The functional properties of dendritic cells (DCs) are strictly dependent on their maturational state. To analyze the influence of the maturational state of DCs on priming and differentiation of T cells, immature CD83− and mature CD83+ human DCs were used for stimulation of naive, allogeneic CD4+ T cells. Repetitive stimulation with mature DCs resulted in a strong expansion of alloreactive T cells and the exclusive development of T helper type 1 (Th1) cells. In contrast, after repetitive stimulation with immature DCs the alloreactive T cells showed an irreversibly inhibited proliferation that could not be restored by restimulation with mature DCs or peripheral blood mononuclear cells, or by the addition of interleukin (IL)-2. Only stimulation of T cells with mature DCs resulted in an upregulation of CD154, CD69, and CD70, whereas T cells activated with immature DCs showed an early upregulation of the negative regulator cytotoxic T lymphocyte–associated molecule 4 (CTLA-4). These T cells lost their ability to produce interferon γ, IL-2, or IL-4 after several stimulations with immature DCs and differentiated into nonproliferating, IL-10–producing T cells. Furthermore, in coculture experiments these T cells inhibited the antigen-driven proliferation of Th1 cells in a contact- and dose-dependent, but antigen-nonspecific manner. These data show that immature and mature DCs induce different types of T cell responses: inflammatory Th1 cells are induced by mature DCs, and IL-10–producing T cell regulatory 1–like cells by immature DCs.

A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-γ on either protein or mRNA levels. The anergic state of CD4+CD25+ T cells is not reversible by the addition of anti-CD28, anti–CTLA-4, anti–transforming growth factor β, or anti–IL-10 antibody. However, the refractory state of CD4+CD25+ T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties.

Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.

Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 × 106 DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 × 106 and 12 × 106 DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1–specific CD8+ cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8+ T cell infiltration, whereas nonregressing lesions lacked CD8+ T cells as well as Mage-3 mRNA expression. This study proves the principle that DC “vaccines” can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8+ CTL–tumor cell interaction in situ as well as escape by lack of tumor antigen expression.

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-gamma (IFN-gamma), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-gamma bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 by murine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-gamma production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.