PIM kinase inhibitor AZD1208 in conjunction with Th1 cytokines potentiate death of breast cancer cellsin vitrowhile also maximizing suppression of tumor growthin vivo when combined with immunotherapy.

PIM kinases are over-expressed by a number of solid malignancies including breast cancer, and are thought to regulate proliferation, survival, and resistance to treatment, making them attractive therapeutic targets. Because PIM kinases sit at the nexus of multiple oncodriver pathways, PIM antagonist drugs are being tested alone and in conjunction with other therapies to optimize outcomes. We therefore sought to test the combination of pharmacological PIM antagonism and Th1-associated immunotherapy. We show that the pan PIM antagonist, AZD1208, when combined in vitro with Th1 cytokines IFN-γ and TNF-α, potentiates metabolic suppression, overall cell death, and expression of apoptotic markers in human breast cancer cell lines of diverse phenotypes (HER-2pos/ERneg, HER-2pos/ERpos and triple-negative). Interestingly, AZD1208 was shown to moderately inhibit IFN-γ secretion by stimulated T lymphocytes of both human and murine origin, suggesting some inherent immunosuppressive activity of the drug. Nonetheless, when multiplexed therapies were tested in a murine model of HER-2pos breast cancer, combinations of HER-2 peptide-pulsed DCs and AZD1208, as well as recombinant IFN-γ plus AZD1208 significantly suppressed tumor outgrowth compared with single-treatment and control groups. These studies suggest that PIM antagonism may combine productively with certain immunotherapies to improve responsiveness.

MEETING HIGHLIGHTS: THE THIRD MARIE SKLODOWSKA-CURIE SYMPOSIUM ON CANCER RESEARCH AND CARE AT ROSWELL PARK COMPREHENSIVE CANCER CENTER, BUFFALO, NY, SEPTEMBER 20-22, 2023.

Marie Sklodowska-Curie Symposia on Cancer Research and Care (MSCS-CRC) promote collaborations between cancer researchers and care providers in the United States, Canada and Central and Eastern European Countries (CEEC), to accelerate the development of new cancer therapies, advance early detection and prevention, increase cancer awareness, and improve cancer care and the quality of life of patients and their families. The third edition of MSCS-CRC, held at Roswell Park Comprehensive Cancer Center, Buffalo, NY, in September 2023, brought together 137 participants from 20 academic institutions in the US, Poland, Ukraine, Lithuania, Croatia and Hungary, together with 16 biotech and pharma entities. The key areas of collaborative opportunity identified during the meeting are a) creating of a database of available collaborative projects in the areas of early-phase clinical trials, preclinical development, and identification of early biomarkers; b) promoting awareness of cancer risks and efforts at cancer prevention; c) laboratory and clinical training; and d) sharing experience in cost-effective delivery of cancer care and improving the quality of life of cancer patients and their families. Examples of ongoing international collaborations in the above areas were discussed. Participation of the representatives of the Warsaw-based Medical Research Agency, National Cancer Institute (NCI) of the United States, National Cancer Research Institutes of Poland and Lithuania, New York State Empire State Development, Ministry of Health of Ukraine and Translational Research Cancer Center Consortium of 13 cancer centers from the US and Canada, facilitated the discussion of available governmental and non-governmental funding initiatives in the above areas.

Identification of Immunogenic MHC Class II Human HER3 Peptides that Mediate Anti-HER3 CD4+ Th1 Responses and Potential Use as a Cancer Vaccine.

The HER3/ERBB3 receptor is an oncogenic receptor tyrosine kinase that forms heterodimers with EGFR family members and is overexpressed in numerous cancers. HER3 overexpression associates with reduced survival and acquired resistance to targeted therapies, making it a potential therapeutic target in multiple cancer types. Here, we report on immunogenic, promiscuous MHC class II-binding HER3 peptides, which can generate HER3-specific CD4+ Th1 antitumor immune responses. Using an overlapping peptide screening methodology, we identified nine MHC class II-binding HER3 epitopes that elicited specific Th1 immune response in both healthy donors and breast cancer patients. Most of these peptides were not identified by current binding algorithms. Homology assessment of amino acid sequence BLAST showed >90% sequence similarity between human and murine HER3/ERBB3 peptide sequences. HER3 peptide-pulsed dendritic cell vaccination resulted in anti-HER3 CD4+ Th1 responses that prevented tumor development, significantly delayed tumor growth in prevention models, and caused regression in multiple therapeutic models of HER3-expressing murine tumors, including mammary carcinoma and melanoma. Tumors were robustly infiltrated with CD4+ T cells, suggesting their key role in tumor rejection. Our data demonstrate that class II HER3 promiscuous peptides are effective at inducing HER3-specific CD4+ Th1 responses and suggest their applicability in immunotherapies for human HER3-overexpressing tumors.

Murine Dendritic Cells Grown in Serum-Free Culture Show Potent Therapeutic Activity when Loaded with Novel Th Epitopes in an Orthotopic Model of HER2pos Breast Cancer.

Preferred methods for generating mouse dendritic cells (DC) would encompass qualities of consistency, high yield, and potent function. Serum-free culture is also highly desirable, since this is the standard for cell-based therapies used in humans. We report here a serum-free modification of a culture method generating mature, activated DCs from bone marrow precursors. This is achieved through a two-stage culture comprised of 6-day expansion in Flt3 ligand and IL-6 followed by brief differentiation in a medium containing GM-CSF and IL-4, with subsequent activation using TLR ligands ODN1826 and LPS. The serum-free DCs achieve yields and surface phenotype including IL-12p70 secretion similar to standard serum-replete cultures, display a capacity to sensitize in vivo against both MHC class I- and Class II-restricted antigens, and exhibit some aspects of "killer DC" function against tumor cells. We used these DCs to help identify novel CD4pos Th epitopes on the rat ErbB2/HER-2 protein and demonstrated a subset of these as effective immunogens in a DC-based therapeutic model of HER-2pos breast cancer in Balb/c mice, where they induced powerful Th1-polarized immune responses. This method represents a useful way to efficiently produce large numbers of murine dendritic cells with excellent in vivo function well-suited for use in experimental vaccine studies.

Sunitinib Combined with Th1 Cytokines Potentiates Apoptosis in Human Breast Cancer Cells and Suppresses Tumor Growth in a Murine Model of HER-2pos Breast Cancer.

Although immune-based therapies have made remarkable inroads in cancer treatment, they usually must be combined with standard treatment modalities, including cytotoxic drugs, to achieve maximal clinical benefits. As immunotherapies are further advanced and refined, considerable efforts will be required to identify combination therapies that will maximize clinical responses while simultaneously decreasing the unpleasant and sometimes life-threatening side effects of standard therapy. Over the last two decades, evidence has emerged that Th1 cytokines can play a central role in protective antitumor immunity and that combinations of Th1 cytokines can induce senescence and apoptosis in cancer cells. To explore the possibility of combining targeted drugs with Th1-polarizing vaccines, we undertook a study to examine the impact of combining Th1 cytokines with the relatively broad-spectrum receptor tyrosine kinase antagonist, sunitinib. We found that when a panel of five phenotypically diverse human breast cancer cell lines was subjected to treatment with sunitinib plus recombinant Th1 cytokines IFN-γ and TNF-α, synergistic effects were observed across a number of parameters including different aspects of apoptotic cell death. Interestingly, sunitinib was found to have a profoundly suppressive effect of T cell's capacity to secrete IFN-γ, indicating that in vivo use of this drug may hinder robust Th1 responses. Nonetheless, this suppression was circumvented in a mouse model of HER-2pos breast disease by supplying recombinant interferon-gamma to achieve a combination therapy significantly more potent than either agent.

Th1 cytokines in conjunction with pharmacological Akt inhibition potentiate apoptosis of breast cancer cells in vitro and suppress tumor growth in vivo.

Targeted drug approaches have been a major focus for developing new anticancer therapies. Although many such agents approved in the last 20 years have improved outcomes, almost all have underperformed expectations. The full potential of such agents may yet be obtained through novel combinations. Previously, we showed that anti-estrogen drugs combined with a dendritic cell-based anti-HER-2 vaccine known to induce strong Th1-polarized immunity dramatically improved clinical response rates in patients with HER-2pos/ERpos early breast cancer. Here, we show that the small molecule Akt antagonist MK-2206, when combined with the Th1 cytokines IFN-gamma and TNF-alpha, maximize indicators of apoptotic cell death in a panel of phenotypically-diverse human breast cancer lines. These findings were mirrored by other, structurally-unrelated Akt-targeting drugs that work through different mechanisms. Interestingly, we found that MK-2206, as well as the other Akt antagonist drugs, also had a tendency to suppress Th1 cytokine expression in stimulated human and murine lymphocytes, potentially complicating their use in conjunction with active immunotherapy. After verifying that MK-2206 plus IFN-gamma could show similar combined effects against breast cancer lines, even in the absence of TNF-alpha, we tested in a rodent HER-2pos breast cancer model either a HER-2-based DC vaccine, or recombinant IFN-gamma with or without MK-2206 administration. We found that for MK-2206, co-administration of recombinant IFN-gamma outperformed co-administration of DC vaccination for slowing tumor growth kinetics. These findings suggest a combined therapy approach for Akt-targeting drugs that incorporates recombinant Interferon-gamma and is potentially translatable to humans.

Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2pos Disease.

A dendritic cell-based, Type 1 Helper T cell (Th1)-polarizing anti-Human Epidermal Growth Factor Receptor-2 (HER-2) vaccine supplied in the neoadjuvant setting eliminates disease in up to 30% of recipients with HER-2-positive (HER-2pos) ductal carcinoma in situ (DCIS). We hypothesized that drugs with low toxicity profiles that target signaling pathways critical for oncogenesis may work in conjunction with vaccine-induced immune effector mechanisms to improve efficacy while minimizing side effects. In this study, a panel of four phenotypically diverse human breast cancer lines were exposed in vitro to the combination of Th1 cytokines Interferon-gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-α) and lipophilic statins. This combination was shown to potentiate multiple markers of apoptotic cell death. The combination of statin drugs and Th1 cytokines minimized membrane K-Ras localization while maximizing levels in the cytoplasm, suggesting a possible means by which cytokines and statin drugs might cooperate to maximize cell death. A combined therapy was also tested in vivo through an orthotopic murine model using the neu-transgenic TUBO mammary carcinoma line. We showed that the combination of HER-2 peptide-pulsed dendritic cell (DC)-based immunotherapy and simvastatin, but not single agents, significantly suppressed tumor growth. Consistent with a Th1 cytokine-dependent mechanism, parenterally administered recombinant IFN-γ could substitute for DC-based immunotherapy, likewise inhibiting tumor growth when combined with simvastatin. These studies show that statin drugs can amplify a DC-induced effector mechanism to improve anti-tumor activity.

T-helper 1-type cytokines induce apoptosis and loss of HER-family oncodriver expression in murine and human breast cancer cells.

A recent neoadjuvant vaccine trial for early breast cancer induced strong Th1 immunity against the HER-2 oncodriver, complete pathologic responses in 18% of subjects, and for many individuals, dramatically reduced HER-2 expression on residual disease. To explain these observations, we investigated actions of Th1 cytokines (TNF-α and IFN-γ) on murine and human breast cancer cell lines that varied in the surface expression of HER-family receptor tyrosine kinases. Breast cancer lines were broadly sensitive to the combination of IFN-γ and TNF-α, as evidenced by lower metabolic activity, lower proliferation, and enhanced apoptosis, and in some cases a reversible inhibition of surface expression of HER proteins. Apoptosis was accompanied by caspase-3 activation. Furthermore, the pharmacologic caspase-3 activator PAC-1 mimicked both the killing effects and HER-2-suppressive activities of Th1 cytokines, while a caspase 3/7 inhibitor could prevent cytokine-induced HER-2 loss. These studies demonstrate that many in vivo effects of vaccination (apparent tumor cell death and loss of HER-2 expression) could be replicated in vitro using only the principle Th1 cytokines. These results are consistent with the notion that IFN-γ and TNF-α work in concert to mediate many biological effects of therapeutic vaccination through the induction of a caspase 3-associated cellular death mechanism.

Th1 cytokines sensitize HER-expressing breast cancer cells to lapatinib.

The HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members have therefore become thefocus of many drug and immune-based therapy innovations. The targeted anti-cancer agent, lapatinib, is a small molecule inhibitor that directly interferes with EGFR (HER-1)and HER-2 signaling, and indirectly reduces HER-3 signaling, thus suppressing important downstream events. A recently-developed dendritic cell-based vaccine against early breast cancer (ductal carcinoma in situ; DCIS) that generates strong Th1-dominated immunity against HER-2 has induced pathologic complete response in about one-third of immunized individuals. In vitro studies suggested cytokines secreted by Th1 cells could be major contributors to the vaccine effects including induction of apoptosis and suppression of HER expression. With a view toward improving complete response rates, we investigated whether the principle Th1 cytokines (IFN-γ and TNF-α) could act in concert with lapatinib to suppress activity of breast cancer lines in vitro. Lapatinib-sensitive SKBR3, MDA-MB-468 and BT474 cells were incubated with Th1 cytokines, lapatinib, or both. It was found that combined treatment maximized metabolic suppression(Alamar Blue assay), as well as cell death (Trypan Blue) and apoptosis(Annexin V/Propidium Iodide and TMRE staining). Combined drug plus cytokine treatment also maximized suppression of both total and phosphorylated forms of HER-2 and HER-3. Interestingly, when lapatinib resistant lines MDA-MB-453 and JIMT-1 were tested, it was found that the presence of Th1 cytokines appeared to enhance sensitivity for lapatinib-induced metabolic suppression and induction of apoptotic cell death, nearly abrogating drug resistance. These studies provide pre-clinical data suggesting the possibility that targeted drug therapy may be combined with vaccination to enhance anti-cancer effects, and furthermore that robust immunity in the form of secreted Th1 cytokines may have the capacity to mitigate resistance to targeted drugs.

Addition of anti-estrogen therapy to anti-HER2 dendritic cell vaccination improves regional nodal immune response and pathologic complete response rate in patients with ERpos/HER2pos early breast cancer.

HER2-directed therapies are less effective in patients with ERpos compared to ERneg breast cancer, possibly reflecting bidirectional activation between HER2 and estrogen signaling pathways. We investigated dual blockade using anti-HER2 vaccination and anti-estrogen therapy in HER2pos/ERpos early breast cancer patients. In pre-clinical studies of HER2pos breast cancer cell lines, ERpos cells were partially resistant to CD4+ Th1 cytokine-induced metabolic suppression compared with ERneg cells. The addition of anti-estrogen treatment significantly enhanced cytokine sensitivity in ERpos, but not ERneg, cell lines. In two pooled phase-I clinical trials, patients with HER2pos early breast cancer were treated with neoadjuvant anti-HER2 dendritic cell vaccination; HER2pos/ERpos patients were treated with or without concurrent anti-estrogen therapy. The anti-HER2 Th1 immune response measured in the peripheral blood significantly increased following vaccination, but was similar across the three treatment groups (ERneg vaccination alone, ERpos vaccination alone, ERpos vaccination + anti-estrogen therapy). In the sentinel lymph nodes, however, the anti-HER2 Th1 immune response was significantly higher in ERpos patients treated with combination anti-HER2 vaccination plus anti-estrogen therapy compared to those treated with anti-HER2 vaccination alone. Similar rates of pathologic complete response (pCR) were observed in vaccinated ERneg patients and vaccinated ERpos patients treated with concurrent anti-estrogen therapy (31.4% vs. 28.6%); both were significantly higher than the pCR rate in vaccinated ERpos patients who did not receive anti-estrogen therapy (4.0%, p = 0.03). Since pCR portends long-term favorable outcomes, these results support additional clinical investigations using HER2-directed vaccines in combination with anti-estrogen treatments for ERpos/HER2pos DCIS and invasive breast cancer.

Calcium mobilizing treatment acts as a co-signal for TLR-mediated induction of Interleukin-12 (IL-12p70) secretion by murine bone marrow-derived dendritic cells.

We sought to determine whether pharmacological calcium-mobilizing agents could act in cooperation with Toll-like receptor (TLR) signals to induce high-level IL-12 production from murine bone marrow-derived dendritic cells. We found that calcium mobilization alone induced no IL-12, yet dramatically enhanced IL-12p70 secretion elicited by TLR ligands. Enhanced IL-12 production induced by calcium ionophore plus single TLR ligands, but not through dual TLR ligands, was inhibited by the calcineurin antagonist cyclosporine A, suggesting divergent mechanisms of IL-12 induction. Dendritic cells activated with calciumionophore plus the TLR9 ligand ODN1826 could induce Th1 polarization in naïve murine CD4pos T cells at levels equal or superior to dendritic cells activated with the most efficient TLR ligand pairing; ODN1826 plus bacterial lipopolysaccharide. Parallel analysis of 38 inflammation-associated soluble products showed calciumionophore enhancement was restricted to a small set of factors. These data demonstrate previously undocumented activation co-signals for IL-12 production by dendritic cells.

Progressive loss of anti-HER2 CD4+ T-helper type 1 response in breast tumorigenesis and the potential for immune restoration.

Genomic profiling has identified several molecular oncodrivers in breast tumorigenesis. A thorough understanding of endogenous immune responses to these oncodrivers may provide insights into immune interventions for breast cancer (BC). We investigated systemic anti-HER2/neu CD4+ T-helper type-1 (Th1) responses in HER2-driven breast tumorigenesis. A highly significant stepwise Th1 response loss extending from healthy donors (HD), through HER2pos-DCIS, and ultimately to early stage HER2pos-invasive BC patients was detected by IFNγ ELISPOT. The anti-HER2 Th1 deficit was not attributable to host-level T-cell anergy, loss of immune competence, or increase in immunosuppressive phenotypes (Treg/MDSCs), but rather associated with a functional shift in IFNγ:IL-10-producing phenotypes. HER2high, but not HER2low, BC cells expressing IFNγ/TNF-α receptors were susceptible to Th1 cytokine-mediated apoptosis in vitro, which could be significantly rescued by neutralizing IFNγ and TNF-α, suggesting that abrogation of HER2-specific Th1 may reflect a mechanism of immune evasion in HER2-driven tumorigenesis. While largely unaffected by cytotoxic or HER2-targeted (trastuzumab) therapies, depressed Th1 responses in HER2pos-BC patients were significantly restored following HER2-pulsed dendritic cell (DC) vaccinations, suggesting that this Th1 defect is not "fixed" and can be corrected by immunologic interventions. Importantly, preserved anti-HER2 Th1 responses were associated with pathologic complete response to neoadjuvant trastuzumab/chemotherapy, while depressed responses were observed in patients incurring locoregional/systemic recurrence following trastuzumab/chemotherapy. Monitoring anti-HER2 Th1 reactivity following HER2-directed therapies may identify vulnerable subgroups at risk of clinicopathologic failure. In such patients, combinations of existing HER2-targeted therapies with strategies to boost anti-HER2 CD4+ Th1 immunity may decrease the risk of recurrence and thus warrant further investigation.

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.

A novel dendritic cell-based immunization approach for the induction of durable Th1-polarized anti-HER-2/neu responses in women with early breast cancer.

Twenty-seven patients with HER-2/neu overexpressing ductal carcinoma in situ of the breast were enrolled in a neoadjuvant immunization trial for safety and immunogenicity of DC1-polarized dendritic cells (DC1) pulsed with 6 HER-2/neu promiscuous major histocompatibility complex class II-binding peptides and 2 additional human leukocyte antigen (HLA)-A2.1 class I-binding peptides. DC1 were generated with interferon-γ and a special clinical-grade bacterial endotoxin (lipopolysaccharide) and administered directly into groin lymph nodes 4 times at weekly intervals before scheduled surgical resection of ductal carcinoma in situ. Patients were monitored for the induction of new or enhanced antipeptide reactivity by interferon-γ ELISPOT and enzyme-linked immunosorbentassays performed on Th cells obtained from peripheral blood or excised sentinel lymph nodes. Responses by cytotoxic T lymphocyte against HLA-A2.1-binding peptides were measured using peptide-pulsed T2 target cells or HER-2/neu-expressing or nonexpressing tumor cell lines. DC1 showed surface phenotype indistinct from "gold standard" inflammatory cocktail-activated DC, but displayed a number of distinguishing functional characteristics including the secretion of soluble factors and enhanced "killer DC" capacity against tumor cells in vitro. Postimmunization, we observed sensitization of Th cells to at least 1 class II peptide in 22 of 25 (88%; 95% exact confidence interval, 68.8%-97.5%) evaluable patients, whereas 11 of 13 (84.6%; 95% exact confidence interval, 64%-99.8%) HLA-A2.1 patients were successfully sensitized to class I peptides. Perhaps most importantly, anti-HER-2/neu peptide responses were observed up to 52-month postimmunization. These data show that even in the presence of early breast cancer such DC1 are potent inducers of durable type I-polarized immunity, suggesting potential clinical value for development of cancer immunotherapy.

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.

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.

Toll-like receptor agonists as third signals for dendritic cell-tumor fusion vaccines.

BACKGROUND: The aim of the present study was to evaluate the therapeutic efficacy of dendritic cell (DC)-tumor fusion hybrids with Toll-like receptor (TLR) agonists. METHODS: DC-tumor fusion hybrids were generated by electrofusion and injected into the inguinal lymph nodes of C57BL/6 mice with 3-day established pulmonary metastases. Paired TLR agonists polyinosine:polycytadilic acid [poly(I:C)] and cytosine-phosphate-guanine (CpG) were then injected intraperitoneally. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate interleukin (IL)-12 production from the DC-tumor fusion hybrids in vitro. RESULTS: Fusion + TLR agonists (60 metastases) had significantly fewer metastases than did the untreated control (262 metastases, p = .0001) and fusion alone (150 metastases, p = .02). ELISA showed that the DC-tumor fusion hybrids yielded 90 pg of IL-12 after TLR stimulation compared with 1610 pg from dendritic cells alone. CONCLUSIONS: CpG and poly(I:C) administered as a third signal with fusion hybrids as described significantly reduce melanoma metastasis compared with fusion hybrids alone. Fusion hybrids do not appear to be a significant source for IL-12 secretion.

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.

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.

Reengineering dendritic cell-based anti-cancer vaccines.

Despite initial enthusiasm, dendritic cell (DC)-based anti-cancer vaccines have yet to live up to their promise as one of the best hopes for generating effective anti-tumor immunity. One of the principal reasons for the generally disappointing results achieved thus far could be that the full potential of DCs has not been effectively exploited. Here, we argue that dramatic improvements in vaccine efficacy will probably require a careful re-evaluation of current vaccine design. The formulation of new strategies must take into account the natural history of DCs, particularly their role in helping the immune system deal with infection. Equally critical is the emerging importance of soluble factors, notably interleukin-12, in modulating the quality of immune responses. Vaccines should also be designed to recruit helper T cells and antibody-producing B cells rather than simply cytotoxic T lymphocytes. Finally, the judicious selection of tumor, target antigen, and disease stage best suited for treatment should serve as the foundation of trial designs. Our discussion addresses a recent clinical vaccine trial to treat early breast cancer, where many elements of this new strategy were put into practice.