MUC1 mucin-mediated regulation of human T cells
Babita Agrawal and
B. Michael Longenecker
611-Heritage Medical Research Centre, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2S2, Canada
Correspondence to: B. Agrawal; E-mail: bagrawal{at}ualberta.ca
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Abstract
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MUC1 mucin is expressed by normal human epithelial cells and is overproduced in underglycosylated form by malignant epithelial cells. A number of anticancer immunotherapeutic strategies are being designed with the goal of inducing humoral and cellular immune responses against MUC1 mucin. Newly synthesized MUC1 mucin is also expressed on polyclonally stimulated human T cells. An immunoregulatory role has been postulated for MUC1 mucin expressed by activated T cells. We now show that several MUC1 peptide and glycopeptide epitope specific antibodies bind to activated T cells and inhibit their proliferation. Inhibition by antibody B27.29 could be reversed by glycopeptide haptens specific for the antibody. Intact antibody B27.29 and its divalent F(ab')2 fragment inhibited the proliferation of T cells undergoing T cell activation but the monovalent Fab' fragment did not, indicating that cross-linking of the MUC1 antigen on T cells is required for the inhibitory effect. MUC1 expression on activated T cells was increased in the presence of IL-12 but was not affected by IFN-
, IL-2, IL-4, IL-5, IL-10, IL-13 or TNF-
. Treatment of T cells inhibited by monoclonal antibody (MAb) B27.29 with either IL-2 or costimulatory anti-CD28 antibody restored proliferation to a level equivalent to that of control cultures. These results provide further support for the hypothesis that the expression of MUC1 on the activated T cell surface is associated with the regulation of T cell responses.
Keywords: activation molecule, antibodies, immune response, inhibitory coreceptor, T lymphocyte
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Introduction
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Mucins are large (>200 kDa) glycoproteins with a high carbohydrate content (5090% by weight) expressed by a variety of normal and malignant secretory epithelial cells (1). Twelve mucin genes have been at least partially cloned, MUC1, 2, 3, 5AC, 5B, 6, 7, 8 (2), and more recently cloned MUC 9, 11 and 12 (3). Mucin proteins are characterized by a variable but large number of tandem repeat (VNTR) amino acid sequences in the extracellular domains. Serine and threonine residues constitute a large proportion of mucin VNTR sequences, giving rise to numerous potential O-glycosylation sites. The MUC1 mucin (CD227) gene has been cloned (4) and characterized very well. MUC1 has been used most extensively as a tumor marker for monitoring the disease status of breast cancer patients, as the expression of MUC1 is dramatically increased in breast cancer cells (5). MUC1, previously known as episialin, PEM, H23Ag, EMA, CA15-3, MCA and others, is a polymorphic mucin like protein that contains a large extracellular domain consisting of a glycosylated polypeptide made up of 30100 tandem repeats of a 20-amino acid sequence, a transmembrane domain, and a cytoplasmic tail of 72 amino acids (6). During the biosynthesis, MUC1 protein is modified to a large extent by O-linked sugar moieties. After translation, MUC1 protein undergoes a proteolytic cleavage event generating two cleavage products (7). These form a heterodimeric complex that is composed of a large tandem repeat array containing an extracellular domain linked by non-covalent, SDS sensitive bonds to the much smaller protein molecule, which includes the transmembrane and cytoplasmic domains. The MUC1 extracellular domain can be shed by the cells by yet unknown mechanisms. Recently, a supplementary and unique MUC1 isoform MUC1/Y, generated by differential splicing, has been identified. MUC1/Y is a transmembrane protein that shares identical transmembrane and cytoplasmic domains with MUC1, but does not contain the repeat and flanking extracellular domains. MUC1/Y has been shown to be exclusively expressed by human epithelial secretory tumors, but not detectable by adjacent normal tissue. MUC1/Y has been suggested to be involved in tumor initiation and progression in vivo, and intracellular signalling as it undergoes tyrosine phosphorylation and potentially binds to a second messenger, such as GRB2 (8). Secreted MUC1 has been suggested to be a ligand for MUC1/Y leading to signalling events and changes in cell morphology in tumor cells (9). MUC1 has also been suggested to be a ligand for intercellular adhesion molecule-1 (ICAM-1) expressed on T cells and endothelial cells (10).
MUC1 mucin is a tumor antigen of choice for various vaccine immunotherapeutic studies targeted at generating B and T cell responses. MUC1 is a self-molecule that is normally expressed, but it is also a target for immunotherapy because it is significantly altered in expression during tumorigenesis, in terms of abundance and aberration in expression (11). MUC1 derived peptides have been suggested to be targets of cytotoxic T lymphocytes (CTLs) capable of antitumor killing (1214). Several clinical trials are underway examining the potential of MUC1 derived vaccine candidates in various formulations as new candidates of immunotherapy of adenocarcinomas.
Many functions have been proposed for MUC1. The extensive expression of MUC1 from mid-gestation throughout adulthood in secretory epithelial tissues and the elevated level of expression found in carcinomas and metastatic lesions suggest important functions in epithelial morphogenesis and tumor progression (4). MUC1 could function at several levels: (i) by steric hindrance by the large glycosylated extracellular domain, (ii) by remodelling the cytoskeletal network (15,16), or (iii) by down-regulating the activities of other molecules such as catenins, cadherins or integrins via signal transduction events (17,18). Its cytoplasmic tail is phosphorylated, consistent with a transmembrane signal transduction function (8,19).
Paradoxically, MUC1 has been proposed to act both as an anti-adhesive and as an adhesive molecule. The extended conformation may contribute to its anti-adhesive properties, resulting in reduced cellcell aggregation and decreased adherence to extracellular matrix components in vitro adhesion assays (20, 21).
Our working hypothesis is that, along with the other possible functions, MUC1 mucin is involved in normal immune regulation. Evidence supporting this hypothesis includes the observation that (i) newly synthesized MUC1 mucin is rapidly induced and appears on the cell surface of the majority of activated human T cells, (ii) there is down-regulation of MUC1 mucin expression after the mitogenic stimulus is removed, (iii) anti-MUC1 MAb B27.29 inhibits the T cell proliferative response, (iv) MUC1 mucin is either shed or secreted into the supernatants of cultures of PHA activated human T cells, and (v) soluble MUC1 mucin derived from cancer cells inhibits T cell proliferation and induces an anergy-like state that is reversible by IL-2 or anti-CD28 antibody (22). The observation that cancer-associated MUC1 mucin inhibits T cell proliferation has been investigated by several other research groups as well (2326).
Our original finding that activated human T cells express MUC1 on the surface (27) has been reproduced by several laboratories around the world (2830). It has also been demonstrated that tyrosines on the cytoplasmic tail of MUC1 mucin expressed on activated T cells are phosphorylated supporting our original hypothesis (31). Further, it has been demonstrated that in response to chemokines, MUC1 molecules were concentrated in the leading edge of polarized activated T cells suggesting that MUC1 could be involved in early interactions between T cells and endothelial cells at inflammatory sites (2830).
The exact role and the mechanism of immune regulation by MUC1 mucin are not clear. However, the observations that MUC1 mucin can present multiple functional domains, i.e. anti-adhesion, pro-adhesion as well as inhibition of T cell proliferation, support the hypothesis that MUC1 expression on T cells may play an important homeostatic function. It is possible that MUC1 mucin expressed by activated T cells acts as an inhibitory coreceptor and also serves a role in lymphocyte trafficking due to its adhesion and/or anti-adhesion properties.
The regulation of T cell activation involves a dynamic equilibrium between activating and inhibitory signals (32). T cell responses are initiated through cellular interactions between T cells and antigen presenting cells (APCs). The specificity of interaction is dictated by the TCR-MHC complex, whereas optimal T cell responses are dependent upon costimulatory and/or coinhibitory signals induced by interaction of accessory cell surface molecules. The multiplicity of interactions represents the fine tuning of regulatory mechanisms of T cell activation during specific stages of activation and deactivation, involving sequential interactions and activation of individual surface antigens and their pathways.
In this paper we have further examined the role of MUC1 in T cell activation and regulation. We have tested antibodies specific for carbohydrate epitopes, glycopeptide epitopes or tandem repeat peptide core epitopes for binding to activated T cells and for inhibiting T cell proliferation responses. We have determined whether cytokines regulate MUC1 expression on activated human T cells. We have examined whether inhibition of T cell proliferation by anti-MUC1 MAb requires the cross-linking of the MUC1 expressed on activated T cells and whether this inhibition can be reversed by certain cytokines. Our results suggest that expression of MUC1 on the surface of activated T cells is associated with modulation of T cell responses.
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Methods
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Antibodies, cytokines, haptens and media
Anti-MUC1 antibodies BC3, DF3, SM3, BC4E549, DF3, M29, 3E1-2, DH-1 and M26 were obtained from the TD4.1 MUC1 workshop participation (Dr. P.D. Rye, Oslo, Norway). MAb B27.29 (anti-MUC1, isotype IgG1) and isotype control B80.3 (anti-prostate specific antigen) were raised and purified by Biomira Inc. (Edmonton, Canada). MAb B27.29 was raised against a partially purified mucin fraction from the ascites fluid of a cancer patient. Its reactivity with MUC1 has been localized to the SAPDTRPA sequence of the tandem repeat domain (33), but its reactivity is specific towards glycopeptides with glycosylation of the initial S or the preceding T residue (33, 34). The cross-linking goat anti-mouse second antibody was obtained from Sigma Chemical Co. (St. Louis, MO). Anti-CD3 MAb OKT3 and anti-Pan class I MAb W6/32 were purified from cell supernatants of hybridomas obtained from American Type Culture Collections (ATCC, Rockville, MD). Anti-CD30 MAb HRS-4 was obtained from Immunotech (Beckman Coulter Canada, Mississauga, ON). PE-labeled anti-CD69 and anti-CD25 were obtained from BD Biosciences (Mississauga, ON). Recombinant human cytokines used in these studies IFN-
, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and TNF-
were obtained from Pharmingen (San Diego, CA, USA). The haptens GVTSAPDTRPAPGSTA, GVTS(Tn)APDTRPAPGSTA and GVT(Tn)SAPDTRPAPGSTA were synthesized at Biomira Inc. as reported previously (34). The cell culture media AIM-V was obtained from Life Technologies (Burlington, ON, Canada). Peripheral blood samples were obtained from donors 3060 years of age of both sexes after informed consent. Use of human blood samples was approved by the institutional Health Research Ethics Board (HREB) at the University of Alberta, Canada.
Preparation of F(ab')2 and Fab' fragments of MAb B27.29
The F(ab')2 fragment of MAb B27.29 was prepared by digestion of antibody with 2% (w/w, enzyme/substrate) pepsin (Sigma, cryst.) in 0.1 M citrate buffer, pH 4.0. After incubation for 18 h at 37°C, the reaction was stopped by raising the pH to 7.5. The F(ab')2 fragment was separated from other fragments and traces of undigested antibody by chromatography on a column (1.6 x 90 cm) of Sephacryl S-200. The Fab' fragment was prepared by digestion of antibody with 1% (w/w, enzyme/substrate) papain (Sigma, 2 x cryst.) in phosphate buffered saline, pH 7.2, containing 10 mM EDTA and 10 mM L-cysteine. After incubation for 6 h at 37°C, the reaction was stopped by adding iodoacetamide to a final concentration of 30 mM. Any residual intact Fc fragment was removed by passing the digest through Protein A Sepharose (Pharmacia, Uppsala, Sweden). The solution was concentrated by diafiltration (Amicon stirred cell, YM3 membrane) and the Fab' fragment was separated from other fragments on Sephacryl S-200 as described above. Reactivity of the Fab' fragment with antigen was confirmed by competition with intact MAb in ELISA.
Cell surface immunofluorescence staining
For detection of cell surface antigens, normal human peripheral blood mononuclear cells (PBMCs) cultured as indicated in each experiment were stained essentially as previously described (35). Anti-MUC1 MAb B27.29 (1 µg/5 x 105 T cells) (33) or isotype control antibody B80.3 were used with indirect labeling with FITC or PE conjugated second antibody (goat anti-mouse IgG1). Isotype control groups had <2% positive cells. The samples were analyzed by flow cytometry using FACSort® (Becton & Dickinson, Franklin Lakes, NJ). Percent positive cells were defined as the fraction of cells exhibiting fluorescence intensities beyond a region set to exclude at least 98% of the isotype-matched control antibody stained cells.
Proliferation assay
Enriched T cell populations were purified from peripheral blood obtained from normal donors by using nylon wool columns according to previously published procedures (35, 36). Nylon wool purification provided >93% CD3+ T cells in repeated experiments (data not shown). T cells were cultured in the presence or absence of PHA (5 µg/ml) or OKT3 (5 µg/ml), MAb B27.29 or isotype control MAb B80.3 (amounts shown in figures), and goat anti-mouse (5 µg/ml) in 96-well plates in quadruplicate. On the third or fourth day, the wells were pulsed with 1 µCi/well 3H-thymidine ([3H]TdR) (specific activity 89.0 Ci mmol1) (Amersham Canada Limited, Oakville, ON, Canada). Incorporation of [3H]TdR into DNA of proliferating T cells was measured after harvesting the plates after 1824 h and counting in a liquid scintillation counter (Beckman LS 60001C, Mississauga, ON, Canada). In various experiments, reagents were added to cultures as indicated in each figure. Each experiment was performed with a new blood donor; therefore, there is variability in the proliferation in response to the mitogen used.
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Results
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Inhibition of T cell proliferation by surface MUC1 cross-linking is epitope dependent
MUC1 mucin is highly glycosylated; however, its glycosylation characteristics appear to be highly variable and seem to depend upon the tissue of origin. In tumors, MUC1 is aberrantly glycosylated such that the peptide core is accessible to antibodies and T cells (2123). Initially, we used antibody B27.29 (reactive with cancer-associated MUC1 mucin and glycosylated MUC1-derived peptides, specificity localized within the sequence SAPDTRPA) (33, 34) and detected the expression of MUC1 on the activated T cell surface (27). In order to further prove the molecule-specific interaction of anti-MUC1 antibodies with activated T cell surface and to prove that the observed inhibition of T cell proliferation is physiologically significant, we used various available anti-MUC1 antibodies directed against peptide, glycopeptide or carbohydrate epitopes of MUC1 to examine whether they bind to the activated T cell surface and inhibit T cell proliferation upon cross-linking surface MUC1. There are >56 mAb against MUC1 with known specificities against MUC1 peptide, glycopeptide or carbohydrate epitopes of cancer-associated MUC1. We examined 10 different antibodies (different isotypes) specific for MUC1 peptide, glycopeptide or carbohydrate epitopes. All of the tested antibodies bound to the activated T cells and inhibited T cell proliferation upon cross-linking surface MUC1 through these antibodies to various degrees (496% inhibition by different antibodies) (Table 1). We also used pan anti-class I (W6/32) and anti-CD30 antibodies as negative control antibodies that bind to activated T cells and found that upon cross-linking with second antibody, these mAbs did not inhibit T cell proliferation (Table 1). For each of the antibodies, we used isotype-matched antibodies as specificity controls and did not observe any binding (<1% cells positive) or inhibition of T cell proliferation at the same concentration of the antibodies (data not shown).
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Table 1. Anti-MUC1 antibodies reactive against carbohydrate, tandem repeat peptide core or non-VNTR region bind to activated human T cells and induce an inhibition of proliferation
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Glycopeptide haptens specific for anti-MUC1 antibody B27.29 block its effect on T cell proliferation
In order to determine the specificity of the effect of anti-MUC1 antibody B27.29 on T cell proliferation, specific hapten-inhibition experiments using one peptide and two glycopeptide haptens containing the B27.29 epitope (33, 34) were performed. It has been shown previously that the antibody B27.29 does not bind or binds weakly to the peptide GVTSAPDTRPAPGSTA whereas it binds strongly to the glycopeptides GVTS(Tn)APDTRPAPGSTA [abbreviated as GVT(4 Tn)] and GVT(Tn)SAPDTRPAPGSTA [abbreviated as GVT(3 Tn)]. It was also shown previously that the specificity of B27.29 is towards the glycopeptide epitope and not the carbohydrate epitope only (33, 34). The inhibitory effect of the B 27.29 antibody on T cell proliferation was reversed and/or prevented by the presence of glycopeptide haptens but not the corresponding peptide hapten (Fig. 1), where significant inhibition in T cell proliferation was observed. None of these haptens had any effect on T cell proliferation in the corresponding cultures containing the isotype control (Fig. 1).

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Fig. 1. Haptens specific for anti-MUC1 antibody B27.29 block its effect on T cell proliferation. A total of 2 x 105 human peripheral blood T lymphocytes/well were stimulated with anti-CD3 (OKT3, polyclonal stimulus, 5 µg ml1) in the presence of anti-MUC1 antibody (B27.29, 5 µg ml1) or isotype control and second cross-linking goat anti-mouse antibody with or without the stated concentrations of MUC1 synthetic peptide or glycopeptide haptens for 3 days in a flat 96-well microtiter plate. After this time, the wells were pulsed with [3H]TdR overnight and the cells were harvested. Incorporation of [3H]TdR in the DNA of proliferating T cells was determined by liquid scintillation counting. The sequence of MUC1 haptens used in this experiment are GVTSAPDTRPAPGSTA, BP.16GVT; GVTS(Tn)APDTRPAPGSTA, BP.16GVT(4Tn); and GVT(Tn)SAPDTRPAPGSTA, BP.16GVT(3Tn). The data shown are representative of three repeated experiments.
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Cross-linking of T cell surface MUC1 is required for inhibition of proliferation by anti-MUC1 mAb
In order to confirm that down-regulation of T cell proliferation in the presence of anti-MUC1 antibody requires cross-linking, the effects of purified monovalent Fab' and divalent F(ab')2 fragments of B27.29 were compared with that of the intact antibody (Fig. 2). In these experiments, the second antibody goat anti-mouse was only added in the third group shown in the figure, otherwise the experiment was performed in the absence of the second cross-linking antibody. Human peripheral blood-purified T cells were activated with the polyclonal stimulus OKT3 (anti-CD3 antibody). In the presence of the monovalent Fab' fragment of B27.29, there was no significant inhibition (<10%) of the T cell proliferation response at antibody concentrations up to 250 µg ml1. With the dimeric F(ab')2 fragment of B27.29 over the range of 5250 µg ml1, the T cell proliferation response was inhibited in a dose-dependent manner (4090% inhibition). The inhibition in T cell proliferation that was observed with intact mAb B27.29 was increased when the goat anti-mouse second antibody was added to cross-link the B27.29 antibody (Fig. 2).

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Fig. 2. Binding of a univalent Fab' fragment of anti-MUC1 antibody does not inhibit T cell proliferation. A total of 2 x 105 human peripheral T lymphocytes per well were stimulated with anti-CD3 (OKT3, 5 µg ml1) in the presence of anti-MUC1 antibody B27.29, Fab' or F(ab')2 fragments of B27.29, or isotype control antibody B80 at different concentrations, and goat anti-mouse (G m) antibody at 1 µg ml1 (only in specified group), for 3 days in a flat 96-well microtiter plate. After this time, the wells were pulsed with [3H]TdR overnight and the cells were harvested. Incorporation of [3H]TdR in the DNA of proliferating T cells was determined by liquid scintillation counting. The experiment was reproduced three times with three independent donors' T cells.
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MUC1 appears on the T cell surface late in activation and its cross-linking does not affect CD25 or CD69 expression
T cells were activated with a mitogen (PHA) and double stained with either anti-CD69 or anti-CD25 and anti-MUC1 mAb at various times as shown in Fig. 3(a). After one day of activation, the CD69 expression had increased from <20% of cells (no PHA control) to nearly 100%, while MUC1 had appeared on only 14% of cells. By day 3, 82% of the cells were double positive for CD69 and MUC1. The staining pattern with CD25 was similar to that of CD69 with most of the cells becoming positive for CD25 after one day of stimulation, while CD25/MUC1 double positives increased from 15% at one day to 79% at 3 days. Since MUC1 appeared significantly later than activation markers CD69 and CD25, it seemed unlikely that cross-linking MUC1 on the T cell surface by the anti-MUC1 antibody would affect CD69 or CD25 expression. The expression of CD69 and CD25 in T cells stimulated with PHA was then examined in the presence of the cross-linking anti-MUC1 antibody and, as expected, there was no reduction in the expression of CD69 or CD25 (Fig. 3b). However, there was significant inhibition of T cell proliferation stimulated by PHA in the presence of B27.29 and the second goat anti-mouse antibody (Fig. 3c).

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Fig. 3. (a) MUC1 is co-expressed with other T cell activation markers. Human peripheral blood T lymphocytes were activated in the presence or absence of PHA for 4 h, 1 day and 3 days. At each time point, the T cells were stained with anti-CD69 or anti-CD25 and anti-MUC1 antibodies. As controls, isotype-matched antibodies were used to stain cells and gave <2% positive cells. The media (no PHA) controls gave <25% of T cells positive for CD25, <30% positive for CD69 and <3% positive for MUC1. (b) CD25 and CD69 expression is not down-regulated when T cell proliferation is inhibited by MUC1 cross-linking. T cells were stimulated with PHA in the presence of anti-MUC1 antibody or isotype-matched control antibody and goat anti-mouse antibody for 3 days. At this time, the T cells were stained for CD69 or CD25 by single staining with direct PE-labeled anti-CD25 or anti-CD69 antibodies. The histogram plots show cells gated to include only the activated lymphocytes. (c) T cells have reduced proliferation in the presence of MUC1 cross-linking antibody despite normal expression of CD25 and CD69. The experiments were repeated three times with similar results.
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Effect of cytokines on MUC1 expression on activated T cells
An immunoregulatory role for the expression of MUC1 on activated T cells has been suggested (27). Therefore, we determined whether various cytokines would affect MUC1 expression on T cells. Purified human T lymphocytes were activated with a mitogen PHA in the absence or presence of cytokines IFN-
, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 and TNF-
at various concentrations for 72 h. At this time, the cells present in the culture were double stained for CD3 and MUC1 expression. As shown in Fig. 4(a) and (b), various concentrations of IFN-
, IL-2, IL-4, IL-5, IL-10, IL-13 or TNF-
did not affect the level of MUC1 expression on T cells. However, in the presence of recombinant human IL-12 at 0.1100 ng ml1, expression of MUC1 was increased significantly (P = 0.012) (Fig. 4a and b). The graph in Fig. 4(b) depicts the summary of the mean of the percent increase in MUC1 expression by cytokines from three different experiments, using three different donors (standard deviation from these experiments was within 10% of the mean values).
Anti-CD28 mAb and IL-2 prevent and/or reverse the effect of anti-MUC1 mAb on T cell proliferation
In order to determine whether the co-stimulation by anti-CD28 or IL-2 would abrogate the effect of MUC1 cross-linking on T cell proliferation, we added exogenous IL-2 or anti-CD28 mAb to human T cell cultures stimulated with OKT3 and treated with anti-MUC1 mAb plus goat anti-mouse second antibody. As shown in Fig. 5, the effect of anti-MUC1 mAb on T cell proliferation was abrogated in the presence of IL-2 or anti-CD28 mAb. In these cultures, we did not observe significant cell death with or without IL-2 or anti-CD28 mAb. Also, when the IL-2 or anti-CD28 was added in these cultures at 72 h post-initiation of the culture, we still observed the reversal of inhibition of T cell proliferation further suggesting that T cell death or apoptosis was not induced in these cultures.

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Fig. 5. IL-2 and anti-CD28 antibody reverse or prevent the T cell inhibition induced by anti-MUC1 cross-linking. A total of 2 x 105 human peripheral blood T lymphocytes per well were stimulated with PHA (polyclonal stimulus, 5 µg ml1) in the presence of anti-MUC1 antibody (B27.29, 5 µg ml1) or the appropriate isotype control only, or with the addition of the stated concentration of IL-2 or anti-CD28 mAb [added at the beginning (b) of the culture or at 72 h (c) after initiation of the culture] for 4 days in a flat 96-well microtiter plate. After this time, the wells were pulsed with [3H]TdR overnight and the cells were harvested. Incorporation of [3H]TdR in the DNA of proliferating T cells was determined by liquid scintillation counting. These data were reproduced in three separate experiments.
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Discussion
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MUC1 mucin, initially described as a secretory epithelial cell antigen and also highly expressed on various adenocarcinomas, has attracted attention as a potential target antigen for immunotherapy of various cancers. We have been intrigued by the possibility that cancer-associated MUC1 mucin might have immunomodulatory properties as well (11, 27).
We postulated a normal immunoregulatory role for MUC1 mucin (11, 27) and observed that activated human T cells express MUC1 mucin on their surface and shed or secrete MUC1 in the supernatant (11, 27). MUC1 mucin has been implicated to have (i) anti-adhesion, (ii) signal transduction and (iii) cell growth regulation properties, through studies in tumor cells or cell lines (19, 3639). We postulate that MUC1 mucin transiently expressed on the T cell surface might serve all of these three functions. In the present study, we have focused our work on MUC1 expression on T cells with respect to its role in T cell proliferation.
MUC1 mucin expressed by normal secretory epithelial cells comprises a high molecular weight glycoprotein with a protein core and highly branched carbohydrate side chains (40). However, MUC1 mucin molecules expressed by adenocarcinomas are underglycosylated and cryptic peptide and glycopeptide epitopes are exposed (41). Ten different antibodies specific against the MUC1-exposed peptide core or the carbohydrate epitopes were examined for binding on activated T cells (Table 1) by flow cytometry. They all bound to activated T cells in the 2880% range, further suggesting that the MUC1 expressed by activated T cells may have exposed peptide core epitopes available for binding to antibodies. However, the exact glycosylation status of T cell-expressed MUC1 in relation to carcinoma-expressed MUC1 needs further investigation. Also, upon cross-linking with these antibodies T cell proliferation was inhibited to varying degrees. The differential ability of various mAbs to inhibit T cell proliferation upon cross-linking surface MUC1 may be dependent upon their relative binding affinities, differential signaling upon binding to cell surface MUC1 epitopes or they may react with different subsets of MUC1 molecules on the cell surface. The inhibition does not seem to depend upon particular peptide or glycopeptide epitope-specific binding. All of the peptide-reactive antibodies against tandem repeat region used in our studies are directed towards the PDTRP region of the MUC1 tandem repeat which is immunodominant and has been show to protrude out in a knob-like structure (42). The specificity of binding of anti-MUC1 antibodies has been substantiated by several laboratories (43).
The specificity of interaction of one of the anti-MUC1 mAbs with the T cell surface MUC1 was demonstrated by the results shown in Fig. 1. Addition of glycopeptide haptens to mAb B27.29 abrogated its effect on proliferative response. The same amino acid sequence but without a carbohydrate side chain, to which the B27.29 mAb does not bind with high affinity, had no effect. Therefore, it seems likely that inhibition in proliferation response by anti-MUC1 antibody B27.29 is due to a specific interaction with epitopes present on cell surface MUC1. However, the fact that several peptide-specific antibodies (Table 1) also inhibited the proliferative response suggests that the inhibition is not totally dependent on carbohydrate epitopes or the glycopeptide epitopes, but rather is a function of antibodies binding and cross-linking T cell surface MUC1.
In order to support our hypothesis that cross-linking surface MUC1 is necessary to inhibit T cell proliferation, we prepared Fab' (monovalent) and F(ab')2 (divalent) fragments of B27.29. T cells stimulated in the presence of the monovalent Fab' fragment of B27.29 did not have significant inhibition in proliferation up to 250 µg ml1 concentration of the Fab' (Fig. 2). In contrast, F(ab')2, which has a capability of cross-linking T cell surface MUC1, provided dose-dependent inhibition of T cell proliferation (Fig. 2). Similarly, whole B27.29 mAb, or the B27.29 mAb in the presence of goat anti-mouse antibody (providing optimum cross-linking conditions), inhibited T cell proliferation significantly.
The inhibition of T cell proliferation response upon cross-linking surface MUC1 is in agreement with our hypothesis (11, 27, 39) that MUC1 mucin which is expressed on activated T cells is involved with signaling and may function to down-regulate an ongoing T cell response under appropriate conditions. The role of MUC1 mucin in signal transduction and cell adhesion is further supported by recent studies, where MUC1 was found to be enriched with various signal transducer molecules in a buoyant low-density membrane fraction termed as the glycolipid-enriched microdomain (GEM) (31). The GEM as a membrane unit has been implicated in cell adhesion coupled with signal transduction, and signal transduction molecules characteristic of T cells, such as CD45, lck56 and Src family kinases, are all associated with the same fraction (44). Although we have not yet investigated the putative ligand or receptor for MUC1 expressed on T cells, it is possible that (i) MUC1 mucin interacts with ICAM-1 (9), (ii) MUC1 mucin released from activated T cells or expressed on the surface engages in a homotypic-like interaction (10) or (iii) there is yet another receptor, not implicated from any previous studies, that is involved in interaction with T cell surface expressed MUC1 mucin. However, irrespective of the ligand/receptor for MUC1, it seems important that the interaction with MUC1 may need to be of a cross-linking type in order to provide a negative growth regulatory signal to T cells.
In the experiment designed to examine the time course of MUC1 expression relative to other activation markers, such as CD69 and CD25, we observed that MUC1 is expressed later than both of these markers (Fig. 3a). Therefore, as expected, we did not observe any effect of anti-MUC1-mediated cross-linking on the levels of CD25 and CD69 expression (Fig. 3b) despite the inhibition of T cell proliferation in the same cultures (Fig. 3c). In separate experiments, CD4+CD25+ (Treg) and CD4+CD25 T cells were sorted from PBMCs and examined for MUC1 expression. The CD4+CD25 T cells, upon stimulation with mitogen PHA, expressed both CD25 and MUC1 (data not shown), further suggesting MUC1 expression on activated T cells expressing induced CD25. In addition, MUC1 was expressed on both CD4+ and CD8+ T cells upon mitogen stimulation (Agrawal B, unpublished results), suggesting that it is not specifically expressed by CD4+ regulatory T cells.
Since we observed (27) that MUC1 is expressed on T cells activated by mitogenic stimulus and that MUC1 may be involved with regulation of cell growth (proliferation) of T cells, we sought to determine whether various cytokines, which are induced after an infection or antigenic challenge, would have any effect on MUC1 expression. We examined the effect of IFN-
, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 and TNF-
on MUC1 expression on polyclonally stimulated T cells. Except for IL-12, none of the tested cytokines had any effect on MUC1 expression (Fig. 4). The effect of IL-12 is presumably not due to induction of IFN-
because IFN-
did not affect MUC1 expression. IL-12 has been shown to function as an immune response activator in both IFN-
-dependent and IFN-
-independent mechanisms. Furthermore, MUC1 expression does not seem to be modulated by either type 1 or type 2 biased cytokines. IL-10, which has been shown to suppress a Th1 response and also IFN-
production from Th1 cells, did not have any effect on MUC1 expression. Interestingly, IL-2 also did not have any effect on MUC1 expression on T cells. MUC1 expression may be related to T cell activation by an initial activator of T cell immune responses, such as IL-12, and may be independent of type of T cell response. However, it is possible that polyclonal stimuli used in our experiments provide a strong mitogenic stimulus that any smaller effects of cytokines are not obvious in these experiments. We will be looking at these possibilities in our future studies with less strong antigen-specific responses.
In order to investigate whether surface MUC1 cross-linking induced inhibition in T cell proliferation is preventable and/or reversible, we added exogenous IL-2 or anti-CD28 antibody to the polyclonally stimulated T cells cultured in the presence of cross-linking MUC1 mAb or isotype control mAb. As shown in Fig. 5, we observed that addition of IL-2 or anti-CD28 antibody prevented and/or reversed the inhibition of T cell proliferation induced by anti-MUC1 mAb cross-linking. It is not clear whether the cells inhibited by anti-MUC1 antibody are the ones released by anti-CD28 or IL-2. Also, in the experiment where IL-2 or anti-CD28 was added 72 h after the initiation of the culture (Fig. 5), the inhibitory effect of anti-MUC1 antibody was abrogated, suggesting that the cross-linking of MUC1 on the T cell surface by mAb did not lead to cell death, but rather some kind of anergy-like status which could be reversed by providing a second signal through IL-2 or anti-CD28. These results also suggest that in the presence of optimal co-stimulation, anti-MUC1 antibody mediated inhibition may not be evident, or the anti-MUC1 antibody mediated inhibition is observed in sub-optimally activated T cell cultures.
In conclusion, our findings have further supported our initial hypothesis and observation that MUC1 mucin is involved in normal T cell responses and in the regulation of cell growth (proliferation) of T cells. It is likely that this function of MUC1 on T cells is exerted in conjunction with several other regulatory molecules. Further studies are required to elucidate the intricate intracellular mechanisms which link MUC1 mucin with other T cell surface or intracellular molecules leading to a complete cascade of T cell regulation.
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Acknowledgements
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The authors would like to acknowledge the excellent technical assistance by Teri Tan, Ewa Witkos, Barbara Goudreau and Ivy Ma. Alberta Heritage Foundation for Medical Research (AHFMR) and University Hospital Foundation are greatly acknowledged for providing an establishment grant and pilot project grant to B.A. B.A. is a recipient of an AHFMR Medical Scholar Award.
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Abbreviations
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GEM | glycolipid-enriched microdomain |
[3H]TdR | [3H]thymidine |
TNF- | tumor necrosis factor- |
VNTR | variable but large number of tandem repeat |
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Notes
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Transmitting editor: A. Falus
Received 23 September 2004,
accepted 12 January 2005.
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References
|
---|
- Devine, P. L. and McKenzie, I. F. C. 1992. Mucins: structure, function, and associations with malignancy. Bioessays 14:619.[ISI][Medline]
- Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J. et al. 1990. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J. Biol. Chem. 265:15286.[Abstract/Free Full Text]
- Williams, S. J., McGuckin, M. A., Gotley, D. C., Eyre, H. J., Sutherland, G. R. and Antalis, T. M. 1999. Two novel mucin genes down-regulated in colorectal cancer identified by differential display. Cancer Res. 59:4083.[Abstract/Free Full Text]
- Gendler, S. J. and Spicer, A. P. Epithelial mucin genes. 1995. Annu. Rev. Physiol. 57:607.[CrossRef][ISI][Medline]
- McGuckin, M. A., Walsh, M. D., Hohn, B. G., Ward, B. G. and Wright, R. G. 1995. Prognostic significance of MUC1 epithelial mucin expression in breast cancer. Hum. Pathol. 26:432.[ISI][Medline]
- Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J. et al. 1990. Molecular cloning and expression of the human tumor associated polymorphic epithelial mucin PEM. J. Biol. Chem. 265:15286.[Abstract/Free Full Text]
- Lightenberg, M. J. L. 1992. Cell associated episialin is a complex, containing two proteins derived from a common precursor. J. Biol. Chem. 267:6171.[Abstract/Free Full Text]
- Zrihan-Licht, S., Baruch, A., Elroy-Stein, O., Keydar, I. and Wreschner, D. H. 1994. Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins-cytokine receptor-like molecules. FEBS Lett. 356:130.[CrossRef][ISI][Medline]
- Baruch, A., Hartmann, M., Yoeli, M. et al. 1999. The breast cancer-associated MUC1 gene generates both a receptor and its cognate binding protein. Cancer Res. 59:1552.[Abstract/Free Full Text]
- Regimbald, L. H., Pilarski, L. M., Longenecker, B. M., Reddish, M. A., Zimmermann, G. and Hugh, J. C. 1996. The breast cancer mucin MUC1 as a novel adhesion ligand for endothelial ICAM-1 in breast cancer. Cancer Res. 56:4244.[Abstract]
- Agrawal, B., Gendler, S. J. and Longenecker, B. M. 1998. The biological role of mucins in cellular interactions and immune regulation: prospects for cancer immunotherapy. Mol. Med. Today 4:397.[CrossRef][ISI][Medline]
- Agrawal, B., Reddish, M. A. and Longenecker, B. M. 1996. In vitro induction of MUC-1 peptide-specific type 1 T lymphocyte and cytotoxic T lymphocyte responses from healthy multiparous donors. J. Immunol. 157:2089.[Abstract]
- Agrawal, B., Reddish, M. A., Christian, B. et al. 1998. The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences. Cancer Res. 58:5151.[Abstract]
- Mukherjee, P., Ginardi, A. R., Madsen, C. S. et al. 2003. Mucin 1-specific immunotherapy in a mouse model of spontaneous breast cancer. J. Immunother. 26:47.[CrossRef][ISI][Medline]
- Yamamoto, M., Bharti, A., Li, Y. and Kufe, D. 1997. Interaction of the DF3/MUC1 breast carcinoma-associated antigen and beta-catenin in cell adhesion. J. Biol. Chem. 272:12492.[Abstract/Free Full Text]
- Parry, G., Beck, J. C., Moss, L., Bartley, J. and Ojakian, G. K. 1990. Determination of apical membrane polarity in mammary epithelial cell cultures: the role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions. Exp. Cell Res. 188:302.[CrossRef][ISI][Medline]
- Li, Y., Bharti, A., Chen, D., Gong, J. and Kufe, D. 1998. Interaction of glycogen synthase kinase 3ß with the DF3/MUC1 carcinoma-associated antigen and ß-catenin. Mol. Cell. Biol. 18:7216.[Abstract/Free Full Text]
- Kondo, K., Kohno, N., Yokiyama, A. and Hiwada, K. 1998. Decreased MUC1 expression induces E-cadherin-mediated cell adhesion of cancer cell lines. Cancer Res. 58:2014.[Abstract]
- Mockensturm-Gardner, M. and Gendler, S. J. 1996. The role of MUC1 at the membrane may involve transduction of a signal. Mol. Biol. Cell. 7:434a.
- Wesseling, J., Van der Valk, S. W., Vos, H. L., Sonnenberg, A. and Hilkens, J. 1995. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components. J. Cell. Biol. 129:255.[Abstract]
- Wesseling, J., van der Valk, S. W. and Hilkens, J. 1996. A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1. Mol. Biol. Cell. 7:565.[Abstract]
- Agrawal, B., Krantz, M. J., Reddish, M. A. and Longenecker, B. M. 1998. Cancer-associated MUC1 mucin inhibits human T cell proliferation, which is reversible by IL-2. Nature Med. 4:43.[ISI][Medline]
- Chen, D., Koido, S., Li, Y., Gendler, S. and Gong, J. 2000. T cell suppression as a mechanism to MUC1 antigen in MUC1 transgenic mice. Breast Cancer Res. Treat. 60:107.[CrossRef][ISI][Medline]
- Kim, J. A., Dayton, M. A., Aldrich, W. and Triozzi, P. L. 1999. Modulation of CD4 cell cytokine production by colon cancer-associated mucin. Cancer Immunol. Immunother. 48:525.[CrossRef][ISI][Medline]
- Chan, A. K., Lockhart, D. C., von Bernstorff, W. et al. 1999. Soluble MUC1 secreted by human epithelial cancer cells mediates immune suppression by blocking T-cell activation. Int. J. Cancer 82:721.[CrossRef][ISI][Medline]
- Paul, S., Bizouarne, N., Price, M. R., Hansson, G. C., Kieny, M. P. and Acres, R. B. 1999. Lack of evidence for an immunosuppressive role for MUC1. Cancer Immunol. Immunother. 48:22.[CrossRef][ISI][Medline]
- Agrawal, B., Krantz, M. J., Parker, J. and Longenecker, B. M. 1998. Expression of MUC1 mucin on activated human T-cells: implications for a role of MUC1 in normal immune regulation. Cancer Res. 58:4079.[Abstract]
- Correa, I., Plunkett, T., Vlad, A. et al. 2003. Form and pattern of MUC1 expression on T cells activated in vivo or in vitro suggests a function in T-cell migration. Immunology 108:32.[CrossRef][ISI][Medline]
- Fattorossi, A., Battaglia, A., Malinconico, P. et al. 2002. Constitutive and inducible expression of the epithelial antigen MUC1 (CD227) in human T cells. Exp. Cell Res. 280:107.[CrossRef][ISI][Medline]
- Wykes, M., MacDonald, K. P., Tran, M. et al. 2002. MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells. J. Leukoc. Biol. 72:692.[Abstract/Free Full Text]
- Handa, K., Jacobs, F., Longenecker, B. M. and Hakomori, S.-I. 2001. Association of MUC-1 and SPGL-1 with low-density microdomain in T-lymphocytes: a preliminary note. Biochem. Biophys. Res. Comm. 285:788.[CrossRef][ISI][Medline]
- Mustelin, T., Rahmouni, S., Bottini, N. and Alonso, A. 2003. Related role of protein tyrosine phosphatases in T cell activation. Immunol. Rev. 191:139.[CrossRef][ISI][Medline]
- Reddish, M., Black, N., Almeida, A., Suresh, M. R. and Longenecker, B. M. 1992. Epitope mapping of MAb B27.29 within the peptide core of the malignant breast carcinoma-associated mucin antigen coded for the human MUC1 gene. J. Tumor Marker Oncol. 7:19.
- Liu, X., Sejbal, J., Kotovych, G. et al. 1995. Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides. Glycoconj. J. 12:607.[ISI][Medline]
- Agrawal, B., Reddish, M. and Longenecker, B. M. 1996. CD30 expression on human CD8+ T cells isolated from peripheral blood lymphocytes of normal donors. J. Immunol. 157:3229.[Abstract]
- Pandey, P., Kharbanda, S. and Kufe, D. 1995. Association of the DF3/MUC1 breast cancer antigen with the Brb2 and the sos/ras exchange protein. Cancer Res. 55:4000.[Abstract]
- Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A. and Maas, M. C. 1995. Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extra cellular matrix components. J. Cell. Biol. 129:255.[Abstract]
- Baruch, A., Hartmann, M., Zrihan-Licht, S. et al. 1997. Preferential expression of novel MUC1 tumor antigen isoforms in human epithelial and their tumor-potentiating function. Int. J. Cancer 71:741.[CrossRef][ISI][Medline]
- Spicer, A. P., Rowse, G. J., Lidner, T. K. and Gendler, S. J. 1995. Delayed tumor progression in MUC-1 null mice. J. Biol. Chem. 50:30093.[CrossRef]
- Hanisch, H.-G., Uhlenbruck, G., Peter-Katalinic, J., Egge, H., Dabrowski, J. and Dabrowski, U. 1989. Structure of O-linked polyacrosaminoglycans on human skim milk mucin: a novel type of linearly extended poly-N-acetyl-lactosamine back-bones with Gal ß (1-4) GlcNAc ß (1-6) repeating units. J. Biol. Chem. 264:872.[Abstract/Free Full Text]
- Burchell, J., Gendler, S., Taylor-Papadimitriou, J. et al. 1987. Development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin. Cancer Res. 47:5476.[Abstract]
- Fontenot, J. D., Mariappan, S. V. S., Catasti, P., Domenech, N., Finn, O. J. and Gupta, G. 1995. Structure of a tumor associated antigen containing a tandemly repeated immunodominant epitope. J. Biomol. Struct. Dyn. 13:245.[ISI][Medline]
- Price, M. R., Rye, P. D., Petrakou, E., Murray A., Brady, K. et al. 1997. Summary report on the ISOBM TD-4 workshop: analysis of 56 monoclonal antibodies against MUC1 mucin. Tumor Biol. 19(Suppl. 1):1.
- Hakomori, S., Handa, K., Iwabuchi, K., Yamamura, S. and Prinetti, A. 1998. New insights in glycosphingolipid function: "glycosignaling domain," a cell surface assembly of glycosphingolipids with signal transducer molecules, involved in cell adhesion coupled with signaling. Glycobiology 8:xi.[ISI][Medline]