Immunoglobulin isotype regulation by antigen-presenting cells in vivo

The isotype and magnitude of the B cell response clearly depends on the in vivo activation of T helper (Th) cells which secrete different lymphokines. Since Th are activated by the presentation of the antigen on specialized cells, we wished to test whether the nature of the antigen-presenting cells (APC) influences the isotypic profile of the humoral response. Data are presented showing that antigen-pulsed dendritic cells (DC) and peritoneal macrophages induce the synthesis of specific antibodies when injected in syngeneic animals. By contrast, a single injection of antigen-pulsed resting B cells does not prime the mice in vivo. Moreover, the injection of antigen-pulsed DC induces the synthesis of specific IgG2a and IgG1 antibodies, whereas peritoneal macrophages favor the production of IgG1 and IgE antibodies specific for the antigen. These data show that the isotype and the amplitude of the B cell response can be regulated by the nature of the APC, and indirectly suggest that Th cell differentiation is controlled at the level of antigen presentation.     

The secretory IFN-gamma response of single CD4 memory cells after activation on different antigen presenting cell types

It is unknown to what extent the heterogeneity of antigen presenting cells (APC) influences the IFN-gamma response of CD4 memory cells. We re-stimulated DO11.10 T cell receptor (TCR)-transgenic cells and wild-type CD4 memory cells with OVA-peptide 323-339 presented on purified dendritic cells (DC), macrophages, and B cells. Using IFN-gamma ELISPOT assays, we measured the number of cytokine producing T cells and the amount of cytokine produced by individual T cells at different time points after antigen encounter. The data showed that, when CD4 cells recognized antigen on DC, the induction of cytokine production was accelerated compared to macrophages and B cells. In contrast, the per-cell cytokine productivity was independent of the type of APC by which the T cells were re-stimulated. Moreover, the peptide concentration required for CD4 cell activation was comparable for the different APC. The data suggest that DC induce cytokine production in memory cells with accelerated activation kinetics, whereas 24 h of antigen stimulation on DC, macrophages, and B cells results in comparable levels of T cell activation. These data have implications for the understanding of T cell memory responses when T cells re-encounter antigen on different APC as well as for the monitoring of memory T cell responses ex vivo.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1 / Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extra corporeally with antigen, induced the development of cells secreting IL-2, IFN-γ and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-γ. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to co-stimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/ B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-γ-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Role of MHC class II expressing CD4+ T cells in proteolipid protein(91-110)-induced EAE in HLA-DR3 transgenic mice

MHC class II molecules play a central role in the control of adaptive immune responses through selection of the CD4(+) T cell repertoire in the thymus and antigen presentation in the periphery. Inherited susceptibility to autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and IDDM are associated with particular MHC class II alleles. Advent of HLA transgenic mice has helped us in deciphering the role of particular HLA DR and DQ class II molecules in human autoimmune diseases. In mice, the expression of class II is restricted to professional antigen-presenting cells (APC). However, in humans, class II is also expressed on T cells, unlike murine T cells. We have developed new humanized HLA class II transgenic mice expressing class II molecules not only on APC but also on a subset of CD4(+) T cells. The expression of class II on CD4(+) T cells is inducible, and class II(+) CD4(+) T cells can present antigen in the absence of APC. Further, using EAE, a well-established animal model of MS, we tested the functional significance of these class II(+) CD4(+) T cells. DR3.AEo transgenic mice were susceptible to proteolipid protein(91-110)-induced EAE and showed CNS pathology accompanied by widespread inflammation and demyelination seen in human MS patients, suggesting a role for class II(+) CD4(+) T cells in the pathogenesis.     

Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor

We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1-dominated B cell and CD4(+) T cell responses. In this study we show that vaccination with DC pre-pulsed ex vivo with CT-conjugated OVA (OVA-CT) gives rise to OVA-specific CD8(+) T cells that produce IFN-gamma and are cytotoxic for OVA-expressing E.G7 tumor cells both in vitro and in vivo. The induction of specific CD8(+) CTL by OVA-CT-treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC. Vaccination of mice with OVA-CT-pulsed DC resulted in rejection of already established MHC class I-positive, MHC class II-negative, OVA-expressing E.G7 tumors in an antigen-specific, CD8(+) T cell-dependent fashion and was associated with high numbers of tumor-infiltrating CD8(+) T cells. Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor-binding B subunit and induced strong activation-maturation signals through the biologically active A subunit. These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.     

Wortmannin,an inhibitor of phospholipase D activation,selectively blocks major histocompatibility complex class II-restricted antigen presentation

Wortmannin, a fungal metabolite, is a specific inhibitor of phospholipase D (PLD) activation. Presentation of defined exogenous soluble proteins to specific T cell hybridomas was studied by using different antigen-presenting cells (APC): IA-positive peritoneal macrophages (MΦ?), B lymphoma cells (B) or dendritic cells (DC). Major histocompatibility complex class II-restricted antigen presentation by MΦ? was blocked when cells were pretreated with wortmannin. However, when cells constitutively expressing IA molecules (B. DC) were used as APC, no inhibition was observed. Additionally, MHC class I antigen presentation was not impaired by wortmannin. Moreover, wortmannin does not block either peptide presentation or presentation to autoreactive T cells. This effect was time and dose dependent and occurred at the level of intracellular handling of the antigen. Mainly because it was not a toxic inhibition, it was reversible with time and neither antigen uptake and catabolism, nor IA synthesis were affected. Because MΦ, but not B or DC, express PLD activity and only the former were blocked by wortmannin in antigen presentation, our results strongly suggest that a differential antigen-processing pathway exists in these disparate APC, which could be based essentially on a wortmannin-sensitive, PLD-dependent step present in MΦ but absent and/or unnecessary in both B lymphoma cells and DC.     

B cells can prime naive CD4+ T cells in vivo in the absence of other professional antigen-presenting cells in a CD154-CD40-dependent manner

The role of B cells as APC is well established. However, their ability to prime naive T cells in vivo has been difficult to examine because of the presence of dendritic cells. The current studies were undertaken to examine this issue in a model of adoptive transfer of antigen-specific B cells and T cells into histoincompatible Rag2(-/-) mice. By means of this system, we were able to demonstrate that antigen-specific B cells are competent APC for naive CD4(+) T cells specific for the same antigen. In vivo antigen presentation resulted in expansion of both CD4(+) T cells and B cells. The antigen-presenting function of the transferred B cells was dependent on the CD154-CD40 interaction, as transfer of CD154-deficient antigen-specific CD4(+) T cells or CD40-deficient B cells failed to induce T and B cell expansion in response to immunization. These results indicate that antigen-specific B cells have the capacity to induce primary T cell responses in the absence of other competent APC.     

Freshly isolated mouse 4F7+ splenic dendritic cells process and present exogenous antigens to T cells

The antibody 4F7 was reported to recognize an epitope expressed on dendritic cells (DC) from various tissues. To study the ability of splenic 4F7⁺ dendritic cells to process antigen for presentation to CD4⁺ T cells, DC were enriched using a separation procedure avoiding overnight culture which could lead to an altered phenotype. These DC were used as antigen-presenting cells (APC) in stimulation cultures of major histocompatibility complex class II-restricted T cells. It was found that they induce antigen-dependent lymphokine production by T cells and therefore could present exogenous antigens. These processing takes place intracellularly, because fixation abrogates presentation to T cells. Moreover, antigen presentation needs intracellular processing within endo- or lysosomes as chloroquine-treatment prevents T cell activation. Titration of APC numbers revealed that contaminating APC most likely did not account for antigen-specific T cell activation by DC. No evidence was found for release of antigenic peptides or for partial antigen processing possibly done by cell surface located enzymes on DC. In conclusion, these results indicate that freshly enriched DC are able to process antigens similarly to other APC.     

Generation of CD4 and CD8 T Lymphocyte Responses by Dendritic Cells Armed with PSA/Anti-PSA (Antigen/Antibody) Complexes

Dendritic cells (DC) acquire antigens through a number of cell surface structures including receptors for the Fc portion of immunoglobulins and mannose. Little is known about the effects of antigen uptake via these receptors on antigen processing and presentation. We compared the capacity of DC to generate CD4(+) and CD8(+) T cell responses after exposure to prostate-specific antigen (PSA) alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47). Autologous CD3(+) T cells were added to monocyte-derived immature DC that had been cultured with GM-CSF/IL-4 for 4 days, exposed to antigen, and matured with CD40L or TNFalpha/IFN-alpha. After several rounds of stimulation, T cell responses were assessed by intracellular IFN-gamma production using flow cytometry. Both CD4(+) and CD8(+) T cell responses were observed after stimulation with DC exposed to the PSA/anti-PSA complexes, whereas CD4(+) predominated over CD8(+) T cell responses after stimulation with PSA-armed DC or PSA-m. These CD8(+) T cells responded when rechallenged with DC pulsed with HLA allele-restricted PSA peptides. These results indicate that PSA and PSA-m are processed primarily through pathways that favor HLA Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived DC. These findings have potential applications in designing more effective cancer vaccines for prostate cancer.     

Dendritic cells, but not macrophages or B cells, activate major histocompatibility complex class II-restricted CD4+ T cells upon immune-complex uptake in vivo

Professional antigen-presenting cells (APC) are able to process and present exogenous antigen leading to the activation of T cells. Antigen-immunoglobulin (Ig)G complexes (IC) are much more efficiently processed and presented than soluble antigen. Dendritic cells (DC) are known for their ability to take up and process immune complex (IC) via FcgammaR, and they have been shown to play a crucial role in IC-processing onto major histocompatibility complex (MHC) class I as they contain a specialized cross-presenting transport system required for MHC class I antigen-processing. However, the MHC class II-antigen-processing pathway is distinct. Therefore various other professional APC, like macrophages and B cells, all displaying FcgammaR, are thought to present IC-delivered antigen in MHC class II. Nonetheless, the relative contribution of these APC in IC-facilitated antigen-presentation for MHC class II in vivo is not known. Here we show that, in mice, both macrophages and DC, but not B cells, efficiently capture IC. However, only DC, but not macrophages, efficiently activate antigen-specific MHC class II restricted CD4(+) T cells. These results indicate that mainly DC and not other professional APC, despite expressing FcgammaR and MHC class II, contribute significantly to IC-facilitated T cell activation in vivo under steady-state conditions.     

Dendritic cells,T cells and their interaction in rheumatoid arthritis

Dendritic cells (DCs) are the key professional antigen-presenting cells which bridge innate and adaptive immune responses, inducing the priming and differentiation of naive to effector CD4⁺ T cells, the cross-priming of CD8⁺ T cells and the promotion of B cell antibody responses. DCs also play a critical role in the maintenance of immune homeostasis and tolerance. DC–T cell interactions underpin the generation of an autoimmune response in rheumatoid arthritis (RA). Here we describe the function of DCs and review evidence for DC and T cell involvement in RA pathogenesis, in particular through the presentation of self-peptide by DCs that triggers differentiation and activation of autoreactive T cells. Finally, we discuss the emerging field of targeting the DC–T cell interaction for antigen-specific immunotherapy of RA.     

HLA-DM negatively regulates HLA-DR4-restricted collagen pathogenic peptide presentation and T cell recognition

Rheumatoid arthritis, an autoimmune disease, is significantly associated with the HLA class II allele HLA-DR4. While the etiology of rheumatoid arthritis remains unknown, type II collagen (CII) is a candidate autoantigen. An immunodominant pathogenic epitope from this autoantigen, CII(261-273), which binds to HLA-DR4 and activates CD4+ T cells, has been identified. The non-classical class II antigen, HLA-DM, is also a key component of class II antigen presentation pathways influencing peptide presentation by HLA-DR molecules expressed on professional antigen-presenting cells (APC). Here, we investigated whether the HLA-DR4-restricted presentation of the pathogenic CII(261-273) epitope was regulated by HLA-DM expression in APC. We show that APC lacking HLA-DM efficiently display the CII(261-273) peptide/epitope to activate CD4+ T cells, and that presentation of this peptide is modulated dependent on the level of HLA-DM expression in APC. Mechanistic studies demonstrated that the CII(261-273) peptide is internalized by APC and edited by HLA-DM molecules in the recycling pathway, inhibiting peptide presentation and T cell recognition. These findings suggest that HLA-DM expression in APC controls class II-mediated CII(261-273) peptide/epitope presentation and regulates CD4+ T cell responses to this self epitope, thus potentially influencing CII-dependent autoimmunity.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells

Cell-based immunotherapy, in which antigen-loaded antigen-presenting cells (APC) are used to elicit T cell responses, has become part of the search for alternative cancer and infectious disease treatments. Here, we report on the feasibility of using mRNA-electroporated CD40-activated B cells (CD40-B cells) as alternative APC for the ex vivo induction of antigen-specific CD8(+) T cell responses. The potential of CD40-B cells as APC is reflected in their phenotypic analysis, showing a polyclonal, strongly activated B cell population with high expression of MHC and co-stimulatory molecules. Flow cytometric analysis of EGFP expression 24 h after EGFP mRNA-electroporation showed that CD40-B cells can be RNA transfected with high gene transfer efficiency. No difference in transfection efficiency or postelectroporation viability was observed between CD40-B cells and monocyte-derived dendritic cells (DC). Our first series of experiments show clearly that peptide-pulsed CD40-B cells are able to (re)activate both CD8+ and CD4(+) T cells against influenza and cytomegalovirus (CMV) antigens. To demonstrate the ability of viral antigen mRNA-electroporated CD40-B cells to induce virus-specific CD8+ T cell responses, these antigen-loaded cells were co-cultured in vitro with autologous peripheral blood mononuclear cells (PBMC) for 7 days followed by analysis of T cell antigen-specificity. These experiments show that CD40-B cells electroporated with influenza M1 mRNA or with CMV pp65 mRNA are able to activate antigen-specific interferon (IFN)-gamma-producing CD8(+) T cells. These findings demonstrate that mRNA-electroporated CD40-B cells can be used as alternative APC for the induction of antigen-specific (memory) CD8(+) T cell responses, which might overcome some of the drawbacks inherent to DC immunotherapy protocols.     

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.     

Role of antigen-presenting cells in the polarized development of helper T cell subsets: evidence for differential cytokine production by Th0 cells in response to antigen presentation by B cells and macrophages

Immune challenges can elicit polarized responses skewed towards the development of T helper type 1 (Th1) or Th2 T cell subsets. To determine if distinct antigen-presenting cells (APC) populations might selectively influence Th subset development, we studied the role of two key APC populations, B cells and macrophages, in the differentiation of effector Th populations from naive precursor Th in vitro. Antigen (Ag)-specific, naive CD4⁺ T cells were enriched from a mouse strain, AND, bearing a transgenic α/β T cell receptor (TCR) encoding reactivity with pigeon cytochrome c peptide 88-104. Peptide Ag was used throughout these studies so that differences in the uptake and processing by the two APC populations would not influence the results. Both APC populations, activated B cells and bone marrow-derived macrophages, supported the development of effector Th having the capacity to secrete high levels of cytokines when restimulated. Regardless of APC population present during effector development, exogenous interferon-γ (IFN-γ) and interleukin-4 (IL-4) had dominant effects on Th subset development. Thus, with both APC populations, effector Th generated in the presence of IFN-γ acquired a Th1-type cytokine profile, Th generated with IL-4 acquired a Th2-type cytokine profile, and Th generated without IFN-γ or IL-4 acquired a Th0-type cytokine profile. B cells and macrophages also had equivalent APC function in the restimulation of Th1 and Th2-like effectors, since only minor differences in cytokine production were noted for these effector populations when restimulated with the two APC populations. However, in 8 of 19 experiments, the Th0-like effector population generated in the presence of IL-2 differentially responded to restimulation with B cells and macrophages, secreting significantly more IFN-γ when restimulated with B cells, and significantly more IL-4 when restimulated with macrophages. We also found that Th effector populations recultured in IFN-γ or IL-4 assumed a more Th1 or Th2-like phenotype, respectively, regardless of their initial cytokine profile. We conclude that through a subtle capacity to skew cytokine production by a Th0 subset, different APC may selectively influence Th subset development under conditions of prolonged or chronic stimulation in an autocrine fashion.     

Helicobacter pylori-infected macrophages induce Th17 cell differentiation

Th17 cells represent a novel subset of CD4(+) T cells, which is associated with chronic inflammation. The present study evaluated Th17 cell responses to Helicobacter pylori infection in mouse model and CD4(+) T cell differentiation in response to H. pylori-infected macrophages. Th17 cells were observed in the H. pylori-infected gastric tissue. Co-culture of CD4(+) T cells with H. pylori-infected macrophages elevated IL-17 and IFN-γ secretion, up-regulated retinoid-related orphan receptor gamma t (RORγt) and T box expressed in T cells (T-bet) expression and increased the numbers of Th17 and Th1 cells. The expression of CD40, CD80, and CD86 and the secretion of IL-6, TGF-β1, IL-23, and CCL20 were significantly increased in H. pylori-stimulated macrophages. NF-κB pathway participated in the production of IL-6, IL-23, and CCL20 from macrophages in response to H. pylori, and inhibition of NF-κB pathway of macrophages resulted in less Th17 cell differentiation. Taken together, these results suggest that H. pylori induces Th17 cell differentiation via infected macrophages.     

Relative efficiency of microglia, astrocytes, dendritic cells and B cells in naive CD4+ T cell priming and Th1/Th2 cell restimulation.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.     

Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4(+) T cells

Successful antigen presentation by antigen-presenting cells is governed by a number of factors including the efficiency of antigen capture by cell-surface receptors, targeting to compartments of antigen processing, surface expression of MHC II-peptide complexes and presence of co-stimulatory signals. Ganglioside GM1 is an important component of membrane glycosphingolipids, and has been implicated in cell differentiation, apoptosis and signal transduction pathways. Using the B subunit of Escherichia coli enterotoxin (EtxB), a potent immunogen that binds GM1 with high affinity, and a non-binding mutant of EtxB, EtxB(G33D), we demonstrate that GM1 is intimately involved in several aspects of antigen presentation. Thus, GM1-mediated presentation of EtxB by B cells and CD11c(+) dendritic cells (DC) significantly enhanced the proliferation and cytokine expression of EtxB-specific CD4(+) T cells. Investigation regarding potential mechanisms revealed that EtxB binding directly augments the expression of MHC class II on B cells, and fractionation of B cells demonstrated that EtxB binding to GM1 results in rapid internalization and targeting to class II-rich compartments. GM1-mediated uptake of antigens and access to class II compartments in B cells can be exploited to significantly enhance the presentation of ovalbumin-conjugated to EtxB. These results demonstrate that GM1 can play an important role in antigen presentation via the MHC II pathway.