Spontaneous immune responses against glioma-associated antigens in a long term survivor with malignant glioma

<p>Abstract</p> <p>Background</p> <p>In patients with high grade glioma, little is known regarding existence of naturally occurring adaptive T cell reactivity against glioma-associated antigens (GAAs). In this report, we characterized GAA-specific CD8⁺T cells and innate immune cells in a patient who has survived with anaplastic astrocytoma (AA) for over 12 years without recurrence.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells (PBMCs) derived from the long term survivor with AA were evaluated for the frequency, cytotoxic T lymphocyte (CTL) activity and differentiation status of CD8⁺cells recognizing GAA-derived epitopes as well as relative numbers of other immune cell subsets. This patient's AA tissue was evaluated for expression of two GAAs EphA2 and interleukin-13 receptor α2 subunit (IL-13Rα2) by immunohistochemistry.</p> <p>Results</p> <p>The patient's tumor expressed both EphA2 and IL-13Rα2, and <it>in vitro </it>stimulated PBMC demonstrated superior EphA2_883–891and IL-13Rα2_345–353-specific CTL reactivity compared to PBMC samples from two other patients with progressing malignant glioma. Unstimulated EphA2_883–891-reactive CD8⁺T cells contained high numbers of CD45RA^-/CCR7^-late effector and CD45RA^-/CCR7⁺central memory cells. Among other leukocyte subsets, elevated numbers of NK-T cells were found.</p> <p>Conclusion</p> <p>To our knowledge, the current study is one of the first demonstrating the presence of antigen-experienced, GAA-reactive CD8⁺T cells in a patient who has survived with AA for over 12 years without recurrence. Further studies are warranted to determine whether the status of GAA-reactive CD8⁺T cells dictates survival of patients and/or response to therapeutic vaccines.</p

Differential activity of interferon-α8 promoter is regulated by Oct-1 and a SNP that dictates prognosis of glioma

We have previously reported that the single nucleotide polymorphism (SNP) rs12553612 in IFNA8 is associated with better overall survival of glioma patients with the AA-genotype compared with patients with the AC-genotype. As rs12553612 is located in the IFNA8 promoter, we hypothesized that the A-allele allows for an enhanced IFNA8 promoter activity compared with the C-allele. Reporter assays in the human monocyte derived THP-1 cell line demonstrated a superior promoter activity of the A-allele compared with the C-allele. Electrophoretic mobility shift assays (EMSA) further demonstrated that the A-genotype specifically binds to more nuclear proteins than the C-genotype, including the transcription factor Oct-1. Further, co-transfection of plasmids encoding Oct-1 and the reporter constructs revealed that Oct-1 enhanced the promoter activity with the A- but not the C-allele. Taken together, our data demonstrate that the A-allele in the rs12553612 SNP, which is associated with better glioma patient survival, allows for IFNA8 transcription by allowing for Oct-1 binding, which is absent in patients with C allele, and suggests a molecular mechanism of IFNA8 mediated immune-surveillance of glioma progression

Toll like receptor-3 ligand poly-ICLC promotes the efficacy of peripheral vaccinations with tumor antigen-derived peptide epitopes in murine CNS tumor models

BACKGROUND: Toll-like receptor (TLR)3 ligands serve as natural inducers of pro-inflammatory cytokines capable of promoting Type-1 adaptive immunity, and TLR3 is abundantly expressed by cells within the central nervous system (CNS). To improve the efficacy of vaccine strategies directed against CNS tumors, we evaluated whether administration of a TLR3 ligand, polyinosinic-polycytidylic (poly-IC) stabilized with poly-lysine and carboxymethylcellulose (poly-ICLC) would enhance the anti-CNS tumor effectiveness of tumor peptide-based vaccinations. METHODS: C57BL/6 mice bearing syngeneic CNS GL261 glioma or M05 melanoma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes- mEphA2 (671–679), hgp100 (25–33) and mTRP-2 (180–188) for GL261, or ovalbumin (OVA: 257–264) for M05. The mice also received intramuscular (i.m.) injections with poly-ICLC. RESULTS: The combination of subcutaneous (s.c.) peptide-based vaccination and i.m. poly-ICLC administration promoted systemic induction of antigen (Ag)-specific Type-1 CTLs expressing very late activation antigen (VLA)-4, which confers efficient CNS-tumor homing of vaccine-induced CTLs based on experiments with monoclonal antibody (mAb)-mediated blockade of VLA-4. In addition, the combination treatment allowed expression of IFN-γ by CNS tumor-infiltrating CTLs, and improved the survival of tumor bearing mice in the absence of detectable autoimmunity. CONCLUSION: These data suggest that poly-ICLC, which has been previously evaluated in clinical trials, can be effectively combined with tumor Ag-specific vaccine strategies, thereby providing a greater index of therapeutic efficacy

miR-17-92 expression in differentiated T cells - implications for cancer immunotherapy

<p>Abstract</p> <p>Background</p> <p>Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated.</p> <p>Methods</p> <p>We performed miR microarray analyses on <it>in vitro </it>differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells.</p> <p>Results</p> <p>Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4⁺T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-γ production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD).</p> <p>Conclusion</p> <p>The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy.</p

Bioinformatics Analyses Determined the Distinct CNS and Peripheral Surrogate Biomarker Candidates Between Two Mouse Models for Progressive Multiple Sclerosis

Previously, we have established two distinct progressive multiple sclerosis (MS) models by induction of experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) in two mouse strains. A.SW mice develop ataxia with antibody deposition, but no T cell infiltration, in the central nervous system (CNS), while SJL/J mice develop paralysis with CNS T cell infiltration. In this study, we determined biomarkers contributing to the homogeneity and heterogeneity of two models. Using the CNS and spleen microarray transcriptome and cytokine data, we conducted computational analyses. We identified up-regulation of immune-related genes, including immunoglobulins, in the CNS of both models. Pro-inflammatory cytokines, interferon (IFN)-γ and interleukin (IL)-17, were associated with the disease progression in SJL/J mice, while the expression of both cytokines was detected only at the EAE onset in A.SW mice. Principal component analysis (PCA) of CNS transcriptome data demonstrated that down-regulation of prolactin may reflect disease progression. Pattern matching analysis of spleen transcriptome with CNS PCA identified 333 splenic surrogate markers, including Stfa2l1, which reflected the changes in the CNS. Among them, we found that two genes (PER1/MIR6883 and FKBP5) and one gene (SLC16A1/MCT1) were also significantly up-regulated and down-regulated, respectively, in human MS peripheral blood, using data mining