Granulocyte macrophage colony-stimulating factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and CD83+ cells in cancer patients.

Antigen-presenting cells (APCs) are essential for stimulating antigen-specific immunity, including immunity against tumor cells. We hypothesized that systemic administration of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, which promote monocytes to differentiate into dendritic cells in vitro, might enhance the number and antigen-presenting activity of CD14+ cells in vivo. Patients with metastatic solid malignancies were treated with daily s.c. injections of either GM-CSF alone (2.5 microg/kg/day) or GM-CSF in combination with IL-4 (0.5-6.0 microg/kg/day) in a multicohort study. When given alone, GM-CSF increased the number of CD14+ cells but did not enhance the cells' expression of APC markers or antigen-presenting activity. In contrast, combination therapy with GM-CSF and IL-4 stimulated CD14+ cells to acquire several APC characteristics including increased expression of HLA-DR and CD11c, decreased CD14, increased endocytotic activity, and the ability to stimulate T cells in a mixed leukocyte reaction. Combination therapy also induced a dose-dependent increase in the number of CD14-/CD83+ cells with APC activity. Clinically significant and sustained tumor regression was observed in one patient. Systemic therapy with GM-CSF and IL-4 may provide a mechanism for increasing the number and function of APCs in patients with cancer.

Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4.

The combination of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) induces the growth of antigen-presenting cells (APC) from adherent peripheral blood leukocytes. These cells have been characterized as dendritic cells (DC), yet many questions exist regarding their relationship to other DC populations and the nature of their progenitors. To address these issues, we utilized a combination of immunomagnetic depletion, cell sorting, and cell culture to isolate four distinct APC populations; macrophages expressing high levels of CD14 (CD14bright macrophages), DC produced by culturing adherent cells in GM-CSF and IL-4 (cultured DC), and two different subsets of fresh DC that express low levels of CD14 (CD14dim DC). Each population exhibited a unique morphology and a unique profile of cell surface markers. In contrast to macrophages,all three DC populations expressed the DC marker CD83, as well as highlevels of MHC molecules and the costimulatory molecules B7-1 (CD80) and B7-2 (CD86). In addition. all three DC populations presented soluble tetanus toxin antigen and stimulated T cell proliferation to levels far superior to that of macrophages. Blocking studies demonstrated a costimulatory role for B7-1, B7-2, and CD40 in antigen presentation, although B7-2 expression was the single most important factor. To identify the progenitors of cultured DC, we sorted the adherent fraction of PBMC into discrete subpopulations prior to exposure to GM-CSF and IL-4. DC activity derived entirely from CD14+ precursors and was equally demonstrable using either the CD14dim or CD14bright subsets. Although these DC precursors lost expression of CD14 in culture, they maintained most of their other myeloid features. We conclude that human CD14+ leukocytes acquire the phenotype and function of DC when cultured in GM-CSF and IL-4.

Interferon-alpha and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells.

The diverse roles of interferon-alpha (IFN-alpha) in regulating the immune response to infectious agents suggested that it might affect dendritic cell (DC) development. Peripheral blood mononuclear cells cultured with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) developed a dendritic morphology and expressed high levels of the class I and II human leukocyte antigens (HLA), B7 co-stimulatory molecules, adhesion proteins, and CD40. Elevated DC expression of B7-2 and HLA-DR was observed with increasing IFN-alpha concentrations up to 5000 U/mL. The effects of IFN-alpha on DC immunophenotype were not reversed by adding neutralizing antibodies against interleukin-4 (IL-4) or tumor necrosis factor alpha to the cell cultures or by eliminating lymphocytes from the cultures. The addition of IFN-alpha to cultures containing optimal concentrations of IL-4 and GM-CSF significantly increased the B7-2 and HLA-DR levels above those present on DCs grown in two cytokines. The DCs generated with IFN-alpha and GM-CSF were potent antigen-presenting cells in allogeneic mixed leukocyte reactions. They also were capable of taking up, processing, and presenting tetanus toxin to autologous T lymphocytes. These results demonstrate an important role for IFN-alpha in the generation of DCs with potent antigen-presenting capabilities from peripheral blood monocytes.

A comparison of gene transfer methods in human dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen-specific T-cell activation. DCs may be highly enriched from peripheral blood-adherent leukocytes by short-term (7-day) culture in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. Various methods of gene transfer were studied, including DNA/liposome complexes, electroporation, CaPO4 precipitation, and recombinant adenovirus (AdV) vectors. Low levels of expression were obtained with the physical methods tested. In contrast, AdV vectors expressing luciferase, beta-galactosidase, IL-2, and IL-7 all readily transduced human DCs. Increasing levels of gene expression were observed over a range of multiplicity of infection (MOI) of 10:1 to 10,000:1, with transduction efficiencies exceeding 95% at higher MOI. Although levels of maximal gene expression in DCs were significantly lower than those obtained using human tumor cell lines, IL-2 and IL-7 production of up to 5 x 10(2) ng/10(6) DC were achieved. These results suggest that AdV vectors are a promising vehicle for genetically engineering human DCs.

Interleukin-7 gene transfer in non-small-cell lung cancer decreases tumor proliferation, modifies cell surface molecule expression, and enhances antitumor reactivity.

Cytokine gene transfer to tumor cells can augment host antitumor responses and modify tumor phenotype. To evaluate the immunoregulatory and antitumor capacities of lung tumor-derived interleukin-7 (IL-7), we transduced non-small-cell lung cancer (NSCLC) cell lines with the IL-7/HyTK internal ribosomal entry site (IRES) retroviral vector and evaluated modifications in tumor phenotype and cocultured effector activities. In vitro proliferation of IL-7-transduced tumor cells was significantly less than control vector-transduced and parental tumor cells. The decreased proliferation rates of IL-7-transduced cells could be reproduced by adding high concentrations of recombinant IL-7 to the parental cells. Anti-IL-7 monoclonal antibody significantly increased the proliferation of the IL-7-transduced cells (P < .05). Parental NSCLC cells were found to express the IL-7 receptor, and IL-7 gene transduction did not alter expression of the IL-7 receptor. IL-7 transduction significantly altered tumor cell expression of intracellular adhesion molecule 1, major histocompatibility complex 1, lymphocyte function-related antigen 3, very late activation antigen beta 1, and p185neu. Peripheral blood lymphocytes cocultured with either IL-7-transduced tumor cells or tumor supernatants had enhanced cytolytic and proliferative capacities compared with coculture with control vector-transduced or parental cells. Our findings indicate that IL-7 gene transfer in NSCLC significantly augments cocultured effector activities in vitro, inhibits tumor cell proliferation, and modifies tumor cell surface phenotype. These findings suggest that IL-7 gene therapy may be effective in modifying host antitumor responses in NSCLC.

Systemic IL-1 and adjuvant treatment of an experimental tumor. II. Immune status during primary tumor challenge.

In this investigation, systemic administration of interleukin-1 (IL-1) and local adjuvant therapy were shown to modify immunological parameters associated with the lymphatics draining the site of experimental tumor inoculation. These immunological parameters were shown to be modified early (within 7 days) following tumor inoculation and within the time period of IL-1 administration. IL-1 induced a marked increase in the number of lymphocytes within the brachial and axillary lymph nodes associated with the tumor inoculation site. This increase was characterized by an overall augmentation in the number of CD8+ and CD4+ lymphocytes. In vitro, these lymph node cells showed enhanced proliferation in response to interleukin-2 (IL-2) when compared to non-IL-1 treated animals, and were capable of mounting a potentially greater cytotoxic response for both NK sensitive and NK resistant tumor targets. Without IL-1 administration, temporal and sequential lymph node cellular changes were observed, but were diminished and delayed when compared to the IL-1 treated animals. By adoptive transfer of tumor resistance, lymph node cells from IL-1 treated animals were demonstrated to be tumor-protective in vivo. These results demonstrate that systemic IL-1 induces regional changes in the lymphatics of mice undergoing primary tumor challenge with adjuvant therapy and that these changes result in tumor protection for the host.

Systemic IL-1 and adjuvant treatment of an experimental tumor. I. Immune status following tumor rechallenge.

Local adjuvant therapy of weakly immunogenic tumors protects against primary tumor challenge. However, this form of therapy does not produce long-lasting immunity to the tumor. In this study, local adjuvant therapy combined with systemic IL-1 administration produced not only primary tumor protection, but also long lasting immunity to the tumor. IL-1 and adjuvant protected animals resisted rechallenge with tumor as much as 180 days after initial tumor administration. Resistance to tumor rechallenge was IL-1 dose dependent. IL-1 and adjuvant protected animals also exhibited delayed type hypersensitivity reactions which were tumor-specific. Splenic and lymph node cell populations from IL-1 and adjuvant protected animals mounted tumor-specific lymphoproliferative responses. No such responses were observed in animals which had been administered either IL-1 or adjuvant alone. These results demonstrate that systemic IL-1 functions to augment specific immune protection when administered in conjunction with local adjuvant, resulting in long-lasting tumor immunity.

Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells.

Tumors produce a number of immunosuppressive factors that block the maturation of CD34+ stem cells into dendritic cells (DC). We hypothesized that tumors might also interfere with the maturation and/or function of human monocyte-derived DC. In contrast to stem cells, we found that CD14+ cells responded to tumor culture supernatant (TSN) by increasing expression of APC surface markers, up-regulating nuclear translocation of RelB, and developing allostimulatory activity. Although displaying these characteristics of mature DC, TSN-exposed DC lacked the capacity to produce IL-12, did not acquire full allostimulatory activity, and rapidly underwent apoptosis. The effects of TSN appeared to be specific for maturing DC, and were not reversed by Abs against known DC regulatory factors including IL-10, vascular endothelial growth factor, TGF-beta, or PGE2. Supernatants collected from nonmalignant cell sources had no effect on DC maturation. The altered maturation and early apoptosis of monocyte-derived DC may represent another mechanism by which tumors evade immune detection.

Vaccination with helper-dependent adenovirus enhances the generation of transgene-specific CTL.

Recombinant adenoviral vectors (AdV) have been used experimentally as vaccines to present antigenic transgenes in vivo. However, administration of first-generation vectors (FG-AdV) is often limited by their induction of antiviral immunity. To address this limitation, helper-dependent vectors (HD-AdV) were developed that lack viral coding regions. While the administration of HD-AdV results in long-term gene expression in vivo, their utility as immunogens has never been examined. Direct vaccination with 10(8) blue-forming units (BFU) of HD-AdV injected into C57BL/6 mice lead to superior transgene-specific CTL and antibody responses when compared to the same amount of a FG-AdV. The antibody responses to viral antigens were high in response to both the vectors. As a mechanism to reduce viral exposure, dendritic cells (DC) were transduced with HD-AdV in vitro and then used as a cell-based vaccine. DC transduced with HD-AdV expressed higher levels of transgene-specific mRNA and up to 1200-fold higher levels of transgene protein than did DC transduced with a FG-AdV. In addition, HD-AdV-transduced DC stimulated superior transgene-specific CTL responses when administered in vivo, an effect that was further enhanced by maturing the DC with LPS prior to administration. In contrast to direct immunization with HD-AdV, vaccination with HD-AdV-transduced DC was associated with limited antibody responses against the AdV. We conclude that HD-AdV stimulates superior transgene-specific immune responses when compared to a FG-AdV, and that immunization with a DC-based vaccine maintains this efficacy while limiting antiviral reactivity.

Pulmonary macrophages suppress the proliferation and cytotoxicity of tumor-infiltrating lymphocytes.

Adoptive immunotherapy with interleukin-2 and tumor-infiltrating lymphocytes (TIL) is rarely effective in primary lung cancer. We hypothesize that pulmonary macrophages (PM), which are increased substantially in the lungs of smokers, might suppress TIL function. The addition of PM into the TIL cytotoxicity assay produced a concentration-dependent suppression of TIL cytotoxicity with up to 71% inhibition of autologous tumor killing at the 1:1 PM:TIL ratio. Inhibition was not target-specific, as killing of NK-sensitive (K562), NK-resistant (M14), and autologous tumor targets were equally suppressed. Nor was inhibition specific for lung TIL, as similar inhibition was observed with melanoma and renal TIL. Using a model system, we demonstrated that both CD3+ antigen-specific and CD56+ nonspecific lymphocytes are susceptible to the suppressive effects of the PM. Direct co-incubation of PM and TIL for 4 to 44 h resulted in progressive suppression of TIL proliferation and cytotoxicity. TIL cytotoxicity remained suppressed even if PM were removed from the co-culture after 24 h, but was restored if the separated TIL were re-incubated in interleukin-2. These results suggest that PM may locally regulate the proliferative and cytotoxic function of adoptively transferred TIL.

Microbial lipopeptides stimulate dendritic cell maturation via Toll-like receptor 2.

The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.

Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10.

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.