Intercellular and Intracellular Events Following the MHC-Unrestricted TCR Recognition of a Tumor-Specific Peptide on the Epithelial Antigen MUC1 This information is current as of September 28, 2021. Julie Magarian-Blander, Pawel Ciborowski, Shyuan Hsia, Simon C. Watkins and Olivera J. Finn J Immunol 1998; 160:3111-3120; ; http://www.jimmunol.org/content/160/7/3111 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Intercellular and Intracellular Events Following the MHC-Unrestricted TCR Recognition of a Tumor-Specific Peptide Epitope on the Epithelial Antigen MUC11

Julie Magarian-Blander,2* Pawel Ciborowski,* Shyuan Hsia,‡ Simon C. Watkins,† and Olivera J. Finn3*

We examined the functional and molecular parameters involved in direct TCR recognition of a tumor-specific peptide epitope on the tumor Ag MUC1. This peptide epitope is tandemly repeated and recognized on the native molecule rather than processed and bound to the MHC. Even though the TCR was not MHC restricted, intercellular interactions found to facilitate this recognition included intercellular adhesion molecule-1/LFA-1, LFA-3/CD2, and class I/CD8. Intracellular parameters of MHC-unrestricted CTL activation were examined to compare the recognition of the MUC1 epitope presented on synthetic microspheres, with the Downloaded from recognition of the native epitope in the context of other molecules on the target cells. The epitope on microspheres induced a ,transient influx of Ca2؉ that was not accompanied by detectable tyrosine phosphorylation of the ␨-associated ZAP-70 whereas recognition of MUC1 on tumor cells caused a sustained Ca2؉ influx and ZAP-70 phosphorylation. The transient influx of Ca2؉ was not sufficient to cause translocation of the nuclear factor of activated T cells (NF-AT) into the nucleus or CTL proliferation. In contrast, recognition of the MUC1 epitope on tumor cells resulted in full activation of the CTL, nuclear trans- http://www.jimmunol.org/ location of NF-AT, and proliferation. MHC-unrestricted TCR triggering, therefore, involves similar intercellular and intracellular events that participate in the conventional, MHC-restricted Ag recognition. Direct recognition of the MUC1 peptide epitope by the TCR in the absence of presentation by the MHC induces a partial signal that is completed by further interactions of other receptor/ligand pairs on the surface of the CTL and their target cells. The Journal of Immunology, 1998, 160: 3111–3120.

lymphocytes typically recognize antigenic peptides that rheumatoid arthritis (4), and from mice immunized with Mycobac- are presented to them within the groove of self MHC terium tuberculosis (5). MHC-unrestricted ␥␦ T cells have also T molecules on the surface of APC (1). This MHC-re- been isolated that are specific for Ags such as Ig Ids on stricted recognition is mediated through the TCR/CD3 complex. tumors (6), a herpesvirus (7), and nonpeptide prenyl by guest on September 28, 2021 Recognition of the antigenic peptide by the TCR/CD3 complex pyrophosphates (8). Ag-specific, MHC-unrestricted, ␣␤ T cells results in the activation of the via a cascade of signal-trans- have also been described for complex such as and duction events (2, 3). Early activation events include tyrosine myelin basic protein (9), as well as for a nonpeptide Ag such as the phosphorylation of membrane and cytoplasmic proteins, hydroly- heme moiety of hemoglobin (10). Several studies have also de- sis of membrane inositol phospholipids, activation of protein ki- scribed arsonate- and fluorescein-specific T cells that can recog- nase C, and increases in the cytoplasmic concentration of calcium. nize Ag in the absence of MHC molecules (11–13). More recently, Activation of the T cell ultimately results in proliferation and tran- carbohydrate-specific MHC-unrestricted T cells were generated scriptional activation of a variety of that lead to the release that were specific for the carbohydrate moiety on glycosylated of cytokines, expression of new surface molecules, and maturation peptides derived from the vesicular stomatitis virus of effector function. nucleoprotein (14). Ag-specific MHC-unrestricted recognition has also been de- We have reported previously MHC-unrestricted ␣␤ T cells that ␥␦ scribed. MHC-unrestricted T cells specific for mycobacterial recognize a peptide epitope on the molecule MUC1 (15, 16), a Ags have been isolated from the synovial fluid of patients with type I transmembrane glycoprotein that is expressed on the surface of ductal epithelial cells as well as carcinomatous cells of the same origin Departments of *Molecular Genetics and Biochemistry, and †Cell Biology and Phys- (17). The bulk of its extracellular domain is composed of a tandemly iology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and ‡De- partment of Pathology, Allegheny General Hospital, Pittsburgh, PA 15212 repeated 20-amino acid sequence that contains the T cell epitope. The MHC-unrestricted recognition of the MUC1 tandem repeat epitope is Received for publication August 27, 1997. Accepted for publication November 26, 1997. blocked by Abs to the TCR and CD3 complex, showing that this The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance recognition is TCR mediated. We have proposed that MUC1 tandem with 18 U.S.C. Section 1734 solely to indicate this fact. repeats present a dense array of unprocessed epitopes directly to the 1 This work was supported by National Institutes of Health Grants RO1 CA56103 and TCR as rigid structures that bypass the need for presentation by MHC RO1 CA57820, and a grant from Corixa Corporation to O.J.F. The Cell Structure and Imaging Facility was supported by University of Pittsburgh Comprehensive Cancer molecules (15, 18). Structural studies of the MUC1 tandem repeat Center Core Grant from National Institutes of Health, 2P30CA47904. protein core using synthetic peptide analogues have confirmed that the 2 Current address: Yale University School of Medicine, Section of Immunobiology, T cell epitope assumes a stable ordered structure that forms a loop 310 Cedar Street, FMB402, P.O. Box 208011, New Haven, CT 06520-8011. protruding past the extended ␤-turn helix structure of the polypeptide 3 Address correspondence and reprint requests to Dr. Olivera J. Finn, Department of core (19). Molecular Genetics and Biochemistry, W1142 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail address: Recognition of a native antigenic epitope directly by the TCR ojfi[email protected] without presentation within the groove of the MHC is not very

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 3112 MUC-1-SPECIFIC MHC-UNRESTRICTED TCR SIGNALING common and therefore not well understood. Nothing is known nologies) supplemented with 10% human AB serum (Gemini Bioproducts, about the T cell activation events that occur upon MHC-unre- Calabasas, CA) and 20 U/ml IL-2, at 1 ϫ 106 cells/ml, and stimulated with stricted recognition of Ag via the TCR, nor about the role that irradiated (6000R) allogeneic breast cell lines BT-20 and CAMA-1 in an alternating fashion every 7 days. The CTL lines were cul- coreceptor and/or adhesion molecules play in this type of recog- tured on tumor cells for 9 wk, and the CTL were further expanded on 10 nition. In this study, we have characterized for the first time, some ng/ml anti-CD3 mAb OKT-3 (American Type Culture Collection (ATCC), of the cell surface and intracellular molecules that contribute to a Rockville, MD) in the presence of irradiated allogeneic PBMCs and 10 productive MHC-unrestricted recognition of MUC1. We describe ng/ml IL-2 for 2 wk, then switched to 0.1 ng/ml OKT-3 in the presence of PBMCs and 2 ng/ml IL-2 every 2 wk. MA MUC1-specific CTL became two types of MUC1-specific MHC-unrestricted CTL, one depen- monoclonal, expressing one V␣ and one V␤ chain, as evident by PCR dent on MHC class I/CD8 coreceptor engagement, and the other amplification and sequencing (M. Alter et al., manuscript in preparation). independent of this interaction. Both types are dependent on the The cultures were kept on OKT-3 for a period not exceeding 6 to 8 wk. ϩ additional interactions of adhesion molecules ICAM-1 and LFA-3 Tyrosine phosphorylation, NF-AT translocation, and some of the Ca2 with LFA-1 and CD2, but to differing degrees. We further explored influx measurements were performed on CTL that were in the second week after 0.1 ng/ml OKT-3 stimulation. To generate an allospecific (anti-HLA- the activation of MHC-unrestricted MUC1-specific CTL in re- A2) CTL line, peripheral blood of a donor with HLA type A23, 24; B7, 57 sponse to MUC1 epitopes expressed in the presence or absence of (A2ϪB7ϩ) was applied to a sodium diatrizoate-Ficoll gradient, lymphocyte these molecules. We show that similar to the MHC-restricted pep- separation medium (Organon Technica, Durham, NC), and the resulting ϩ tide recognition, the MHC-unrestricted CTL recognition of the na- mononuclear cell fraction was stimulated weekly with irradiated HLA-A2 JY cells. tive MUC1 epitope on the surface of tumor cells induces sustained Ca2ϩ mobilization, phosphorylation of the ␨-associated protein Cytotoxicity assays Downloaded from ZAP-70, translocation of the nuclear factor of activated T cells Target cells were labeled for1hbyincubating 1 ϫ 106 cells in 50 ␮Ci of 4 51 (NF-AT) to the nucleus, and CTL proliferation. When these CTL Na2 CrO4 (Amersham, Arlington Heights, IL) at 37°C. Labeled cells were were stimulated by MUC1 peptides on the surface of micro- washed and seeded at 2 ϫ 103/100 ␮l/well in a 96-well V-bottom plate spheres, in the absence of any other cell surface molecules, only a with varying numbers of effector T cells. The plates were centrifuged and incubated for4hin5%CO at 37°C. All determinations were performed partial CTL response was observed characterized by a transient 2 2ϩ in triplicates. Supernatants were harvested using a Skatron harvesting press Ca influx, lack of detectable ZAP-70 tyrosine phosphorylation, (Skatron Instruments, Sterling, VA), and counted in a gamma counter (Co- cytoplasmic NF-AT, and no proliferation. These data show that bra II, auto gamma; Packard Instrument, Downer’s Grove, IL). Maximum http://www.jimmunol.org/ native MUC1 tandem repeat peptides can directly bind the TCR of release was obtained by adding 100 ␮l of 1 N HCl to the labeled target MHC-unrestricted CTL and induce their activation, but this acti- cells. Spontaneous release was obtained by incubating the labeled targets with medium in the absence of T cells. Percentage of release was calculated vation is only partial. When MUC1 tandemly repeated epitopes are using the following equation: percentage of release ϭ 100 ϫ (experimental recognized by CTL in the company of additional molecules on the release Ϫ spontaneous release)/(maximum release Ϫ spontaneous release). surface of tumor cells, full CTL activation results. For blocking experiments with anti-CD3 mAb OKT-3 (ATCC), anti-CD58 (ATCC), anti-CD54 (ICAM-1) (PharMingen, San Diego, CA), anti-CD2 (LFA-2) (ATCC), anti-CD11a (LFA-1) (ATCC), and anti-class I mAb Materials and Methods W6/32 (ATCC), 2 ϫ 103 labeled targets/100 ␮l were incubated for 30 min

Reagents and cell lines at 37°C in the presence of the indicated concentrations of Abs. T cells were by guest on September 28, 2021 then added at the indicated E:T ratio. The 100-amino acid-long MUC1 peptide ((GVTSAPDTRPAPGSTAP PAH) ϫ 5) was synthesized in Peptide Synthesis Facility, Department of Fluorescence spectroscopy Molecular Genetics and Biochemistry, University of Pittsburgh, Pitts- 6 burgh, PA employing F-moc chemistry, on an advanced Chemtech 200 A quantity amounting to 1 ϫ 10 T cells/ml was labeled for 20 min at room synthesizer. The purified product was characterized by electron mass spec- temperature in 50 ␮M of Calcium Green-1 acetoxymethyl (AM) ester so- trometry and by ELISA using anti-MUC1 mAbs. Polylactide-coglycolide lution (Molecular Probes, Eugene, OR) prepared according to the manu- (PLGA) microspheres have been described previously (20). The MUC1 facturer’s instructions. Measurements were performed on a Perkin-Elmer peptide or OVA peptide was conjugated to PLGA microspheres at South- (Norwalk, CT) luminescence spectrometer LS50B equipped with a 96-well ern Research Institute (Birmingham, AL), in collaboration with Corixa plate reader. Labeled cells were resuspended in prewarmed 37°C simplified Corp. (Seattle, WA). The average number of MUC1 peptides per micro- medium (145 mM NaCl, 5 mM KCl, 1 mM Na2PO4, 1 mM CaCl2, 0.5 mM 9 sphere was 2.8 ϫ 10 . The breast tumor cell lines BT-20 (A24, A31; B15, MgSO4, 5 mM glucose, and 10 mM HEPES, pH 7.4) and supplemented 5 7; Bw4, 6) and CAMA-1 (A2, 3; B40/4701, B15) were previously de- with 1% inactivated human serum. A quantity amounting to 5 ϫ 10 T cells scribed (16). The pancreatic tumor cell lines T3 M4 (A2, 7; B40/4701, per experiment was added to white flat-bottom Microfluor 96-well plate B51/52/7801; Bw4, 6) and HPAF (A1, 2601; B5401/55/56/5901, B8; Bw6) (Dynatech Labs., Biotechnology Products, Chantilly, VA) with excitation were previously described (15). The MUC-1-transfected cell line T2 was at 506 nm and emission at 534 nm. An equal number of tumor cells (BT- previously described (21). The lymphoblastoid cell line JY (A2, 2; B7, 7) 20, CAMA-1, or DM-6) were briefly centrifuged with the T cells. PLGA was obtained from Dr. V. Engelhard, University of Virginia (Charlottes- beads (0.1 mg) conjugated to either MUC1 or OVA were centrifuged 5 ville, VA). The cell line DM-6 (A2, 2; B13, 44; Bw4) was briefly with 5 ϫ 10 T cells. Ionomycin (Sigma Chemical Co., St. Louis, obtained from Dr. C. Slingluff, University of Virginia. MO) was added directly to the well at a 10 ␮M concentration. For anti- CD3 cross-linking, T cells were incubated for 30 min at 4°C in the presence Collection and culture of T lymphocytes of 50 ␮g/ml OKT-3 (ATCC), and ␥-chain-specific goat anti-mouse IgG (Zymed Laboratories) was then added at a 20 ␮g/ml concentration. The To generate MUC1-specific CTL lines, the tumor-draining lymph node ϩ increase in fluorescence intensity was monitored over time relative to from a patient with was used as the source of the CD8 T background. lymphocytes. Cells (108) were stained on ice for 1 h with Leu3a (anti-CD4) Ab (gift from Dr. E. Engelman, Stanford University, Stanford, CA) and Confocal microscopy washed in Dulbecco’s PBS (Life Technologies, Grand Island, NY). Two A quantity amounting to 5 ϫ 105 Calcium Green-1-loaded T cells was 100-mm petri dishes (Nunc, LabTek, Naperville, IL) were precoated with ␮ 10 ␮g/ml goat anti-mouse IgG, ␥-chain specific (Zymed Laboratories, San briefly centrifuged with 40 g of MUC1- or OVA-conjugated PLGA Francisco, CA) in 0.05 M Tris, pH 9.5, at room temperature for1hand beads, or incubated for 30 min at 37°C on BT-20, HPAF, or DM-6 tumor washed. A quantity amounting to 5 ϫ 107 cells was added to each plate and monolayers grown on sterile coverslips. T cells were observed under a incubated at 4°C for 1 h. The nonadherent CD8ϩ T cell-enriched popula- Molecular Dynamics (Sunnyvale, CA) Multiprobe 2001 confocal laser mi- tion was collected, resuspended in AIM-V lymphocyte medium (Life Tech- croscope (CLSM) to quantify distribution and intensity of labeling. Images through the midplane of the cells were collected at 20-s intervals using a Nikon 20 or ϫ60 objective, 1024 ϫ 1024 pixels, 512 illuminating laser 4 Abbreviations used in this paper: NF-AT, nuclear factor of activated T cells; line, 535 primary dichroic, and 570 barrier filter. All images were collected 2ϩ ␣ [Ca ]i, intracellular calcium concentration; phenyl-GalNAc, phenyl-N-acetyl- - under exactly the same conditions of laser intensity to ensure consistent galactosaminide; PLGA, polylactide coglycolide. sampling between conditions. Three-dimensional image stacks were also The Journal of Immunology 3113 collected with no time delay, through the entire thickness of the cells, as rapidly as possible with a Z interval of 0.5 ␮m (15 images total).

ZAP-70 immunoprecipitations and immunoblots T cells and stimulator cells were used at a ratio of 10:1. The T cells and stimulator cells or MUC1-conjugated microspheres were mixed and cen- trifuged briefly to facilitate contact. For T cell stimulations with anti-CD3 Ab, T cells were first stained on ice for 30 min with anti-CD3 mAb OKT-3 (ATCC) and washed, and the surface OKT-3 was cross-linked with goat anti-mouse Ab at 50 ␮g/ml (Zymed Laboratories). Stimulations were per- formed in microcentrifuge tubes at 37°C for the indicated times. The cells were lysed immediately in a final 1% Brij-97 (Sigma Chemical Co.), 150 mM NaCl, 10 mM Tris, pH 7.5, 1 mM Na3VO4 (Sigma Chemical Co.), 10 ␮g/ml aprotinin (Sigma Chemical Co.), 10 ␮g/ml leupeptin (Sigma Chem- ical Co.), and 1 mM PMSF (Sigma Chemical Co.). ZAP-70 was immuno- precipitated from postnuclear lysates of 2 ϫ 107 to 3 ϫ 107 T cell equiv- alents by adding 1 to 1.5 ␮l of anti-ZAP-70 Ab (kind gift from Dr. A. Weiss, University of San Francisco, San Francisco, CA). The lysates were rotated with anti-ZAP-70 Ab for 24 h, followed by an overnight incubation ϩ FIGURE 1. Cytotoxicity of the MUC1-specific CTL toward MUC1 with 100 ␮l protein G-Sepharose slurry (4 Fast Flow; Pharmacia Biotech, Downloaded from Uppsala, Sweden). The protein G-Sepharose beads were then washed twice tumor cell lines is mediated through the CD3/TCR complex. The tumor cell lines used as targets are melanoma DM-6 (squares), breast tumor BT-20 in cold PBS/0.01% Brij-97/1 mM Na3VO4 (Sigma Chemical Co.), and once in cold PBS. The immunoprecipitated proteins were eluted off protein (diamonds), breast tumor CAMA-1 (circles), and pancreatic tumor T3M4 G by boiling for 4 min in 75 ␮l1ϫ SDS/sample buffer in the presence of (triangles). Cytotoxicity of the clonal MA MUC1-specific CTL (A) and 5% 2-ME (Kodak, Rochester, NY). The eluted proteins were loaded onto allo-specific CTL (C). Blocking with anti-CD3 mAb OKT-3 at an E:T ratio 12 to 15% SDS/polyacrylamide gels, electrophoresed, transferred onto ni- of 10:1 for the MA MUC1-specific (B) and allo-specific CTL (D). The trocellulose membrane (BioBlot, NC, Corning Costar, Corning, NY), and open bars represent the percentage of specific killing in the absence of http://www.jimmunol.org/ blocked in 5% nonfat powdered milk (Carnation). The membrane was OKT-3, and the filled bars represent the percentage of specific killing in the probed with either anti-phosphotyrosine Ab (4G10) (Upstate Biotechnol- presence of 10 ␮g/ml OKT-3. Similar results were obtained in at least three ogy, Placid Lake, NY) or anti-ZAP-70 mAb (Upstate Biotechnology) over- experiments. night at 4°C, followed by the secondary peroxidase-conjugated anti-mouse IgG (whole molecule) (Sigma Chemical Co.), and developed by enhanced chemiluminescence (ECL) method per manufacturer’s instructions (Amersham). Results The MHC-unrestricted CTL recognition of MUC1 is dependent Proliferation assays to varying degrees on the expression of class I, CD54, and CD58 on tumor cells by guest on September 28, 2021 T cells were seeded at 2 ϫ 105/100 ␮l/well in round-bottom 96-well plates (Costar, Cambridge, MA). Anti-CD3 mAb OKT-3 (ATCC) was immobi- Figure 1A shows the function of the clonal CTL line MA as an lized to the wells at 30 ␮g/ml in sterile Dulbecco’s PBS overnight at room example of the function of all MUC1-specific MHC-unrestricted ␥ temperature following immobilization of goat anti-mouse IgG, -chain CTL we have derived, and compares it with the function of allo- specific (Zymed Laboratories) at 30 ␮g/ml. Irradiated (6000 R) stimulator reactive, HLA-A2-specific CTL. At 8 wk of culture, when MA was tumor cells DM-6, BT-20, and CAMA-1 were added to the appropriate ϩ ϩ wells in the presence or absence of T cells, at a T cell:stimulator ratio of 100% CD8 , it killed MUC1 breast tumor lines BT-20 and 3:1. A quantity amounting to 0.1 mg MUC1 tandem repeat peptide-con- CAMA-1 and also MUC1ϩ pancreatic tumor cell line T3 M4. The jugated or OVA-conjugated PLGA beads was added to the appropriate MUC1Ϫ melanoma cell line DM-6 was not killed. The cytotoxicity wells in sterile PBS. All cells were resuspended in AIM-V lymphocyte of MA toward the MUC1ϩ tumors was blocked with anti-CD3 medium (Life Technologies) supplemented with 10% inactivated human AB serum (Gemini Bioproducts) in the presence of 5 U/ml IL-2. The wells mAb (Fig. 1B). The reactivity of the HLA-A2-specific CTL were pulsed with [3H]TdR (Amersham, Life Science) for the last 18 h of against the same tumor targets was quite different, corresponding a 3-day period. The plates were harvested by a Skatron semiautomatic cell only to their HLA type. The MUC1ϪHLA-A2ϩ melanoma cell harvester (Skatron Instruments) and counted on a Wallac 1205 betaplate line DM-6 was killed, as were the MUC1ϩHLA-A2ϩ cell lines liquid scintillation counter (Gaithersburg, MD). The results are expressed T3M4 and CAMA-1. CAMA-1 was killed at low levels in the as mean values of duplicate determinations. experiment shown, but has been observed to be recognized at higher level in other experiments. The MUC1ϩ but HLA-A2Ϫ NF-AT translocation (HLA-A24, 31) tumor cell line BT-20 was not killed (Fig. 1C). The HLA-A2-specific killing of this T cell line was blocked by the NF-AT translocation into the nucleus was observed by fluorescence mi- anti-HLA-A2 mAb MA2.1 (not shown). The cytotoxicity of the croscopy, as previously described (22). T cells and stimulator cells, as ϩ indicated, at a ratio of 10:1, or T cells and 0.1 mg MUC1- or OVA-con- HLA-A2-specific T cell line toward the HLA-A2 tumors was jugated microspheres were incubated for 4, 18, 24, or 48 h at 37°C in sterile also blocked in the presence of anti-CD3 mAb (Fig. 1D). 96-well V-bottom plates. The cells were then centrifuged onto slides (Cy- We wanted to further show the lack of MHC restriction in tospin 3; Shandon, Pittsburgh, PA) for 3 min at 250 rpm. The slides were MUC1-specific CTL function by the ability of MUC1-specific fixed immediately in 0.5% paraformaldehyde (10 min), permeabilized in cold methanol (2 min), and rehydrated in PBS (10 min). The cells were CTL to recognize and lyse T2 cells transfected with MUC-1 cDNA (T2 MUC1) (Fig. 2). The advantage of using T2 is that it is a stained with anti-NF-ATc mAb 7A6 (1:1,000) (kind gift from Dr. Gerald Crabtree, Stanford University), followed by anti-mouse biotin-conjugated mutant cell line, deficient in TAP-mediated transport of peptides goat anti-mouse IgG (H ϩ L) rat-adsorbed Ab (1:1,000) (Caltag Labora- from the cytoplasm into the (ER) (23), and tories, So. San Francisco, CA), and streptavidin-conjugated Cy-3-labeled thus not expected to present MUC1 peptides efficiently. The dis- Ab (1:12,000) (Jackson ImmunoResearch Laboratories, West Grove, PA). Hoescht dye was added for 30 s and washed. Separate red and blue sections advantage is that T2 MUC1 cells express a fully glycosylated were collected and overlaid for each field. Final image processing was MUC1 molecule (21) that does not express the unglycosylated performed using Adobe Photoshop (Mountain View, CA). epitope recognized by the MUC1-specific MHC-unrestricted CTL 3114 MUC-1-SPECIFIC MHC-UNRESTRICTED TCR SIGNALING

FIGURE 2. Cytotoxicity of the MUC1-specific CTL against muc-1- transfected T2 cells. MUC1-specific MA CTL were used. T2 MUC1 cells were treated with the O- inhibitor phenyl-GalNAc every 24 h for a period of 72 h (72 h gi). BT-20 and HPAF are MUC1ϩ tumor cells.

(24). This epitope, however, can be generated by extensive treat- ment with phenyl-N-acetyl-␣-D-galactosaminide (phenyl-Gal- NAc), a compound that inhibits elongation of the O-linked sac- Downloaded from charide chains (25). We have published previously that treatment of many EBV-immortalized B cells transfected with MUC1 can FIGURE 4. The dependence of MHC-unrestricted MUC1-specific CTL sensitize them to killing by MUC1-specific CTL, and that untrans- effector function on CD54 and CD58. MUC1ϩ tumor cell target is fected parental cells treated the same way do not gain susceptibil- CAMA-1. A, Differential dependence on anti-CD54 or anti-CD58 corre- ity to CTL lysis (16). In Figure 2, we show that following this lating with MHC dependency. The CTL (MHC dependent or independent)/ treatment, specific lysis of T2 MUC1 cells at an E:T of 40:1 is mAb combination is indicated in the legend. B, Blocking effector function indeed observed, although at lower levels than the specific lysis of of two MUC1-specific CTL lines (MHC dependent (MA) or independent, http://www.jimmunol.org/ the MUC1ϩ breast tumor cell line BT-20 and the MUC1ϩ pan- as indicated) shown on the x-axis with anti-CD54 and anti-CD11a mAbs, as indicated in the legend at 50 ␮g/ml each. Data are representative of at creatic tumor cell line HPAF. least three independent experiments. The question we next addressed was whether MHC class I mol- ecules that do not present MUC1 peptides could still have an ac- cessory role in MHC-unrestricted CTL recognition of this Ag. For each line, an E:T ratio was chosen in which ϳ50% maximal lysis these experiments, three MUC1-specific CTL lines were used. For was observed. One CTL line was equally cytotoxic against various MUC1ϩ tumors in the presence or absence of mAb to MHC class

I molecules (Fig. 3A). The cytotoxicity of the other two CTL lines, by guest on September 28, 2021 including the MA CTL described above, toward the same MUC1ϩ tumor targets was inhibited significantly in the presence of mAb to class I molecules. The results for one of these targets, T3M4, are presented in Figure 3B. Inhibition of lysis was also seen in the presence of mAb to CD8, and the presence of both anti-class I and anti-CD8 mAbs had an additive blocking effect. These three CTL are good examples of two types of MHC-unrestricted MUC1-spe- cific CTL we have observed consistently: MHC (and CD8) inde- pendent and MHC (and CD8) dependent. Several different adhesion molecules are crucial in the MHC- restricted recognition of Ag by T cells (26), and we examined their role in MHC-unrestricted recognition of MUC1. We compared the MHC-unrestricted cytotoxic function of MHC-dependent and the MHC-independent MUC1-specific CTL for their dependence on CD54 (ICAM-1) and CD58 (LFA-3) molecules expressed on the tumor cell targets. The levels of these molecules were comparable on all tumor cell targets that were tested, as detected by FACS analysis. Dependence of the CTL-target interaction on these ad- hesion molecules corresponded well to their degree of dependence on MHC class I molecules. We found that the MHC-independent unrestricted CTL were also less dependent on CD54 and CD58 (Fig. 4A). The presence of anti-CD54 or anti-CD58 mAb at dif- FIGURE 3. The dependence of MHC-unrestricted MUC1-specific CTL ferent concentrations of 5, 25, and 50 ␮g/ml inhibited ϳ20%. In effector function on MHC class I. A, An MHC-independent CTL line. The Ϫ contrast, the same Abs were able to block the cytotoxic function of x-axis shows the tumor cell targets, DM-6 (MUC1 ) and T3M4, CAMA-1, the MHC-dependent CTL (Fig. 4A). Dependence on CD11a and and BT-20 (MUC1ϩ). B, Two MHC-dependent CTL lines. On the x-axis CD2, ligands for CD54 and CD58, respectively, expressed on the are shown two MUC1-specific CTL lines, MA CTL shown on the left. The tumor cell target is MUC1ϩCAMA-1. The percentage of specific killing is CTL, also showed correlation with the CTL dependence on MHC represented in A and B on the y-axis in the presence or absence of anti-class molecules. Figure 4B shows that anti-CD11a blocked the MHC- I mAb W6/32 or anti-CD8 mAb OKT-8 at 10 ␮g/ml, as indicated in the unrestricted cytotoxicity of the MHC-dependent MUC1-specific legend, and at an E:T ratio of 10:1. Data are representative of at least three CTL, while no significant inhibition was observed for the MHC- independent experiments. independent MUC1-specific CTL. The presence of both The Journal of Immunology 3115 anti-CD11a and anti-CD54 almost completely blocked the cyto- PDTRP (not shown). As a control, microspheres conjugated with toxicity of the MHC-dependent CTL, but not the MHC-indepen- OVA were used. We first examined changes in intracellular cal- 2ϩ dent CTL (Fig. 4B). The same results were obtained with anti-CD2 cium concentration ([Ca ]i) in response to CTL stimulation. Fig- Ab and the combination of anti-CD2 and anti-CD58 (not shown). ure 5A shows that the MUC1-specific, MHC-unrestricted clonal 2ϩ ϩ MA CTL undergo a large increase in [Ca ]i when incubated on MHC-unrestricted, MUC1-specific CTL influx Ca2 in response ϩ ϩ a monolayer of the MUC1 breast tumor cell line BT-20. Similar to MUC1 tumor cells and MUC1 peptide conjugated to results were obtained with a monolayer of the pancreatic MUC1ϩ microspheres tumor cell line HPAF (not shown). The white pseudocolor indi- 2ϩ The observation that the effector function of some MHC-unre- cates an increased level of [Ca ]i. The CTL were found to form stricted CTL lines is critically dependent on different molecules pseudopods over the tumor monolayer, a change in shape that is expressed at the surface of tumor cells prompted us to examine the indicative of CTL activation. T cell shape has been shown to be 2ϩ full extent to which a native MUC1 tandem repeat epitope can sensitive to a rise in [Ca ]i (27). stimulate CTL in the absence of these molecules. We compared the When MA CTL were incubated with MUC1-conjugated PLGA ϩ signal-transduction events that take place following the MHC-un- microspheres, they also underwent a similar influx of Ca2 . Figure restricted recognition of MUC1 expressed on tumor cells with the 5B shows a three-dimensional image reconstruction of a MUC1- events that follow recognition of the MUC1 Ag alone. A synthetic specific CTL interacting with a large MUC1-conjugated micro- 100-amino acid-long (five tandem repeats) MUC1 peptide was sphere. Several image slices on multiple planes were acquired as a ϩ conjugated to the surface of 20 to 40 ␮m PLGA microspheres. representation of the Ca2 influx at one time point, and they Downloaded from These microspheres were stained with anti-MUC1 mAb, and were showed that the CTL had almost fused to the microsphere on one shown to express the relevant MUC1 immunodominant epitope http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Ca2ϩ influx profile in MUC1-specific CTL in response to FIGURE 5. MUC1-specific CTL influx Ca2ϩ in response to MUC1. A, MUC1ϩ tumor cell lines. Response of the MA CTL is shown. A,Inre- MUC1-specific CTL MA showing Ca2ϩ influx in response to a monolayer sponse to cross-linking OKT-3 with anti-mouse IgG. B, In response to the of MUC1ϩBT-20 tumor cells at ϫ10 magnification. The data are repre- MUC1ϩCAMA-1 cells. C, In response to the MUC1ϩBT-20 cells. D,In sentative of 35 separate fields. B, MUC1-specific CTL MA showing Ca2ϩ response to the MUC1ϪDM6 cells. The intensity of fluorescence expressed influx in response to a MUC1ϩ microsphere. An enlarged three-dimen- in arbitrary units is shown on the y-axis. Each tick mark represents 2 ar- sional reconstruction of the image at ϫ60 magnification is shown. The bitrary units. The time of measurement in seconds is shown on the x-axis. color scale on the right is for both A and B. It shows the range of fluores- Time t ϭ 0 represents the beginning of measurement after a 5-s centrifu- cence intensity from 0 to 255, in which white represents the highest flu- gation of CTL with tumor cells, and immediate measurement after the orescence intensity of Calcium Green-1, and dark blue represents no flu- addition of cross-linking Ab. Data are representative of at least three orescence. The data were similar in two other independent experiments. experiments. 3116 MUC-1-SPECIFIC MHC-UNRESTRICTED TCR SIGNALING side, in contrast to the two CTL toward the top that were separating change in fluorescence intensity upon the addition of an equal from the bead. The CTL still interacting with the microsphere was amount of MUC1ϩ microspheres (Fig. 7B). Similarly, the MUC1- 2ϩ white, representing an increased [Ca ]i. In addition, this CTL had specific CTL did not respond to OVA conjugated to microspheres undergone a change in shape, in which the cell was now elongated (Fig. 7C), and neither did the control allo-specific CTL (Fig. 7D). along the microsphere surface, with pseudopodia extending around The MHC-unrestricted recognition of MUC1ϩ tumor cells, but a small section of the perimeter. The two round CTL toward the ϩ top are presumed to have interacted with the microsphere earlier, not MUC1 microspheres, results in tyrosine phosphorylation of 2ϩ ZAP-70 and their [Ca ]i level, while still high, is not as high as the CTL 2ϩ still in contact with the microsphere. No such changes in shape or Along with increases in [Ca ]i, we measured another early pa- 2ϩ [Ca ]i levels were observed when MUC1-specific MA CTL were rameter of T cell activation manifested by the tyrosine phosphor- incubated with OVA-conjugated microspheres (not shown). ylation of the syk family protein tyrosine kinase ZAP-70 (28).

2ϩ MHC-unrestricted, MHC-dependent MA CTL were stimulated for Specific CTL undergo a transient Ca influx in response to ϩ 2ϩ different times with either the MUC1 pancreatic tumor cell line MUC1 conjugated to microspheres, but a sustained Ca influx Ϫ ϩ ϩ T3M4, the MUC1 melanoma cell line DM-6, or the MUC1 in response to MUC1 tumor cells microspheres. Figure 8A shows the tyrosine phosphorylation status We further examined the pattern of Ca2ϩ influx over a period of of immunoprecipitated ZAP-70 molecules. Upon stimulation of time. Measurements were made in microtiter plate wells by fluo- MUC1-specific MA CTL with MUC1ϩT3M4 tumor cells, there rescence spectroscopy. The background fluorescence was stable, was a detectable increase in tyrosine phosphorylation of ZAP-70 Downloaded from and when ionomycin, a calcium ionophore, was added to the CTL, (lane 3), compared with unstimulated CTL. The signal was not as a 10-U or greater increase in fluorescence intensity was observed. strong as that detected after OKT-3 cross-linking (lane 2). No Cross-linking the TCR/CD3 complex on MUC1-specific MA CTL phosphorylated ZAP-70 was detected after stimulation with MUC- resulted in a slow and sustained increase in fluorescence intensity 1ϪDM-6 melanoma cells either at 5 min (lane 7) or 1 min (not that began at ϳ20 s (Fig. 6A). No such increase in fluorescence shown). This result shows that MHC-unrestricted recognition of intensity was observed in the absence of cross-linking anti-mouse MUC1 on tumor cells allows successful tyrosine phosphorylation IgG (not shown). Stimulation of MA CTL with MUC1ϩ breast of ZAP-70, a key player in signal transduction in MHC-restricted http://www.jimmunol.org/ tumor cell lines CAMA-1 (Fig. 6B) or BT-20 (Fig. 6C) also re- T cells. When MUC1 epitopes were presented to MA CTL at the sulted in a slow stepwise sustained increase in fluorescence inten- surface of microspheres and in the absence of other signals from sity that began at 50 to 60 s. Stimulation of MA CTL with the additional receptor/ligand interactions, no ZAP-70 phosphoryla- MUC1Ϫ melanoma cell line DM-6 showed no increases in fluo- tion was detected at either 1 min (lane 8)or5min(lane 9). rescence intensity over background levels (Fig. 6D). Immediate lysis of MA CTL following a brief 5-s centrifugation Stimulation of MUC1-specific MA CTL with MUC1 conjugated with MUC1-conjugated microspheres did not reveal ZAP-70 phos- to microspheres showed a large increase in fluorescence intensity phorylation, and no phosphorylation was detected at 30 s either

that began at 10 s of measurement and continued only for 100 s, by guest on September 28, 2021 after which the fluorescence intensity declined to background lev- els (Fig. 7A). This transient response was specific to MUC1-spe- cific MA CTL, in as much as allo-specific CTL did not elicit a

FIGURE 8. ZAP-70 tyrosine phosphorylation in MUC1-specific CTL upon stimulation with MUC1ϩ tumor cells, but not with MUC1ϩ micro- spheres. A, SDS-PAGE of ZAP-70 immunoprecipitates from 2 ϫ 107 MUC1-specific MA CTL equivalents immunoblotted with anti- phosphotyrosine mAb. Unstimulated MA CTL (lane 1); OKT-3 cross- linking at 5 min (lane 2); with MUC1ϩ tumor cells T3M4 at 1 min (lane FIGURE 7. MUC1-specific CTL influx Ca2ϩ following interaction with 3), 5 min (lane 4), 15 min (lane 5), and 30 min (lane 6); with MUC1- 100-mer MUC1 peptide conjugated to PLGA microspheres, but not OVA negative melanoma cells DM-6 at 5 min (lane 7); with MUC1 conjugated conjugated to PLGA microspheres. Response to MUC1-conjugated micro- to microspheres at 1 min (lane 8) and 5 min (lane 9). B, Immunoblot of the spheres by MUC1-specific MA CTL (A) and by allo-specific CTL (B). same gel as in A with anti-ZAP-70 Ab. C, Constitutively phosphorylated Response to OVA conjugated to microspheres by MUC1-specific MA CTL ␨-chains coimmunoprecipitated with ZAP-70 using anti-ZAP-70 Ab, and (C) and by allo-specific CTL (D). Intensity of fluorescence expressed in immunoblotted with anti-phosphotyrosine mAb. Unstimulated MUC1-spe- arbitrary units is shown on the y-axis. Each tick mark represents 2 arbitrary cific MA CTL (lane 1); OKT-3 cross-linking at 5 min (lane 2); with units. The time of measurement in seconds is shown on the x-axis. Time MUC1ϩ tumor cells T3M4 at 1 min (lane 3) and 5 min (lane 4); with t ϭ 0 represents the beginning of measurement after a 5-s centrifugation of MUC1-conjugated microspheres at 1 min (lane 5) and 5 min (lane 6); with CTL with microspheres. Similar results were obtained in another indepen- MUC1-negative melanoma cells DM-6 at 5 min. Data are representative of dent experiment. three independent experiments. The Journal of Immunology 3117

The MHC-unrestricted recognition of MUC1ϩ tumor cells results in NF-AT translocation to the nucleus, while recognition of MUC1ϩ microspheres does not NF-AT nuclear translocation is a consequence of MHC-restricted T cell activation, and has been shown to be necessary for prolif- eration and IL-2 production (29). We examined whether the dif- ferent proliferative responses we observed would correspond to differential translocation of NF-AT to the nucleus. MUC1-specific

and allo-specific CTL were stained with the anti-NF-ATc mAb 7A6. In unstimulated allo-specific and MUC1-specific MHC-de-

pendent MA CTL, NF-ATc staining was predominantly cytoplas- mic, excluded from the blue nuclear staining visualized by the Hoechst dye (Fig. 10, A and B). After stimulation with ionomycin and phorbol dibutyrate as positive controls, NF-AT staining was predominantly nuclear (Fig. 10, C and D). When CTL were incu- bated with tumor cells, the allo-specific HLA-A2-reactive CTL translocated NF-AT in response to the HLA-A2ϩT3M4, but not to Ϫ

the HLA-A2 HPAF (Fig. 10, E and G). MHC-unrestricted Downloaded from MUC-1-specific MA CTL translocated NF-AT to the nucleus in ϩ FIGURE 9. MUC1-specific CTL proliferate in the presence of MUC1ϩ response to both MUC1 T3 M4 and HPAF (Fig. 10, F and H). tumor cell lines, but not MUC1ϩ microspheres. The proliferative response When both CTL lines were tested in response to the HLA- ϩ Ϫ for MUC1-specific MA CTL is shown, and was measured by labeling with A2 MUC1 tumor DM-6, only the allo-specific CTL translocated [3H]thymidine for 18 h at the end of a 3-day culture. A, Background pro- NF-AT to the nucleus (not shown). liferation of MA CTL (T), and proliferation in the presence of immobilized

The MUC1 peptide alone conjugated to microspheres was not http://www.jimmunol.org/ anti-CD3 (T/anti-CD3). B, Proliferation of irradiated DM-6 cells (DM-6), sufficient to cause NF-AT translocation when examined at three CTL in the presence of irradiated DM-6 (T/DM-6), irradiated BT-20 cells different time points, 4 h, 24 h, and 48 h after interaction with the (BT-20), CTL in the presence of irradiated BT-20 (T/BT-20), irradiated CTL (24-h time point shown in Fig. 10I). No nuclear translocation CAMA-1 cells (CAMA-1), and CTL in the presence of irradiated CAMA-1 ϩ of NF-AT was observed even in the layer of MUC1-specific MA (T/CAMA-1). C, Proliferation of CTL in the presence of MUC1 micro- spheres (T/MUC1 beads), and proliferation of CTL in the presence of OVA CTL still in intimate contact with the MUC1-conjugated micro- conjugated to microspheres (T/OVA beads). Data were reproduced in at spheres (4 h, Fig. 10J). As expected, no NF-AT translocation was least three independent experiments. observed in the control allo-specific CTL line in response to either MUC1 or OVA conjugated to microspheres (not shown). by guest on September 28, 2021

(not shown). All lanes contained equal amounts of immunopre- Discussion cipitated ZAP-70 (Fig. 8B). MHC-unrestricted recognition is a direct TCR recognition of an The ␨-chain coimmunoprecipitated with ZAP-70, and it was antigenic epitope that is neither processed by proteosomes nor pre- phosphorylated constitutively in both stimulated and unstimulated sented within the groove of MHC molecules (30). Most of the cells (Fig. 8C). Both and p23 phosphorylated forms of the described MHC-unrestricted ␣␤ T cells have been specific for hap- ␨-chain were detected in these cells, and this was due to the ex- tens, nonpeptide Ags, and carbohydrates. The MHC-unrestricted pansion of MUC1-specific MA CTL on OKT-3. The data further ␣␤ T cell recognition of the tumor-specific Ag MUC1 is unique in suggest that ZAP-70 in these CTL is already recruited to the CD3 that the epitope targeted on this Ag is a peptide sequence (15, 16, complex because of its coimmunoprecipitation with the ␨-chain. 31). Lack of MHC restriction has been described for Ags that have Specific phopshorylation of ZAP-70, however, was extensive distinct properties such as multivalency, high level of expression, enough to be detected only after the CTL were specifically stim- and an ordered conformational structure (8, 10, 12, 32). The ϩ ulated with MUC1 tumor cells or after CD3 cross-linking (Fig. MUC1 peptide epitope possesses all of these properties, in that it 8A, lanes 3 and 2, respectively). is tandemly repeated, it is overexpressed on the surface of tumor cells, and it has a rigid ordered structure (30, 19). The presence of The MHC-unrestricted recognition of MUC1ϩ tumor cells, but ϩ this repeated rigid MUC1 epitope at high density is expected to not MUC1 microspheres, induces proliferation of MUC1- engage multiple specific TCRs without the need for anchoring that specific CTL an MHC molecule provides to a short processed peptide sequence, To examine a downstream event in successful T cell activation, we and with an overall high avidity. chose MUC1-specific CTL proliferation in response to MUC1 Unlike MHC-restricted recognition of peptides, MHC-unre- conjugated to microspheres, or in response to the MUC1ϩ tumor stricted recognition is not well understood. We have taken advan- cell lines (Fig. 9). The positive control was CTL proliferation in tage of the fact that MUC1-specific T cells are MHC unrestricted, response to immobilized anti-CD3 mAb (Fig. 9A). Consistent with and have used them to study the mechanism and parameters in- their cytotoxic function, the MUC1-specific MA CTL proliferated volved in this less common recognition of peptide Ags by T cells. in response to MUC1ϩ tumor cells BT-20 and CAMA-1, but not The experiments that we have described in this work demonstrate in response to the MUC1Ϫ melanoma DM-6 (Fig. 9B). There was for the first time that MHC-unrestricted CTL recognition involves no proliferation in response to the MUC1-conjugated micro- the same activation events that follow conventional MHC-re- spheres, nor in response to the negative control, OVA-conjugated stricted recognition of peptide/MHC complexes. Importantly, we microspheres (Fig. 9C). The CTL did not produce IL-2 or IFN-␥ showed that MUC1-specific MHC-unrestricted CTL can specifi- in response to MUC1 conjugated to microspheres, whereas they cally influx Ca2ϩ in response to the native unprocessed MUC1 did in response to MUC1ϩ tumor cells (not shown). tandemly repeated peptide sequence by itself conjugated to the 3118 MUC-1-SPECIFIC MHC-UNRESTRICTED TCR SIGNALING Downloaded from FIGURE 10. NF-AT nuclear translocation in MUC- 1-specific CTL in response to MUC1 expressed on tu- mor cells but not on microspheres. All stimulations shown were performed for 18 h, and the MUC1-specific CTL used were the clonal MA. Allo-specific CTL (A) and MUC1-specific CTL (B) unstimulated; allo-specific CTL (C) and MUC1-specific CTL (D) after ionomycin/ http://www.jimmunol.org/ phorbol esters; allo-specific CTL (E) and MUC1-spe- cific CTL (F) after T3M4; allo-specific CTL (G) and MUC1-specific CTL (H) after HPAF; MUC1-specific CTL after stimulation with MUC1-conjugated micro- spheres (I); and MUC1-specific CTL in contact with MUC1-conjugated microspheres (J). NF-AT staining is represented by the red Cy-3 label, and nuclear staining is blue with the Hoechst dye. Cells were observed under

oil immersion. Similar results were obtained in another by guest on September 28, 2021 independent experiment.

surface of microspheres. These results provide for the first time reported previously, in which successful NF-AT nuclear translo- direct evidence that MHC-unrestricted CTL can be triggered by a cation was directly shown to be linked to the sustained nature of peptide sequence in its native conformation and without its pre- the Ca2ϩ signal (22). We have demonstrated that MUC1-specific sentation by MHC molecules. CTL do undergo a sustained Ca2ϩ influx in response to MUC1 Upon further characterization of the Ca2ϩ influx in response to expressed at the surface of tumor cells. This prolonged Ca2ϩ mo- the MUC1 tandemly repeated peptide in isolation, we found that it bilization resulted in NF-AT translocation to the nucleus and sub- was transient in nature and it did not result in NF-AT translocation sequent CTL proliferation. Therefore, the MHC-unrestricted rec- to the nucleus or in CTL proliferation. The lack of NF-AT trans- ognition of MUC1 also involves a regulated link between the Ca2ϩ 2ϩ location to the nucleus, after a transient influx of [Ca ]i, has been signal and downstream NF-AT activation. The Journal of Immunology 3119

Our inability to detect ZAP-70 tyrosine phosphorylation despite full response. It should be emphasized that the MHC-unrestricted the presence of a Ca2ϩ influx in response to the MUC1 peptide TCR signaling in this study was examined in MHC-dependent epitope alone at the surface of microspheres is indicative of partial MUC1-specific CTL. signaling. Such partial signaling in T cells has been described by In another model of T cell activation, sustained signaling that several groups (33, 34). In these reports, partial signaling was de- productively results in T cell proliferation has been shown to re- scribed postengagement of the TCR with altered peptide TCR li- quire a prolonged occupancy of the TCR by its peptide/MHC li- gands presented at the surface of APCs that provide the full range gand through the formation of stable T cell/APC conjugates (39– of costimulatory and accessory molecules. Partial signaling was 41). In these T cell/APC conjugates, continuous signaling through manifested in the lack of detectable ZAP-70 phosphorylation, de- the TCR by serial engagements of up to 200 TCRs is maintained spite the presence of a transient Ca2ϩ influx (35). In the case of by the formation of a large changing area of membrane contact MHC-unrestricted recognition of MUC1, several possibilities may between the T cell and the APC. A number of adhesion molecules explain our inability to detect ZAP-70 phosphorylation in response play a crucial role in maintaining the area of intimate contact be- to MUC1-conjugated microspheres. Because the Ca2ϩ influx in tween the two opposing membranes (42). Molecules such as CD8 response to these microspheres was transient, it may be possible and CD2 on T cells have been shown to have signaling capabilities that the tyrosine phosphorylation of ZAP-70 was also of short on their own (43–45). A synthetic surface such as PLGA beads is duration. It is also possible that ZAP-70 does undergo tyrosine devoid of these molecules, which may deprive CTL of prolonged phosphorylation, but at levels below the sensitivity of immuno- conjugate formation and subsequent generation of costimulatory blotting. This is very likely, since when compared with the levels signals that allow a complete T cell response. Furthermore, the Downloaded from of ZAP-70 phosphorylation upon CD3 cross-linking, phosphory- surface of PLGA beads is different in nature than the fluid bilipid lation of ZAP-70 after stimulation with MUC1ϩ tumor cells ap- membranes of cells. This may preclude rolling of CTL along the peared to be at a much lower level. Yet another, albeit less likely surface and achieving the minimal threshold level of TCR occu- possibility is that when presented to CTL in isolation, MUC1 tan- pancy necessary for the end result of proliferation (46). dem repeat peptides may induce a Ca2ϩ influx through a pathway At the extracellular level of MHC-unrestricted recognition, we other than that initiated by ZAP-70 tyrosine phosphorylation. An found that adhesion molecules such as LFA-1 and CD2 on the important difference between our studies and studies with altered CTL are indeed important for proper effector function. We ob- http://www.jimmunol.org/ peptide ligands is that we compared the signaling elicited by the served two patterns of dependence on these adhesion molecules same MUC1 epitope recognized in different contexts. Our results correlating with the CTL dependence on MHC, and that may re- suggest that the same epitope can cause either a partial or complete flect the relative affinity of their TCRs. We had reported previously signal through the TCR, depending on the participation or lack of that human MUC1-specific CTL can kill either porcine or human participation of other accessory molecules. On the other hand, the muc-1-transfected target cells, depending on the density of MUC1 same MUC1 epitope on tumor cells is surrounded by severely epitope expression and the simultaneous presence of MHC class I truncated saccharide chains (36), and such carbohydrates are ab- molecules (24). This dependence of MHC-unrestricted CTL on

sent on the synthetic 100-mer MUC1 peptide. We have in fact adhesion molecules was at first unexpected. However, as we show by guest on September 28, 2021 shown that the complexity of the saccharide chain surrounding the in this study, full CTL activation necessitates the presence of these CTL epitope dramatically affects CTL effector function.5 It is thus molecules. Studies supporting our reported dependency of MUC1 possible that the presence of adjacent carbohydrate residues may recognition on adhesion molecules show that MUC1 is extensively contribute to either proper conformation of the MUC1 CTL sialylated, imparting negative charges on tumor cells (47). This epitope or the affinity of the TCR for the epitope. hinders cell-cell interactions and CTL cytotoxicity (48, 49). Ad- Studies with altered peptide ligands have also reported a distinct ditional interactions of adhesion molecules at the cell surface are pattern of ␨-chain phosphorylation (33, 34). ␨-Chain was consti- thus important to decrease repulsion between CTL and MUC1ϩ tutively phosphorylated in our CTL cultures because of their ex- tumor cells, and to result in effective signaling through the TCR. pansion on low levels of OKT-3 mAb. We consistently observed Such interactions for overcoming cell surface net negative charges constitutive phosphorylation of the ␨-chain by the appearance of are just as important during MHC-restricted recognition of both p21 and p23 forms, and thus could not address the effect that peptides (50). MUC1 recognition in isolation has on ␨-chain phosphorylation. In conclusion, the MHC-unrestricted recognition of the native Furthermore, ZAP-70 and ␨-chain have consistently coimmuno- MUC1 peptide involves the same events of T cell activation as the precipitated even in unstimulated cells, indicating that in these MHC-restricted recognition of a nominal peptide Ag. Only the CTL cultures ZAP-70 is already associated with the ␨-chain. initial recognition step differs in the MHC-unrestricted recogni- Therefore, a signal that phosphorylates ZAP-70 in association with tion, in which the MUC1 epitope directly engages the TCR rather the ␨-chain is missing or weak in the MHC-unrestricted recogni- than being presented as a processed peptide bound to MHC. tion of MUC1 epitope in isolation, but is present and strong when this epitope is recognized on the surface of tumor cells. We can Acknowledgments speculate that the best candidate for this signal is the src protein We thank Dr. Helena Chang and Dr. Ming Liu from Brown University tyrosine kinase lck, which associates with the cytoplasmic tail of (Providence, RI) for providing us with a constant supply of lymph node CD8 molecules (37) and phosphorylates ZAP-70 (28). These data cells from patients with breast cancer. emphasize that even though the recognition of MUC1 is MHC unrestricted, this recognition becomes more efficient in the pres- References ence of CD8 molecules (MHC dependency), as well as other ac- 1. Germain, R. N. 1994. MHC-dependent antigen processing and peptide presenta- cessory molecules. These molecules may augment the MHC-un- tion: providing ligands for T lymphocyte activation. Cell 76:287. restricted signal through the TCR (38), allowing it to proceed to a 2. Janeway, C. A., Jr., and K. Bottomly. 1994. Signals and signs for lymphocyte responses. Cell 76:275. 3. Qian, D., and A. Weiss. 1997. T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 9:205. 5 J. Magarian-Blander, J. Schmilau, S. Itzkowitz, and O. J. Finn. Cytotoxic T cells 4. Holoshitz, J., F. Koning, J. E. Coligan, J. De Bruyn, and S. Strober. 1989. Iso- recognize MUC1 molecules expressed exclusively on epithelial tumors and not on lation of CD4ϪCD8Ϫ mycobacteria-reactive T lymphocyte clones from rheuma- normal cells transfected with MUC1 cDNA. Submitted for publication. toid arthritis synovial fluid. Nature 339:226. 3120 MUC-1-SPECIFIC MHC-UNRESTRICTED TCR SIGNALING

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