ABSTRACT
The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.
Subject(s)
Adenoviridae/genetics , Cancer Vaccines/administration & dosage , Genetic Therapy/methods , Immunotherapy/methods , Oligodeoxyribonucleotides/administration & dosage , Thymoma/therapy , Adenoviridae/immunology , Adjuvants, Immunologic/administration & dosage , Animals , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Combined Modality Therapy , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Oligodeoxyribonucleotides/immunology , T-Lymphocytes, Regulatory/immunology , Thymoma/drug therapy , Thymoma/immunologyABSTRACT
We set out to develop a PSA peptide-loaded tetramer for enumeration of PSA-specific CD8(+) T cells in the Balb/c mouse model. A candidate major histocompatibility complex (MHC) class I PSA peptide (HPQKVTKFML(188-197)) was selected on the basis of its ability to restimulate PSA-specific CD8(+) T cells to secrete interferon-γ in our assays. Next, H-2L(d)-restricted peptide-loaded and fluorescently labeled tetramers were produced in conjunction with the NIH Tetramer Core Facility, Atlanta, GA, USA. This tetramer was then tested for staining specificity and optimized for detection of PSA-specific CD8(+) T cells induced by our PSA-encoding adenovirus tumor vaccine. The MHC class I PSA peptide demonstrated successful restimulation of CD8(+) T cells isolated from mice previously vaccinated with a PSA-encoding adenovirus tumor vaccine, with no restimulation observed in control-vaccinated mice. The peptide-loaded H-2L(d) tetramer exhibited the desired binding specificity and allowed for detection and frequency determination of PSA-specific CD8(+) T cells by flow cytometry. We have successfully designed and validated a PSA peptide tetramer for use in the Balb/c mouse model that can be used to test PSA-based prostate cancer vaccines. Until now, PSA-specific CD8(+) T cells in the mouse have only been detectable via cytotoxic T-lymphocyte assays or intracellular cytokine staining, which primarily assess antigen-specific functional activity and not their absolute number. This research tool provides laboratories the ability to directly quantitate CD8(+) T cells elicited by PSA-specific immunotherapies and cancer vaccines that are tested in mouse models.
