The relationship between antigen concentration, antigen internalization, and antigenic complexes: modeling insights into antigen processing and presentation

Native antigen is processed and subsequently presented on the surface of antigen-presenting cells, an important step in the elicitation of an immune response. The early events of antigen processing and presentation include: ingestion of a native antigen, intracellular degradation to expose an antigenic peptide fragment, binding of this fragment with an MHC class II molecule, and display of this newly formed complex on the cell surface. Through the development of a mathematical model, a set of mathematical equations which describes the time-dependent appearance, disappearance, and movement of individual molecules, quantitative insight can be gained into the pathways and rate-limiting steps of antigen presentation. The credibility of the model has been verified by comparison to literature data. For example, it has been shown experimentally that macrophages require 60 min for effective antigen presentation, whereas B cells require 6-8 h. The mathematical model predicts these presentation times and identifies the difference in the cell's respective pinocytic rates and sizes as important parameters. B cells capture antigen in their environment through nonspecific fluid-phase pinocytosis as well as by binding antigen to their surface immunoglobulin, allowing receptor-mediated uptake. Uptake of antigen via receptor-mediated endocytosis has been reported to require 1,000-fold less antigen than uptake via nonspecific pinocytosis. The mathematical model clearly predicts this decrease in concentration. The model also makes quantitative predictions for the number of MHC class II-antigen complexes needed to produce T cell stimulation.     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.     

HSP70 natively and specifically associates with an N-terminal dermcidin-derived peptide that contains an HLA-A*03 antigenic epitope

Tumor cells very often have elevated expression of HSP70, the anti-apoptotic properties of which contribute to overall tumor survival. Independent of its anti-apoptotic properties, HSP70 was also suggested to be involved in the antigen presentation process by chaperoning cytosolic peptides, thus protecting them from rapid degradation and securing the peptide pool for further processing. In this study, we identified a 33-amino acid N-terminal dermcidin (DCD)-derived peptide from the repertoire of in vivo HSP70-associated peptides isolated from a leukemic cell line, K562. The DCD peptide has been previously shown to be involved in tumorigenesis, to increase tumor survival rate, to improve tumor stress resistance, and to aid growth. We show that HSP70 is a specific binding partner for the DCD prosurvival peptide and define an ATP-dependent DCD-binding site (GNPCH). We also identify an HLA-A*03 antigenic epitope within the DCD peptide, which follows and partially overlaps the HSP70-binding site (CHEASAAQK). This study describes the interaction between HSP70 and the DCD-derived prosurvival peptide, an interaction that may direct the peptide toward antigen presentation and independently contribute to the prosurvival mechanism mediated by DCD.     

TAP-independent antigen presentation on MHC class I molecules: lessons from Epstein-Barr virus

For recognition by CD8(+) lymphocytes, peptides derived from cytosolically processed antigen need to access MHC class I molecules en route to the target cell surface. This normally requires peptide transport into the endoplasmic reticulum via the transporter associated with antigen presentation (TAP) complex. However, as recent work with Epstein-Barr virus illustrates, TAP-independent presentation pathways also exist and are growing in number.     

Activation of T Cells by Autoantigen Immobilized by Specific Antibodies

A convenient microtiter-plate assay that uses immobilized antibody to capture specific antigens for presentation to T cells has been developed. Initial experiments used KLH as the antigen, immune antisera and draining lymph node cells from immunized NOD mice as the source of antibody and T cells, and spleen cells from naive NOD mice as the source of antigen-presenting cells (APCs). The resulting proliferation of the T cells was shown to be antibody- and antigen-specific, suggesting that the APCs had internalized and processed the captured antigen, presenting it to the T cells in the form of peptide/MHC complexes. The approach was also tested for an autoimmune disease as part of an effort to identify autoantigens responsible for the proliferation of T cells in the synovial fluid of rheumatoid arthritis patients. When immunoglobulin from autologous synovial fluid was captured on plates coated with anti-human immunoglobulin antibodies, the addition of HLA-DR4 peripheral blood mononuclear cells as APCs and synovial fluid-reactive HLA-DR4-restricted T-cell clones resulted in significant proliferation, indicating that the specific antigen in the crude synovial fluid was human immunoglobulin. This response was also shown to be antigen-specific and HLA-DR4-restricted. This assay format should permit the definition of autoantigens by capturing with antibodies to crude autoantigen extracts, followed by the addition of the appropriate APC and T-cell populations.     

Newly synthesized class II MHC chains are required for VSV G presentation to CTL clones.

Mechanisms of antigen processing and presentation to thymus-derived lymphocytes have been under intense study for several years, focusing on both Class I-restricted antigen presentation and Class II-MHC restricted responses. The studies described here examine the processing and presentation of exogenously provided soluble glycoprotein of the vesicular stomatitis virus and as well as newly synthesized viral glycoprotein. Evidence is provided that newly synthesized Class II MHC chains are required for cell surface expression of processed glycoprotein determinants irrespective of the origin of the viral antigen. Inhibitors of distinct cellular processes, including ammonium chloride, emetine, and Brefeldin A, have been used to dissect the pathways utilized.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.     

Role of B cell antigen processing and presentation in the humoral immune response

In the 25 years since it was first indicated that lymphocyte subpopulations must interact during the generation of a humoral immune response, there has been an explosion of data on the molecular mechanism of this interaction. It has been demonstrated that T cells recognize a processed antigen fragment presented by a major histocompatibility complex molecule on the surface of an antigen-presenting cell. The minimal peptides required for T cell recognition of several proteins have been determined, the molecular genetics of many of the cell surface molecules involved have been defined, and the three-dimensional structure of the T cell receptor and the major histocompatibility antigens have been deduced. Several cell types have been found to act as antigen-presenting cells, although the roles of these populations in vivo remain unclear. However, it is clear that there must be a physical interaction between a B cell and a T cell before the B cell can respond to a T-dependent antigen. This interaction requires processing and presentation of the antigen by the B cell. Therefore this review focuses on antigen processing and presentation by resting B cells, one of the key steps in initiation of a humoral immune response.     

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.     

Importance of the COOH terminal of angiotensin in antigenicity and in the formation of an antigen-containing complex with cellular membrane structures

To more carefully determine how a peptide antigen interacts with the antigen-presenting cell (APC), we have begun an analysis of the fate of APC-associated peptide antigens. These studies have shown that a stable cell-bound form of APC-associated peptide exists, which is a complex of the peptide with surface membrane structures (peak A). In the experiments described here, we have begun to examine the chemical mechanism of this peak A complex formation. By modifying either the carboxyl terminal or amino terminal group of the octapeptide antigen angiotensin II we have established that the terminal carboxyl group, but not the terminal amino group, was critical for forming the peak A complex with APC membrane structures. In addition, blocking the carboxyl but not the amino terminal dramatically reduced the antigenicity of the peptide for AII-immune T cell in vitro proliferation. These results show that the carboxyl terminal of AII is essential for both peak A formation and antigenicity, and suggest that peak A is critical for antigen presentation to T cells.     

Use of antibody/peptide constructs of direct antigenic peptides to T cells: evidence for T cell processing and presentation.

Human T cells can express MHC-class II products and were shown to be potential antigen-presenting cells. However, they are unable to capture the antigen and only antigens, which bind to T cell membranes such as the gp120 glycoprotein of HIV, are internalized, processed, and presented by T cells. To better understand the role of T cells as antigen-presenting cells, we established a method which overcomes the lack of antigen capture by T cells. Antigen (tetanus toxoid, TT) or an antigenic peptide of TT (residue 830-843, P2) was coupled to antibodies directed to T cell surface molecules such as CD2, CD4, CD8. Antibody/TT and antibody/P2 constructs stimulated P2-specific T cell clones in the absence of accessory cells, if the antibody recognized a T cell surface structure. Compared to the peptide alone, a 100-500 times lower molar concentration of the antibody/peptide construct was required to achieve a similar proliferative response. T cell stimulation via the constructs involved intracellular processing, as nonspecific, glutaraldehyde fixed T cell lines pulsed with the constructs could present the peptide and processing inhibitors like Leupeptin or Chloroquine inhibited the development of a proliferative response to the constructs. Our data underline the ability of T cells to function as antigen-processing and -presenting cells and show that antibody/antigen or antibody/peptide constructs are able to direct a certain antigen or peptide to a T cell. Antibody/peptide constructs may be interesting tools to better understand antigen processing and to study the consequences of antigen presentation by different cells.     

Activated mouse T-cells synthesize MHC class II, process, and present morbillivirus nucleocapsid protein to primed T-cells

A pivotal step in the initiation of T-cell immunity is the presentation of antigenic peptides by major histocompatibility complex (MHC) expressed on antigen presenting cells. The expression of MHC class II molecules by mouse T-cells has not been shown unequivocally. In the present work, we demonstrate that activated mouse T-cells synthesize MHC class II molecules de novo and express them on their surface. Further, we have demonstrated that in vitro activated T-cells take up extra-cellular soluble nucleocapsid protein of a morbillivirus. The internalized antigen goes to antigen processing compartment as shown by co-localization of antigen and LAMP-1 using confocal microscopy. We show that activated T-cells express H2M, a chaperone molecule known to have a role in antigen presentation. Further, we demonstrate that activated T-cells process and present internalized extra-cellular antigen to primed T-cells as shown by IL-2 secretion and in vitro proliferation. The presentation of antigen by T-cells may have implications in immuno-regulation, control of infection by lymphotropic viruses and maintenance of immunological memory.     

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.     

Processing and presentation of insulin. I. Analysis of immunogenic peptides and processing requirements for insulin A loop-specific T cells

The processing and presentation of insulin by B hybridoma cells to insulin A loop-specific T cell hybridomas was investigated. We found that the activation of these T cells requires insulin to be processed in a manner that permits unfolding of the molecule and prevents extensive proteolysis. An analysis of insulin peptides formed by either enzymatic digestion in vitro or solid phase synthesis revealed that a conformational determinant comprised of residues A1-A14 disulfide-linked to B7-B15 is most immunogenic to these T cells. Reduction and/or proteolysis of this peptide markedly decreases its immunogenicity. The pork insulin A1-A14/B7-B15 peptide differs only at residue A4 from its mouse insulin homolog. Thus, Glu A4 forms part of the antigenic site recognized by a pork insulin/I-Ad-specific mouse T cell. This insulin peptide can be induced to assume an alpha-helical configuration in a hydrophobic environment. In addition, virtually all of the residues of this peptide are identical with those predicted to be situated in amphipathic regions of the native insulin molecule. N-Ethylmaleimide and bacitracin, which inhibit the activity of two cytosolic enzymes that cleave insulin, enhance the antigen presentation of insulin. This suggests that these enzymes may participate in the nonlysosomal antigen processing of insulin by a B lymphocyte. A comparison of the relative avidity of several T cell hybridomas, which have the same apparent specificity for this insulin peptide, showed that an increase in their avidity was associated with a degeneracy in their fine specificity. Our data demonstrate that the efficiency of processing and presentation of a given antigenic determinant is related to the conformation of the determinant and the specificity and avidity of the T cell.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells

The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.     

Improvement of the enzymatic stability of a cytotoxic T-lymphocyte-epitope model peptide for its oral administration

Oral administration of peptide antigens, to provide proper mucosal and/or systemic immunity, is largely ineffective. This is mainly due to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal absorption membrane. The present study focuses on the improvement of the enzymatic stability of a 13 amino acid long peptide containing a cytotoxic T-lymphocytes (CTL)-epitope. Within this study, it is shown, that simple chemical modification at the N- and C-terminus of the peptide can provide significant stability towards enzymatic attack by intestinal exopeptidases. Around 50% of the modified peptide resisted enzymatic attack on native porcine intestinal mucosa within 3h of incubation at pH 6.8 and 37 degrees C, whereas unmodified control peptide was almost completely degraded within the same time period. Additionally, a mucoadhesive drug carrier matrix with specific inhibitory properties towards luminally secreted endopeptidases has been generated. The incorporation of the simply modified peptide in this delivery system should enhance the amount of biologically active antigen being available at the mucosal site for further presentation to immunomodulating systems. This might open the door for a successful oral immunotherapy.     

Nitration of the pollen allergen bet v 1.0101 enhances the presentation of bet v 1-derived peptides by HLA-DR on human dendritic cells

Nitration of pollen derived allergens can occur by NO(2) and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.     

Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes

Peptides that are presented by class I MHC (MHC-I) molecules derive from cytosolic Ags processed via the conventional MHC-I pathway or exogenous Ags processed via alternate MHC-I processing mechanisms. Alternate MHC-I processing by macrophages and dendritic cells allows presentation of peptides from particulate Ags, including bacteria. Despite the established phagocytic activity of neutrophils, MHC-I processing and presentation of phagocytosed Ags by neutrophils has not been investigated. Murine neutrophils from peritoneal exudates were shown to express MHC-I molecules and tested for the ability to process HB101.Crl-OVA, Escherichia coli transfected to express a fusion protein containing the 257-264 epitope of OVA. Neutrophils were found to process HB101.Crl-OVA and present OVA(257-264)-K(b) complexes to CD8OVA T hybridoma cells via a pathway that was resistant to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport, and lactacystin, a proteasome inhibitor. These results suggest that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway that does not involve cytosolic processing. In addition, neutrophils were found to secrete or "regurgitate" processed peptide that was subsequently presented by neighboring prefixed macrophages or dendritic cells. Thus, neutrophils may influence T cell responses to bacteria, either by directly presenting peptide-MHC-I complexes or by delivering peptides to other APCs for presentation. Hypothetically, neutrophils may directly present peptide to effector T cells in vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to initiate T cell responses.