Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.     

Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for antitumor vaccination

In cancer, mucins are aberrantly O-glycosylated, and consequently, they express tumor-associated antigens such as the Tn determinant (alpha-GalNAc-O-Ser/Thr). As compared with normal tissues, they also exhibit a different pattern of expression. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins, and as such, they may be used as potential targets for immunotherapy. To develop anticancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in Escherichia coli, using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies that are specific to human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce immunoglobulin G (IgG) antibodies that recognized a human tumor cell line. In conclusion, the large-scaled production of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anticancer vaccines, and further studies of their immunological properties are warranted.     

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.     

Role of glycosylation on the ensemble of conformations in the MUC1 immunodominant epitope

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O‐glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O‐glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn‐antigen (GalNAc‐O‐Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding‐competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution‐state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre‐selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies.     

Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR polymorphism

Aberrant glycosylation of mucins is a common phenomenon associated with oncogenic transformation. We investigated the association between expression of the tumor-associated antigens T, Tn, and sialyl-Tn and polymorphism in the length of the MUC1 mucin tandem repeat in a series of gastric carcinomas. We further evaluated the relevance of MUC1 tandem repeat length on the expression of these tumor-associated carbohydrate antigens (TACAs) using a gastric carcinoma cell line model expressing recombinant MUC1 constructs carrying 0, 3, 9, and 42 repeats. Gastric carcinomas showed a high prevalence of Tn and sialyl-Tn antigens, whereas T antigen was less frequently expressed. The expression of T antigen was significantly higher in gastric carcinomas from patients homozygous for MUC1 large tandem repeat alleles. No significant associations were found for Tn and sialyl-Tn antigens. This novel association was reinforced by the gastric carcinoma cell line model experiments, where de novo expression of T antigen was detected in clones transfected with larger VNTR regions. Our results indicate that polymorphism in the MUC1 tandem repeat influences the expression of TACAs in gastric cancer cells and may therefore allow the identification of subgroups of patients that develop more aggressive tumors expressing T antigen.     

Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines.

Mucin O-glycosylation in cancer is characterized by aberrant expression of immature carbohydrate structures leading to exposure of simple mucin-type carbohydrate antigens and peptide epitopes. Glycosyltransferases controlling the initial steps of mucin O-glycosylation are responsible for the altered glycosylation observed in cancer. We studied the expression in gastric cell lines of six UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T1, T2, T3, T4, T6, T11) that catalyze the initial key step in the regulation of mucin O-glycosylation, the transfer of GalNAc from UDP-GalNAc to serine and threonine residues. We also studied the expression of ST6GalNAc-I, the enzyme responsible for the synthesis of Sialyl-Tn antigen (NeuAcalpha2,6GalNAc) and the ST3Gal-I, the enzyme responsible for the synthesis of Sialyl-T antigen (NeuAcalpha2,3Galbeta1,3GalNAc). This study was done using specific monoclonal antibodies, enzymatic assays, and RT-PCR. Our results showed that GalNAc-T1, -T2, and -T3 have an ubiquitous expression in all gastric cell lines, whereas GalNAc-T4, -T6, and -T11 show a restricted expression pattern. The immunoreactivity with MAb VU-2-G7 suggests that, apart from GalNAc-T4, another GalNAc transferase is involved in the glycosylation of the Thr in the PDTR region of the MUC1 tandem repeat. The expression of ST3Gal-I correlates with the expression of the Sialyl-T antigen in gastric cell lines and in the control cell lines studied. The expression of ST6GalNAc-I is low in gastric cell lines, in accordance with the low/absent expression of the Sialyl-Tn antigen.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Probing the conformational and dynamical effects of O-glycosylation within the immunodominant region of a MUC1 peptide tumor antigen.

MUC1 mucin is a large transmembrane glycoprotein, the extracellular domain of which is formed by a repeating 20 amino acid sequence, GVTSAPDTRPAPGSTAPPAH. In normal breast epithelial cells, the extracellular domain is densely covered with highly branched complex carbohydrate structures. However, in neoplastic breast tissue, the extracellular domain is under-glycosylated, resulting in the exposure of a highly immunogenic core peptide epitope (PDTRP in bold above), as well as in the exposure of normally cryptic core Tn (GalNAc), STn (sialyl alpha2-6 GalNAc) and TF (Gal beta1-3 GalNAc) carbohydrates. Here, we report the results of 1H NMR structural studies, natural abundance 13C NMR relaxation measurements and distance-restrained MD simulations designed to probe the structural and dynamical effects of Tn-glycosylation within the PDTRP core peptide epitope. Two synthetic peptides were studied: a nine-residue MUC1 peptide of the sequence, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6-Arg7-Pro8-Ala9, and a Tn-glycosylated version of this peptide, Thr1-Ser2-Ala3-Pro4-Asp5-Thr6(alphaGalNAc)-Arg7-Pro8-Ala9. The results of these studies show that a type I beta-turn conformation is adopted by residues PDTR within the PDTRP region of the unglycosylated MUC1 sequence. The existence of a similar beta-turn within the PDTRP core peptide epitope of the under-glycosylated cancer-associated MUC1 mucin protein might explain the immunodominance of this region in vivo, as the presence of defined secondary structure within peptide epitope regions has been correlated with increased immunogenicity in other systems. Our results have also shown that Tn glycosylation at the central threonine within the PDTRP core epitope region shifts the conformational equilibrium away from the type I beta-turn conformation and toward a more rigid and extended state. The significance of these results are discussed in relation to the possible roles that peptide epitope secondary structure and glycosylation state may play in MUC1 tumor immunogenicity.     

Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes

We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.     

Aberrant O‐glycosylation contributes to tumorigenesis in human colorectal cancer

Aberrant O‐glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O‐glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O‐glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio‐functional investigations showed that T‐synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss‐of‐function and a consequent inactive T‐synthase. Transfection of LS174T cells with WT Cosmc restored mature O‐glycosylation, which subsequently down‐regulated cancer cell proliferation, migration and apoptotic‐resistant ability. Significantly, the expression of MUC2, a heavily O‐glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O‐glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells.     

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn,Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.     

Carbohydrate antigens as targets for active specific immunotherapy

 Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewis^y and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of tumor cells in vitro and associated with prolonged disease-free and overall survival in patients. Received: 6 August 1996 / Accepted: 20 September 1996     

High Immunogenicity of the Human Leukocyte Antigen Peptidomes of Melanoma Tumor Cells

Human leukocyte antigens (HLA) bind peptides generated by limited proteolysis in cells and present them at the cell surfaces for recognition by T cells. Through this antigen presentation function they control the specificity of T cell responses and thereby adaptive immune responses. Knowledge of HLA-bound peptides is thus key to understanding adaptive immunity and to the development of vaccines and other specific immune intervention strategies. To gain insight into the antigenicity of melanomas, peptides were extracted from HLA isolated from the tumor cells, separated by two-dimensional HPLC, and sequenced by mass spectrometry. The spectra were analyzed by database-dependent MASCOT searches and database-independent de novo sequencing and, where required, confirmed with synthetic peptides, which were also used to determine their immunogenicity. Comparing four different melanoma cell lines, little overlap of the HLA-bound peptides was found, suggesting a high degree of individualization of the HLA peptidomes. This notwithstanding, the peptidomes were highly immunogenic in the patients from whom the tumor cells had been established and in unrelated patients. This broad cross-patient immunogenicity was only exceptionally related to individual peptides. The majority of the identified epitopes were derived from low to medium abundance proteins, mostly involved in sensitive cellular processes such as cell cycle control, DNA replication, control of gene expression, tumor suppressor function, and protein metabolism. The peptidomes thus provide insights into processes potentially related to tumorigenesis. Furthermore, analyses of the peptide sequences yield information on the specificity of peptide selection by HLA applicable to the developing prediction algorithms for T cell epitopes.     

Solid-phase synthesis of a pentavalent GalNAc-containing glycopeptide (Tn antigen) representing the nephropathy-associated IgA hinge region

Incomplete or aberrant glycosylation leading to Tn antigen (GalNAcα1-Ser/Thr) expression on human glycoproteins is strongly associated with human pathological conditions, including tumors, certain autoimmune diseases, such as the idiopathic IgA nephropathy, and may modulate immune homeostasis. In addition, the Tn antigen is highly expressed by certain pathogens and plays a role in host-pathogen interactions. To enable experimental approaches to study interactions of the Tn antigen with the immune system and analyze anti-Tn antibody responses in infection or disorders, we generated a Tn-expressing resource that can be used for high-throughput screening. In consideration of IgA nephropathy in which the hinge region is incompletely glycosylated, we used this hinge sequence that encodes five potential glycosylation sites as the ideal template for the synthesis of a Tn antigen-expressing glycopeptide. Inclusion of an N-terminal biotin in the peptide enabled binding to streptavidin-coated ELISA plates as monitored using Helix pomatia agglutinin or anti-Tn monoclonal antibody. We also found that the biotinylated IgA-Tn peptide is a functional acceptor for β1-3-galactosylation using recombinant T-synthase (β1-3-galactosyltransferase). Besides its immunochemical functionality as a possible diagnostic tool for IgA nephropathy, the peptide is an excellent substrate for glycan elongation and represents a novel template applicable for glycan-antigen-associated diseases.     

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.     

Lymphocyte specific heterogeneity in the rat leucocyte common antigen (T200) is due to differences in polypeptide sequences near the NH2-terminus.

The leucocyte-common antigen (L-CA, T200 or CD45) consists of a family of heavily glycosylated glycoproteins of apparent Mr 180,000-240,000 which are restricted to lymphoid and myeloid cells. Forms of L-CA which differ in their apparent Mr, antigenicity and glycosylation are expressed on different lymphocyte types. One specific antigenic determinant called MRC OX-22 is of particular interest because it distinguishes two sets of T helper cells that have different functions. From the sequence of different L-CA cDNA clones we now conclude that there is sequence heterogeneity such that at least four forms of L-CA exist with sequences in the range 1118-1250 amino acids. All the sequence variation occurs at a point starting 6 residues from the NH2-terminus and the last 1112 residues of all forms are identical. Two of the variants can be directly related to the antigenic variation because they include sequence that was determined for a peptide that carries the MRC OX-22 determinant. Analysis of glycopeptides from thymocyte L-CA identified only one non-glycosylated position out of 14 possible N-glycosylation sites and established that all O-glycosylation was within the first 32 amino acids. The extra protein sequence in the longer forms was also suggestive of extensive O-glycosylation.     

The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer

The functional properties of glycoproteins are strongly influenced by their profile of glycosylation, and changes in this profile are seen in malignancy. In mucin-type O-linked glycosylation these changes can result in the production of mucins such as MUC1, carrying shorter sialylated O-glycans, and with different site occupancy. Of the tumor-associated sialylated O-glycans, the disaccharide, sialyl-Tn (sialic acid alpha2,6GalNAc), is expressed by 30% of breast carcinomas and is the most tumor-specific. The ST6GalNAc-I glycosyltransferase, which can catalyze the transfer of sialic acid to GalNAc, shows a highly restricted pattern of expression in normal adult tissues, being largely limited to the gastrointestinal tract and absent in mammary gland. In breast carcinomas, however, a complete correlation between the expression of RNA-encoding ST6GalNAc-I and the expression of sialyl-Tn is evident, demonstrating that the expression of sialyl-Tn results from switching on expression of hST6GalNAc-I. Endogenous or exogenous expression of hST6GalNAc-I (but not ST6GalNAc-II) always results in the expression of sialyl-Tn. This ability to override core 1/core 2 pathways of O- linked glycosylation is explained by the localization of ST6GalNAc-I, which is found throughout the Golgi stacks. The development of a Chinese hamster ovary (CHO) cell line expressing MUC1 and ST6GalNAc-I allowed the large scale production of MUC1 carrying 83% sialyl-Tn O-glycans. The presence of ST6GalNAc-I in the CHO cells reduced the number of O-glycosylation sites occupied in MUC1, from an average of 4.3 to 3.8 per tandem repeat. The availability of large quantities of this MUC1 glycoform will allow the evaluation of its efficacy as an immunogen for immunotherapy of MUC1/STn-expressing tumors.     

B cells as antigen presenting cells

Several characteristics confer on B cells the ability to present antigen efficiently: (1) they can find T cells in secondary lymphoid organs shortly after antigen entrance, (2) BCR-mediated endocytosis allows them to concentrate small amounts of specific antigen, and (3) BCR signaling and HLA-DO expression direct their antigen processing machinery to favor presentation of antigens internalized through the BCR. When presenting antigen in a resting state, B cells can induce T cell tolerance. On the other hand, activation by antigen and T cell help converts them into APC capable of promoting immune responses. Presentation of self antigens by B cells is important in the development of autoimmune diseases, while presentation of tumor antigens is being used in vaccine strategies to generate immunity. Thus, detailed understanding of the antigen presenting function of B cells can lead to their use for the generation or inhibition of immune responses.