Influence of stage of the reproductive cycle and estradiol on thymus cell antigen presentation

The objective of this study was to determine whether thymus cells present antigen and if endocrine balance influences antigen presentation. We report here that antigen presenting cells (APC) from the thymus glands of male and female rats, when incubated with ovalbumin (OVA)-specific T cells and OVA, are functionally able to present antigen via MHC class II. To determine whether antigen presentation in the thymus is under hormonal control, tissues from female rats at different stages of the estrous cycle were analyzed. Antigen presentation was higher at estrus and proestrus than that seen at diestrus when estradiol levels are low. Estradiol given to ovariectomized animals for 3 days stimulated antigen presentation by adherent thymus cells compared to saline controls. Flow cytometry studies indicated that the adherent thymus cell preparations consisted of DC, T cells, B cells and cells of the myeloid lineage all of which expressed MHC class II, as did a small population of non-leukocytes. Antibody neutralization studies indicated that thymus cell antigen presentation involves the expression of transmembrane proteins B7.1 and B7.2. These studies demonstrate that sex hormones play a central role in regulating antigen presentation in the thymus.     

Immunological responses and actin dynamics in macrophages are controlled by N-cofilin but are independent from ADF

Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation.     

A deterministic model for the processing and presentation of bacteria-derived antigenic peptides

The amount and the dynamics of antigen supply to the cellular antigen processing and presentation machinery differ largely among diverse microbial antigens and various types of antigen presenting cells. The precise influence, however, of antigen supply on the antigen presentation pattern of cells is not known. Here, we provide a basic deterministic mathematical model of antigen processing and presentation of microbial antigens. The model predicts that different types of antigen presenting cells e.g. cells presenting or cross-presenting exogenous antigens, cells infected with replicating microbes, or cells in which microbial antigen synthesis is blocked after a certain period of time have inherently different antigen presentation patterns which are defined by the kinetics of antigen supply. The reevaluation of existing experimental data [Sijts, A.J., Pamer, E.G., 1997. Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes. J. Exp. Med. 185, 1403-1411] describing the processing and presentation of two antigenic peptides derived from the p60 proteins of the facultatively intracellular bacterium Listeria monocytogenes shows that p60 proteins accumulating intracellularly during bacterial infection of cells play no measurable role as substrate for the cytosolic antigen presentation pathway.     

Hydrocortisone-mediated inhibition of monocyte antigen presentation: Dissociation of inhibitory effect and expression of DR antigens

The suppressive effects of hydrocortisone (HC) on the human immune system are well known. The mediation of the immunosuppressive effects of HC on lymphocyte responses via inhibition of monocyte function has been examined by monocyte-dependent, antigen-induced lymphocyte proliferation. Monocytes that were first treated with HC and then washed were unaffected in their subsequent ability to present antigen. However, there was a dramatic inhibition of lymphocyte proliferative responses if HC was present while monocytes were pulsed with antigen. This was directly related to the dose of HC present. HC-mediated inhibition of monocyte antigen presentation could not be overcome by the addition of interleukin-1 (IL-1) to the cultures, and thus inhibition of monocyte IL-1 secretion cannot totally account for the inhibition of monocyte antigen presentation. Although HC inhibits monocyte antigen presentation, HC increases the expression of HLA-DR antigens on monocytes. Other monocyte stimulants, including lipopolysaccharide (LPS), lymphokine, and gamma interferon, were examined for their effect on monocyte DR expression and their effect on monocyte antigen presentation. No correlation was found between the ability to increase monocyte DR antigen expression and the effect on antigen presentation. While HC, lymphokine, and gamma interferon all increased the expression of DR antigens on monocytes, HC, LPS, and lymphokine, but not gamma interferon, inhibited monocyte antigen presentation. Although HC can exert profound immunosuppressive effects via monocytes, it is not the only mechanism of inhibition. HC added to cultures after monocytes had been pulsed with antigen was also inhibitory.     

Preserved MHC-II antigen processing and presentation function in chronic HCV infection

Individuals with chronic HCV infection have impaired response to vaccine, though the etiology remains to be elucidated. Dendritic cells (DC) and monocytes (MN) provide antigen uptake, processing, presentation, and costimulatory functions necessary to achieve optimal immune responses. The integrity of antigen processing and presentation function within these antigen presenting cells (APC) in the setting of HCV infection has been unclear. We used a novel T cell hybridoma system that specifically measures MHC-II antigen processing and presentation function of human APC. Results demonstrate MHC-II antigen processing and presentation function is preserved in both myeloid DC (mDC) and MN in the peripheral blood of chronically HCV-infected individuals, and indicates that an alteration in this function does not likely underlie the defective HCV-infected host response to vaccination.     

Label-free proteomics and systems biology analysis of mycobacterial phagosomes in dendritic cells and macrophages

Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.     

Antigen-specific. I region-restricted interactions in vitro between tumor cell lines and T cell hybridomas

A series of H-2d B cell tumor lines and one monocytic tumor cell line were shown to be capable of I region-restricted antigen presentation to I-A-d- and I-Ed- restricted, antigen-specific cloned T cell hybridomas. For the most part, antigen presentation correlated with the present of Ia antigens on the presenting cells, although in a few interesting cases Ia-expression lines failed to present antigen. These T cell hybridomas, together with the B cell and to monocyte tumor cell lines, offer a unique set of tools to study the phenomenon of I region-restricted antigen presentation.     

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut-derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co-culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA-specific CD4(+) T-cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA-specific T-cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.     

Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction.

The B-cell antigen receptor (BCR) internalizes bound antigen such that antigen-derived peptides become associated with emigrating major histocompatibility complex (MHC) class II molecules for presentation to T cells. Experiments with B-cell transfectants reveal that BCR confers a specificity of intracellular targeting since chimeric antigen receptors which internalize antigen by virtue of a heterologous cytoplasmic domain do not necessarily give rise to presentation. In contrast, however, previous studies have shown that antigen binding to irrelevant cell surface molecules (e.g. transferrin receptor, MHC class I) can ultimately lead to presentation. The solution to this paradox appears to be that the intracellular targeting by BCR actually reflects an acceleration of antigen delivery. Depending on the nature of the BCR-antigen interaction, this accelerated targeting can be essential in determining whether or not internalization leads to significant presentation. Physiologically, the accelerated delivery of antigen by BCR could prove of particular importance early in the immune response when antigen-BCR interaction is likely to be poor.     

Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity

We developed antibody conjugates by covalently coupling antibodies against mouse mu-chain and monoclonal antibodies against nominal antigen, myoglobin, as a tool for antigen presentation and as a model of specific presentation of antigen by antigen-specific B cells and T-B interaction. In the presence of the antibody conjugates, myoglobin-specific Iad-restricted cloned T cells proliferated at 1000-fold lower concentration of myoglobin than the stimulatory concentration without the conjugates. This enhanced presentation was observed only when Iad spleen cells were 1000 R-irradiated but not 3300 R-irradiated, consistent with B cell presentation. The simple mixture of each component of the conjugates had no enhancement effects. The conjugates per se had no mitogenic effects on either splenic B cells or the cloned T cells at concentrations employed for antigen presentation. The conjugates reduced the number of antigen-presenting cells required for the maximal response but did not change the kinetics of response. The enhanced presentation by the conjugates required a genetically restricted interaction with B cells. Antigen specificity of the enhanced presentation was confirmed by using various T cell clones or lines with different antigen specificities and different conjugates constructed with monoclonal antibodies of known epitope specificity. The enhanced presentation was significantly inhibited by competition with exogenous mouse IgM or anti-mouse mu-chain but was not significantly inhibited by monoclonal antibodies against Fc receptor. Thus, conjugate-coated B cells serve as models for myoglobin-specific B cells in that they can take up specific antigens at extremely low concentration and can present the antigen to specific T cells. This model system can be applied to any antigen and any species without the need to develop antigen-specific B cell clones, which is not yet possible for most antigens and species of experimental animals. This system allowed us to investigate the relationship between T cell epitope and B cell epitope when these cells interact with each other in an antigen-specific and Ia-restricted manner. Experiments using antibody conjugates of different monoclonal antibodies against myoglobin and various myoglobin-specific cloned T cells of known antigen specificity revealed that there are some particular combinations in which much more limited enhancement of antigen presentation is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-presentation of tumour antigens: evaluation of threshold, duration, distribution and regulation

The development of technology to measure antigen presentation in the secondary lymphoid system has provided the opportunity of analysing components of the host antitumour immune response that have, until now, been unavailable for study. In particular, this technology has enabled us to evaluate threshold levels of tumour antigen required for cross-presentation in draining lymph nodes, the duration of this antigen presentation and processes that regulate tumour antigen presentation. Thus, we have been able to dissect out the relationship between antigen presentation and the resultant development of effector function in class I-restricted T cells, as well as the role of regulatory CD4 cells. We have also used this technology to evaluate the effects of antitumour therapy on local antigen cross-presentation.     

B cells extract and present immobilized antigen: implications for affinity discrimination

Binding of antigen to B-cell antigen receptor (BCR) leads to antigen internalization and presentation to T cells, a critical process in the initiation of the humoral immune response. However, antigen internalization has been demonstrated for soluble antigen, in vivo antigen is often encountered in insoluble form or tethered to a cell surface. Here, we show that not only can B cells internalize and present large particulate antigen (requiring a signalling-competent BCR to drive antigen uptake), but they can also extract antigen that is tethered tightly to a non-internalizable surface. The form in which the antigen is displayed affects the B cell's ability to discriminate antigen-BCR affinity. Thus, arraying an antigen on a particle or surface allows efficient presentation of low affinity antigens. However, the presentation efficiency of antigen arrayed on an internalizable particle plateaus at low affinity values. In contrast, extraction and presentation of antigen from a non-internalizable surface depends on antigen-BCR affinity over a wide affinity range. The results have implications for understanding both the initiation and affinity maturation of the immune response.     

Generation of MHC class I peptide antigens by protein processing in the secretory route by furin

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans -Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.     

Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS*

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.     

Targeted antigen presentation using crosslinked antibody heteroaggregates

We have targeted protein antigens to antigen-presenting cells in vitro by using antibody heteroaggregates containing an antibody against a protein antigen covalently crosslinked to an antibody against a target structure on the surface of the antigen-presenting cells. Antigen presentation was assessed by measurement of lymphokine released by antigen-specific T cell hybridomas. Depending on the experimental conditions, the crosslinked antibodies decreased the amount of antigen required to give a response by the hybridomas by factors of 10(2) to 10(3). Enhanced presentation occurred when antigen was targeted to MHC class I and class II molecules, surface immunoglobulin, or Fc gamma receptors on the surface of the murine B cell lymphoma-hybridoma, TA3. An enhancement of antigen presentation also occurred when antigen was targeted to surface IgD, or class I and class II MHC molecules on murine splenic B cells, and when antigen was targeted to class I and class II molecules on irradiated adherent spleen cells. No response was seen when antigen was targeted to Fc gamma R on B cells or adherent spleen cells. The ability of each crosslinked antibody to enhance presentation paralleled the total amount of each that bound to the surface of the antigen-presenting cells. Antigen presentation, mediated by crosslinked antibody, was antigen-specific and I-A restricted. The presentation of one antigen by using crosslinked antibody did not result in enhanced presentation of a second, bystander antigen. These results suggest that a novel means of stimulating immune responses may be possible in vivo, by targeting antigen to surface structures on antigen-presenting cells.     

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4⁺ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulum-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4⁺ T cells that would not otherwise be activated.     

AKAPs in lipid rafts are required for optimal antigen presentation by dendritic cells

Dendritic cell (DC) maturation and antigen presentation are regulated by activation of protein kinase A (PKA) signaling pathways, through unknown mechanisms. We have recently shown that interfering with PKA signaling through the use of anchoring inhibitor peptides hinders antigen presentation and DC maturation. These experiments provide evidence that DC maturation and antigen presentation are regulated by A-kinase anchoring proteins (AKAPs). Herein, we determine that the presence of AKAPs and PKA in lipid rafts regulates antigen presentation. Using a combination of western blotting and immuno-cytochemistry, we illustrate the presence of AKAP149, AKAP79, Ezrin and the regulatory subunits of PKA in DC lipid rafts. Incubation of DCs with the type II anchoring inhibitor, AKAP-in silico (AKAP-IS), removes Ezrin and RII from the lipid raft without disrupting raft formation. Addition of a lipid raft disruptor, methyl-β-cyclodextrin, blocks the efficacy of AKAP-IS, suggesting that the lipid raft must be intact for AKAP-IS to inhibit antigen presentation. Ezrin and AKAP79 are present in the lipid raft of stimulated KG1 cells, but Ezrin is not present in the lipid raft of unstimulated KG1 cells and AKAP79 levels are greatly diminished, suggesting that Ezrin and AKAP79 may be the key AKAPs responsible for regulating antigen presentation.     

Protein kinase C-alpha and -delta are required for FcalphaR (CD89) trafficking to MHC class II compartments and FcalphaR-mediated antigen presentation

Studies have demonstrated that receptor-mediated signaling, receptor/antigen complex trafficking, and major histocompatibility complex class II compartments (MIIC) are critically related to antigen presentation to CD4+ T cells. In this study, we investigated the role of protein kinase C (PKC) in FcalphaR/gammagamma (CD89, human IgA receptor)-mediated internalization of immune complexes and subsequent antigen presentation. The classical and novel PKC inhibitor, Calphostin C, inhibits FcalphaR-mediated antigen presentation and interaction of MIIC and cargo vesicle (receptor and antigen). PKC-alpha, PKC-delta, and PKC-epsilon were recruited to lipid rafts following FcalphaR crosslinking, the extent of which was determined by the phenotype of the gamma chain. Mutant gamma chain with an FcgammaRIIA ITAM (immunoreceptor tyrosine-based activation motif) insert was less able to recruit PKC and trigger antigen presentation. Both PKC isoform-specific peptide inhibitors and short interfering RNA (siRNA) showed that PKC-alpha and PKC-delta, but not PKC-epsilon, were required for association of cargo vesicle and MIIC and for FcalphaR-mediated and soluble antigen presentation. Inhibition of PKC (classical and novel) did not alter major histocompatibility class II biosynthesis, assembly, transport, or plasma membrane stability. PKC's role in facilitating interaction of cargo vesicle and MIIC is likely due to regulation of vesicle biology required for fusion of cargo vesicles to MIIC.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.