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.

Interleukin-10 (IL-10)-treated dendritic cells (DCs) induce an alloantigen-or peptide-specific anergy in various CD4 ؉ and CD8 ؉ T-cell populations. In the present study, we analyzed whether these anergic T cells are able to regulate antigenspecific immunity. Coculture experiments revealed that alloantigen-specific anergic CD4 ؉ and CD8 ؉ T cells suppressed proliferation of syngeneic T cells in a dosedependent manner. The same effect was observed when the hemagglutininspecific CD4 ؉ T-cell clone HA1.7 or tyrosinase-specific CD8 ؉ T cells were cocultured with anergic T cells of the same specificity. Anergic T cells did not induce an antigen-independent bystander inhibition. Suppression was dependent on cellto-cell contact between anergic and responder T cells, required activation by antigen-loaded DCs, and was not mediated by supernatants of anergic T cells. IntroductionThe task of eliminating autoreactive T cells is mediated in part by clonal deletion in the thymus. Autoreactive T cells that did not undergo negative selection in the thymus are subject to several peripheral mechanisms controlling unwanted T-cell activation. 1 These include immunological ignorance, deletion, immunoregulation, and anergy. [2][3][4][5] Maintenance of tolerance might be of clinical importance not only for self-tolerance but also for the control of pathogen-induced immune processes. 6,7 Anergy is immunologically defined as the inability of antigenspecific T cells to produce interleukin (IL)-2 and to clonally expand on rechallenge with fully competent antigen-presenting cells (APCs). 8 Induction of anergy is an active process that occurs when T-cell receptors (TCRs) are ligated by antigen in the absence of costimulation. 9 Anergy can also be induced in the presence of costimulation when the TCR is ligated by superantigen or by altered peptide ligands that bear a single amino acid substitution in the sequence of the agonistic peptide. 10,11 Although quite distinct, these approaches to induce anergy appear to share common biochemical events characterized by hypophosphorylation of several signal transduction-related proteins. [12][13][14][15] One important aspect of this type of regulation is that alloreactive T cells, tolerant to a specific antigen (alloantigen/autoantigen), can potentially downregulate the response of other naive allogeneic or antigen-specific T cells. 16,17 Dendritic cells (DCs) are highly specialized APCs of the immune system. 18 Fully mature DCs are potent activators of naive T cells and are regarded as important initiators of primary T-cell immune responses. In contrast to modulators inducing the maturation of DCs, the immunosuppressive properties of IL-10 on DCs have been well documented in several studies. 19 The inhibitory influence of IL-10 on APC function of DCs may be due to several phenotypic and functional alterations, such as down-regulation of major histocompatibility complex class II and costimulatory molecules and the reduced secretion of a variety of inflammatory cytokines. [20][21][22][23] Additionall...

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