Restoration by IL-15 of MHC class I antigen-processing machinery in human dendritic cells inhibited by tumor-derived gangliosides

We have recently reported that MHC class I Ag-processing machinery (APM) component expression in dendritic cells (DC) might be down-regulated by tumor cells. However, the tumor-derived factors responsible for inhibition of the APM component expression in DC generated in the tumor microenvironment as well as potential protective mechanism have not yet been investigated. In this article, we demonstrate that expression of several MHC class I APM components, including MB1 (beta5), LMP2, LMP7, LMP10, and ERp57, is significantly down-regulated in human DC generated in the presence of primary oral squamous cell carcinoma cell lines or coincubated with purified gangliosides. Suppression of MHC class I APM component expression in DC generated in the presence of tumor cells was significantly attenuated by the inhibition of glucosyl transferase in tumor cells, suggesting that tumor-induced MHC class I APM component down-regulation in DC was mediated in part by oral squamous cell carcinoma-derived gangliosides. Furthermore, rIL-15 restored both tumor cell-induced and ganglioside-induced MHC class I APM component expression in DC, as well as their ability to present Ags to autologous Ag-specific T cells. These results demonstrate that IL-15 restores MHC class I APM component expression in DC down-regulated by tumor-derived gangliosides.     

Interferon regulatory factor 8 mediates tumor-induced inhibition of antigen processing and presentation by dendritic cells

Introduction  Suppression of dendritic cells (DCs) is a crucial mechanism by which tumor cells escape immune recognition and elimination. We have recently reported that MHC class I antigen processing machinery (APM) component expression in human DCs is down-regulated by tumor-derived gangliosides. However, the molecular mechanisms underlying this abnormality were not identified. Thus, the aim of this work was to analyze the role of interferon regulatory factor 8 (IRF-8) in APM protein expression and the antigen presenting capacity of DCs developed in the tumor microenvironment. Results  We demonstrate that the expression of several MHC class I APM components, including delta, MB-1, LMP-10, ERp57, and tapasin, is significantly decreased in murine DCs generated in the presence of prostate cancer cells. APM component down-regulation was associated with decreased ability of DCs to present model antigen to antigen-specific T cells. Notable, impaired antigen-presenting activity of DCs co-cultured with tumor cells was accompanied by decreased levels of IRF-8. Transduction of DCs with the silencing RNA for the IRF-8 gene also led to reduced expression of APM components in DCs and decreased antigen presenting function. Conclusion  Together, our data suggest that tumor-induced inhibition of antigen processing and presenting function of DCs is mediated by IRF-8, a member of the interferon regulatory factor family. These results provide a new molecular target for optimizing the generation of efficient DC vaccines for cancer therapy.     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Tumor-derived MUC1 mucins interact with differentiating monocytes and induce IL-10highIL-12low regulatory dendritic cell

Dendritic cells (DC) initiate immunity by the activation of naive T cells and control immunity through their ability to induce unresponsiveness of lymphocytes by mechanisms that include deletion and induction of regulatory cells. An inadequate presentation to T cells by tumor-induced "regulatory" DC, among several mechanisms, can explain tolerance to tumor-associated Ags. In this study, we show that tumor-derived mucin profoundly affects the cytokine repertoire of monocyte-derived DC and switch them into IL-10(high)IL-12(low) regulatory APCs with a limited capacity to trigger protective Th1 responses. In fact, DC cocultured with pancreatic tumor cell lines in a Transwell system did not reach full maturation, had low immunostimulatory functions, did not produce IL-12, and released high levels of IL-10. The involvement of known tumor-derived immune-suppressive factors (e.g., vascular endothelial growth factor, TGF-beta, IL-6, and IL-10) was considered and excluded. We provide evidence that tumor-derived MUC1 mucins are responsible for the impaired DC maturation and function. DC obtained in the presence of tumor microenvironment preferentially polarized IL-4(+) response. Moreover, T cells primed by these regulatory DC became anergic and behaved as suppressor/regulatory cells. These findings identify mucin secretion as a novel mechanism of tumor escape from immune surveillance and provide the basis for the generation of potentially tolerogenic DC.     

A novel tumor-associated pancreatic glycoprotein is internalized by human dendritic cells and induces their maturation

Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.     

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.     

Cooperative induction of a tolerogenic dendritic cell phenotype by cytokines secreted by pancreatic carcinoma cells

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123- myeloid DC (MDC)) or immunosuppressive T cell development (CD11c-,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-gamma. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-beta, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.     

Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity

Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-gamma by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.     

Role of antigen-processing machinery in the in vitro resistance of squamous cell carcinoma of the head and neck cells to recognition by CTL

Squamous cell carcinoma of the head and neck (SCCHN) cells are poorly recognized in vitro by CTL despite expressing the restricting HLA class I allele and the targeted tumor Ag (TA). Several lines of evidence indicate that the lack of SCCHN cell recognition by CTL reflects defects in targeted TA peptide presentation by HLA class I Ag to CTL because of Ag-processing machinery (APM) dysfunction. First, lack of recognition of SCCHN cells by CTL is associated with marked down-regulation of the IFN-gamma-inducible APM components low-m.w. protein 2, TAP1, TAP2, and tapasin. Second, SCCHN cell recognition by CTL is restored by pulsing cells with exogenous targeted TA peptide. Third, the restoration of CTL recognition following incubation of SCCHN cells with IFN-gamma is associated with a significant (p = 0.001) up-regulation of the APM components TAP1, TAP2, and tapasin. Lastly, and most conclusively, SCCHN cell recognition by CTL is restored by transfection with wild-type TAP1 cDNA. Our findings may explain the association between APM component down-regulation and poor clinical course of the disease in SCCHN. Furthermore, the regulatory nature of the APM defects in SCCHN cells suggests that intralesional administration of IFN-gamma may have a beneficial effect on the clinical course of the disease and on T cell-based immunotherapy of SCCHN by restoring SCCHN cell recognition by CTL.     

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.     

Plasmacytoid dendritic cells are activated by Toxoplasma gondii to present antigen and produce cytokines

Infection with the parasite Toxoplasma gondii leads to the induction of a Th1-type response dominated by IFN-gamma production and control of this pathogen. Cells of the innate immune system are essential in initiating this response both through the production of IL-12 as well as the presentation of parasite-derived Ags to MHC-restricted T cells. Although dendritic cells (DCs) have been implicated in these events, the contribution of individual DC populations remains unclear. Therefore, multiparameter flow cytometry was used to identify and characterize subsets of murine DCs during acute toxoplasmosis. This approach confirmed that infection leads to the expansion and activation of conventional DC (cDC) subsets. Unexpectedly, however, this analysis further revealed that plasmacytoid DCs are also expanded and that these cells up-regulate MHC class II and costimulatory molecules associated with their acquired ability to prime naive CD4(+) T cells. Furthermore, T. gondii-activated plasmacytoid DCs produce high levels of IL-12 and both plasmacytoid DC maturation and cytokine production are dependent on TLR11. Together these studies suggest that pDCs are a prominent DC subset involved in the initial stages of T. gondii infection, presenting parasite Ags and producing cytokines that are important for controlling infection.     

Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.     

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.     

Phagocytosis induces lysosome remodeling and regulated presentation of particulate antigens by activated dendritic cells

Immunization with particulate Ag effectively induces antitumor and antiviral T cell-mediated immunity. Immature dendritic cells (DCs) efficiently internalize, process, and present a variety of particulate Ags; however, previously published data suggest that both the uptake of soluble Ag through micropinocytosis, and phagocytosis of particulates are significantly curtailed in activated DC populations. In this study, we demonstrate that although macropinocytosis of soluble Ag is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags. Live cell imaging of activated DCs reveals that phagocytosis of particulates can result in cytoskeletal remodeling and perinuclear lysosome cluster disruption in a time-dependent manner. Interestingly, our results suggest that in activated DC populations, presentation of phagocytosed particulate Ags is dependent on the nature of the activation signal. These results provide direct evidence of functional heterogeneity in DC populations and contribute to the development of particle-based immunization strategies.     

Reovirus activates human dendritic cells to promote innate antitumor immunity

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-alpha, TNF-alpha, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-kappaB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-gamma production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-gamma) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus's direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.     

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.     

Provision of continuous maturation signaling to dendritic cells by RIG-I-stimulating cytosolic RNA synthesis of Sendai virus

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors, but the functional capacity of DC must be assessed in detail, especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant, providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol, resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors, or TLRs, do not show these functional features. Therefore, SeV-infected DC have the potential for DC-directed immunotherapy.     

Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines.

Two types of dendritic cells (DC) are circulating in human blood and can be identified by their differential expression of the myeloid Ag CD11c. In this study, we show that CD11c- peripheral blood (PB)-DC correspond to plasmacytoid DC of lymphoid tissue not only by their surface Ag expression profile but, more impressively, by their peculiar ultramorphology. We also demonstrate that CD11c- and CD11c+ DC differ in the quality of their response to and in their requirement for certain cytokines. Freshly isolated CD11c- cells depend on IL-3 for survival and use autocrine or exogenous TNF-alpha as maturation signal, leading to the appearance of a highly dendritic phenotype, the up-regulation and redistribution of MHC class II from lysosomal compartments to the plasma membrane, the increased expression of costimulatory molecules, and the switch from a high Ag-processing to a low Ag-processing/potent accessory cell mode. Surprisingly, IL-4 efficiently killed freshly isolated CD11c- PB-DC, but did not impair the viability of CD11c+ PB-DC and, together with GM-CSF, induced maturation of these cells. A direct functional comparison revealed that neo-Ag-modified and subsequently matured CD11c- but to a lesser extent CD11c+ DC were able to prime naive Ag-specific CD4+ T cells. Our findings show that two diverse DC types respond to certain T cell-derived cytokines in a differential manner and, thus, suggest that suppression or activation of functionally diverse DC types may be a novel mechanism for the regulation of the quantity and quality of immune responses.