Presence Of Soluble Antigen Research Articles

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells.

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-Mø), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-Mø from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-Mø population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.

CD40 stimulation in vivo does not inhibit CD4+ T cell tolerance to soluble antigens

Anergy, or T cell unresponsiveness to antigen, is one mechanism of T cell tolerance. However, the signaling events that lead to immune tolerance are not well understood. A common assumption is that soluble antigens, such as food antigens, are poor immunogens and induce tolerance because they fail to upregulate co-stimulatory molecules on the professional APC. Engagement of CD40 through a stimulatory antibody causes the upregulation of these costimulatory molecules. Using a CD4+ T cell adoptive transfer model specific to ovalbumin (OVA), we show that after upregulation of CD86 on APC through CD40 stimulation in vivo, T cells from OVA-fed mice remain refractory to proliferation, interleukin (IL)-2 and interferon (IFN)-γ production. We conclude that upregulation of CD86 alone does not inhibit oral tolerance induction of CD4+ T cells, indicating that additional signals are involved in the decision process for CD4+ T cells to commit to tolerance versus sensitization. Our data challenge the belief that reconstitution of a costimulatory signal in the presence of soluble antigen is sufficient to override T cell tolerance and anergy.

Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)

Dendritic cells are a complex group of mainly bone marrow derived cells that possess significant ability to take up, process, and present soluble antigens to responder cells in the lymphoid tissues. Characterization of DC surface markers has been a difficult task owing to the lack of appropriate reagents of sufficient specificity. Molecules closely associated with dendritic cells are being identified and investigated and flow cytometric approaches to evaluation of monoclonal antibodies against DC populations are being developed. This informative unit describes assays for determination of the existence of subsets within two DC populations, the kinetics of antigen expression, and the identification of specific markers for DC subsets. A Support Protocol presents an approach for generating dendritic cells and Langerhans cells (a skin-derived DC) from blood monocytes.

Inhibition of Dendritic Cell Differentiation by Fumaric Acid Esters

Fumaric acid esters have proved to be effective for the systemic treatment of severe psoriasis vulgaris. These compounds have been shown to induce a Th2-like cytokine secretion pattern in T cells and to reduce keratinocyte proliferation in vitro. Dendritic cells seem to be of major importance as regulatory cells driving the psoriatic tissue reaction. Monocytes or CD34-positive myeloid progenitor cells are precursors of dendritic cells that can be generated in vitro by culture with granulocyte-macrophage colony-stimulating factor and interleukin-4. Using this model the effect of fumaric acid esters on granulocyte-macrophage colony-stimulating factor/interleukin-4-induced differentiation of monocyte-derived dendritic cells was investigated. The results of this study show that dimethylfumarate as well as methylhydrogenfumarate-calcium-salt (0.01-100 microg per ml) concentration-dependently inhibit monocyte-derived dendritic cell differentiation. This was reflected by an inhibition of CD1a, CD40, CD80, CD86, and HLA-DR expression as well as by a reduced capacity of dimethylfumarate-treated monocyte-derived dendritic cells to stimulate lymphocytes in the allogeneic mixed lymphocyte reaction. Other fumaric acid esters showed no effect on monocyte-derived dendritic cell-differentiation. At higher concentrations (30-100 microg per ml) dimethylfumarate, but not methylhydrogenfumarate calcium-salt induced apoptosis in monocyte-derived dendritic cells as measured by expression of Apo 2.7 and DNA fragmentation (TUNEL assay). These data point to a high susceptibility of the monocyte/dendritic cell system to dimethylfumarate and its main metabolite methylhydrogenfumarate. Other fumaric acid esters investigated were without effect. As the effects of fumarates on monocyte-derived dendritic cells observed occur at concentrations 20-fold lower compared with lymphocytes, our data seem to be of relevance in explaining the possible mode of action of these compounds in psoriasis.

CLIP-derived self peptides bound to MHC class II molecules of medullary thymic epithelial cells differ from those of cortical thymic epithelial cells in their diversity, length, and C-terminal processing.

Medullary thymic epithelial cells (mTEC) are able to present soluble antigens to CD4+ helper T cell lines, whereas cortical thymic epithelial cells (cTEC) are not (Mizuochi, T., et al., J. Exp. Med. 1992. 175: 1601-1605). In addition, class II heterodimers from mTEC migrated with apparently less relative molecular mass in SDS-PAGE than those from cTEC (Kasai, M., et al., Eur. J. Immunol. 1998. 28:1867-1876). To investigate the cause of the distinct migration profiles of class II heterodimers in both TEC types, class II heterodimer-associated peptides were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Self peptides from cTEC were shown to vary moderately in length and to be highly diverse, including low amounts of CLIP (class II-associated invariant chain peptide) variants. On the other hand, self peptides from two mTEC consisted predominantly of two CLIP variants with exceptional C-terminal extensions. C-terminally overhanging residues of CLIP in mTEC may be responsible for the distinct migration of class II heterodimers in SDS-PAGE. Differences in migration of class II heterodimers on SDS gels was also observed in H2-DM+ vesicles isolated from both TEC. The possible contribution of self peptides bound to class II heterodimers in TEC to positive or negative selection of T cells in the thymus is discussed.

The role of aquaporins in dendritic cell macropinocytosis.

Immature dendritic cells (DCs) constitutively take up large volumes of fluid by macropinocytosis and concentrate the macrosolutes in the endocytic compartment. This concentration mechanism that is the basis of their high capacity to present soluble antigens requires that DCs be capable of rapidly exchanging water across their membranes. We report that two members of the aquaporin family, AQP3 and AQP7, are expressed in immature DCs and are downregulated after maturation. Treatment of DCs with p-chloromercuribenzenesulphonate (pCMBS), a mercuric drug that blocks aquaporins, inhibited uptake and concentration of macrosolutes taken up by fluid phase endocytosis and led to dramatic cell swelling. In contrast, pCMBS did not affect receptor-mediated endocytosis via the mannose receptor. These findings indicate that aquaporins represent essential elements of a volume control mechanism that allows DCs to concentrate macrosolutes taken up via macropinocytosis.

Glucocorticoids increase the endocytic activity of human dendritic cells.

We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10(-8) M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC-dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex. The expression of receptors involved in antigen capture was investigated by FACS analysis. Dex up-regulates MR, CD16 and CD32 expression on DC. After maturation with tumor necrosis factor-alpha or CD40 ligand in Dex-treated DC, despite a reduction induced by maturation the endocytic activity of FITC-dextran and LY, the expression of MR, CD16 and CD32 remained higher than in control DC. In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting tetanus toxin to specific autologous T cell lines. In conclusion our data suggest a new mechanism by which GC may influence immune responses. In fact with the increase in endocytic activity, Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibiting the capacity to stimulate T cells.

Dendritic cells in allergy.

Dendritic cells in allergy.

IL-13 can substitute for IL-4 in the generation of dendritic cells for the induction of cytotoxic T lymphocytes and gene therapy.

Immunization with tumor-associated antigen pulsed dendritic cells (DC) has been shown to elicit both protective and therapeutic antitumor immunity in a variety of animal models and is currently being investigated for the treatment of cancer patients in clinical trials. In this study we show that DC can be generated from peripheral blood mononuclear cells of healthy donors as well as breast and melanoma cancer patients using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-13 (IL-13) and that these DC have many of the same characteristics as DC differentiated using GM-CSF and IL-4. The DC generated in GM-CSF and IL-13 are CD14- and express high levels of the cell surface markers CD86, HLA-DR, and CD58, as do DC generated in GM-CSF and IL-4. The purity and yield of both DC populations are not significantly different. Furthermore, both populations of DC are effective at presentation of alloantigen as determined in a mixed lymphocyte response, and both are able to process and present soluble tetanus toxoid antigen to CD4+ T cells. Because we are interested in the generation of DC for antigen-specific cytotoxic T lymphocyte (CTL) generation, we compared the ability of peptide-pulsed DC differentiated in GM-CSF and IL-4 versus GM-CSF and IL-13 for the generation of influenza and MART-1 specific CTL. Both populations of DC induced CD3+ CD8+ CD4- and CD56- CTL, which could lyse the appropriate targets in an antigen-specific manner. Finally, both GM-CSF and IL-4 DC and GM-CSF and IL-13 DC yielded similar beta galactosidase expression levels after transduction with recombinant adenovirus containing the LacZ gene. These results suggest that DC generated in GM-CSF and IL-13 may be useful for immunotherapy and gene therapy protocols.

Potent immunoregulatory effects of Salmonella typhi flagella on antigenic stimulation of human peripheral blood mononuclear cells.

A key function of monocytes/macrophages (Mphi) is to present antigens to T cells. However, upon interaction with bacteria, Mphi lose their ability to effectively present soluble antigens. This functional loss was associated with alterations in the expression of adhesion molecules and CD14 and a reduction in the uptake of soluble antigen. Recently, we have demonstrated that Salmonella typhi flagella (STF) markedly decrease CD14 expression and are potent inducers of proinflammatory cytokine production by human peripheral blood mononuclear cells (hPBMC). In order to determine whether S. typhi and soluble STF also alter the ability of Mphi to activate T cells to proliferate to antigens and mitogens, hPBMC were cultured in the presence of tetanus toxoid (TT) or phytohemagglutinin (PHA) and either killed whole-cell S. typhi or purified STF protein. Both whole-cell S. typhi and STF suppressed proliferation to PHA and TT. This decreased proliferation was not a result of increased Mphi production of nitric oxide, prostaglandin E2, or oxygen radicals or the release of interleukin-1beta, tumor necrosis factor alpha, interleukin-6, or interleukin-10 following exposure to STF. However, the ability to take up soluble antigen, as determined by fluorescein isothiocyanate-labeled dextran uptake, was reduced in cells cultured with STF. Moreover, there was a dramatic reduction in the expression of CD54 on Mphi after exposure to STF. These results indicate that whole-cell S. typhi and STF have the ability to alter in vitro proliferation to soluble antigens and mitogens by affecting Mphi function.

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.

The biology and clinical applications of dendritic cells.

Dendritic cells (DC) have an essential role in the induction of immune responses to antigen by naive T cells. As 'professional' antigen-presenting cells they are specialized to take up, process and present soluble antigens in complexes with either class I or class II MHC molecules. They are present in only trace numbers in most tissues and in a relatively immature state but, in the presence of inflammatory signals, they rapidly take up foreign antigens and undergo maturation into potent antigen-presenting cells that migrate to secondary lymphoid tissue where they initiate an immune response. It is now possible to expand populations of DC in vitro both from primitive haemopoietic progenitors as well as from more mature peripheral blood mononuclear cells. This has shed light on many developmental aspects of DC biology and furthered our knowledge of the mechanisms of antigen processing and presentation. It is clear that there is more than one pathway of DC differentiation and that some DC may actually induce immunological unresponsiveness--a possible mechanism for tolerance to self-antigens. For clinicians the most exciting prospect is of their use as cellular adjuvants to generate beneficial responses to antigens of low immunogenicity such as tumour antigens. This review outlines aspects of human DC development and the way in which a greater understanding of their biology may lead to promising clinical applications.

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.

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA

Backvround: Chimeric T cell recptors with specificity for defined tumor associated antigens are valuable tools to target T cells to tumor cells. The function of these receptors in the presence of soluble antigen, however, is crucial because the tumor associated antigen is often shed into the serum of cancer patients. Methods: For this reason we have generated a chimeric T cell receptor with specificity for the membrane bound form of the carcinoembryonic antigen (CEA). This receptor (humBW431/26-CH2/3-T) is composed of a humanized single chain antibody (humBW431/26), that was fused to the Fc domains (CH2/3) of the human lgG1 and the signaling unit of the Fc~R1 receptor t chain ('/). Results: After transfection this chimeric receptor converted MD45 T cells to specificity for CEA+ tumor cells. Only immobilized, especially membrane-bound CEA, could crosslink the chimeric receptor resulting in cellular activation with IL-2 release and cytolytic activity towards CEA+ tumor cells. Cellular activation by membrane bound antigen was also efficient in the presence of high amounts of soluble CEA (up to 25 lag/ml whereas an anti-idiotypic antibody could completely block T cell activation even at low concentrations (0,1 lag/ml). Conclusion: Our data demonstrate, that the chimeric T cell receptor humBW431/26-CH2/3-y has selective specificity for membrane-bound CEA and that targeting of tumor cells by chimeric T cell receptors can be performed even in the presence of high amounts of soluble antigen.

Characterisation of two human dendritic cell-lines that express CD1a, take-up, process and present soluble antigens and induce MLR

Dendritic cells (DC) are bone marrow derived cells present in diverse tissues and organs including the skin, mucosae and blood. DC have a capital role in the afferent pathway of the immune response because of its role in up-take, processing and presenting antigens to immune cells. Human DC are usually identified by the expression of surface CD1a and HLA-DR. Despite the significant recent developments for in vitro generation of DC derived from blood by using cytokines like GM-CSF and IL-4, the studies on DC and specially on human Langerhans cells (LC) have been hampered by the laborious isolation procedure and the small yield of cells obtained by the various methods of isolation used so far. Therefore, a priority has been a search for monoclonal dendritic cell-lines with LC characteristics in order to facilitate the research in this area. The present study reports on the generation of two stable, self-replicant, adherent, dendritic, CD1a +, HLA-DR +, CD45RO +, CD23/FcERII + cell-lines that up-take and process soluble antigens but also inducing MLR and antigen-dependent T-cell response.

Generation of dendritic cells from peripheral blood adherent cells in medium with human serum.

Dendritic cells (DC) provide an effective pathway for presenting antigens to T cells, both self-antigens during T-cell development and foreign antigens during immunity. As such, these cells may be promising adjuvants for immunotherapy. Thus, it is important to establish simple and fast method(s) to generate sufficient numbers of human DC in medium free of calf serum so that the cells can be used for both experimental and clinical purposes. In this report, we used peripheral blood adherent cells, without laborious cell purification or depletion, as the starting population and cultured them in medium supplemented with granulocyte/macrophage colony-stimulating factor and interleukin-4. Substantial numbers of cells with the phenotypical and functional characteristics of immature DC were obtained in a 7-day culture. We then compared DC cultured in medium supplemented with either fetal calf serum or pooled human ABRh+ serum and found no difference in cell yields and in their ability to stimulate alloreactive T cells or to present soluble antigens to T cells. Irradiated cells were less efficient than non-irradiated cells in antigen presentation and stimulation of T cells. Finally, we have examined DC with or without additional tumour necrosis factor-alpha treatment and found that antigen-pulsed mature cells could as efficiently present antigen to T cells as did immature cells. This method is suitable for the generation of DC in studies of large clinical materials.

Cytotoxic T lymphocytes in resistance to tuberculosis.

Recent experimental evidence has suggested T cells recognizing antigens in the context of both classical MHC class I and nonclassical class I-like molecules contribute to protective responses against Mycobacterium tuberculosis (MTB) infection. Our aims were to characterize both types of T cells, and to explore the basis of communication between the tubercle bacilli and the MHC class I pathway of the host macrophage. A model system was developed using exogenously added ovalbumin as a surrogate antigen to study presentation by MTB-infected macrophages. Viable, virulent MTB and closely related mycobacterial species facilitated the presentation of ovalbumin on MHC class I molecules to CD8+ cytolytic T cells that was dependent upon the cytosolic transport of peptides, implying communication between the MTB phagosome and the host cell cytoplasm. MHC class I presentation of soluble antigens was mimicked by Listeria monocytogenes, which grows within the host cell cytoplasm, as well as its purified hemolysin. We have also characterized T cells that recognize nonpeptide MTB antigens presented by CD1 molecules. CD1-restricted T cells demonstrated to lyse macrophages infected with virulent MTB were divided into distinct subsets based on surface phenotype (CD4-CD8- versus CD8-) and cytotoxicity mechanism (Fas receptor-mediated versus granule exocytosis). A functional consequence of these two mechanisms was observed that while both subsets lysed infected macrophages, only those T cells utilizing the granule exocytosis pathway were able to reduce viability of intracellular MTB.

Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations

Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion. A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk. All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney. At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58%. The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion. Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD). Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD. Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling. The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments.

The role of dendritic cells in T cell activation.

Dendritic cells (DC) are distinguishable from other antigen-presenting cells by their potent antigen-presenting capacity. They are not only efficient at presenting peptide antigen but can also process and present soluble protein antigen sto antigen-specific T cells and cloned T cell lines. They are very strong stimulators of both allogeneic and syngeneic mixed lymphocyte reactions and have a unique capacity to stimulate naive T cells. The potent functional capacity of DC is related to a high-level expression of major histocompatibility complex class I/II molecules and constitutive expression of costimulatory molecules, such as CD80/CD86, as well as heat stable antigen, CD40 and the leucocyte function antigen (LFA) family of adhesion molecules. Recent studies have shown that DC are also involved in regulation of the immune response via induction of both central and peripheral tolerance.