Tumor cells present MHC class II-restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.     

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

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

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.     

Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen.

Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.     

Endocytic recycling is required for the presentation of an exogenous peptide via MHC class II molecules

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin-derived peptide in the context of HLA-DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N-terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II-restricted peptide presentation via the endocytic pathway.     

The class II MHC-restricted presentation of endogenously synthesized ovalbumin displays clonal variation, requires endosomal/lysosomal processing, and is up-regulated by heat shock.

LB27.4 cells (a B lymphoblastoid APC) were transfected with a plasmid containing an OVA cDNA. Functional analysis of six independent clones yielded three patterns of MHC-restricted presentation of the endogenously synthesized OVA. A clone displayed either: 1) strong class I and class II-restricted presentation, 2) strong class I but little or no class II-restricted presentation or, 3) only a modest class I-restricted presentation. There was no clonal variation in class II-restricted presentation of exogenous Ag or in the amount of surface class I or II molecules. Heat shock increased the presentation of endogenous but not exogenous Ag with class II. These results indicate that an endogenously synthesized Ag both constitutively and during heat shock can gain access to the class II, MHC-restricted, presentation pathway. The amount of OVA synthesized by a cell correlated with whether OVA-class II complexes were detected. However, the amount of OVA secreted into the extracellular fluid was not sufficient to sensitize APC, which suggests that endogenously synthesized OVA enters the class II pathway of Ag presentation by an intracellular route rather than by an extracellular/reuptake route. Also, the functional and quantitative analysis of the clones suggests that endogenously synthesized OVA was presented more efficiently with class I as compared to class II-MHC molecules. Leupeptin and chloroquine inhibited the class II-restricted presentation of endogenously synthesized OVA. Together these results indicate that endogenously synthesized OVA can gain access to an endosomal/lysosomal compartment via an intracellular route and be processed and presented in association with class II-MHC molecules.     

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.     

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.     

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.     

Efficient delivery of T cell epitopes to APC by use of MHC class II-specific Troybodies

A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91-101 lambda2(315), which is presented by I-E(d); 110-120 hemagglutinin (I-E(d)); 323-339 OVA (I-A(d)); and 46-61 hen egg lysozyme (I-A(k)). We denote such APC-specific, epitope-containing Ab "Troybodies." When mixed with APC, all four class II-specific Troybodies were approximately 1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000-10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100-100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development.     

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.     

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.     

Invariant chain and the MHC class II cytoplasmic domains regulate localization of MHC class II molecules to lipid rafts in tumor cell-based vaccines

Cell-based tumor vaccines, consisting of MHC class I+ tumor cells engineered to express MHC class II molecules, stimulate tumor-specific CD4+ T cells to mediate rejection of established, poorly immunogenic tumors. Previous experiments have demonstrated that these vaccines induce immunity by functioning as APCs for endogenously synthesized, tumor-encoded Ags. However, coexpression of the MHC class II accessory molecule invariant chain (Ii), or deletion of the MHC class II cytoplasmic domain abrogates vaccine immunogenicity. Recent reports have highlighted the role of lipid microdomains in Ag presentation. To determine whether Ii expression and/or truncation of MHC class II molecules impact vaccine efficacy by altering MHC class II localization to lipid microdomains, we examined the lipid raft affinity of MHC class II molecules in mouse M12.C3 B cell lymphomas and SaI/A(k) sarcoma vaccine cells. Functional MHC class II heterodimers were detected in lipid rafts of both cell types. Interestingly, expression of Ii in M12.C3 cells or SaI/A(k) cells blocked the MHC class II interactions with cell surface lipid rafts. In both cell types, truncation of either the alpha- or beta-chain decreased the affinity of class II molecules for lipid rafts. Simultaneous deletion of both cytoplasmic domains further reduced localization of class II molecules to lipid rafts. Collectively, these data suggest that coexpression of Ii or deletion of the cytoplasmic domains of MHC class II molecules may reduce vaccine efficacy by blocking the constitutive association of MHC class II molecules with plasma membrane lipid rafts.     

Mutation of the invariant chain transmembrane region inhibits II degradation, prolongs association with MHC class II, and selectively disrupts antigen presentation.

The invariant chain (Ii) is a key player in regulating the MHC Class II antigen presentation pathway. Here we used site-directed mutagenesis to identify functionally important regions of the invariant chain in regulating antigen presentation function in transfected cells. Mutation of Ii residues 42-53 caused a defect in the presentation of the ovalbumin 247-265/A(k) epitope, but not in the inhibition of presentation of two hen egg lysozyme epitopes, HEL34-45/A(k) and HEL74-88/A(b), from endogenously expressed antigens. The mutation did not prevent ER translocation, trimerization, or association with MHC Class II molecules and had no obvious effect on endosomal targeting of Ii. It did, however, increase the half-life of the invariant chain, suggesting that sequences in this region influence the degradation of the invariant chain and as a consequence its function in antigen presentation.     

Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages

Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4(+) T cells and MHC class II(+) APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4(+) dendritic cells (DC) and macrophages (Mphi) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and Mphi. DC efficiently presented partially degraded, but not native CII alpha-chains, while Mphi presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and Mphi presented soluble HCgp39, and activated Mphi from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and Mphi to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.     

Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes.

We find that expression of the membrane dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) enhances presentation of certain endogenously synthesized peptides to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes. ACE appears to function only in an intracellular secretory compartment of antigen-presenting cells. ACE-enhanced antigen presentation requires the expression of the putative antigenic peptide transporters, TAP1 and TAP2. These findings demonstrate that a protease can influence the processing of endogenously synthesized antigens and strongly suggest that longer peptides can be transported from the cytosol to a secretory compartment where trimming of antigenic peptides to the lengths preferred by MHC class I molecules can occur if the appropriate protease is present.     

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.     

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.     

Abrogation of tumorigenicity by MHC class II antigen expression requires the cytoplasmic domain of the class II molecule

Transfection of syngeneic MHC class II genes into the lethal mouse SaI tumor abrogates the malignancy of the tumor in the autologous host, and protects the host against subsequent challenges with the wild type class II- tumor. We have hypothesized that the transfectants induce protective immunity by functioning as APC for tumor peptides, and stimulating tumor-specific Th cells. Recent in vitro studies suggest that Ag presentation by class II-restricted APC requires the cytoplasmic domain of the class II molecule, and may involve intracellular signaling via the cytoplasmic domain. To determine if the class II cytoplasmic domain is required for enhanced tumor-specific immunity, SaI mouse sarcoma cells were transfected with syngeneic Aak and Abk genes with truncated cytoplasmic domains. These transfectants are as malignant as wild type class II- SaI cells in autologous A/J mice. Stimulation of tumor-specific immunity by class II+ tumor cells is therefore dependent on the class II cytoplasmic region, and may involve intracellular signaling events.     

Cutting edge: H-2DM is responsible for the large differences in presentation among peptides selected by I-Ak during antigen processing

We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-A(k) molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.A(k) line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.