Human tumor antigen MUC1 is chemotactic for immature dendritic cells and elicits maturation but does not promote Th1 type immunity

The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DCs) has been well characterized. There are many fewer examples of similar interactions between DCs and self-molecules, especially the abnormal self-proteins such as many tumor Ags, and their effects on DC function and the immune response. We show that human epithelial cell Ag MUC1 mucin is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DCs as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). On encounter with MUC1, similar to the encounter with LPS, immature DCs increase cell surface expression of CD80, CD86, CD40, and CD83 molecules and the production of IL-6 and TNF-alpha cytokines but fail to make IL-12. When these DCs are cocultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our data suggest that, in vivo in cancer patients, MUC1 attracts immature DCs to the tumor through chemotaxis and subverts their function by negatively affecting their ability to stimulate type 1 helper T cell responses important for tumor rejection.     

Impaired tumor antigen processing by immunoproteasome-expressing CD40-activated B cells and dendritic cells

Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1_26?C34 (M26) and Cyp1B1_239?C247 (Cyp239)] and one derived from the influenza A viral antigen [FluM1_58?C66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.     

Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.     

Endosomal sorting of MHC class II determines antigen presentation by dendritic cells

Dendritic cells (DCs) initiate primary immune responses by presenting pathogen-derived antigens in association with major histocompatibility Class II molecules (MHC II) to T cells. In DCs, MHC II is constitutively synthesized and loaded at endosomes with peptides from hydrolyzed endogenous proteins or exogenously acquired antigens. Whether peptide loaded MHC II (MHC II-p) is subsequently recruited to and stably expressed at the plasma membrane or degraded in lysosomes is determined by the status of the DC. In immature DCs, MHC II-p is ubiquitinated after peptide loading, driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles, and the MHC II-p they carry, are delivered to lysosomes for degradation. MHC II-p is inefficiently ubiquitinated in DCs that are activated by pathogens or inflammatory stimuli, thus allowing its transfer to and stable expression at the plasma membrane.     

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.     

Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendritic cells

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-1(27-35) that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-1(27-35) also protected mice against growth of B16-A2/K(b) tumors in HLA-A2.1/K(b) transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.     

Recombinant human tumor antigen MUC1 expressed in insect cells: structure and immunogenicity

MUC1, a member of the mucin family of molecules, is a transmembrane glycoprotein abundantly expressed on human ductal epithelial cells and tumors originating from those cells. MUC1 expressed by malignant cells is aberrantly O-glycosylated. Differences in O-glycosylation of the tandem repeat region of MUC1 make tumor and normal forms of this antigen immunologically distinct. The tumor-specific glycoform is, therefore, expected to be a good target for immunotherapy and a good immunogen for generation of antitumor immune responses. We have generated a renewable source of this glycoform by expressing MUC1 cDNA in Sf-9 insect cells using a baculovirus vector. This form of MUC1 (BV-MUC1) is O-glycosylated at a very low level, approximately 0.3% (w/w), and this is not due to the lack of appropriate glycosylotransferases in insect cells. Peptidyl GalNAc-transferases isolated from Sf-9 cells were able to glycosylate in vitro a synthetic MUC1 peptide as efficiently as the transferases isolated from human milk. Neither preparation of peptidyl GalNAc-transferases, however, was able to glycosylate BV-MUC1. This underglycosylated recombinant MUC1 mimics underglycosylated MUC1 on human tumor cells and could serve as an immunogen to stimulate responses that would recognize MUC1 on tumor cells. To test this we immunized mice with Sf-9 cells expressing BV-MUC1. Sera from immunized mice recognized MUC1 on human tumor cells. We also generated MUC1-specific T cells that proliferated in response to synthetic MUC1 peptide.     

Presentation of MUC1 tumor antigen by class I MHC and CTL function correlate with the glycosylation state of the protein taken Up by dendritic cells.

We previously reported that the glycosylated MUC1 tumor antigen circulating as soluble protein in patients' serum is not processed by dendritic cells and does not elicit MHC-Class II-restricted T helper responses in vitro. In contrast, a long synthetic peptide from the MUC1 tandem repeat region is presented by Class II molecules, resulting in the initiation of T helper cell responses. Here we addressed the ability of dendritic cells to present various glycosylated or not glycosylated forms of MUC1 by MHC Class I. We found that three different forms of MUC1, ranging from glycosylated and underglycosylated protein to unglycosylated synthetic peptide, were able to elicit MUC1-specific, Class-I-restricted CTL responses. The efficiency of processing and the resulting strength of CTL activity were inversely correlated with the degree of glycosylation of the antigen. Furthermore, the more efficiently processed 100mer peptide primed a broader repertoire of CTL than the glycosylated protein.     

The actin regulatory protein HS1 is required for antigen uptake and presentation by dendritic cells

The hematopoietic actin regulatory protein hematopoietic lineage cell-specific protein 1 (HS1) is required for cell spreading and signaling in lymphocytes, but the scope of HS1 function in Ag presentation has not been addressed. We show that dendritic cells (DCs) from HS1(-/-) mice differentiate normally and display normal LPS-induced upregulation of surface markers and cytokines. Consistent with their normal expression of MHC and costimulatory molecules, HS1(-/-) DCs present OVA peptide efficiently to CD4(+) T cells. However, presentation of OVA protein is defective. Similarly, MHC class I-dependent presentation of VSV8 peptide to CD8(+) T cells occurs normally, but cross-presentation of GRP94/VSV8 complexes is defective. Analysis of Ag uptake pathways shows that HS1 is required for receptor-mediated endocytosis, but not for phagocytosis or macropinocytosis. HS1 interacts with dynamin 2, a protein involved in scission of endocytic vesicles. However, HS1(-/-) DCs showed decreased numbers of endocytic invaginations, whereas dynamin-inhibited cells showed accumulation of these endocytic intermediates. Taken together, these studies show that HS1 promotes an early step in the endocytic pathway that is required for efficient Ag presentation of exogenous Ag by DCs.     

The kinetics of in vivo priming of CD4 and CD8 T cells by dendritic/tumor fusion cells in MUC1-transgenic mice

Previous work has demonstrated that dendritic/tumor fusion cells induce potent antitumor immune responses in vivo and in vitro. However, little is known about the migration and homing of fusion cells after s.c. injection or the kinetics of CD4+ and CD8+ T cell activation. In the present study, fluorescence-labeled dendritic/MUC1-positive tumor fusion cells (FC/MUC1) were injected s.c. into MUC1-transgenic mice. The FC/MUC1 migrated to draining lymph nodes and were closely associated with T cells in a pattern comparable with that of unfused dendritic cells. Immunization of MUC1-transgenic mice with FC/MUC1 resulted in proliferation of T cells and induced MUC1-specific CD8+ CTL. Moreover, CD4+ T cells activated by FC/MUC1 were multifunctional effectors that produced IL-2, IFN-gamma, IL-4, and IL-10. These findings indicate that both CD4+ and CD8+ T cells can be primed in vivo by FC/MUC1 immunization.     

Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.     

The mechanism of unresponsiveness in lymphocytes specifically selected by antigen in normal rats.

The immunological potentialities of populations of thoracic duct lymphocytes collected from rats recently challenged with sheep erythrocytes were compared with those of lymphocytes from rats immunologically tolerant of this antigen. Both "selected" and tolerant populations fail to mount immune responses to sheep erythrocytes in irradiated syngeneic hosts but can do so if transferred to semi-allogeneic recipients. If co-cultivated with normal lymphocytes in irradiated hosts, both types of population can modulate the responsiveness of the former to sheep erythrocytes. Supplementation of normal lymphocytes with "selected" populations collected earlier than 28 hours after challenge of the donors tends to result in augmented responses while later collections are more likely to be associated with suppression. Consequently, it is suggested that "selected" populations are unresponsive because of the presence of specifically activated cells rather than as a result of their depletion. The influence of "selected" populations on the responsiveness of normal lymphocytes is attributed to this content of activated cells.     

Cerulenin is a potent inhibitor of antigen processing by antigen-presenting cells

Cerulenin is an antibiotic that inhibits eukaryotic lipid and sterol synthesis and blocks lipid modification of proteins. The effect of cerulenin on the ability of accessory cells to present antigen to T cells was investigated. This antibiotic strongly inhibits the ability of accessory cells to present antigen to murine T-T hybrids. This effect is observed for multiple distinct antigens including L-glutamic acid60-L-alanine30-L-tyrosine10, bovine insulin, L-glutamic acid56-L-lysine35-L-phenylalanine9, and ovalbumen. Presentation by both macrophage and B lymphoblastoid cell lines is inhibited. The ability to effectively pulse these cells with antigen is inhibited but not the ability of these same cells to present antigen that they have previously processed. Furthermore, this inhibition is selective as it can occur without significant inhibition of the antigen-presenting cell protein or DNA synthesis. Cerulenin does not inhibit antigen uptake or catabolism as assessed with labeled antigen. By these criteria this drug is shown to interfere with an antigen-processing step. The ability of cerulenin to block processing was compared with other known inhibitors. Although cerulenin was effective with all antigens tested, at least one inhibitor was not. Taken together, these results suggest that the effect of cerulenin may define a distinct step in antigen processing and provides evidence that some other processing events are not universally required. The ability of cerulenin to interfere with antigen processing is discussed in the context of the known actions of this antibiotic and events of antigen processing and presentation.     

NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells

To initiate adaptative cytotoxic immune responses, proteolytic peptides derived from phagocytosed antigens are presented by dendritic cells (DCs) to CD8+ T lymphocytes through a process called antigen "crosspresentation." The partial degradation of antigens mediated by lysosomal proteases in an acidic environment must be tightly controlled to prevent destruction of potential peptides for T cell recognition. We now describe a specialization of the phagocytic pathway of DCs that allows a fine control of antigen processing. The NADPH oxidase NOX2 is recruited to the DC's early phagosomes and mediates the sustained production of low levels of reactive oxygen species, causing active and maintained alkalinization of the phagosomal lumen. DCs lacking NOX2 show enhanced phagosomal acidification and increased antigen degradation, resulting in impaired crosspresentation. Therefore, NOX2 plays a critical role in conferring DCs the ability to function as specialized phagocytes adapted to process antigens rather than kill pathogens.     

Oxidative stress impairs intracellular events involved in antigen processing and presentation to T cells

For T cells to recognize foreign antigens, the latter must be processed into peptides and associated to major histocompatibility complex (MHC) class II molecules by antigen-presenting cells (APC). APCs frequently operate under stress conditions induced by tissue damage, antigens, or inflammatory reactions. We analyze the effects of oxidative stress on intracellular processing using APC B cell lines. Before being tested for APC function, B cells (IIA1.6) were exposed for 2 hours to hydrogen peroxide (H2O2), a treatment that impairs their capacity to stimulate specific T cell clones. Because paraformaldehyde-fixed H2O2-treated B cells can still present extracellular peptides to T cell clones, the intracellular events of processing were investigated. Purified lysosomes from H2O2-treated B cells show increased proteolytic activity and increased generation of antigenic peptides. In addition, H2O2 treatment targets antigens to compartments that express low levels of MHC II and proteins (H-2M, H-2O) required for peptide loading onto this molecule. Finally, we suggest that impairment of antigen processing by oxidative stress reduces the induction of a T cell's response because H2O2 decreases the activation of naive T lymphocytes by dendritic cells. Together, these data indicate that oxidative stress inhibits the capacity of APCs to process antigens and to initiate a primary T cell response. The role of such modifications on the outcome of the specific immune response is discussed.     

The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma

The epithelial mucin MUC1 is a high molecular weight membrane glycoprotein frequently overexpressed and aberrantly glycosylated in adenocarcinoma. Mucins normally contain high amounts of O-linked carbohydrate structures that may influence immune reactions to this antigen. During malignant transformation, certain glyco-epitopes of MUC1, such as Tn-antigen, TF-antigen and their sialylated forms become exposed. The role of these glycan structures in tumor biology is unknown, but their presence is known to correlate with poor prognosis in several adenocarcinomas. We analyzed the potency of MUC1 containing Tn-antigens (MUC1-Tn) to target C-type lectins that function as carbohydrate recognition and uptake molecules on dendritic cells (DC). We identified the macrophage galactose type C-type lectin (MGL), expressed by both DC and macrophages, as the receptor for recognition and binding of MUC1-Tn. To validate the occurrence of MGL–MUC1 interactions in situ, we studied the binding of MGL to MUC1 in primary colon carcinoma tissue. Isolation of MUC1 out of colon carcinoma tissue showed strong binding activity to MGL. Interestingly, MGL binding to MUC1 was highly correlated to binding by the lectin Helix pomatia agglutinin (HPA), which is associated with poor prognosis in colorectal cancer. The detection of MGL positive cells in situ at the tumor site together with the modified glycosylation status of MUC1 to target MGL on DC suggests that MGL positive antigen presenting cells may play a role in tumor progression.     

Modulation of MUC1 and blood group antigen expression in gastric adenocarcinoma cells by cytokines

Immunohistological studies demonstrated that MUC1 expression in gastric cancer is associated with a poor prognosis. As a mediator of cell-cell interactions, MUC1 may also be involved in metastasis. However, these aspects are of relevance since cytokine levels are locally increased as a consequence of peritumorous inflammatory response and coexisting chronic gastritis. Therefore we analyzed the potential influence of several cytokines on the expression of tumor-associated MUC1 and Lewis blood group antigens in gastric carcinoma cells. Gastric cancer cell lines AGS and KATOIII were incubated with the cytokines interleukin-1beta, interferon-gamma, tumor necrosis factor-alpha (TNF-alpha), and hepatocyte growth factor over a period of 72 h. Expressions of mucin antigens and cytokine secretion were measured by immunocytochemistry and/or enzyme-linked immunosorbent assay (ELISA). Analysis by fluorescence-activated cell sorter (FACS) demonstrated that MUC1 and sialyl Lewis A reactivities of AGS cells were increased significantly following TNF-alpha stimulation but not by other cytokines. Expression of mucin-associated antigens by cell line KATOIII was not affected by any of the employed cytokines. These data provide evidence that TNF-alpha can raise the expression of important mucin peptide as well as mucin-associated carbohydrate antigens and thereby potentially influence the progression of gastric carcinomas.     

Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.