Presentation of antigens internalized through the B cell receptor requires newly synthesized MHC class II molecules

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their alpha- or beta-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak beta-chain is required for the presentation of the hen egg lysozyme determinant (46-61) following BCR internalization, but that dependence is not seen for the (34-45) determinant derived from the same protein. The tail of the beta-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II beta-chains control the loading of determinants produced after extensive Ag processing.     

Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding.

MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.     

Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway

The intracellular sites in which Ags delivered by the B cell receptor (BCR) are degraded and loaded onto class II molecules remain poorly defined. To address this issue, we generated wild-type and invariant chain (Ii)-deficient H-2k mice bearing BCR specific for hen egg lysozyme. Our results show that, 1) unlike Ags taken up from the fluid phase, Ii is required for presentation of hen egg lysozyme internalized through the BCR in a manner independent of the peptide analyzed; 2) BCR ligation induces intracellular accumulation of MHC class II molecules only in Ii-positive B cells; and 3) these class II molecules reach intracellular compartments where BCR targets exogenous Ag. No differences in expression of adhesion and costimulatory molecules or in the presentation of soluble peptides were detectable between Ii-positive and -negative B cells. Therefore, the BCR delivers its ligand to compartments containing MHC class II-Ii complexes and bypasses the Ii-independent presentation pathway. The linked roles of Ag internalization and B cell activation of the BCR leads to potent Ii-dependent presentation in splenic B cells.     

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.     

The HSC73 molecular chaperone: involvement in MHC class II antigen presentation.

Heat shock proteins (HSP) are conserved proteins, many of which share the ability for indiscriminate peptide binding and ATPase-coupled peptide release. In this paper, we show that heat shock cognate protein (HSC)73, a constitutively expressed member of the HSP70 family, could be a candidate for chaperone activity within the MHC class II presentation pathway. HSC73 expression in macrophages was shown to overlap with expression of MHC class II; overexpression of HSC73 in stable transfectants of a macrophage line markedly enhanced their presentation of exogenous Ag without affecting presentation of processing independent peptide. Ag from an exogenous source was demonstrated to associate with HSC73 in macrophages, and this association was sensitive to ATP treatment and inhibited by deoxyspergualin, an immunosuppressive agent that has previously been shown to bind specifically to HSC73. Furthermore, deoxyspergualin reduced Ag presentation by macrophages in relation to the amount of HSC73 expressed in these cells. The data are consistent with a potential role for HSC73 in binding and protecting peptides from extensive degradation and/or facilitating the kinetics of peptide transfer to MHC class II molecules.     

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.     

Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells.

Phagocytic processing of heat-killed Listeria monocytogenes by peritoneal macrophages resulted in degradation of these bacteria in phagolysosomal compartments and processing of bacterial antigens for presentation to T cells by class II MHC molecules. Within 20 min of uptake by macrophages, Listeria peptide antigens were expressed on surface class II MHC molecules, capable of stimulating Listeria-specific T cells. Within this period, degradation of labeled bacteria to acid-soluble low molecular weight catabolites also commenced. Immunoelectron microscopy was used to evaluate the compartments involved in this processing. Upon uptake of the bacteria, phagosomes containing Listeria fused rapidly with both lysosomes and endosomes. Class II MHC molecules were present in a tubulo-vesicular lysosome compartment, which appeared to fuse with phagosomes, as well as in the resulting phagolysosomes containing internalized Listeria; these compartments were all positive for Lamp 1 and cathepsin D and lacked 46-kD mannose-6-phosphate receptors. In addition, class II MHC and Lamp 1 were co-localized in vesicles of the trans Golgi reticulum, where they were segregated from 46-kD mannose-6-phosphate receptors. Vesicles containing both Listeria-derived components and class II MHC molecules were also observed; some of these may represent vesicles recycling from phagolysosomes, potentially bearing processed immunogenic peptides complexed with class II MHC. These results support a central role for lysosomes and phagolysosomes in the processing of bacterial antigens for presentation to T cells. Tubulo-vesicular lysosomes appear to represent an important convergence of endocytic, phagocytic and biosynthetic pathways, where antigens may be processed to allow binding to class II MHC molecules and recycling to the cell surface.     

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.     

Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation

Liposome-encapsulated protein Ag were used to dissect the roles of various subcellular compartments in Ag processing for class I and class II MHC-restricted presentation. Macrophages exhibited efficient processing of Ag encapsulated in acid-resistant dioleoylphosphatidylcholine/dioleoylphosphatidylserine liposomes, which sequester their contents from potential endosomal processing events and release them only after delivery to lysosomes. Lysosomal processing was demonstrated for all four Ag studied (OVA, murine hemoglobin, bovine ribonuclease A, and hen egg lysozyme), establishing the recycling of immunogenic peptides from lysosomes after Ag processing. These acid-resistant liposomes did not engender class I processing. Ag encapsulated within acid-sensitive dioleoylphosphatidylethanolamine/palmitoylhomocysteine liposomes were also processed via the class II pathway. Of the four Ag encapsulated in liposomes, one, OVA, was tested for ability to stimulate a class I-specific response. OVA in acid-resistant liposomes did not engender a class I-specific response. In contrast, OVA encapsulated in acid-sensitive liposomes was presented by class I molecules, albeit less efficiently than it was presented by class II molecules. We interpret this to be the result of the release of a minor portion of the encapsulated Ag into the cytosol.     

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.     

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.     

The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation

We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.     

Differential antigen presentation by cloned populations of mouse splenic macrophages

Soft agar colonies of mouse splenic macrophages differ in their ability to process and present complex Ag to T cell hybridomas. To determine if the basis for this differential activity was the synthesis of molecules that might interfere with the activity of either the hybridoma or the indicator cells used for the bioassay of IL-2, culture supernatants were compared from Ag-presenting and nonpresenting cultures for their content of suppressor activity, using mitogen-treated mouse SC. No correlation was found between a colony's Ag-presenting activity and its secretion of suppressor factors, nor did colonies unable to present Ag release factors that interfered with the detection of IL-2. In a second approach, paired subcultures from individual colonies were tested for their ability to present, to the same hybridoma, both native Ag and the "preprocessed" peptide of the Ag. The presentation of native Ag was restricted to the progeny of a minority of the cloned macrophage progenitors, but all of the progeny cultures presented the peptide. Together, these results suggest that the basis for differential Ag presentation may be in the manner in which the cloned macrophages degrade and process ingested Ag.     

MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A

The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment. However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process. The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules. An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins. To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A. Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas. No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells. Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins.     

I-Ak polymorphisms define a functionally dominant region for the presentation of hen egg lysozyme peptides

The class II molecules of the MHC not only bind processed antigenic peptides but also interact with the TCR. This latter interaction is thought to be the basis for allele specific "restriction" of Ag presentation to T cells. The specificity of this interaction is likely due to amino acid differences in a small number of polymorphic or "hypervariable" regions located in the amino terminal domains of the alpha- and beta-chains. We have explored the functional significance of these polymorphic regions in an I-Ak-restricted, hen egg lysozyme specific Ag presentation system in which the measurement of IL-2 production by T cell hybridomas was used as the indicator of TCR recognition of the I-A/Ag complex. Chimeric I-A molecules, in which b allelic residues were substituted in one or more of the polymorphic regions of the A alpha k chain or in which d allelic residues were substituted in one or more of the polymorphic regions of the A beta k chain, were used to examine the contribution of each polymorphic region of the molecule to its function. The results obtained demonstrate that the regions between residues 69 to 76 of the A alpha k chain and the regions between residues 63 to 67 and 75 to 78 of the A beta k-chain exert a dominant effect on the presentation of lysozyme peptides by I-Ak to the T cell hybridomas in our panel. These observations were confirmed and extended by the analysis of Ag presentation by seven serologically selected mutants, all of which have amino acid interchanges in or around the dominant polymorphic regions. The results suggest that the serologically selected mutants fail to present Ag not because they fail to bind the peptide Ag but because the amino acid substitutions destabilize the interaction between the Ia/peptide complex and the TCR. Use of the recently published hypothetical model for class II structure to interpret the Ag presentation results suggests that the dominant polymorphic regions lie across from one another near one end of the alpha-helices that form the two walls of the proposed Ag-binding cleft located on the top surface of the class II molecule. Furthermore, the majority of the amino acids which have been changed in the serologically selected mutants have side chains which are postulated to point up toward the exterior of the molecule and would, therefore, be potential contact residues for the TCR.     

Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.

Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited. We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61. This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope. Phagocytosis of E. coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells. Surface-conjugated HEL was processed with even greater efficiency. Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages. Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded. Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response. We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes. Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.     

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.     

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.     

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.