Soluble Protein Antigens Research Articles

Visualizing the T-cell response elicited by oral administration of soluble protein antigen.

Oral administration of soluble protein antigen induces tolerance, while particulate antigens encountered in the intestine provoke active immunity. Although the events that lead to these distinct outcomes are not yet fully characterized, they may reflect differences at the antigen-presenting cell (APC) level. The role of dendritic cells (DC) in regulating responses at mucosal sites has remained largely undefined because of the low frequency of DC in mucosal-associated tissues. In this study we have used the growth factor Flt3-ligand (Flt3L) to expand DC populations in vivo, in combination with an adoptive transfer system, in order to track antigen-specific T cells during oral tolerance induction. We observed rapid T-cell activation, localized particularly in the mucosal tissues, within hours after feeding the soluble protein antigen, ovalbumin (OVA). The response was enhanced in Flt3L-treated mice, indicating an important role for DC during the inductive phase of tolerance.

Novel aggregate structure adjuvants modulate lymphocyte proliferation and Th1 and Th2 cytokine profiles in ovalbumin immunized mice

Cytokines are important mediators of effector lymphoid cell function during an immune response. The principal cytokine producers are the T helper (Th) cells and macrophages. Vaccine strategies need to take into account the balance of Th (Th1/Th2) cytokines they induce. Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. The use of adjuvants has been shown to activate differentially Th1 and Th2 subsets. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment Th1 or Th2 cytokine responses when administered with a soluble protein antigen. Of the three formulations, RAM1 was found to induce predominantly Th1 cytokines; the levels of which were substantially higher than those induced by reference control adjuvants. It was also found that at a late post-vaccinated period, RAM1 can stimulate Th2 responses. In contrast, RAM2 and RAM3 induced cytokine profiles typically associated with Th2 responses.

Unique Features of Dendritic Cells in IFN-γ Transgenic Mice: Relevance to Cancer Development and Therapeutic Implications

Although induction of interferon gamma (IFN-γ) and activation of antigen presenting dendritic cells (DCs) are two vital events during an immune response, the impact of endogenous IFN-γ on DC function has yet to be clarified. The phenotype and function of DCs isolated from mice with high (IFN-γ-transgenic mouse [Tg]) and undetectable levels of circulating IFN-γ (normal mice [NM]) were therefore compared. The capacity to stimulate allogenic (p < 0.05) and antigen-specific T lymphocytes (p < 0.05), as well as the ability to produce IL-12 (p < 0.05) and to process soluble protein antigens (p < 0.05) was found to be significantly higher in DCs from the Tg mice compared to the NM case. The presence of activated DCs in a microenvironment of endogenous IFN-γ suggests that the IFN-γ-Tg mouse is a suitable animal model to study cancer immunotherapy in vivo.

Suppression of antigen-specific T- and B-cell responses by intranasal or oral administration of recombinant Bet v 1, the major birch pollen allergen, in a murine model of type I allergy

Background: Mucosal (nasal or oral) administration of soluble protein antigens induces a state of antigen-specific immunologic hyporesponsiveness. Several studies have shown that induction of mucosal tolerance can prevent the onset or reduce the severity of certain T H1 -mediated experimental autoimmune diseases. Only a few studies describe similar results for type I allergies, which are caused by excessive T H2 cell activities. Objective: We sought to investigate whether mucosal tolerance induction would also be efficient in preventing an allergic type I immune response. Methods: A murine model of inhalative type I allergy, leading to sensitization to birch pollen and its major allergen Bet v 1 in BALB/c mice, was used. Recombinant Bet v 1 was nasally or orally applied in low doses before sensitization. At the time of death, immediate-type skin tests were performed. Blood was taken, and serum was used for measurement of allergen-specific antibodies. Spleen cell cultures were performed to determine cytokine production (IL-4, IL-5, IL-10, and IFN-γ), as well as levels of TGF-β mRNA. Results: Both nasal and oral administration of minimal doses of recombinant Bet v 1 before aerosol sensitization with birch pollen suppressed the allergen-specific antibody production of all isotypes. Consequently, the in vivo type I skin test responses to the allergen were negative in the tolerized, in contrast to the sensitized, group. Moreover, allergen-specific lymphoproliferative responses and cytokine production in vitro (ie, IFN-γ, IL-4, IL-5, and IL-10) were markedly reduced. In contrast, expression of TGF-β mRNA was markedly increased in spleen cells from nasally tolerized animals, indicating regulatory mechanisms for tolerance induction. Conclusion: We conclude from the present study that nasal, as well as oral, administration of recombinant allergen is an effective way to prevent allergen-specific T- and B-cell responses in a T H2 model. (J Allergy Clin Immunol 1999;103:1202-10.)

The adjuvant combination monophosphoryl lipid A and QS21 switches T cell responses induced with a soluble recombinant HIV protein from Th2 to Th1

The induction of protective immunity with recombinant vaccines is dependent on the identification of adjuvant or delivery systems that can augment immune responses, especially cellular immune responses, to soluble protein antigen. In this study we demonstrate that an adjuvant formulation comprising QS21, a purified form of saponin and 3 d-monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS), enhances cellular and humoral immune responses to a recombinant HIV protein. Analysis of cytokine secretion by antigen-specific T-cells from the spleen demonstrated that QS21 augmented Th1 and Th2 responses, whereas addition of 3 d-MPL resulted in preferential induction of type 1 T-cells. Furthermore, analysis of the subclass of the IgG antibody in the serum in mice immunized with gp120 with the combined adjuvant formulation confirmed the selective activation of Th1 cells in vivo. 3 d-MPL was found to enhance B7-1 expression and IL-12 production by macrophages, which are known to be involved in the LPS-induced enhancement of Th1 responses. Thus 3 d-MPL appears to act as an adjuvant, without the toxicity associated with LPS, by controlled and selective potentiating effects on antigen presentation and T-cell activation.

IN VITRO ANTIGEN PRESENTING CELL FUNCTION IS DEFICIENT IN STABLE LUNG TRANSPLANT RECIPIENTS.

143 In vitro peripheral blood mononuclear cells (PBMCs) of stable transplant recipients respond to polyclonal activation, but not to soluble protein antigens (ag), which have to be processed and presented by self antigen presenting cells (APCs). The aim of the present study was to assess APC function in stable lung transplant (LT) recipients and to elucidate whether defective response to soluble ag is due to T cell or APC dysfunction. We obtained PBMCs from 29 stable LT recipients with normal pulmonary function a mean of 3 years after transplantation. Patients received a triple CsA-based immunosuppressive regimen. We also obtained PBMCs from 29 healthy volunteers, which served as controls. LT recipients' and control PBMCs were stimulated with a) allogeneic PBMCs, b) anti-CD3 monoclonal antibody (mAb), c) tetanus toxoid (TT) ± anti-CD28 mAb. APC function of patients' PBMCs was tested in a mixed leukocyte reaction (MLR) using patients' PBMCs as stimulators and control PBMCs as responders. The readout for these experiments was IL-2 production in culture supernatants. APC expression of MHC class II, B7.1, B7.2 and CD40 molecules was determined by flow cytometry. Finally, APC production of IL-10 and IL-12 in response to stimulation with CD40-L transfected fibroblasts was assessed. Patients' T cells produced normal amounts of IL-2 in response to allogeneic PBMCs (500 ± 259 vs 397 ± 236 pg/ml in controls; P = NS) and anti-CD3 mAb (2685 ± 1380 vs 1817 ± 1380 pg/ml in controls; P = NS). Response to TT was completely blunted (203 ± 270 vs 604 ± 574 pg/ml in controls; P < 0.0001) and could not be restored by providing costimulation with anti-CD28 mAb. Patients' APCs were poor stimulators in MLR and this was correlated with a drastic downregulation of MHC class II expression (median fluorescence intensity: 275 ± 66 vs 931 ± 163 in controls; P < 0.0001). Patients' APCs also produced significantly less IL-10 and IL-12 than control APCs subsequent to CD40 engagement. In conclusion, stable LT recipients' T cells cannot be activated in vitro by soluble ag such as TT. Deficient ag presentation by dysfunctional APCs probably contributes to this defect.

Evaluation of novel aggregate structures as adjuvants: composition, toxicity studies and humoral responses

Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. There are numerous pathogens for which there are no protective vaccines and since alum is the only adjuvant licensed for use in humans, there is a clear need for more effective adjuvant preparations. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment antigen-specific antibody responses when administered with a soluble protein antigen. All three adjuvants were shown to be nontoxic to mice and elicited antigen-specific antibody responses. Of the three formulations, RAM1 was found to induce the highest titers of antibody; these were substantially higher than those induced by reference control adjuvants. RAM1 elicited antibodies of the IgG1 and IgG2a subclasses indicating, indirectly, that this adjuvant can stimulate Th2 and Th1 type immunity.

Enteric viral infections of pigs and strategies for induction of mucosal immunity

This chapter discusses the enteric viral infections of pigs and strategies for the induction of mucosal immunity. Enteropathogenic viruses—such as transmissible gastroenteritis virus (TGEV) and rotavirus—replicate and induce lesions only in the gastrointestinal tract. The susceptible target cell is the villous enterocyte. Thus, active immunity against enteropathogenic viral infections depends on stimulation of local immune responses within the intestine. To date, only limited success has been achieved in the development of oral vaccines to prevent neonatal viral diarrheas, and commercial vaccines show limited efficacy in the field. Currently, only oral vaccines containing live replicating organisms have been highly effective in inducing mucosal immune responses, especially secretory, (S)IgA antibodies. The oral administration of soluble or killed antigens generally induces immunity of short duration or even systemic tolerance. Whether the problems encountered with oral administration of soluble protein antigens can be overcome by the use of improved mucosal adjuvants (such as muranyl dipeptide, immune stimulating complexes [ISCOMs], cholera or Escherichia coli enterotoxins, avridine, proteosomes, and cytokines] or new and novel delivery systems (liposomes, live recombinant vectors, microspheres, DNA plasmids, virus-like particles) requires further investigation, and specific examples are given in this chapter.

Generation of Functional Human Dendritic Cells From Adherent Peripheral Blood Monocytes by CD40 Ligation in the Absence of Granulocyte-Macrophage Colony-Stimulating Factor

AbstractRecently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide–specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor-α (TNF-α) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I–restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.

Generation of Functional Human Dendritic Cells From Adherent Peripheral Blood Monocytes by CD40 Ligation in the Absence of Granulocyte-Macrophage Colony-Stimulating Factor

Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide-specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I-restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.

Alterations in Cytokine Production Following Intraocular Injection of Soluble Protein Antigen: Impairment in IFN-γ and Induction of TGF-β and IL-4 Production

AbstractImmune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-γ production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-γ production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-γ production required IL-4. Interestingly, significant levels of TGF-β were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-β was produced primarily by the splenic CD4 and non-T cells and was of the TGF-β1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-β at several different (including the effector) phases during the response.

Acute and chronic phases of Toxoplasma gondii infection in mice modulate the host immune responses.

Murine antibody responses to soluble proteins are generally restricted to the immunoglobulin G1 (IgG1) isotype. When mice were infected with Toxoplasma gondii Beverley and concomitantly immunized with a soluble unrelated protein antigen, a modification in the isotypic distribution of antibodies directed against this nonparasite antigen was observed, with a preferential production of IgG2a. Interestingly, when mice were immunized with a soluble protein antigen during the chronic phase (day 40) of infection with T. gondii Beverley, a similar modification in the isotypic distribution of antiprotein antibodies was observed.

Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen.

Encapsulation of soluble protein antigens in liposomes was previously shown to result in processing of antigen via the major histocompatibility complex class I pathway, as evidenced by costaining of the trans-Golgi region of murine bone marrow-derived macrophages (BMs) by fluorophore-labeled liposomal antigen and by a trans-Golgi-specific fluorescent lipid. Evidence is presented here that free or liposome-encapsulated RTS,S, a particulate malaria antigen consisting of hepatitis B particles coexpressed with epitopes from the Plasmodium falciparum circumsporozoite protein, also was localized in the trans-Golgi after incubation with BMs, suggesting processing by the class I pathway. An in vivo cytotoxic T-lymphocyte (CTL) response was detected, however, only after immunization with RTS,S encapsulated in liposomes containing lipid A and not after immunization with free RTS,S or with RTS,S encapsulated in liposomes lacking lipid A. Therefore, intracellular delivery of antigen containing CTL epitopes to the Golgi of BMs does not necessarily result in a CTL response in vivo unless an additional adjuvant, such as liposomes containing lipid A, is utilized. Encapsulation of RTS,S in liposomes containing monophosphoryl lipid A (MPL) resulted in a dose-dependent enhancement of the NANP-specific immunoglobulin G (IgG) antibody response compared to that of free RTS,S. The IgG1 and IgG2a subclasses predominated after immunization with RTS,S encapsulated in liposomes containing MPL. These results demonstrate that encapsulation of a lipid-containing particulate antigen, such as RTS, S, in liposomes containing lipid A can enhance both humoral and cellular immune responses.

A novel concept in mucosal adjuvanticity: the CTA1-DD adjuvant is a B cell-targeted fusion protein that incorporates the enzymatically active cholera toxin A1 subunit.

A promising novel concept in mucosal adjuvant research is demonstrated here. The adjuvant and toxic effects of the cholera toxin (CT) have been successfully separated in a gene fusion protein, CTA1-DD. This protein consists of the ADP-ribosylating A1 subunit of CT linked to a synthetic analogue of protein A. The CTA1-DD protein was found to exert comparable adjuvant activity to that of CT after systemic as well as mucosal immunizations with soluble protein antigens, such as KLH or ovalbumin (OVA). However, contrary to CT it was completely non-toxic. The CTA1-DD approach to the construction of a potential vaccine adjuvant is unique and highly promising. Conceptually, the CTA1-DD fusion protein demonstrates that: (i) contrary to CT the CTA1-DD is a highly targeted adjuvant, directed to B cells and possibly other antigen-presenting cells; (ii) it is possible to introduce ADP-ribosyltransferase activity into cells via an alternative pathway to the GM1 receptor pathway used by CTB; (iii) the adjuvant effect of CTA1-DD, and possibly also of CT, depend on the enzymatic activity; and (iv) one possible mechanism, shared by CT, that may explain the adjuvant effect of CTA1-DD is its ability to induce expression of the costimulatory molecule CD86 on B cells.

Human Dermal Dendritic Cells Process and Present Soluble Protein Antigens

Recently, a novel type of dendritic antigen-presenting cell has been identified in the dermis of normal human and mouse skin. These dermal dendritic cells (DDC) occur in higher numbers than epidermal Langerhans cells, represent a distinct differentiation pathway of dendritic cells, and are as potent as Langerhans cells in the activation of superantigen specific T cells. As yet, nothing is known about their capacity to take up, process, and present soluble protein antigens. We used the model of tetanus toxoid (TT) driven T cell proliferation to address these questions. To test for active internalization of TT protein, gold labeled TT was incubated with Langerhans cells and DDC and could be traced to multivesicular endo-lysosomal compartments. DDC internalize TT through a receptor-mediated, clathrin-independent pathway, whereas Langerhans cells predominantly use macropinocytosis. To verify that DDC process TT by the exogenous pathway of antigen presentation, we pulsed DDC with TT protein or TT peptide after preincubation with chloroquine. Preincubation with chloroquine diminished the capacity of DDC to induce TT protein specific T cell proliferation (70-80%), but was not effective to suppress TT peptide induced T cell responses. DDC were as potent as Langerhans cells and 5-10 x more potent than plastic adherent monocytes in the presentation of TT to autologous resting T cells. Furthermore, as few as 50 DDC (stimulator:responder ratio of 1:1000) were able to induce a significant TT specific T cell proliferation. Because a subpopulation of DDC expresses low levels of CD1a, a phenotypic marker of Langerhans cells, sorting of CD1a positive and negative DDC was performed. On a per cell basis, CD1a positive and negative DDC were equally potent at mediating TT specific T cell proliferation. Thus, DDC are able to internalize, process, and present soluble protein antigens such as TT and may therefore play an important role in the regulation of skin immune responses.

Oral tolerance in humans: Evidence for active suppression as a mechanism for tolerance production to soluble protein antigens

Oral tolerance in humans: Evidence for active suppression as a mechanism for tolerance production to soluble protein antigens

Oral tolerance in humans: Duration of feeding alters the mechanisms of tolerance production to soluble protein antigens

Oral tolerance in humans: Duration of feeding alters the mechanisms of tolerance production to soluble protein antigens

Mucosal immune responses to fed soluble protein antigen in t cell receptor (TCR) transgenic mice

Background: Oral tolerance defines a state of systemic hyporesponsiveness following oral antigen administration, however, the mechanisms underlying the induction of suppressive T cells are not well understood. We investigated kinetics and dose-dependent features of mucosal and peripheral immune responses following oral antigen application in a TCR transgenic mouse model. Methods: Ovalbumin (OVA)-TCR transgenic mice which have a <80% expression of the clonotype receptor on their peripheral T cells were fed OVA in different doses (5-250 rag) and various frequencies (1-7 times, or continuous feeding in the drinking water). Subsequently, lymphoid tissues were isolated and proliferative and cytokine resonses were determined. Results: Low and medium dose (10, 20, 100 mg) OVA feeding resulted in priming of immune responses, i.e., antigen-specific proliferation as well as interieukin (IL)-2, IL-4, IL-10, and interferon-? (IFN-7) secretion upon in vitro restimulation, both in the mucosal immune system (Peyer's patches [PP]) and in the periphery (spleen and peripheral lymph nodes) as compared to unfed control mice. Proliferative responses in the mesenteric lymph nodes (MLN) were supressed with every feeding regimen. One time high dose feeding (250 mg of OVA) suppressed antigen-specific immune responses in the periphery but not in the PP. Only the highest OVA feeding doses (7 x 250 mg OVA) and continuous feeding (5 mg daily over a 10 week period) diminished the predominant IFN-7 response in the PP and resulted in an occurence of transforming growth factor 13 (TGFI3) production in the PP and the spleen. Conclusions: Our data characterize the differential cytokine production in response to fed soluble protein antigen in the periphery versus the gastrointestinal mucosa. In addition, the occurence of a TGFfl response is based on the counterregulation of Thl cytokines and TGF[I and seems to originate from the PP. These results provide thus further insight into mechanisms involved in regulating suppressive immune functions following antigen feeding. This work was supported by a research grant from the Deutsche Forschungsgemeinschaft to T. M. (DFG Ma 1711/2-1 to 2-3).

Regulation of the IL-12 receptor β2 subunit by soluble antigen and IL-12in vivo

Continuous administration of soluble protein antigen to BALB/c mice inhibits the development of Th1 and induces selective differentiation of Th2 cells. Here we show that interleukin (IL)-12, administered together with soluble protein through a mini-osmotic pump implanted subcutaneously, not only prevents the inhibition of Th1 cell development, but stimulates higher interferon (IFN)-gamma production than in mice receiving IL-12 alone. In parallel to co-stimulation of Th1 cell development, co-administration of IL-12 blocks the Th2 response induced by soluble protein. IL-12 administered in adjuvant with antigen or intraperitoneally 2 days after the immunization does not break the inhibition of Th1 but can still decrease the Th2 response induced by pretreatment with soluble protein antigen. In contrast to IL-12, co-administration of IL-2 or IFN-gamma does not affect the diversion to Th2 induced by soluble antigen. Thus IL-12, but not IL-2 nor IFN-gamma, converts in vivo the inhibitory signal for Th1 cell development delivered by soluble antigen into an immunogenic one, while blocking a positive signal for Th2 cell differentiation. A molecular basis for the co-stimulation of Th1 priming and the prevention of Th2 differentiation by IL-12 in vivo is provided by the observation that transcripts encoding the IL-12 receptor beta2 chain, which is required for IL-12 signaling and Th1 cell development, are selectively inhibited by soluble antigen but are enhanced by IL-12 co-administration.

Role of annexins in endocytosis of antigens in immature human dendritic cells.

We have evaluated the uptake of a soluble protein antigen, denitrophenylated human serum albumin (DNP-HSA), and two different intracellular bacteria; Chlamydia trachomatis serovar L2 and Mycobacterium tuberculosis strain H37Ra, by immature human dendritic cells. These were generated by culturing progenitor cells from blood in the presence of cytokines (granulocyte-macrophage colony-stimulating factor and interleukin-4). Dendritic cells play a crucial part in antigen presentation for the induction of T-cell-dependent immune responses in various tissues. Recently, macropinocytic and phagocytic activity has been shown for immature dendritic cells of mouse, rat and human origin. In the present study, macropinocytosis characterized the uptake of the soluble protein-antigen DNP-HSA, whereas the C. trachomatis were ingested via receptor-mediated endocytosis in coated pits, and opsonized M. tuberculosis via phagocytosis. To follow the intracellular routes of the antigens, their positions were compared with the localization of annexins, a family of Ca(2+)-and phospholipid-binding proteins, involved in membrane fusion, aggregation and transport of different vesicles. To elucidate further the intracellular pathway of the antigens, two other proteins, lysosome-associated membrane protein-1 (LAMP-1) and cathepsin D, were labelled. They are known to colocalize with major histocompatibility complex class II compartments in the immature dendritic cells. We observed a distinct translocation of annexin V to DNP-HSA containing endosomes, and annexin III to vesicles with C. trachomatis. Furthermore, annexin III, IV and V redistributed to phagosomes with M. tuberculosis. Both LAMP-1 and cathepsin D colocalized with DNP-HSA endosomes, and with phagosomes with M. tuberculosis. Thus, immature human dendritic cells have the capacity to phagocytose. Moreover, the handling of these antigens by dendritic cells may represent three distinct intracellular pathways, albeit some properties and compartments are shared.