Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1beta cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mphi), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mphi and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.     

The instructive role of dendritic cells on T-cell responses

Immune responses are initiated in the T-cell areas of secondary lymphoid organs where na?ve T lymphocytes encounter dendritic cells (DCs) that present antigens taken up in peripheral tissues. DCs represent the interface between the universe of foreign and tissue-specific antigens and T lymphocytes, and they are the key players in the regulation of cell-mediated immunity. We discuss how the nature of the DC maturation stimuli and the density and quality of DCs present in the T-cell areas of secondary lymphoid organs determine the magnitude and class of the T-cell response.     

Control of interleukin-18 secretion by dendritic cells: role of calcium influxes

Here we show that dendritic cells accumulate the precursor form of the leaderless secretory protein interleukin-18 (pro-interleukin-18) in the cell cytosol and in organelles co-fractionating with endolysosomes. Upon antigen specific contact with T lymphocytes, particulated pro-interleukin-18 decreases rapidly, and the cytokine appears extracellularly, suggesting that exocytosis of pro-interleukin-18-containing organelles is induced. Exocytosis of secretory lysosomes is modulated by calcium: in agreement with this, calcium influx results in secretion of pro-interleukin-18. In turn, pro-interleukin-18 secretion induced by T cells is prevented by the calcium channel blocker nifedipine. Our results demonstrate a novel, calcium-mediated mechanism of post-translational regulation of secretion for interleukin-18, that allows a fast release of the cytokine.     

Immune responses in hepatitis C virus infection: the role of dendritic cells

Cellular immune responses are critical for the clearance of hepatitis C virus. Persistent infection results from a narrow and weak cellular immune response, in direct contrast to the broad, strong response associated with viral clearance in acute infection. The presence of dendritic cells in the liver facilitates presentation of viral antigens to both CD4+ and CD8+ T cell populations. Exploiting the potent antigen presentation capability of dendritic cells for immunotherapy of chronic hepatitis C is attractive; however, infection or transfection of segments or the entire hepatitis C virus genome appears to impair the allostimulation capacity of dendritic cells. If dendritic cell immunotherapy for hepatitis C virus infection is to become a reality, the mechanism behind the defective allostimulatory capacity needs to be deciphered.     

Killer dendritic cells and their potential role in immunotherapy

Tumor immunosurveillance is mediated by innate and adaptive components of cellular immunity. A complex network of cellular interactions is needed to elicit protective antitumoral CD4+and CD8+T cell responses. Thereby dendritic cells (DCs) play a central role as professional antigen presenting cells (APCs) that take up antigens, process, and present them to prime na?ve T cells. Recognition and lysis of tumor cells has been attributed to innate effectors such as natural killer (NK), NKT and gammadeltaT cells. Recently, novel subsets of cytotoxic DCs, called "killer DCs" (KDCs), have been reported in rodents and humans. Killer dendritic cells could directly link innate and adaptive immunity. This review aims at comparing the different KDC populations, their phenotypes, killer function, and their potential application for anticancer immunotherapy.     

The role of dendritic cells in the innate immune system

Dendritic cells (DCs) are bone-marrow-derived leucocytes that are specialised antigen-presenting cells capable of stimulating a primary T-lymphocyte response to specific antigen. In this chapter we discuss the role DCs play in the innate response acting as a critical link with the adaptive response and the influence of the innate response on dendritic cells.     

Immunogenicity of viable tumor cells

Summary The immunogenicity of KMT-17 fibrosarcoma cells which had been xenogenized by infection with FV was compared to that of KMT-17 cells which had been admixed with BCG. We report here that 10⁵ and 10⁶ KMT-17 cells also grew progressively to kill rats, but when 10⁵ KMT-17 cells were administered with BCG the tumor cells did not grow in the majority of rats. The strength of immunogenicity (ETD₅₀), as measured by the number of immunizing cells required for a suppression of growth of 10⁷ KMT-17 cells in 50% of the rats, was 2.1×10³ for FV-KMT-17 and 36.3×10³ for BCG+KMT-17. The tumor cell dose (LTD₅₀) which was required to kill 50% of the rats immunized with 10⁵ FV-KMT-17 was more than 10,000 times higher than that found in normal rats, whereas the number of tumor cells required to kill 50% of the rats immunized with the same number of BCG+KMT-17 was only 3,680 times higher than the amount found in normal rats. Thus the immunogenicity of FV-KMT-17 is much stronger than that of BCG+KMT-17.The difference in immunogenicity between the two vaccines was also observed in the tumor-neutralizing activities of spleen cells obtained from rats which had been immunized with both vaccines, as measured by a Winn assay. Moreover, the antitumor activity of spleen cells from rats immunized with FV-KMT-17 was concentrated in the carrageenan-resistant and plastic nonadherent cells, while that of spleen cells from rats immunized with BCG+KMT-17 was observed in carrageenan-sensitive and plastic adherent cells as well as in nonadherent cells. The involvement of different effector cells indicates that different mechanisms operate in the antitumor resistance in rats immunized with either FV-KMT-17 or BCG+KMT-17. Abbreviations used: FV, Friend leukemia virus; FV-KMT-17, Friend leukemia virus infected KMT-17 cells; EDT₅₀, a 50% effective tumor dose; LTD₅₀, a 50% lethal tumor dose     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

Fc gamma RIIB regulates nasal and oral tolerance: a role for dendritic cells

Mucosal tolerance prevents the body from eliciting productive immune responses against harmless Ags that enter the body via the mucosae, and is mediated by the induction of regulatory T cells that differentiate in the mucosa-draining lymph nodes (LN) under defined conditions of Ag presentation. In this study, we show that mice deficient in FcgammaRIIB failed to develop mucosal tolerance to OVA, and demonstrate in vitro and in vivo a critical role for this receptor in modulating the Ag-presenting capacity of dendritic cells (DC). In vitro it was shown that absence of FcgammaRIIB under tolerogenic conditions led to increased IgG-induced release of inflammatory cytokines such as MCP-1, TNF-alpha, and IL-6 by bone marrow-derived DC, and increased their expression of costimulatory molecules, resulting in an altered immunogenic T cell response associated with increased IL-2 and IFN-gamma secretion. In vivo we could show enhanced LN-DC activation and increased numbers of Ag-specific IFN-gamma-producing T cells when FcgammaRIIB(-/-) mice were treated with OVA via the nasal mucosa, inferring that DC modulation by FcgammaRIIB directed the phenotype of the T cell response. Adoptive transfer of CD4(+) T cells from the spleen of FcgammaRIIB(-/-) mice to naive acceptor mice demonstrated that OVA-responding T cells failed to differentiate into regulatory T cells, explaining the lack of tolerance in these mice. Our findings demonstrate that signaling via FcgammaRIIB on DC, initiated by local IgG in the mucosa-draining LN, down-regulates DC activation induced by nasally applied Ag, resulting in those defined conditions of Ag presentation that lead to Tr induction and tolerance.     

The role of dendritic cells, B cells, and M cells in gut-oriented immune responses

Although induction of T cell responses to fed Ag (oral tolerance) is thought to happen within the organized lymphoid tissue of the gut, we found that mice lacking Peyer's patches, B cells, and the specialized Ag-handling M cells had no defect in the induction of T cell responses to fed Ag, whether assayed in vitro by T cell proliferation or cytokine production, or in vivo by delayed-type hypersensitivity or bystander suppression against mycobacterial Ags in CFA. Feeding of Ag had a major influence on dendritic cells from fed wild-type or muMT mice, such that these APCs were able to elicit a different class of response from naive T cells in vitro. These results suggest that systemic immune responses to soluble oral Ags do not require an organized gut-associated lymphoid tissue but are most likely induced by gut-conditioned dendritic cells that function both to initiate the gut-oriented response and to impart the characteristic features that discriminate it from responses induced parenterally.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

The role of dendritic cells in the pathogenesis of systemic lupus erythematosus

The etiology of the autoimmune disease systemic lupus erythematosus is not known, but aberrant apoptosis and/or insufficient clearance of apoptotic material have been assigned a pivotal role. During apoptosis, nucleosomes and several endogenous danger-associated molecular patterns are incorporated in blebs. Recent data indicate that apoptotic blebs induce maturation of myeloid dendritic cells, resulting in IL-17 production by T cells. In this review we summarize current knowledge on the role of dendritic cells in the pathogenesis of systemic lupus erythematosus with special emphasis on the uptake of apoptotic blebs by dendritic cells, and the subsequent induction of Th17 cells.     

Immunogenicity of human embryonic stem cells

Human embryonic stem cells (HESC) are pluripotent stem cells isolated from the inner cell mass of human blastocysts. With the first successful culturing of HESC, a new era of regenerative medicine was born. HESC can differentiate into almost any cell type and, in the future, might replace solid organ transplantation and even be used to treat progressive degenerative diseases such as Parkinson’s disease. Although this sounds promising, certain obstacles remain with regard to their clinical use, such as culturing HESC under well-defined conditions without exposure to animal proteins, the risk of teratoma development and finally the avoidance of immune rejection. In this review, we discuss the immunological properties of HESC and various strategic solutions to circumvent immune rejection, such as stem cell banking, somatic cell nuclear transfer and the induction of tolerance by co-stimulation blockade and mixed chimerism.     

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1–4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.     

New insight into the role of dendritic cells in malaria immune pathogenesis

The mechanism by which the host develops protective immunity to malaria remains poorly understood. Dendritic cells (DCs) are central to the initiation and regulation of the adaptive immune response. Modulation of DC function might enable Plasmodium to evade the immune system. Millington et al. propose one mechanism by which malaria inhibits DC-T-cell interactions without interfering directly with T-cell receptor engagement. The consequence is a decrease in the co-stimulation required to develop an effective immune response.     

Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.     

Reciprocal activating interaction between dendritic cells and pamidronate-stimulated gammadelta T cells: role of CD86 and inflammatory cytokines

We investigated the interactions between human monocyte-derived dendritic cells (DCs) and Ag-activated circulating TCR-gammadelta-expressing lymphocytes (Vdelta2). Coculture of immature DCs (iDCs) with peripheral blood Vdelta2 T cells activated with either pyrophosphomonoesters (isopentenyl pyrophosphate; IPP) or aminobiphosphonates (pamidronate; PAM) led to a significant up-modulation of CD86 and MHC class I molecules and to the acquisition of functional features typical of activated DCs. DC activation induced by both IPP- and PAM-stimulated gammadelta T cells was mostly mediated by TNF-alpha and IFN-gamma secreted by activated lymphocytes. However, the effect of PAM-activated gammadelta T cells, but not that of IPP-activated cells, required cell-to-cell contact. Reciprocally, activation of Vdelta2 T cells by PAM, but not by IPP, was dependent on cell contact with iDCs. In fact, when PAM-stimulated DC-gammadelta T cell cocultures were separated by a semipermeable membrane or treated with blocking anti-CD86 Abs, induction of CD25 and CD69 as well as IFN-gamma and TNF-alpha secretion by Vdelta2 cells were strongly reduced. These results demonstrate for the first time a bidirectional activating interaction between iDCs and PAM-stimulated gammadelta T lymphocytes, thus suggesting a potential adjuvant role of this early cross-talk in the therapeutic activity of aminobiphosphonate drugs.     

Cutting edge: Th2 response induction by dendritic cells: a role for CD40.

We investigated the influence of dendritic cell (DC) CD40 expression on Th2 and Th1 development by in vivo transfer of Ag-pulsed bone marrow-derived DC generated from wild-type (WT) or CD40(-/-) mice. Contrary to expectation, CD40(-/-) DC primed with Ag that inherently induce a Th2 response (soluble egg Ag from Schistosoma mansoni) failed to induce a Th2 response or any compensatory Th1 response, whereas CD40(-/-)DC primed with Ag that inherently induce a Th1 response (Propionibacterium acnes) generated a competent Th1 response. Thus, DC expression of CD40 is a prerequisite for initiation of Th2, but not Th1, responses by these Ag. Consistent with this, CD154(-/-) mice, unlike WT mice, failed to mount a Th2 response when directly injected with schistosome eggs but mounted a normal Th1 response after challenge with P. acnes. CD40-CD154 interaction can therefore play a major role in Th2 response induction.