Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory T cells.

BACKGROUND: Autophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles (Defective ribosomal products in blebs), were excellent sources of antigens for efficient cross priming of tumor-specific CD8⁺ T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported. METHODS: DRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus (CMV) pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus (EBV), and Influenza (Flu) epitopes (CEF) were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4⁺ and CD8⁺ memory T cells was detected by intracellular staining. The effect of cytokines (GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ) TLR agonists (Lipopolysaccharide, Polyinosinic-polycytidylic acid (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored. RESULTS: In this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8⁺ memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8⁺ T cell responses. CONCLUSIONS: DRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with Poly(I:C), CD40 ligand, and GM-CSF. This study provides proof-of-concept for applying this strategy to activate memory T cells against other antigens, including tumor-specific T cells ex vivo for immunological monitoring and adoptive immunotherapy, and in vivo as vaccines for patients with cancer.

Maturation of dendritic cell precursors into IL12-producing DCs by J-LEAPS immunogens.

LEAPS (ligand epitope antigen presentation system) vaccines consist of a peptide containing a major histocompatibility antigen binding peptide conjugated to an immune cell binding ligand (ICBL) such as the 'J' peptide from beta-2-microglobulin. Treatment of monocytes, monocytes plus GMCSF, or monocytes plus GMCSF and IL4 with JgD (containing a peptide from gD of herpes simplex virus type 1) or JH (with a peptide from HIV p17 gag protein) was sufficient to promote their maturation into Interleukin 12 producing dendritic cells. JgD-dendritic cells supported allotypic activation of T cells to produce Th1-related cytokines.

STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells.

The clinical outcomes of dendritic cell (DC)-based immunotherapy remain disappointing, with DCs often displaying a tenuous capacity to complete maturation and DC1 polarization in the tumor host. Surprisingly, we observed that the capacity for successful DC1 polarization, including robust IL12p70 production, could be regulated by STAT-dependent events even prior to DC differentiation. Exposure of CD34(pos) cells to single-agent granulocyte-macrophage colony-stimulating factor (GMCSF) induced multilineage, STAT5-dependent differentiation, including DCs that failed to mature in the absence of further exogenous signals. In contrast, Flt3L induced nearly global differentiation of CD34(pos) cells into spontaneously maturing DCs. IL-6 synergized with Flt3L to produce explosive, STAT3-dependent proliferation of phenotypically undifferentiated cells that nevertheless functioned as committed DC1 precursors. Such precursors not only resisted many tumor-associated immunosuppressants, but also responded to tumor contact or TGFbeta with facilitated DC maturation and IL12p70 production, and displayed a superior capacity to reverse tumor-induced T-cell tolerance. GMCSF preempted Flt3L or Flt3L plus IL-6 licensing by blocking STAT3 activation and promoting STAT5-dependent differentiation. Paradoxically, following overt DC differentiation, STAT5 enhanced whereas STAT3 inhibited DC1 polarization. Therefore, nonoverlapping, sequential activation of STAT3 and STAT5, achievable by sequenced exposure to Flt3L plus IL-6, then GMCSF, selects for multilog expansion, programming, and DC1 polarization of tumor-competent DCs from CD34(pos) cells.

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer.

The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous - single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85-22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.

A pilot study of an autologous tumor-derived autophagosome vaccine with docetaxel in patients with stage IV non-small cell lung cancer.

BACKGROUND: Tumor-derived autophagosome vaccines (DRibbles) have the potential to broaden immune response to poorly immunogenic tumors. METHODS: Autologous vaccine generated from tumor cells harvested from pleural effusions was administered to patients with advanced NSCLC with the objectives of assessing safety and immune response. Four patients were vaccinated and evaluable for immune response; each received two to four doses of vaccine. Study therapy included two cycles of docetaxel 75 mg/m2 on days 1 and 29 to treat the tumor, release hidden antigens and produce lymphopenia. DRibbles were to be administered intradermally on days 14, 43, 57, 71, and 85, together with GM-CSF (50 µg/d x 6d, administered via SQ mini pump). Peripheral blood was tested for immune parameters at baseline and at each vaccination. RESULTS: Three of four patients had tumor cells available for testing. Autologous tumor-specific immune response was seen in two of the three, manifested by IL-5 (1 patient after 3 doses), and IFN-γ, TNF-α, IL-5, IL-10 (after 4 doses in one patient). All 4 patients had evidence of specific antibody responses against potential tumor antigens. All patients came off study after 4 or fewer vaccine treatments due to progression of disease. No significant immune toxicities were seen during the course of the study. CONCLUSIONS: DRibble vaccine given with GM-CSF appeared safe and capable of inducing an immune response against tumor cells in this small, pilot study. There was no evidence of efficacy in this small poor-prognosis patient population, with treatment not feasible. Trial registration NCT00850785, initial registration date February 23, 2009.

Extracellular ATP and Toll-like receptor 2 agonists trigger in human monocytes an activation program that favors T helper 17.

Strategically-paired Toll-like receptor (TLR) ligands induce a unique dendritic cell (DC) phenotype that polarizes Th1 responses. We therefore investigated pairing single TLR ligands with a non TLR-mediated danger signal to cooperatively induce distinct DC properties from cultured human monocytes. Adenosine triphosphate (ATP) and the TLR2 ligand lipoteichoic acid (LTA) selectively and synergistically induced expression of IL-23 and IL-1ß from cultured monocytes as determined by ELISA assays. Flow cytometric analysis revealed that a sizable sub-population of treated cells acquired DC-like properties including activated surface phenotype with trans-well assays showing enhanced migration towards CCR7 ligands. Such activated cells also preferentially deviated, in an IL-23 and IL-1-dependent manner, CD4(pos) T lymphocyte responses toward the IL-22(hi), IL-17(hi)/IFN-γ(lo) Th17 phenotype in standard in vitro allogeneic sensitization assays. Although pharmacological activation of either ionotropic or cAMP-dependent pathways acted in synergy with LTA to enhance IL-23, only inhibition of the cAMP-dependent pathway antagonized ATP-enhanced cytokine production. ATP plus atypical lipopolysaccharide from P. gingivalis (signaling through TLR2) was slightly superior to E. coli-derived LPS (TLR4 ligand) for inducing the high IL-23-secreting DC-like phenotype, but greatly inferior for inducing IL-12 p70 production when paired with IFN-γ, a distinction reflected in activated DCs' ability to deviate lymphocytes toward Th1. Collectively, our data suggest TLR2 ligands encountered by innate immune cells in an environment with physiologically-relevant levels of extracellular ATP can induce a distinct activation state favoring IL-23- and IL-1ß-dependent Th17 type response.

Effect of multiple activation stimuli on the generation of Th1-polarizing dendritic cells.

It was originally reported that only a small fraction of total matured dendritic cells (DCs) produced interleukin (IL)-12, but it has never been determined whether different combinations of activating signals now shown to maximize secreted IL-12 do so through increasing output by the same IL-12 producers, or by recruiting additional cytokine-secreting cells. We therefore tested all combinations of bacterial lipopolysaccharide (LPS) (TLR4 ligand), R848 (TLR8 ligand), interferon (IFN)-γ, and CD40L for activating human monocyte-derived dendritic cells (DC), and determined by intracellular flow cytometry that enhanced IL-12 secretion was accomplished in large part by markedly increasing the proportion of cells producing IL-12, with the triple and quadruple combinations recruiting the most DC. This optimization requirement for multiple signals was not reflected in differential Toll-like receptor (TLR) expression by the cells. Interestingly, DCs activated with single TLR ligands plus IFN-γ were capable of responding with a second burst of IL-12 upon later CD40L stimulation, whereas DCs activated with R848 plus LPS were not, despite the trend of the latter for superior polarization of naive T cells toward IFN-γ-secreting Th1. These results have implications for the biology of IL-12-secreting DCs and choice of activation regimen for prospective use in DC-based immunotherapy.

Paired Toll-like receptor agonists enhance vaccine therapy through induction of interleukin-12.

Minimal requirements for generating effective immunity include the delivery of antigenic (signal 1) and costimulatory (signal 2) signals to T lymphocytes. Recently, a class of third signals, often delivered by antigen-presenting dendritic cells, has been shown to greatly enhance immune responses, especially against tumors. Among signal 3 factors, interleukin (IL)-12 is particularly effective and can be conditionally induced by agonists of Toll-like transmembrane receptors (TLR). In this study, we assessed the therapeutic effect of adjuvant TLR agonist administration upon the capacity of dendritic cell (DC)-tumor electrofusion hybrids to eradicate established MCA205 sarcomas in syngeneic mice. Paired, but not solitary combinations of polyinosine:polycytadilic acid (P[I:C]; TLR3 agonist) and CpG DNA (ODN1826l; TLR9 agonist) stimulated IL-12 secretion from DCs in vitro and synergized with vaccination to achieve potent tumor rejection. Therapeutic effects, however, required coadministration of paired TLR agonists and DC-tumor fusion hybrids. The administration of TLR agonists alone or with fusion vaccine induced transient splenomegaly but without apparent toxicity. The therapeutic effects of this immunization regimen were significantly abrogated through the neutralization of IL-12p70, indicating that production of this third signal was essential to the observed tumor regression. These results show the profound functional consequences of TLR cooperativity and further highlight the critical role of IL-12 in antitumor immunity.