Lymph node-targeted immunotherapy mediates potent immunity resulting in regression of isolated or metastatic human papillomavirus-transformed tumors.

PURPOSE: The goal of this study was to investigate the therapeutic potential of a novel immunotherapy strategy resulting in immunity to localized or metastatic human papillomavirus 16-transformed murine tumors. EXPERIMENTAL DESIGN: Animals bearing E7-expressing tumors were coimmunized by lymph node injection with E7 49-57 antigen and TLR3-ligand (synthetic dsRNA). Immune responses were measured by flow cytometry and antitumor efficacy was evaluated by tumor size and survival. In situ cytotoxicity assays and identification of tumor-infiltrating lymphocytes and T regulatory cells were used to assess the mechanisms of treatment resistance in bulky disease. Chemotherapy with cyclophosphamide was explored to augment immunotherapy in late-stage disease. RESULTS: In therapeutic and prophylactic settings, immunization resulted in a considerable expansion of E7 49-57 antigen-specific T lymphocytes in the range of 1/10 CD8(+) T cells. The resulting immunity was effective in suppressing disease progression and mortality in a pulmonary metastatic disease model. Therapeutic immunization resulted in control of isolated tumors up to a certain volume, and correlated with antitumor immune responses measured in blood. In situ analysis showed that within bulky tumors, T-cell function was affected by negative regulatory mechanisms linked to an increase in T regulatory cells and could be overcome by cyclophosphamide treatment in conjunction with immunization. CONCLUSIONS: This study highlights a novel cancer immunotherapy platform with potential for translatability to the clinic and suggests its potential usefulness for controlling metastatic disease, solid tumors of limited size, or larger tumors when combined with cytotoxic agents that reduce the number of tumor-infiltrating T regulatory cells.

TLR-9 signaling and TCR stimulation co-regulate CD8(+) T cell-associated PD-1 expression.

Elevated Programmed Death-1 (PD-1) expression can inhibit T cell activity and is a potential barrier to achieving persisting and optimal immunity via therapeutic vaccination. Using a direct lymph node-targeted vaccination procedure that enabled uncoupling of synthetic peptide (signal 1, TCR-mediated) and adjuvant (signal 2, non-TCR-mediated), we evaluated the impact of varied doses of Toll-like receptor (TLR)-9 ligand CpG oligodeoxynucleotide (ODN) adjuvant on epitope-specific CD8(+) T cell-associated PD-1 expression. Peptide vaccination without adjuvant yielded CD8(+) T cells with significantly elevated PD-1 expression. This conferred impaired function ex vivo, but was reversible by antibody-mediated PD-1 blockade. By comparison, peptide vaccination with escalating doses of CpG ODN adjuvant yielded higher magnitudes of CD8(+) T cells with progressively lower PD-1 expression and greater ex vivo function. CpG ODN adjuvant in context of titrated peptide doses for vaccination yielded the lowest overall PD-1 expression levels, demonstrating that fine-tuning both TCR-independent (adjuvant dose) and -dependent (antigen dose) stimuli can synergize to co-regulate PD-1 expression on epitope-specific CD8(+) T cells. These data hint at strategies to elicit PD-1(low) CD8(+) T cells using TLR-9 ligand adjuvants, and also shed light on the PD-1-regulated homeostasis of CD8(+) T cells.

Enhancing DNA vaccination by sequential injection of lymph nodes with plasmid vectors and peptides.

DNA vaccines or peptides are capable of inducing specific immunity; however, their translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein, we demonstrate that a novel immunization strategy, encompassing sequential exposure of the lymph node milieu to plasmid and peptide in a heterologous prime-boost fashion, results in considerable MHC class I-restricted immunity in mice. Plasmid-primed antigen expression was essential for the generation of a population of central memory T cells, expressing CD62L and low in PD-1, with substantial capability to expand and differentiate to peripheral memory and effector cells, following subsequent exposure to peptide. These vaccine-induced T cells dominated the T cell repertoire, were able to produce large amounts of chemokines and pro-inflammatory cytokines, and recognized tumor cells effectively. In addition to outlining a feasible and effective method to transform plasmid DNA vaccination into a potentially viable immunotherapeutic approach for cancer, this study sheds light on the mechanism of heterologous prime-boost and the considerable heterogeneity of MHC class I-restricted T cell responses.