Disruption of MHC class II-restricted antigen presentation by vaccinia virus

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.     

Efficiency of antigen presentation after antigen targeting to surface IgD, IgM, MHC, Fc gamma RII, and B220 molecules on murine splenic B cells

Bispecific heteroconjugate antibodies can bind soluble protein Ag to APC and thereby enhance Ag presentation. We used such antibodies to bind hen egg lysozyme (HEL) to various structures on the surface of normal splenic B cells to determine which structures would provide the best targets for enhanced presentation. We found that HEL was presented efficiently to hybridoma T cells if bound to sIgD, sIgM, or class I or II MHC molecules, but not at all if bound to Fc gamma RII, or B220 molecules on B cells. The efficiency of presentation of HEL was measured as a function of the amount of 125I-HEL bound per cell. HEL was presented with 5 to 10 times greater efficiency when bound to sIg, than when bound to MHC molecules. When compared on the basis of the amount of HEL bound, sIgD and sIgM functioned equally as target structures, as did class I and class II MHC molecules. Large amounts of HEL bound to B220, but no presentation resulted, indicating that focusing HEL to the APC surface was not sufficient for presentation to occur. HEL was internalized rapidly and in large amounts when bound to sIgD or sIgM, but slowly and in small amounts, when bound to class I or class II MHC molecules. Thus, a rapid rate of internalization may in part explain the high efficiency of Ag presentation after binding to sIg. However, the small amount of HEL internalized via MHC molecules was utilized efficiently for presentation. These results indicate that sIgM and sIgD serve equally on normal B cells to focus and internalize Ag and enhance Ag presentation, but that class I or class II MHC molecules can also be used to internalize Ag and enhance Ag presentation, perhaps by a separate intracellular processing pathway.     

Microbial and T cell-derived stimuli regulate antigen presentation by dendritic cells in vivo

B cells and dendritic cells (DC) internalize and degrade exogenous Ags and present them as peptides bound to MHC class II molecules for scrutiny by CD4(+) T cells. Here we use an Ab specific for a processed form of the model Ag, hen egg lysozyme (HEL), to demonstrate that this protein is not efficiently presented by lymph node DC following s.c. immunization. HEL presentation by the DC can be dramatically enhanced upon coinjection of a microbial adjuvant, which appears to act by enhancing peptide loading onto MHC class II. CD40 cross-linking or the presence of a high frequency of T cells specific for HEL can similarly improve presentation by DC in vivo. For any of these activating stimuli, CD8alpha(+) DC consistently display the highest proportion of HEL-loaded MHC class II molecules. These data indicate that exogenous Ags can be displayed to T cells in lymphoid tissues by a large cohort of resident DC whose presentation is regulated by innate and adaptive stimuli. Our data further reveal the existence of a feedback mechanism that augments Ag presentation during cognate APC-T cell interactions.     

Invariant chain alters the malignant phenotype of MHC class II+ tumor cells.

T lymphocytes usually recognize endogenously encoded Ag in the context of MHC class I molecules, whereas exogenous Ag is usually presented by MHC class II molecules. In vitro studies in model systems suggest that presentation of endogenous Ag by class II molecules is inhibited by the association of class II with its invariant chain (Ii). In the present study we test this hypothesis in an in vivo system in which endogenously encoded tumor peptides are presented by tumor cell MHC class II molecules. In this system, transfection of syngeneic MHC class II genes (Aak and Abk) into a highly malignant, Ii negative, mouse tumor (SaI sarcoma) produces an immunogenic tumor (SaI/Ak) that is rejected by the autologous host. The class II+ transfectants also effectively immunize autologous A/J mice against a subsequent challenge of wild-type class II- tumor cells. We have hypothesized that the SaI/Ak transfectants induce protective immunity because they function as APC for endogenously synthesized tumor peptides, and thereby stimulate tumor-specific Th cells, by-passing the need for professional APC. To test the role of Ii as an inhibitor of presentation of endogenous peptides, SaI/Ak tumor cells were supertransfected with Ii gene (SaI/Ak/Ii cells), and the tumorigenicity of the resulting cells determined. Nine SaI/Ak/Ii clones were tested, and their malignancy compared with that of SaI/Ak and SaI cells. Seven of the nine class II+/Ii+ tumor cells are more malignant than class II+/Ii- tumor cells in autologous A/J mice. Expression of Ii therefore restores the malignant phenotype, presumably by preventing presentation of endogenously synthesized tumor peptides. Ii therefore regulates Ag presentation and can be a critical parameter for in vivo tumor immunity.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Following immunization antigen becomes concentrated in a limited number of APCs including B cells

Immunization with the hen egg-white lysozyme (HEL) protein induces T cells to various of its peptide determinants. The distribution of such T cells, however, does not correlate with the peptide level of each epitope on class II molecules. For this reason, we sought information on the cells responsible for Ag presentation following immunization, hoping to understand the lack of immunodominance in this system. By tracking HEL, and the ensuing peptide/MHC complexes, we find the following: 1) that HEL in the draining lymph node gets concentrated in a limited number of APC, particularly in dendritic cells and macrophages, 2) that these APC are functionally capable of presenting both major and minor determinants of HEL over a 100-fold range of Ag dose, and 3) that B cells present Ag gained at early times after immunization, but only following higher dose immunization. These data indicate that the breadth of a response is maintained over a wide dosage range by concentration of Ag in a limited number of cells presenting high levels and a great diversity of epitopes.     

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.     

Weak base amines can inhibit class I MHC-restricted antigen presentation

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.     

Processing and reactivity of T cell epitopes containing two cysteine residues from hen egg-white lysozyme (HEL74-90)

The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74-88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74-88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74-88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.     

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.     

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.     

Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome

Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4+ T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-Ak genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.     

Dendritic cells process and present antigens across a range of maturation states

We isolated dendritic cells (DC) from lymphoid organs of mice bearing a transgene for a membrane-bound form of the model protein hen egg white lysozyme (HEL). DC from the spleen had a lower representation of costimulatory molecules and class II MHC molecules than those isolated from lymph nodes and thymi. Splenic DC were capable of further maturation by in vivo treatment of mice with LPS. The immature DC from spleen processed HEL and displayed the chemically dominant epitope as evidenced by FACS analysis. These immature DC also presented this epitope to CD4(+) T cells. Splenic DC from another transgenic mouse (ML-5) containing serum HEL also showed the ability to process and present Ag despite low levels of circulating HEL. In vitro-derived DC from the bone marrow (bone marrow-derived DC) of mHEL mice also displayed immature to mature features and in both cases displayed HEL peptides as well as SDS-stable MHC class II molecules. Immature bone marrow-derived DC also processed exogenous HEL. We conclude that the DC sets normally found in tissue show a scale of maturation features but even the most immature process and present peptides by MHC class II molecules.     

Direct cross-priming by th lymphocytes generates memory cytotoxic T cell responses

Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.     

Human T cell clones present antigen

Two human T cells clones are described which react with influenza virus hemagglutinin type H3 and synthetic peptides of H3 when presented by PBMC APC. Both T cell clones also responded to peptide Ag in the absence of additional APC suggesting that T cells can simultaneously present and respond to Ag. T cell clones could only present peptide Ag and not an appropriate strain of inactivated whole influenza virus thus indicating an inability to process Ag conventionally. Peptide presentation by T cells was dose dependent, restricted by MHC class II Ag and was dependent on the number of Ag presenting T cells per culture. Experiments with nested peptides showed that the same epitope was recognized in the presence and absence of PBMC APC. No Ag or IL-2 from the propagation procedure was carried over into assays and two-color fluorescence-activated cell sorter analysis of each clone detected no contaminating cells with the phenotype of monocytes, macrophages or B cells; in each T cell clone, all cells expressing MHC class II Ag co-expressed CD3. These date therefore provide strong evidence that human T cell clones can simultaneously present and respond to appropriate forms of Ag.     

Cutting edge: efficient MHC class I cross-presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells

Priming of CD8(+) T cells requires presentation of short peptides bound to MHC class I molecules of professional APCs. Cross-presentation is a mechanism whereby professional APC present on their own MHC class I molecules peptides derived from degradation of Ags synthesized by other Ag "donor cells." The mechanism of cross-presentation is poorly understood, and the nature of the transferred Ag is unknown. In this report, we demonstrate that the bulk of a cross-presented Ag transferred from donor cells recently infected with vaccinia virus are proteasomal products that are susceptible to peptidases within the donor cell cytosol and not full-length proteins or mature epitopes either free or bound to chaperones.     

Mutations affecting antigen processing impair class II-restricted allorecognition

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.