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-1β from cultured monocytes as determined by ELISA assays.

Immune response to cancer and its regulation in regional lymph nodes.

Regional draining lymph nodes (LNs) play a pivotal role in initiating immune responses. However, the presence of metastases may compromise their normal immunological function. Preclinical studies indicate that despite metastases, early tumor-draining LNs are still a rich source of sensitized T cells.

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.

Immunotherapy using allogeneic squamous cell tumor-dendritic cell fusion hybrids.

Background: Tumor-associated antigens (TAAs) are known to be immunotherapy targets; thus tumor-sharing TAA may be used as a fusion hybrid partner to confer protection against subsequent tumor challenge.

Methods: The squamous cell carcinomas (SCCs), SCCVII and B4B8, were used in C3H/HEN mice: SCCVII (H-2(k)) is syngeneic, B4B8 (H-2(d)) is allogeneic. Experiments using tumor alone included hyperimmunization schedule, subdermal and intranodal routes.

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.

Tumor-induced CD11b+Gr-1+ myeloid cells suppress T cell sensitization in tumor-draining lymph nodes.

Suppression of tumor-specific T cell sensitization is a predominant mechanism of tumor escape. To identify tumor-induced suppressor cells, we transferred spleen cells from mice bearing progressive MCA205 sarcoma into sublethally irradiated mice. These mice were then inoculated subdermally with tumor cells to stimulate T cell response in the tumor-draining lymph-node (TDLN).

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.

Immunotherapy of established murine squamous cell carcinoma using fused dendritic-tumor cell hybrids.

Objective: To investigate the therapeutic efficacy of fused dendritic-tumor cell hybrids against murine squamous cell carcinoma (SCC).

Design: Squamous cell carcinoma VII is a poorly immunogenic murine SCC tumor in C3H/HEN (H-2(K)) mice. Subdermal tumors were established by inoculation in the mid abdomen of mice.

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).

Effective treatment of spontaneous metastases derived from a poorly immunogenic murine mammary carcinoma by combined dendritic-tumor hybrid vaccination and adoptive transfer of sensitized T cells.

Curative immunotherapy against spontaneous metastases of poorly immunogenic tumors has been difficult to demonstrate, but it is highly relevant to clinical disease conditions. The 4T1 mammary carcinoma shares many characteristics of human mammary cancer. Here, mice with 4T1 spontaneous metastases were treated effectively with a combination of dendritic (DC)-tumor hybrid vaccination and adoptive transfer of tumor-draining lymph node-derived immune T cells.

IL-12 is required for anti-OX40-mediated CD4 T cell survival.

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared.

Immunogenicity of dendritic-tumor fusion hybrids and their utility in cancer immunotherapy.

Cancer immunotherapy using fusion hybrid cells generated from dendritic cells (DCs) and tumor cells may be more effective than other DC-based vaccines. DC-tumor fusion potentially confers not only the DCs' antigen-presenting functionality but also a continuing source of endogenous tumor antigens for major-histocompatibility-complex-restricted T-cell sensitization. In animal models, many investigators demonstrated that vaccination with fusion hybrids was protective against tumor challenge and therapeutic, resulting in the regression of established tumors.

RAD inhibition of sarcoma growth: implications for laryngeal transplantation.

Purpose: Laryngeal transplantation has not been widely accepted because of concerns regarding accelerated tumor recurrences in the setting of nonspecific immunosuppression. Allotransplantation could potentially be offered to patients if immunosuppressive therapy could be demonstrated to exert tumor suppressive properties. Preliminary reports have demonstrated an antiproliferative effect of everolimus (RAD), a derivative of the immunosuppressant rapamycin.

Adoptive transfer of tumor-primed, in vitro-activated, CD4+ T effector cells (TEs) combined with CD8+ TEs provides intratumoral TE proliferation and synergistic antitumor response.

The importance of CD4+ Th1 cells during the effector phase of the antitumor response has been overshadowed by emphasis on CD8+ cytotoxic T lymphocytes (CTLs). To determine their respective functions, we purified antigen-primed T cells from tumor-draining lymph nodes and separately activated CD4+ and CD8+ subsets in vitro. Adoptive transfer of CD4+ T effector cells (T(E)s) combined with CD8+ T(E)s provided synergistic therapy for mice bearing subcutaneous, intracranial, or advanced pulmonary metastases.

CXC chemokine ligand 9/monokine induced by IFN-gamma production by tumor cells is critical for T cell-mediated suppression of cutaneous tumors.

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-gamma (Mig) and CXCL10/IFN-gamma-inducible protein 10 following stimulation with IFN-gamma and clones that produce IFN-gamma-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells.

Immune responses in the draining lymph nodes against cancer: implications for immunotherapy.

Regional lymph nodes are the first site for melanoma metastases. The sentinel node (SN), on the direct lymphatic drainage pathway, which usually harbors first metastases, demonstrates significant suppression in its ability to respond to antigenic stimulation. This down-regulation of SN immunity is likely the basis of its susceptibility to tumor metastases, suggesting a potential role of the immune system in the control of malignant tumors.

Host lymphodepletion augments T cell adoptive immunotherapy through enhanced intratumoral proliferation of effector cells.

T-cell adoptive immunotherapy for stringent murine tumor models, such as intracranial, s.c., or advanced pulmonary metastases, routinely uses lymphodepletive conditioning regimens before T-cell transfer, like recent clinical protocols.

Adoptive immunotherapy of cancer with polyclonal, 108-fold hyperexpanded, CD4+ and CD8+ T cells.

T cell-mediated cancer immunotherapy is dose dependent and optimally requires participation of antigen-specific CD4+ and CD8+ T cells. Here, we isolated tumor-sensitized T cells and activated them in vitro using conditions that led to greater than 108-fold numerical hyperexpansion of either the CD4+ or CD8+ subset while retaining their capacity for in vivo therapeutic efficacy. Murine tumor-draining lymph node (TDLN) cells were segregated to purify the CD62Llow subset, or the CD4+ subset thereof.

[Immunotherapeutic reactivity of dendritic cells loaded with a variety of antigen preparations].

The findings summarized here provide a direct comparison of the immunogenicity of various DC loading strategies included pulsing with protein, peptide, tumor cell lysate, irradiated tumor cells and electrofusion of DCs and tumor cells. For the treatment of 3-day established pulmonary metastases, electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. Consistent with their therapeutic activity, fusion hybrids stimulated the release of the largest amount of interferon-gamma from immune T cells.