixes derived from MAGE-A1, MAGE-A3, and NY-ESO-1 and from 7/9 healthy donors using HLA-restricted epitopes. Furthermore, cytotoxic T lymphocyte derived from 4/5 patients displayed specific cytotoxicity of target cells expressing respective cancer germline antigen and HLA partially matched tumor lines. High-density lymphocyte culture prior to stimulation with cancer germline antigen peptides resulted in antigen-specific cytotoxic T lymphocyte from healthy donors and patients from whom cancer germline antigen cytotoxic T lymphocyte culture with conventional methods was not feasible. These data demonstrate that MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific T cells with antigen-specific cytotoxicity can be cultured from healthy donors and patient-derived cells making adoptive immunotherapy with these cytotoxic T lymphocyte feasible.

The DNA methylation inhibitor 5-azacytidine increases regulatory T cells and alleviates airway inflammation in ovalbumin-sensitized mice.

BACKGROUND: Asthma is characterized as a chronic inflammatory disorder of the airways associated with an enhanced TH2 response to inhaled allergens. CD4+ T regulatory (Treg) cells are controlled by the master transcription factor FoxP3 and strictly maintain peripheral immunotolerance. Epigenetic regulation of FoxP3 by DNA methyltransferase inhibitors, such as 5-azacytidine (Aza), can generate a steady supply of functional Treg cells. Therefore, we propose that Aza can augment Treg cells in vivo to prevent the pathogenesis of asthma. METHODS: BALB/c mice were sensitized with chicken ovalbumin (OVA) and treated with different doses of Aza. Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid, circulating titers of OVA-specific IgG1 and IgE, and stimulating levels of TH2 cytokines from splenocytes were then determined. Cellular populations were examined by flow cytometry. PC61 antibody, which depletes CD25+ cells, was used to verify the role of CD25+ cells in Aza-induced tolerance. RESULTS: Administration of Aza to OVA-sensitized mice diminished airway hyperreactivity, pulmonary eosinophilia, levels of OVA-specific IgG1 and IgE in serum, and secretion of TH2 cytokines from OVA-stimulated splenocytes in a dose-dependent manner. Percentages of CD25+ and FoxP3+ cells in the CD4+ cell population were notably increased in Aza-treated mice compared to sensitized control mice. Furthermore, the major symptoms of asthma were exacerbated by depleting CD25+ cells in Aza-treated mice. CONCLUSIONS: Aza may have applications as a novel clinical strategy to increase the production of Treg cells in order to modulate the airway inflammation associated with asthma.

The DNA demethylating agent 5-aza-2'-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma.

Cancer/testis antigens (CTAs) are considered to be suitable targets for the immunotherapy of human malignancies. It has been demonstrated that in a variety of tumors, the expression of certain CTAs is activated via the demethylation of their promoter CpG islands. In our study, we have shown that while the composite expression of 13 CTAs in 30 human glioma specimens and newly established cell lines from the Japanese population was nearly imperceptible, the DNA-demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) markedly reactivated CTA expression in glioma cells but not in normal human cells. We quantified the diminished methylation status of NY-ESO-1-one of the most immunogenic CTAs-following 5-aza-CdR treatment by using a novel Pyrosequencing technology and methylation-specific PCR. Microarray analysis revealed that 5-aza-CdR is capable of signaling the immune system, particularly, human leukocyte antigen (HLA) class I upregulation. (51)Cr-release cytotoxicity assays and cold target inhibition assays using NY-ESO-1-specific cytotoxic T lymphocyte (CTL) lines demonstrated the presentation of de novo NY-ESO-1 antigenic peptides on the cell surfaces. In an orthotopic xenograft model, the systemic administration of 5-aza-CdR resulted in a significant volume reduction of the transplanted tumors and prolonged the survival of the animals after the adoptive transfer of NY-ESO-1-specific CTLs. These results suggested that 5-aza-CdR induces the expression of epigenetically silenced CTAs in poorly immunogenic gliomas and thereby presents a new strategy for tumor immunotherapy targeting 5-aza-CdR-induced CTAs.

HER-2, gp100, and MAGE-1 are expressed in human glioblastoma and recognized by cytotoxic T cells.

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-gamma treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.

Complex relationships between 5-aza-dC induced DNA demethylation and chromosome compaction at mitosis.

A variety of treatments with 5-azadeoxycytidine (5-aza-dC) were applied to cultured human lymphocytes during one to four cell cycles. The effect of 5-aza-dC on DNA methylation was studied by using an antibody against 5-methylcytosine on mitotic chromosomes. 5-Azadeoxycytidine is known to induce strong and permanent demethylation of DNA. Unexpectedly complex relationships were observed between DNA methylation status and chromatid/chromosome compaction. The most dramatic alteration of compaction at mitosis was observed when pre-replicative chromosomes had unifilarly demethylated DNA. The compaction of chromosomes was found to depend only partially on the methylation of their DNA at the time of mitosis. Our results suggest that alteration of DNA methylation prevents the synchronization of chromatin compaction, inducing premature (or delayed) chromosome condensation, and that a crucial step is the DNA methylation status of the pre-replicative chromosome.

Experimental chemotherapy (L1210) with 5-aza-2'-deoxycytidine in combination with pyran copolymer (MVE-4), an immune adjuvant.

The life-span of CDF1 (BALB/c X DBA/2)F1 mice that received intraperitoneal implants with 10(5) L1210 tumor cells was prolonged to 23 days (compared to 8 days in L1210 tumor-implanted, untreated mice) when 5-aza-2'-deoxycytidine (DAC) was given to the mice after the tumor cells were allowed to metastasize (3 days after implant); DAC, however, resulted in no cures (survival beyond 48 days). When the pyran copolymer MVE-4, an immune adjuvant, was given the day after DAC, 25% of the mice treated were cured and the life-span of dying mice was increased by 7 days. When MVE-4 was repeated weekly for 4 weeks, 79% of treated mice were cured. Cured mice were able to resist a subsequent challenge of approximately 2 logs of L1210 cells. This combination of DAC plus MVE-4 was more effective than DAC alone only if the tumor cells and MVE-4 were given intraperitoneally. When this combination was repeated weekly, it became lethally toxic after 3 weeks, but only to L1210-tumor-bearing mice and not to normal mice. When DAC alone was given 2 days before tumor implant, it induced an apparent immune effect so that mice could resist a subsequent challenge of approximately 1.5-2 logs of L1210 cells. Support for part of the antitumor action of DAC exerted through the immune system was given by data that show that later treatment with noncurative doses of DAC is superior to early treatment in mice with large L1210 tumor burdens.