Complex Protein Antigens Research Articles

Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins.

Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning polypeptides, encompass families of proteins that are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels, transporters, enzymes, and adhesion molecules. The high specificity of monoclonal antibodies and the ability to engineer their properties offers a significant opportunity to selectively bind these target proteins, allowing direct modulation of pharmacology or enabling other mechanisms of action such as cell killing. Isolation of antibodies that bind these types of membrane proteins and exhibit the desired pharmacological function has, however, remained challenging due to technical issues in preparing membrane protein antigens suitable for enabling and driving antibody drug discovery strategies. In this article, we review progress and emerging themes in defining discovery strategies for a generation of antibodies that target these complex membrane protein antigens. We also comment on how this field may develop with the emerging implementation of computational techniques, artificial intelligence, and machine learning.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

Deciphering and predicting CD4+ T cell immunodominance of influenza virus hemagglutinin.

The importance of CD4+ T helper (Th) cells is well appreciated in view of their essential role in the elicitation of antibody and cytotoxic T cell responses. However, the mechanisms that determine the selection of immunodominant epitopes within complex protein antigens remain elusive. Here, we used ex vivo stimulation of memory T cells and screening of naive and memory T cell libraries, combined with T cell cloning and TCR sequencing, to dissect the human naive and memory CD4+ T cell repertoire against the influenza pandemic H1 hemagglutinin (H1-HA). We found that naive CD4+ T cells have a broad repertoire, being able to recognize naturally processed as well as cryptic peptides spanning the whole H1-HA sequence. In contrast, memory Th cells were primarily directed against just a few immunodominant peptides that were readily detected by mass spectrometry-based MHC-II peptidomics and predicted by structural accessibility analysis. Collectively, these findings reveal the presence of a broad repertoire of naive T cells specific for cryptic H1-HA peptides and demonstrate that antigen processing represents a major constraint determining immunodominance.

Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).

Humoral immune response to selective mycobacterial antigens and serodiagnosis of active tuberculosis in Bangladeshi patients

Background and objective: Serological test has become an important tool in the diagnosis of TB cases. This study focused on the analysis and comparison of antibody response to two Mycobacterium tuberculosis (MTB) antigens namely Ag85 complex and culture filtrate protein (CFP) in patients with tuberculosis. Antibody response to specific antigen was utilized as a diagnostic tool to detect active tuberculosis (TB) cases.Methods: Sera from 30 patients with active tuberculosis and 30 healthy individuals were tested by enzyme linked immunosorbent assay (ELISA) for the presence of immunoglobulin (Ig) G and IgM antibodies against Ag85 complex and culture filtrate protein (CFP) antigens of MTB.Results: The mean OD values of serum IgM and IgG antibodies against Ag85 and CFP were significantly (p<0.0001) higher in patients than that of healthy control individuals. Among the 30 tuberculosis patients, anti-Ag85 complex IgM and IgG was positive in 66.7% and 70.0% patients respectively. The seropositive rate of anti-CFP IgM and IgG was 33.3% and 56.7% respectively. The sensitivity and specificity of anti Ag85 and anti-CFP IgM and IgG ranged from 60.0% to 96.7%.Conclusion: The study demonstrated that determination of IgM and IgG antibodies against Ag85 complex and CFP could be used as a serological marker for diagnosis of active tuberculosis.IMC J Med Sci 2016; 10(2): 53-57

B-Cell Selection in Germinal Centers Elicited by Complex Antigens

Abstract Germinal center (GC) B cells evolve towards increased affinity by a Darwinian process studied intensively in genetically restricted, hapten-specific responses. While experimentally tractable, genetically restricted humoral responses are atypical as antibodies to complex protein antigens represent genetically diverse, polyclonal humoral responses driven by various epitopes arrayed across the antigen. We have developed a single B-cell culture method that supports the proliferation and plasmacytic differentiation of mature and GC B cells. With this tool, we explore the population dynamics of genetically diverse GC responses to two complex antigens – protective antigen of Bacillus anthracis and influenza hemagglutinin – in which B cells compete both intra- and interclonally for distinct epitopes. Our characterizations begin with antigen-binding, mature naïve B cells and follow clonal selection and affinity maturation in GCs. Preferred VH rearrangements among antigen-binding, naïve B cells were similarly abundant in early GCs but, unlike restricted responses to haptens, clonal diversity increased in GC B cells as early “winners” were replaced by rarer, high affinity clones. Despite affinity maturation, half of GC B cells did not detectably bind immunogen but exhibited genetic selection comparable to antigen-binding cells, as determined by VH usage, mutations, and clonal expansion. In GCs elicited by rPA or rHA, interclonal BCR avidities can differ 100-fold and intraclonal avidity by as much as 40-fold. We propose that intraclonal selection in GCs is permissive for a wide range of BCR affinities and that lower affinity/less fit GC B cells may remain viable in GCs for substantially longer periods than generally thought.

Expanded Human Blood-Derived γδT Cells Display Potent Antigen-Presentation Functions.

Cell-based immunotherapy strategies target tumors directly (via cytolytic effector cells) or aim at mobilizing endogenous anti-tumor immunity. The latter approach includes dendritic cells (DC) most frequently in the form of in vitro cultured peripheral blood monocytes-derived DC. Human blood γδT cells are selective for a single class of non-peptide agonists (“phosphoantigens”) and develop into potent antigen-presenting cells (APC), termed γδT-APC within 1–3 days of in vitro culture. Availability of large numbers of γδT-APC would be advantageous for use as a novel cellular vaccine. We here report optimal γδT cell expansion (>107 cells/ml blood) when peripheral blood mononuclear cells (PBMC) from healthy individuals and melanoma patients were stimulated with zoledronate and then cultured for 14 days in the presence of IL-2 and IL-15, yielding γδT cell cultures of variable purity (77 ± 21 and 56 ± 26%, respectively). They resembled effector memory αβT (TEM) cells and retained full functionality as assessed by in vitro tumor cell killing as well as secretion of pro-inflammatory cytokines (IFNγ, TNFα) and cell proliferation in response to stimulation with phosphoantigens. Importantly, day 14 γδT cells expressed numerous APC-related cell surface markers and, in agreement, displayed potent in vitro APC functions. Day 14 γδT cells from PBMC of patients with cancer were equally effective as their counterparts derived from blood of healthy individuals and triggered potent CD8+ αβT cell responses following processing and cross-presentation of simple (influenza M1) and complex (tuberculin purified protein derivative) protein antigens. Of note, and in clear contrast to peripheral blood γδT cells, the ability of day 14 γδT cells to trigger antigen-specific αβT cell responses did not depend on re-stimulation. We conclude that day 14 γδT cell cultures provide a convenient source of autologous APC for use in immunotherapy of patients with various cancers.

Usage of standardized antigen-presenting cells improves ELISpot performance for complex protein antigens

The enzyme-linked immunospot (ELISpot) assay is a widely used method for immune monitoring in cancer immunotherapy trials. In the ELISpot assay, peripheral blood mononuclear cells (PBMC) are stimulated with specific antigens, and cytokines of interest produced by individual cells are detected. In the standard procedure, T cells rely for antigen presentation on other cells like the monocyte/macrophage population present among the PBMC. Whereas oligopeptides can be added directly to the ELISpot assay without the necessity of a pre-incubation step, protein antigens must be internalized and processed by antigen-presenting cells to accomplish efficient presentation via HLA class I or II. We have studied the impact of sources for different antigen-presenting cell (i.e. PBMC-resident monocytes and monocyte-derived dendritic cells maturated with Poly I:C and PGE-2 based cocktails) on ELISpot assay performance and defined an optimized dendritic cell-based ELISpot protocol. This protocol is suitable for monitoring immune responses directed to protein antigens with higher sensitivity than the standard procedure.

T cell epitopes derived from autoantigens are selected differently than those from exogenous antigens (130.15)

Abstract We have established a novel minimalist cell-free MHC class II antigen-processing system for identifying immunodominant epitopes from protein antigens. The system constitutes HLA-DR1, HLA-DM (DM) and three cathepsins. This system not only identifies the immunogenic epitopes from complex protein antigens, but also provides insight to the mechanism of immunodominance. Understanding of how these immunogenic epitopes are selected certainly provides better strategies for developing effective peptide vaccine designs and therapies against infectious diseases, cancer, autoimmune diseases and allergy. Using our cell-free system, and based on the source of the antigens categorized as exogenous antigens versus autoantigens, we find that both DM and cathepsins are crucial in regulating the selection of immunodominant epitopes. Three types of determinants are generated: first, non-dominant epitopes that are sensitive to both DM mediated dissociation, and cathepsins digestion. These epitopes were eliminated immediately from peptide repertoire. Second, T cell epitopes derived from the exogenous antigen are DM resistant and cathepsin sensitive. Third, T cells epitopes derived from an antoantigen that are sensitive to DM mediated dissociation, but resistant to cathepsin digestion. This shows that generation of immunodominant epitopes is regulated differently depending on the source of proteins antigens.

Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.

The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen dose–response of human naive CD4+ T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 106 naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.

Complexity of Human Immune Response Profiles for CD4+ T Cell Epitopes from the Diabetes Autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplo-types characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.

Uptake and presentation of antigen to T cells by primary colonic epithelial cells in normal and diseased states

Background & Aims: The immunoregulatory properties of primary colonic epithelial cells (CECs) have not been defined. The ability of CECs from wild-type and interleukin 2–deficient (IL-2−/−) mice to take up a complex protein antigen and present peptides via MHC molecules to T cells was assessed and contrasted with that of primary small intestinal epithelial cells (SIECs). Methods: Uptake of fluorescein isothiocyanate (FITC)-labeled ovalbumin (FITC-OVA) by CECs and SIECs from wild-type and IL-2−/− mice was measured by flow cytometry. The effect of disrupting cytoskeleton organization and metabolic activity of CEC on antigen uptake was assessed. An OVA/I-Ab–specific CD4+ T-cell line transfected with an NFAT-lacZ reporter gene construct was used to evaluate the ability of CECs and SIECs as well as CECs from healthy and colitic IL-2−/− mice to present antigen to T cells. Results: Uptake of FITC-OVA by CECs is concentration dependent, is not saturated at physiologic concentrations, and requires metabolically active cells. CECs from IL-2−/− mice take up significantly more antigen than those from wild-type mice. CECs are more efficient APCs than SIECs, and antigen-pulsed CECs from IL-2−/− mice induce the highest levels of T-cell activation. Conclusions: Primary CECs are efficient APCs for CD4 MHC class II–restricted T cells. Antigen uptake and presentation is up-regulated in animals prone to develop intestinal inflammation.GASTROENTEROLOGY 2000;119:1548-1559

B7-1 Overexpression by Thymic Epithelial Cells Results in Transient and Long-Lasting Effects on Thymocytes and Peripheral T Helper Cells but Does Not Result in Immunodeficiency

The B7-1 (CD80) molecule provides costimulatory function for the activation of T helper lymphocytes upon encounter with antigen. To investigate the role of this molecule in thymocyte maturation, we have generated transgenic (Tg) mice in which CD80 expression is driven by the keratin 14 promoter (K14). This overexpression of CD80 resulted in the loss of detectable cell surface CD28 expression on thymocytes and a significant reduction in both the surface T cell receptor expression and the ratio of CD4+ to CD8+ single-positive thymocytes in Tg animals compared to nontransgenic (non-Tg) controls. While many of these defects were transient, the significant decrease in CD4+ versus CD8+ T cell ratio persisted peripherally. Peripheral T cells from these Tg mice were found to be significantly hyporesponsive to T cell mitogens and in mixed leukocyte reaction, effects that our data indicate are due to reduced IL-2 production by Tg T cells upon activation. Despite these functional defects, immunization with both complex and simple protein antigens produced no differences in the proliferative or humoral responses to these antigens between Tg and non-Tg groups. These data indicate that thymic CD80 signaling results in the deletion of significant numbers of CD4+ T cells but does not culminate in antigen-specific immunodeficiency.

Major histocompatibility complex class II–dependent antigen presentation by human intestinal endothelial cells

Background & Aims: In the normal gut, human intestinal microvascular endothelial cells (HIMECs) express major histocompatibility complex (MHC) class II molecules. Enhanced expression is found in chronic inflammation. We examined the cytokine regulation of MHC class II molecules and the associated invariant chain (Ii) in HIMECs and investigated whether such cells can process and present a complex protein antigen to T cells. Methods: Enzyme-linked immunosorbent assay, flow cytometry, immunoelectron microscopy, as well as T-cell activation assay with HIMECs and HLA-DR–restricted T-cell clones were employed. Results: In unstimulated HIMEC monolayers, HLA-DR, -DP, and -DQ and Ii were undetectable at the protein level, but interferon gamma (IFN-γ) (100 U/mL) induced expression that peaked for DR after 2–3 days, for DP after 4–6 days, for DQ after 10–12 days, and for Ii after 2–3 days. Tumor necrosis factor α had no effect alone but enhanced class II expression in combination with IFN-γ, most notably for DQ and DP. HLA-DR3–restricted and Mycobacterium tuberculosis heat shock 65-kilodalton–specific T-cell clones were activated to produce IFN-γ in response to relevant antigen presented by IFN-γ–treated HIMECs. This response was inhibited by blocking monoclonal antibody to HLA-DR and by chloroquine when compared to professional antigen-presenting cells, HIMECs activated T-cell clones quite efficiently. Conclusions: These data suggest that microvascular endothelial cells can present complex protein antigens in the human gut. GASTROENTEROLOGY 1998;114:649-656

Identification and initial characterization of five Cryptosporidium parvum sporozoite antigen genes.

Cryptosporidium parvum, an Apicomplexan parasite of gastrointestinal epithelial cells, causes severe disease in persons with AIDS and is a common cause of self-limited diarrhea in children, animal handlers, and residents of developing countries. No approved therapy exists; in research studies, however, hyperimmune bovine colostrum raised to Cryptosporidium oocysts and sporozoites has eradicated disease or decreased parasite burden in some AIDS patients. Although the protective antigens recognized by bovine hyperimmune colostrum have not been defined, protective antigens of other Apicomplexan parasites frequently have been associated with two unique structures of invasive forms, the trilaminar pellicle and the apical complex. In order to identify immunogenic Cryptosporidium proteins that may be protective antigens for use as recombinant immunogens in passive and/or active immunotherapy, we screened two genomic DNA expression libraries with polyspecific anti-Cryptosporidium antibodies. We used an approach to cloning apical complex and pellicle protein antigens that succeeded despite the lack of large numbers of organisms that would be necessitated for conventional biochemical approaches requiring organelle or membrane purification. We report here the molecular cloning of five C. parvum genes and the characterization of the cognate sporozoite proteins having molecular masses of greater than 500, 68/95, 45, 23, and 15/35 kDa. The light microscopic immunofluorescence pattern of antibodies recognizing these protein antigens suggest that they are located in the pellicle or apical complex of Cryptosporidium sporozoites.

Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides

Recent studies from this laboratory showed that tolerogenic peptide conjugates are very effective reagents for obtaining epitope-specific immunosuppression of antibody responses to immunopathogenic sites on multideterminant complex protein antigens. This paper describes the procedure for synthesis of well-defined conjugates of peptides to monomethoxypolyethylene glycol (mPEG) or to polyvinyl alcohol (PVA). The first step involves succinylation of the hydroxyl groups on the polymers by reaction with succinic anhydride. The polymer is then coupled via the carboxyl of the succinyl group to the alpha-NH2 of the completed peptide on the synthetic resin, while maintaining intact all the side-chain protecting groups on the peptide. The mPEG or PVA-peptide conjugates are cleaved from the resin and purified by standard procedures. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is coupled to the N-terminal of an otherwise unaltered peptide molecule.

Constitutive antigen presentation by mouse splenic macrophages is restricted to the progeny of a distinct progenitor population

Mouse splenic macrophage progenitors differ in their ability to give rise to cloned progeny that constitutively present complex protein antigens to T-cell hybridomas. To determine if the constitutive presentation of diverse antigens is restricted to cells derived from the same subpopulation of progenitors, we expanded macrophage clones into multiple subcultures and compared them for the ability to present different antigens to their respective antigen-specific T-cell hybridomas. Only subcultures derived from the same minority fraction of splenic macrophage progenitors were capable of constitutively presenting the antigens, and the activity of these subcultures was unaffected by the addition of recombinant murine IFN-γ. This suggests that a specialized sub-population of constitutive antigen-presenting macrophages exists in the spleens of mice.

Dominant low responsiveness in the IgG response of mice to the complex protein antigen type 1 fimbriae from Actinomyces viscosus T14V.

Type 1 fimbriae from Actinomyces viscosus T14V, composed of a complex protein of Mr 65,000, mediate the adherence of A. viscosus T14V to the host, whereas type 1 fimbriae-specific antibodies inhibit adherence. Genetic control of the serum IgG response to type 1 fimbriae was evaluated in a series of inbred, hybrid, recombinant inbred, and back-cross mice. Mice were given i.p. injections of 10(8) A. viscosus T14V cells in saline on days 0 and 14, and IgG anti-type 1 fimbriae in sera obtained on day 26 were measured by ELISA. Segregation analysis of the responses of (BALB/cJ x A/J)F1 x A/J backcross mice suggested polygenic control. Linkage analysis in (BALB/cJ x A/J)F1 x A/J backcross and SWXL recombinant inbred mice suggested control by genes linked with H-2 and with Ly-17 and Akp-1. In several F1 hybrid strains derived from H-2-disparate high and low responder parental strains, low responsiveness was dominant. The F1 derived from the H-2-identical high and low responder strains CBA/J and C3H/HeJ was a low responder, suggesting that dominant low responsiveness was mediated by non-H-2-linked genes. A three-gene model is proposed for regulation of the type 1 fimbriae response, including an MHC-linked gene, a gene linked with Ly-17 and Akp-1 on the telomeric portion of chromosome 1, and a background gene whose linkage is unknown.

Production, characterization, and species specificity of monoclonal antibodies to Mycobacterium avium complex protein antigens

The incidence of Mycobacterium avium-Mycobacterium intracellulare complex infections has increased in recent years primarily because a significant proportion of acquired immunodeficiency syndrome patients develop disseminated M. avium complex disease. In an effort to develop new tools to study these infections, we have produced eight monoclonal antibodies directed against M. avium. Western blot (immunoblot) specificity analysis and protease sensitivity assays indicate that four of these antibodies recognize M. avium-specific protein epitopes and two react with M. avium complex-specific peptide determinants. These monoclonal antibodies may be useful clinically in the diagnosis of M. avium complex disease and in the laboratory for isolation and characterization of native and recombinant M. avium complex antigens.

Antigen presenting cells.

A great deal has been learned over the past few years regarding the molecular biology of antigen presentation. These discoveries have been possible in part because of acquisition of protein sequencing data regarding class I and class II MHC molecules and in part because of X-ray crystallographic analysis of the three-dimensional structures of these molecules. These discoveries have merged nicely with detailed immunologic studies delineating the 'minimal antigenic peptides' of complex protein antigens. All of these studies strongly confirm the belief that the antigen-specific interaction of T cells with antigen in the context of antigen presenting cells is exquisitely specific. The process of 'trimolecular complex' formation involves binding interactions between antigenic peptide, class I or class II MHC molecules and the antigen-specific T cell receptor. One of the key functions of antigen presenting cells involves the 'processing' of complex protein antigens so as to allow for the interaction of the 'minimal antigenic peptide' with the appropriate class I or class II MHC molecule. A substantial body of evidence now indicates that the interaction of processed antigenic peptides and class II MHC molecules involves a binding interaction with a significant binding affinity and a slow dissociation constant. In addition to antigen-specific binding interactions which govern antigen presentation, there are a variety of antigen-independent and MHC-independent factors which greatly augment the process of antigen presentation. Along with differences in antigen processing, these factors probably account for the qualitative and quantitative differences seen between the various cell types involved in antigen presentation. There may be a substantial amount of antigen which associates with the antigen presenting cell surface in an MHC-independent fashion associated with so-called 'non-MHC peptide binding structures'. However, if the trimolecular complex theory is to be satisfied, antigen bound to these structures ultimately must become associated with the MHC restricting element in order to effectively engage the antigen-specific T cell receptor. Antigen presenting cells differ in their sensitivity to lymphokines and inflammatory mediators which augment antigen presentation. In addition, antigen presenting cells differ in their capacity to secrete or express membrane-bound costimulatory molecules, such as interleukin 1. Finally, factors which promote the cellular adherence of antigen presenting cells with T cells greatly augment the process of antigen presentation.(ABSTRACT TRUNCATED AT 400 WORDS)