Melanosomes and MHC class II antigen-processing compartments: a tinted view of intracellular trafficking and immunity

Melanosomes are specialized intracellular compartments within melanocytes and retinal pigment epithelial cells that function in the synthesis, storage, and secretion of melanins, which are the major pigments made by mammals. The mechanisms that regulate the formation of melanosomes, and the pathways by which constituent proteins are targeted to them, are related to those involved in the biogenesis of major histocompatibility complex (MHC) class II antigen-processing compartments. Consequently, diseases that affect pigmentation may also affect antigen presentation to T cells. Moreover, many of the tissue-specific proteins that localize to melanosomes and participate in melanin formation double as tumor-associated antigens that are targets for T cells in patients with melanoma. Our studies on melanosome biogenesis are providing new ways of thinking about antigen-processing compartments and the mechanisms regulating presentation of tumor-associated antigens.     

Involvement of autophagy in MHC class I antigen presentation

MHC class I molecules on the cellular surface display peptides that either derive from endogenous proteins (self or viral), or from endocytosis of molecules, dying cells or pathogens. The conventional antigen-processing pathway for MHC class I presentation depends on proteasome-mediated degradation of the protein followed by transporter associated with antigen-processing (TAP)-mediated transport of the generated peptides into the endoplasmic reticulum (ER). Here, peptides are loaded onto MHC I molecules before transportation to the cell surface. However, several alternative mechanisms have emerged. These include TAP-independent mechanisms, the vacuolar pathway and involvement of autophagy. Autophagy is a cell intrinsic recycling system. It also functions as a defence mechanism that removes pathogens and damaged endocytic compartments from the cytosol. Therefore, it appears likely that autophagy would intersect with the MHC class I presentation pathway to alarm CD8⁺ T cells of an ongoing intracellular infection. However, the importance of autophagy as a source of antigen for presentation on MHC I molecules remains to be defined. Here, original research papers which suggest involvement of autophagy in MHC I antigen presentation are reviewed. The antigens are from herpesvirus, cytomegalovirus and chlamydia. The studies point towards autophagy as important in MHC class I presentation of endogenous proteins during conditions of immune evasion. Because autophagy is a regulated process which is induced upon activation of, for example, pattern recognition receptors (PRRs), it will be crucial to use relevant stimulatory conditions together with primary cells when aiming to confirm the importance of autophagy in MHC class I antigen presentation in future studies.     

Peptide exchange in MHC molecules

Summary: Major histocompatibihty complex (MHC)-encoded glycoproteins bind peptide antigens through non-covalent interactions to generate complexes that are displayed on tbe surface of antigen-presenting cells (APC) for recognition by T ceils, Peptide-binding site occupancy is necessary for stable assembly of newly synthesized MHC proteins and export from the endoplasmic reticulum (ER), The MHC class II antigen-processing pathway provides a mechanism for presentation of peptides generated in the endosomal pathway of APC, The chaperone protein, invariant chain, includes a surrogate peptide that stahilizes newly synthesized class II molecules during transport to endosomal compartments. The invariant chain-derived peptide must be replaced through a peptide exchange reaction that is promoted by acidic pH and the MHC-encoded co-factor HLA-DM, Peptide exchange reactions are not required for presentation of antigens by MHC class I molecules because they bind antigens during initial assembly in the ER, However, exchange reactions may play an important role in editing the repertoire of peptides presented by both class II and class I molecules, thus influencing the specificity of immunity and tolerance.     

Protein sorting within the mhc class II antigen-processing pathway

Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.     

Phagocytic antigen processing and effects of microbial products on antigen processing and T-cell responses

Summary: Processing of exogenous antigens and microbes involves contributions by multiple different endocytic and phagocytic compartments. During the processing of soluble antigens, different endocytic compartments have been demonstrated to use distinct antigen-processing mechanisms and to process distinct sets of antigenic epitopes. Processing of particulate and microbial antigens involves phagocytosis and functions contributed by phagocytic compartments. Recent data from our laboratory demonstrate that phagosomes containing antigen-conjugated latex beads are fully competent class U MHC (MHC-II) antigen-processing organelles, which generate peptide:MHC-II complexes. In addition, phagocytosed antigen enters an alternate dass I MHC (MHC-I) processing pathway that results in loading of peptides derived from exogenous antigens onto MHC-I molecules, in contrast to the cytosolic antigen source utilized by the conventional MHC-I antigen-processing pathway. Antigen processing and other Immune response mechanisms may be activated or inhibited by microbial components to the benefit of either the host or the pathogen. For example, antigen processing and T-cell responses (e.g. Th1 vs Th2 differentiation) are modulated by multiple distinct microbial components, including lipopolysaccharide, cholera toxin, heat labile enterotoxin of Escherichia coli , DNA containing CpG motifs (found in prokaryotic and invertebrate DNA but not mammalian DNA) and components of Mycobacterium tuberculosis.     

Bacterial and host factors involved in the major histocompatibility complex class Ib-restricted presentation of Salmonella Hsp 60: novel pathway

Previously, a peptide epitope derived from the Hsp 60 molecule of Salmonella that is presented by the major histocompatibility complex (MHC) class Ib molecule Qa-1 to CD8(+) cytotoxic T cells (CTLs) was described. In the present study we investigated the Salmonella-induced processing and presentation pathway for generating this Qa-1-restricted epitope. Live bacteria and, to a lesser extent, opsonized heat-killed bacteria are able to sensitize target cells for lysis by Salmonella-specific CTL. In contrast, heat-killed bacteria cannot sensitize target cells. Presentation of the Hsp 60 epitope appears independent of bacterial internalization, because cytochalasin D does not affect presentation. Moreover, Salmonella strains defective in the InvA or InvE operon, two critical components of the type III secretion pathway, are as efficient as wild-type Salmonella enterica serovar Typhimurium in sensitizing infected targets to lysis. Collectively, these results suggest the existence of a novel antigen-processing pathway in which exogenous antigens gain access to the cytosolic MHC class I processing machinery. Considering the abundant nature of bacterial Hsp 60 and the upregulation of this protein after Salmonella infection of eukaryotic cells, this mode of antigen presentation may be particularly relevant to understanding the host defense mechanisms against gram-negative bacteria.     

Anti-HER2/neu IgG3-(IL-2) and anti-HER2/neu IgG3-(GM-CSF) promote HER2/neu processing and presentation by dendritic cells: implications in immunotherapy and vaccination strategies

HER2/neu, a transmembrane glycoprotein overexpressed in several types of human cancers, is a potential target for active immunotherapy. However, this protein and especially its extracellular domain (ECD(HER2)), is weakly immunogenic and is poorly processed by dendritic cells (DCs). Previously, we showed that anti-HER2/neu IgG3-(IL-2) and anti-HER2/neu IgG3-(GM-CSF) fusion proteins can enhance the immunogenicity of ECD(HER2) in mice, and that the non-covalent physical association between each antibody fusion proteins and ECD(HER2) was critical to elicit optimal protective immunity against HER2/neu expressing tumors. We now use the professional antigen-presenting DCs to investigate the effect of the antibody fusion protein binding to ECD(HER2) on its trafficking and presentation. We found that when the extracellular domain of HER2/neu fused to ovalbumin (OVA-ECD(HER2)) is bound by HER2/neu-specific antibody-(IL-2) or antibody-(GM-CSF) fusion proteins, the bound antigen is more efficiently processed by murine bone-marrow-derived dendritic cells (BMDCs) and presented to OVA-specific T-cells than the unbound OVA-ECD(HER2). We also found that ECD(HER2) bound by anti-HER2/neu IgG3-(IL-2) is very efficiently internalized and that the internalized ECD(HER2) is not retained in the early endosomal compartments but traffics to the antigen-processing compartments. These results are consistent with our earlier in vivo studies and suggest that both antibody-(IL-2) and antibody-(GM-CSF) fusion proteins can be used to enhance the immune response to poorly immunogenic antigens including tumor-associated antigens (TAAs).     

CD1-Restricted T cells: T cells with a unique immunological niche

Until recently, antigen presentation to T cells was defined only by proteins encoded within the MHC locus. That definition has now been expanded to include proteins encoded outside the MHC locus, most notably the CD1 family of proteins. The pathway of CD1-presented antigens diverges from that of MHC processing, indicating that the CD1 antigen-processing pathway may be complementary to the MHC pathways. The most surprising finding of the CD1 antigen-presenting system is that the antigens presented by CD1 are not peptides, but rather lipid and glycolipid in nature. The most compelling evidence for the role of CD1-restricted T cells in immune homeostasis stems from studies of mycobacterial infection and autoimmunity. These studies suggest that CD1-restricted T cells promote cell-mediated immune responses to intracellular infection and protect against anti-self responses.     

Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation

CD1 proteins present various glycolipid antigens to T cells, but the cellular mechanisms that control which particular glycolipids generate T cell responses are not understood. We show here that T cell recognition of glucose monomycolate antigens with long (C(80)) alkyl chains involves the delivery of CD1b proteins and antigens to late endosomes in a process that takes several hours. In contrast, analogs of the same antigen with shorter (C(32)) alkyl chains are rapidly, but inefficiently, presented by cell surface CD1b proteins. Dendritic cells (DCs) preferentially present long-chain glycolipids, which results, in part, from their rapid internalization and selective delivery of antigens to endosomal compartments. Nonprofessional antigen-presenting cells, however, preferentially present short-chain glycolipids because of their lack of prominent endosomal presentation pathways. Because long alkyl chain length distinguishes certain microbial glycolipids from common mammalian glycolipids, these findings suggest that DCs use a specialized endosomal-loading pathway to promote preferential recognition of glycolipids with a more intrinsically foreign structure.     

Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo

CD8(+) T cells (T(CD8+)) differentiate into effector cells following recognition of specific peptide-major histocompatibility complex (MHC) class I complexes (pMHC-I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC-I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC-I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C-type lectin scavenger receptor A (SR-A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC-I from a donor cell to the pAPC. We demonstrate here that initiation of T(CD8+) responses is normal in mice lacking SR-A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen-processing pathways during the rational design of vaccines aimed at eliciting protective T(CD8+).     

Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.     

Processing of bacterial antigens for peptide presentation on MHC class I molecules

Summary: Professional antigen-presenting cells (pAPC) can process and present exogenous antigens on major histocompatibility complex class 1 (MHC-I) molecules. This unusual pathway for antigen presentation may represent a physiologically important step in the course of priming and tolerance induction of CD8⁺ T cells. In addition, it may play an important role in immunological surveillance for pathogens that survive in vacuolar compartments in APC. The goal of the present review is to discuss recent studies on the processing of bacterial-derived antigens for presentation on MHC-1 molecules. The antigen presentation emphasized will include bacteria that remain confined in vacuolar compartments. This is in contrast to antigens derived from bacteria that have intrinsic properties allowing translocation across membranes and access into the classical MHC-I presentation pathway In particular, presentation of bacterial antigens by dendritic cells (DC) will be emphasized, and MHC-I presentation of antigens derived from apoptotic cells, particularly cells induced to undergo apoptosis by microbial infection, will be presented. Finally, some special aspects of the interaction between bacteria and DC will be discussed as ii relates to DC maturation, antigen presentation and T-cell stimulation.     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Survival of Salmonella enterica serovar Typhimurium within late endosomal-lysosomal compartments of B lymphocytes is associated with the inability to use the vacuolar alternative major histocompatibility complex class I antigen-processing pathway

Gamma interferon (IFN-gamma)-activated macrophages use an alternative processing mechanism to present Salmonella antigens to CD8(+) T lymphocytes. This pathway involves processing of antigen in a vacuolar compartment followed by secretion and loading of antigenic peptides to major histocompatibility complex class I (MHC-I) molecules on macrophage cell surface and bystander cells. In this study, we have shown that B lymphocytes are not able to process Salmonella antigens using this alternative pathway. This is due to differences in Salmonella enterica serovar Typhimurium-containing vacuoles (SCV) when comparing late endosomal-lysosomal processing compartments in B lymphocytes to those in macrophages. The IFN-gamma-activated IC21 macrophage cell line and A-20 B-cell line were infected with live or dead Salmonella enterica serovar Typhimurium. The SCV in B cells were in a late endosomal-lysosomal compartment, whereas SCV in macrophages were remodeled to a non-characteristic late endosomal-lysosomal compartment over time. Despite the difference in SCV within macrophages and B lymphocytes, S. enterica serovar Typhimurium survives more efficiently within the IFN-gamma-activated B cells than in activated macrophage cell lines. Similar results were found during in vivo acute infection. We determined that a lack of remodeling of late endosomal-lysosomal compartments by live Salmonella infection in B lymphocytes is associated with the inability to use the alternative MHC-I antigen-processing pathway, providing a survival advantage to the bacterium. Our data also suggest that the B lymphocyte late endosome-lysosome environment allows the expression of Salmonella virulence mechanisms favoring B lymphocytes in addition to macrophages and dendritic cells as a reservoir during in vivo infection.     

Expression of HLA class I antigens in transporter associated with antigen processing (TAP)-deficient mutant cell lines

Abstract: Cells lacking expression of the transporter associated with antigen processing (TAP) are deficient in surface HLA class I, yet express reduced levels of HLA-A2 antigen through TAP-independent processing pathways. We have analysed the expression of HLA-A, -B and -C antigens on the 721.174 and T2 TAP-deficient mutant cell lines using a panel of monoclonal antibodies specific for the HLA antigens encoded by the genotype of these cells. Our study has shown the constitutive expression of HLA-Cwl molecules on the cell surface of both T2 and 721.174 cells and has confirmed that HLA-A2 and HLA-B51 are expressed at low levels. Transfection of 721.174 cells with cDNAs encoding TAP1 and TAP2 proteins did not fully restore HLA class I antigen expression on these cells, which appeared to be mainly due to a deficiency in expression of the HLA-B51-associ-ated Bw4 epitope. This suggests that additional antigen-processing genes may be required for optimal generation of HLA-B-binding peptides. Our results indicate that TAP-independent pathways of antigen-processing provide peptides for functional expression of all three classical HLA class I molecules.     

Class I MHC presentation of exogenous antigens

Class I MHC (MHC-I) molecules present primarily endogenous antigens, i.e. antigens that are present in the cytosol and are subject to the cytosolic processing mechanisms that comprise the conventional MHC-I processing pathway. However, exogenous antigens can also be presented by MHC-I molecules in certain circumstances, particularly in the case of particulate antigens. Recently, considerable attention has been focused on mechanisms that may contribute to alternate MHC-I processing pathways. Divergent results in several different systems have suggested that more than one alternate processing mechanism may exist. After phagocytic or endocytic uptake, some exogenous antigens can escape the vacuolar system and penetrate into the cytosol, accessing the conventional MHC-I antigen processing mechanisms. In other cases, MHC-I molecules present antigens that have no clear ability to actively escape the vacuolar system. Some results indicate that certain alternate processing mechanisms are quite distinct from the conventional MHC-I pathway and are not dependent on compartments, proteins, or mechanisms that are necessary for the conventional pathway, including the endoplasmic reticulum, the transporter for antigen presentation (TAP) and proteasomes.In vivo, alternate MHC-I processing mechanisms may be expressed primarily by phagocytic antigen presenting cells, i.e., macrophages, and perhaps dendritic cells, although other cell types may contribute in certain circumstances. These mechanisms may play important roles in generating CD8 T cell responses, especially to antigens expressed by vacuolar microorganisms. In addition, they provide a potential avenue for therapeutic immunization to achieve protective CD8 T cell responses with nonviable vaccine preparations, in the absence of the endogenous antigen synthesis that is provided by live viral vaccine preparations.     

Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells

CD4⁺ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the 14.07 macrophage cell line (H-2^k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4⁺ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4⁺ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4⁺ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.     

Distribution of productive antigen-processing activity for MHC class II presentation in macrophages

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigen-presenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.     

Human pathogen subversion of antigen presentation

Summary: Many pathogens have co-evolved with their human hosts to develop strategies for immune evasion that involve disruption of the intra-cellular pathways by which antigens are bound by class I and class II molecules of che major histocompatibility complex (MHC) for presentation ro T cells. Here the molecular events in these pathways are reviewed and pathogen interference is documented for viruses, extracellular and intra-cellular bacteria and intracellular parasites. In addition to a general review, data from our studies of adenovirus, Chlamydia tmchamatis and Coxiella burnetii are summarized. Adenovirus E19 is the first viral gene product described that affects class I MHC molecule expression by two separate mechanisms, intracellular retention of the class I heavy chain by direct binding and by binding to the TAP transporter involved in class I peptide loading. Coxiella and Chlamydia both affect peptide presentation by class II MHC molecules as a result of their residence in endocytic compartments, although the properties of the parasitophorous vacuoles they form are quite different. These examples of active interference with antigen presentation by viral gene products and passive interference by rickettsiae and bacteria are typical of the strategies used by these different classes of pathogens, which need to evade different types of immune responses. Pathogen–host co-evolution is evident in these subversion tactics for which the pathogen crime seems tailored to fit the immune system punishment.     

Vectorial function of major histocompatibility complex class II in a human intestinal cell line.

This study explores the expression and the function of major histocompatibility complex class II in the intestinal epithelial cell line CaCo-2, which has been widely used as a model for the human gastrointestinal epithelium. Human leucocyte antigen (HLA)-DR expression on CaCo-2 cells is induceable by interferon-gamma (IFN-gamma), but responsiveness to IFN-gamma is dependent on cell differentiation and IFN-gamma availability at the basolateral cell surface. HLA-DR expression is concentrated in apical cytoplasmic vesicles and on the basolateral cell surface. Invariant chain is expressed in apical vesicles but is absent from the cell surface. Immunoprecipitation studies show a slow rate of dissociation of HLA-DR from Ii. Double labelling shows some overlap between HLA-DR expression and basolateral endosomal markers but no overlap with apical endosomal markers. Functional studies show processing and presentation of lysozyme endocytosed from the basolateral, but not apical surfaces. CaCo-2 cells may provide a useful model with which to dissect the antigen-processing pathways in polarized epithelial cells. The regulated access of antigens taken up from the gut lumen to the processing compartments may prevent overloading the immune system with antigens derived from normal gut contents.