Inhibition of T Cell Activation by an Autoantibody Induced by Murine Retrovirus Infection

Inhibition of T Cell Activation by an Autoantibody Induced by Murine Retrovirus Infection

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Vol. 82, No. 3, March, pp. 263–273, 1997 Article No. II964316 Inhibition of T Cell Activation by an Autoanti...

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CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 82, No. 3, March, pp. 263–273, 1997 Article No. II964316

Inhibition of T Cell Activation by an Autoantibody Induced by Murine Retrovirus Infection ROBERT M. TOWNSEND,* JOHN L. DZURIS,* IMRAN MIRZA,† THOMAS SIECK,‡ FREDERICK COFFMAN,§ AND KENNETH J. BLANK‡ *Graduate Program in Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140; †Department of Pathology and Laboratory Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102; ‡Department of Microbiology and Immunology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102; §Department of Pathology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07101

This report describes a murine monoclonal IgM antibody, 6E3.C4, induced by retrovirus infection of BALB/ c-H-2b mice which inhibits mitogen stimulation of both mouse and human lymphocytes in vitro. The molecule bound by this antibody appears to be an activation antigen since binding is upregulated by mitogen stimulation. Analysis of the epitope bound by mAb 6E3.C4 revealed that it is associated with a 52-kDa protein with a pI of approximately 5.7 as determined by Western blot analysis. A protein expressing this or a crossreactive epitope was isolated and determined to be atubulin by amino acid sequencing. Reactivity with purified a-tubulin confirms this identification. These findings suggest a potential role for a cell surface molecule that is either a-tubulin or a cross-reactive molecule in the activation processes of T cells. q 1997 Academic Press

INTRODUCTION

Lymphocyte activation antigens are molecules usually absent or weakly expressed in resting lymphocytes but up-regulated upon lymphocyte activation. These molecules are involved in processes associated with the execution of immunological functions of activated cells, such as proliferation, differentiation, adhesion, and trafficking (1–3). Numerous lymphocyte activation antigens have been identified (4–10), including receptors for IL-2 and TNF-a, MHC Class II molecules, and several classes of adhesion molecules, expressed on multiple lineages of lymphocytes and nonlymphoid cells (11, 12). Other cell surface molecules, e.g., receptors for transferrin and insulin, are expressed on a wide variety of cell lines and are involved in processes that regulate cell proliferation (6, 9, 13). Certain activation antigens, e.g., the p55 chain of the IL-2 receptor (IL-2-R), appear on the cell surface within minutes or hours after lymphocyte stimulation, whereas other molecules, e.g., the VLA antigens, may take days to be detected (1, 14). In recent studies, we have examined one of several

autoreactive antibodies generated following retrovirus infection. It has been demonstrated in recent years that infection by retroviruses can lead to the production of autoantibodies both in humans (HIV) (15, 16) and mice (Rausher) (17). Some of the molecules recognized by these autoantibodies are involved in the generation of immune responses. For example, antigens found to interact with these autoantibodies include anti-idiotypic antibodies as well as cell surface molecules such as CD4 and activation antigens such as the IL-2-R (17). It appears possible that these types of autoantibodies may affect the generation of anti-viral immune responses perhaps resulting in the failure to limit virus infection. The antibody generated in our mice (mAb 6E3.C4) reacts with a 52-kDa molecule expressed on the cell surface of activated and/or proliferating cells including T lymphocytes. Incubation of T-lymphocytes with mAb 6E3.C4 inhibits the proliferation of these cells in response to mitogen and antigenic stimulus, indicating that the cell surface expression of this epitope is associated with a function required for cell activation. Binding of mAb 6E3.C4 to the cell surface also results in a rapid increase in intracellular calcium. Further studies have shown that the epitope bound by mAb 6E3.C4 is expressed by a-tubulin. Thus, cell surface expression of a-tubulin or a molecule expressing a cross-reactive epitope may regulate cellular functions by some mechanism involving regulation of intracellular calcium flux. MATERIALS AND METHODS

Mice BALB/c-H-2b (BALB.B) mice were bred in our animal facility from breeding pairs originally obtained from Dr. Frank Lilly (Albert Einstein School of Medicine, Bronx, NY). BALB/c mice were purchased from the National Cancer Institute. Cells The various tumor cell lines used in this study were previously described (18–20). These tumors were de-

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0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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rived from mouse retrovirus-induced lymphomas from BALB/c (CGV), BALB/c-H-2k (KgV, KKA, KKB, KKC), BALB/c-H-2b (BGV, Clone L), and C57/BL6 (RBL5). The lymphomas were induced by inoculating mice with various murine leukemia viruses (MuLVs). Other cell lines used for these studies included the murine T cell clone D10.G4.1 (ATCC TIB224), the murine B cell lymphoma A20 (ATCC TIB208), the feline glial tumor line PG4 (a subline of G355-5; ATCC CRL2033), the monkey kidney cell line Cos-7 (ATCC CRL1651), the hamster ovary cell line CHO (ATCC CRL9618), the human T cell line Jurkat (ATCC CRL8163), the human T cell line Molt-4 (ATCC CRL1582), the murine mastocytoma line P815 (ATCC TIB64), and the murine monocyte–macrophage cell lines P388 D1 (ATCC TIB63) and RAW-264 (ATCC TIB71). Mouse embryo fibroblasts (MEF) were produced by preparing a single cell suspension of BALB.B embryos and growing adherent cells in minimal essential medium supplemented with 10% fetal bovine serum, glutamine, and 50 U/ml penicillin and 50 mg/ml streptomycin. The other fibroblast cell lines including SC-1(ATCC CRL 1404), NIH3T3 (ATCC CRL 1658), and BALB3T3 (ATCC CRL 6587) were maintained in minimal essential medium supplemented with 10% fetal bovine serum, glutamine, and penicillin/streptomycin. The fibroblast lines and MEF were harvested for assay by incubating cells for 10 min at 377C with trypsin–EDTA and washing with phosphate-buffered saline (PBS) three times. Cell Fusion and Production of Monoclonal Antibody 6E3.C4 BALB.B mice were immunized ip with 1 1 106 SC-1 cells that were infected with MuLV. They were boosted ip 2 weeks later with the same inoculum and again 4 days before sacrificing. Fusion of 7 1 107 immune spleen cells and 3.5 1 107 SP2/0 mouse myeloma cells was carried out by first adding the cells to a Con Acoated 60-mm petri dish and incubated for 1 hr at 377C. The petri dish had been coated with 1 ml each of Con A (15 mg/ml Con A in 0.1 M sodium acetate, pH 4.8) and carbodiimide (50 mg/ml in 0.1 M sodium acetate, pH 4.8) and incubated at room temperature for 1 hr with constant rocking followed by three washes with PBS and storage at 0207C until used. Cells were fused by the addition of PEG (Boehringer Mannheim) for no more than 60 sec. Five milliliters of HY medium was slowly added to the plate and then gently aspirated. Cells were washed once more and then incubated overnight at 377C in Kennet’s HY medium supplemented with 20% fetal bovine serum, glutamine, penicillin/ streptomycin, oxaloacetic acid, pyruvate, and insulin. The next day, cells were scraped from the petri dish and plated in 96-well plates prior to addition of HAT. Culture supernatants of hybrid cells were tested by immunofluorescence assay as described below. Positive hybridomas were cloned by limiting dilution.

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Antibodies Hybridoma cell lines HB35, HB36, and HB77, which produce anti-mouse H-2 I-Ab,d, H-2Db, and H-2Dd monoclonal antibodies, respectively, were obtained from ATCC. These monoclonal antibodies were used as controls in immunofluorescence assays. M307, an IgM antibody against murine retrovirus coat protein gp70, was obtained from Dr. Bruce Chesebro (Rocky Mountain Laboratories, Hamilton, MT). Goat anti-mouse Ig peroxidase conjugate was obtained from Sigma (St. Louis, MO). FITC-conjugated goat anti-mouse Ig was obtained from Southern Biotechnology Associates Inc. (Birmingham, AL). Antibody was purified from ascites using E-Z-SEP antibody purification system (Middlesex Sciences, Foxborough MA) according to the manufacturer’s instructions. Cell Activation Single cell suspensions were prepared from spleen and lymph nodes of BALB/c mice. Human PBMC were obtained by Ficoll separation of peripheral blood (21) obtained by venipuncture. Mitogen stimulation of cells was performed by incubating 1 1 106 cells in 24-well tissue culture plates in RPMI 1640 medium supplemented with 10% fetal bovine serum, glutamine, penicillin/streptomycin, 2-mercaptoethanol, and 1 mg/ml concanavalin A (Sigma) for 72 hr. All studies with human cells were performed in AIM V serum-free medium (Gibco, Grand Island, NY). For studies involving the effect of mAb 6E3.C4 on lymphocyte proliferation, mAb’s 6E3.C4 and HB36 were titered into the wells of a 96-well plate containing 4 1 105 BALB/c splenocytes or human PBMC and generally (unless otherwise indicated) 1.0 mg/ml concanavalin A (Sigma), 2 1 105 irradiated C57BL/6 splenocytes, 0.01 mg/ml anti-CD3 antibody (Boehringer Mannheim, 145-2C11), or 40 mg/ml lipopolysaccaride (Sigma). Unless otherwise stated, mAb 6E3.C4 or control antibody was titered in the wells and the cultures were incubated at 377C for 72 hr. After 48 hr, wells were pulsed with 1 mCi [3H]thymidine for the final 24 hr of culture and cells were harvested onto glass fiber filters. The extent of [3H]thymidine incorporation was determined using a liquid scintillation counter and is proportional to cell proliferation. Immunofluorescent Flow Cytometry Cells (2 1 105) were incubated in 100 ml mAb supernatant containing 0.05% NaN3 or PBS containing 1% fetal bovine serum and 0.05% NaN3 for 30 min at 47C. Cells were washed three times with PBS and were suspended in FITC-conjugated goat antibody to mouse Ig diluted 1:30 in PBS. Cells were incubated for 30 min at 47C then washed three times in PBS and fixed overnight at 47C in PBS containing 1% paraformaldehyde.

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Cells were then washed three times in PBS and resuspended in PBS for analysis. Mean fluorescence intensity (MFI) and percent of positive staining cells were determined on FACScan analyzer (Beckton-Dickinson). MFI was calculated on a logarithmic scale based on the comparison with an irrelevant isotype matched mAb used as a negative control. Analysis of Ab-Reactive Molecules Cells were lysed with 1% dodecyl-b-maltoside in 50 mM Tris–Cl with 5 mM EDTA at pH 7.0 and protein concentration was determined with the BCA protein assay reagent (Pierce, Rockford, IL). Lysates (25 mg protein per lane) were electrophoresed on 12% SDS– PAGE using the discontinuous buffer system described by Laemmli (22). Transfer of proteins to Immobilon paper (Millipore, Bedford, MA) was performed overnight at 47C at 30 V in Tris–glycine–methanol buffer. Blots were then blocked with PBS–0.5% Tween and 5% nonfat dry milk. Immunostaining was performed by incubating the blot with primary antibody for 1 hr at room temperature followed by three washes with PBS–Tween. Blots were then incubated with appropriate peroxidase-conjugated secondary antibody for at least 1 hr, washed three times, and developed with 4chloro-1-naphthol. Two-dimensional gel electrophoresis was performed using the Millipore (Bedford, MA) 2D gel system as previously described (23). Briefly, 25 mg of Molt-4 cell lysate was dissolved in 2D sample buffer containing 0.06% SDS, 40 mM DTT, 5.6 mM Tris–HCL, 4.4 mM Tris base, 2.2 mM KCl, 1.2 mM KH2PO4 , and 108 mM NaCl and run in the first-dimension isoelectric focusing tube gel at 2000 V for 17 hr. Gels were removed from tubes, overlaid onto 15% polyacrylamide vertical slab gels, and run at 500 V for 16 hr to separate proteins by size. The proteins from the 2D gel were transferred to Immobilon and developed with monoclonal antibody as described for the Western blot method. Two-dimensional standards (Bio-Rad, Melville, NY) were run in a parallel gel and silver stained to determine approximate pI. Attempts to analyze the presence of molecules on the cell surface that might express a cross reactive epitope with a-tubulin by immunoprecipitation were unsuccessful, although control experiments using other nonIgM antibodies were successful. The failure of IgM antibodies to immunoprecipitate molecules is not unusual. Enzymes To assess the extent of glycosylation, 10 mg of denatured KgV tumor cell lysates was deglycosylated with combinations of 2.0 mU neuraminidase from Vibrio cholerae, 0.4 units recombinant N-glycosidase F, and 2.5 mU O-glycosidase (all purchased from BoehringerMannheim, Indianapolis, IN) at pH 7.2 in 100 ml of 20

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mM sodium phosphate buffer for 20 hr. After incubation, proteins were precipitated with 1 ml ice-cold ethanol at 0207C overnight and resuspended in SDS– PAGE sample buffer. Western blotting was performed as described above to detect changes in banding pattern detected with mAb 6E3.C4 and polyclonal anti-gp70. Digestion of cell extract with Glu-C was performed as previously described by others (24). Briefly, 250 mg of KgV cell extract was boiled for 3 min in the presence of 1% SDS. Sample was diluted 10-fold with phosphatebuffered saline, pH 7.4. One hundred microliters of sample was incubated at 377C with 5 mg/ml enzyme for 4 hr. Western blot analysis was performed as described above to determine the extent of protein digestion. Calcium Flux Cells were labeled with 2.5 mM Fura-2 for 20 min and washed two times with PBS. Cells were resuspended in 3 ml of PBS containing calcium and 5 mM EGTA and incubated at 377C while stirring in the fluorometer. Reagents were added as indicated and changes in cytoplasmic calcium levels were determined using a SPEX fluorolog spectrometer. Membrane Isolation The human T cell line Molt4 was grown in RPMI containing 5% FCS at 377C and 5% CO2 from which membranes were prepared as previously described (25). Approximately 1010 cells were collected and washed two times with PBS by centrifugation at 47C. Cells were resuspended in buffer containing 10 mM Tris, pH 7.6, 1 mM PMSF, 10 mg/ml trypsin inhibitor, 5 mM EDTA at 5 1 108 cells/ml. Cells were disrupted at 47C using a Dounce homogenizer, and nuclei were removed by centrifugation at 500g for 5 min at 47C. The membrane suspension was ultracentrifuged in a Beckman Ti-70.1 rotor at 125,000g, and the membrane pellet was dissolved in 1% dodecyl-b-maltoside, 50 mM Tris, 5 mM EDTA, 200 mM NaCl, pH 7.0. Ion-Exchange FPLC Molt4 membrane extract was subjected to ion-exchange separation using a Pharmacia FPLC as a first step in the isolation of the protein. Extract, 40 mg/run, was loaded onto an HR10/10 MonoQ column (Pharmacia) in 5 ml FPLC buffer (50 mM Tris, 200 mM NaCl, 5 mM EDTA, pH 7.0, 0.05% Tween, 1 mM PMSF, and 10 mg/ml trypsin inhibitor). The column was washed with 15 ml of buffer A to remove unbound proteins and a linear salt gradient from 200 to 700 mM NaCl over 35 ml followed by a 15 ml wash at 1 M NaCl was run to elute bound proteins. Fractions (2 ml) were collected and analyzed for total protein content and immunoreactivity by Western blot as described above. Immunoreactive fractions were collected for further analysis.

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Isolation of Protein by SDS–PAGE Size Exclusion Proteins isolated from the MonoQ column were subjected to separation by size using preparative SDS– PAGE and electroblotting to PVDF membranes. Briefly, 5 mg of protein was loaded per 12% polyacrylamide gel and electrophoresed and electroblotted as described above. Electroblotted PVDF membranes were stained with 0.5% Ponceau S in 1% acetic acid for 5 min. Blots were destained in distilled water for 2 min, and the band of interest was excised. The excised band was placed in microcentrifuge tube, washed with distilled water three to five times. Elution buffer containing 50 mM Tris pH 7.0, 2% SDS, 1% Triton-X was added at room temperature and centrifuged for 10 min. The supernatant containing eluted protein was collected and the elution was repeated if necessary. Internal Sequence Analysis The internal protein sequencing was performed by the Wistar Protein Microsequencing Facility, Philadelphia, to obtain an internal amino acid sequence from the isolated protein. Samples were prepared by SDS– PAGE and electroblotting as described above with the following exceptions. Samples were solublized in buffer containing 0.5 M sucrose, 15% SDS, 312.5 mM Tris, 10 mM EDTA, pH 6.9, and heated for 10 min at 377C. The completely cast gel was allowed to stand for 48 hr at room temperature prior to use. Electrophoresis was carried out with the addition of 0.1 mM thioglycolate in the upper chamber, and the gels were blotted to BioRad’s TransBlot PVDF membranes. Blots were washed three times with distilled water and stained with amido black in 10% acetic acid for 1 min and destained with 5% acetic acid for 1 min, followed by excessive washing with distilled water. RESULTS

Generation of mAb 6E3.C4 and Tissue Distribution The monoclonal antibody 6E3.C4 was initially identified during screening of hybridomas for antibodies which recognize murine retrovirus-induced cell surface proteins. Flow cytometric analysis indicated that mAb 6E3.C4 bound to freshly harvested (unfixed) cells from a MuLV-transformed T-lymphocyte line, KgV, as well as an uninfected fibroblast cell line, SC-1 (Fig. 1). This result suggested that mAb 6E3.C4 was not specific for MuLV-encoded molecules, but reacted with a self molecule. Cells recognized by mAb 6E3.C4 include mitogen-activated lymph node and splenic lymphocytes as well as mouse cell lines representing the T cell, B cell, and the monocyte – macrophage cell lineages (Tables 1 and 2). The finding that freshly isolated spleen cells did not demonstrate surface binding of mAb 6E3.C4 whereas mito-

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gen-activated spleen cells did bind this antibody suggested that the cell surface molecule was differentially expressed on the surface of cells during distinct stages of activation. To examine this possibility, immunofluorescence analysis was performed on freshly isolated mouse and human lymphoid cells that were cultured with or without mitogen (Table 1). The binding of mAb 6E3.C4 was low (MFI Å 1.4) on freshly isolated murine splenocytes and remained low upon culture without mitogen stimulation (MFI Å 1.2). Splenocytes cultured with 1 mg/ml Con A demonstrated a more than fourfold increase in expression of cell surface a-tubulin by Day 3. Murine lymph node cells demonstrated a similar increase in the binding of mAb 6E3.C4 upon mitogen stimulation. In studies using human cells, freshly isolated peripheral blood lymphocytes (PBL) bind little mAb 6E3.C4 (MFI Å 2.0); however, stimulation with Con A in vitro upregulates the binding of mAb 6E3.C4 nearly fourfold in these cells as it did with murine splenocytes and lymph node cells. Thus, mAb 6E3.C4 binding appeared to be related to the activation state of the cells examined. The molecule recognized by mAb 6E3.C4 is also highly expressed on the cellular membranes of neuronal tissue and a wide variety of actively proliferating cells but only weakly expressed by nondividing cells. Inhibition of T Cell Activation The increased binding of mAb 6E3.C4 on the surface of stimulated murine lymphocytes and murine T cell lines relative to unstimulated cells suggested that the antigen bound might play a role in the activation process of these cells (Table 1). To determine if mAb 6E3.C4 binding to the cell surface affected T cell activation, mAb 6E3.C4 was added to cultures of BALB/c splenocytes stimulated with Con A, anti-CD3 mAb, or allogeneic C57BL/6 splenocytes. Control mAb HB36 (anti-H2-Db,d) was added to separate wells as an isotype matched control antibody. As observed in Fig. 2a, addition of 5 and 10 mg/well of mAb 6E3.C4 markedly inhibited the proliferative response of the lymphocytes to Con A (30 and 65%), anti-CD3 (60 and 70%), and alloantigen (90 and 100%) compared to the control antibody (HB36). However, mAb 6E3.C4 had little inhibitory effect on the lymphocyte response to LPS (data not shown). These studies demonstrated that mAb 6E3.C4 had a specific activity against T cell proliferative responses but not B cell proliferative responses. In a similar manner, treatment of human cells with mAb 6E3.C4 also affected human T cell activation. Addition of mAb 6E3.C4 to cultures of human PBMC stimulated with Con A demonstrated that mAb 6E3.C4 markedly inhibited the mitogenic response of human lymphocytes to Con A compared to the isotype

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FIG. 1. Cell surface binding of mAb 6E3.C4 as demonstrated by flow cytometry. (A) SC-1 cells and (B) KgV cells were incubated with culture supernatants from either mAb 6E3.C4 hybridoma (solid line) or mAb HB36 hybridoma (dotted line) as an isotype-matched negative control as described under Materials and Methods. Antibody binding (i.e., mean fluorescence intensity and percentage of positive cells) were determined with a FACScan analyzer (Becton-Dickenson).

matched control antibody (HB36) (Fig. 2b). The inhibitory activity of this antibody on these mitogeninduced responses demonstrate a potential role for this cell surface antigen in the activation of T cells. In the studies described above, addition of mAb 6E3.C4 was found to be inhibitory when antibody was present only at the onset of stimulation. To determine if inhibition was only induced early in the activation process ascites (1%) was added to the cultures of BALB/ c splenocytes at varying time points after the initiation of the Con A response. A high level of inhibition (80%) of the proliferative response was observed when mAb 6E3.C4 ascites was added at the onset of culture. The addition of mAb 6E3.C4 at 1, 2, and 4 hr after stimula-

TABLE 1 Reactivity of mAb 6E3.C4 with Mouse and Human Lymphoid Cells Stimulated in Culture as Detected by Flow Cytometric Analysisa Unstimulatedb (MFI)

Cells BALB/c Day 0 Day 2 Human Day 0 Day 3

SPLN (N Å 3) LN (N Å 3) SPLN (N Å 4) LN (N Å 3)

1.4 2.8 1.2 1.2

PBL (N Å 4) PBL (N Å 4)

2.0 { 0.1 3.2 { 0.7

{ { { {

0.1 1.3 0.1 0.1

Con Ac (MFI)

NAd NA 6.2 { 4.7 5.7 { 2.5 NA 7.5 { 1.7

a

Expression of 6E3-p52 on cells and cell lines was studied by immunofluorescence staining as described in the legend to Fig. 1. Mean fluorescence intensity (MFI) was analyzed on a FACScan analyzer (Beckton-Dickenson). b MFI, mean fluorescence mAb 6E3.C4/mean fluoresence negative control. c Isolated lymphocytes were cultured with 1 mg/ml Con A in RPMI with 10% FCS at 377C. d NA, not applicable.

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tion was progressively less inhibitory: 61, 53, and 47% inhibition, respectively (Fig. 2c). By 24 hr after stimulation, the addition of antibody had no effect on the Con A response of the T cells. Control antibody, HB36, had

TABLE 2 Flow Cytometric Analysis of Selected Cell Lines with mAb 6E3.C4a Tissue tested

% Positiveb

MFIc

Rat embryo caudate nucleus Rat embryo substantia nigra Raw264 Jurkat PC12 D10 A20 RBL5 BGV KgV Molt4 S117 KKB Cos-7 CHO P388 SC-1 Clone L NIH3T3 CGV

81 90 100 76 75 96 99 94 94 82 97 61 77 38 59 50 58 38 47 44

110.6 94.8 41.6 30.3 30.0 21.0 19.3 15.3 15.0 12.0 11.0 10.6 8.4 8.3 5.5 4.3 3.9 3.6 3.5 2.8

Note. Values are representative data from two or three determinations. a Expression of 6E3-p52 on cells and cell lines was studied by immunofluoresence staining as described in the legend to Fig. 1. Mean fluorescence intensity (MFI) and percentage of positive staining cells were analyzed on FACScan analyzer (Beckton-Dickenson). b All cell types tested exhibited a unimodal distribution of reactivity with mAb 6E3.C4. c MFI, mean fluoresence mAb 6E3.C4/mean fluoresence negative control.

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FIG. 2. Inhibition of T cell stimulation by anti-a-tubulin monoclonal antibody (6E3.C4). The ability of mAb 6E3.C4 to inhibit the response of BALB/c splenocytes to various mitogenic stimuli was tested as described under Materials and Methods. (a) Con A (0.2 mg/ml), YCD3 culture supernatants (1:500), and irradiated (2000 cGy) C57BL/6 splenocytes (4 1 105/well) were used as stimulants. As indicated by the figure, purified mAb 6E3.C4 or mAb HB36 was added to appropriate wells at 10 or 5 mg/well. Data are presented as percentages of the untreated mitogen response. The ability of mAb 6E3.C4 to inhibit a human mitogen response to Con A was also tested (b). Human PBMC were cultured with Con A and antibodies were added to the appropriate wells as indicated in the figure. The data are presented as cpm { SEM. MAb 6E3.C4 appears to inhibit an early step in the activation pathway (c). BALB/c splenocytes were stimulated with Con A and cultured as described under Materials and Methods. Antibody (1% ascites) was added to appropriate wells at various time points after stimulation as indicated and the data is presented as cpm.

little effect on the Con A response, regardless of time of addition. These data suggest that inhibition is exerted on a critical early event(s) in the T cell activation pathway induced by mitogen. Calcium Flux Measurements To assess changes in intracellular calcium levels, KgV cells were labeled with Fura-2, and calcium levels

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were measured on a time-resolved fluorometer. Addition of mAb 6E3.C4 to KgV cells induced a dramatic increase in the intracellular calcium concentration ([Ca2/]i) in a dose-dependent manner (Fig. 3a). Addition of HB36 showed little or no effect on the [Ca2/]i level (Fig. 3a). The increase in the [Ca2/]i induced by mAb 6E3.C4 occurred in the presence of 5 mM EGTA, suggesting that calcium was being released from the

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intracellular compartments of the T cell. In similar assays, c709, a murine T cell leukemia which demonstrates no detectable reactivity with mAb 6E3.C4, no changes in [Ca2/]i were observed upon incubation with mAb 6E3.C4 (data not shown). Preincubation of the KgV cells with the phospholipase inhibitor manoalide (10 mM) partially inhibited the release of calcium into the cytoplasm of the cells (Fig. 3b). Preincubation of KgV cells with 0.1 mM staurosporine, a potent inhibitor of PKA and PKC, or 1.0 mM okadaic acid, a potent inhibitor of protein phosphatases, had no effect on the increase in [Ca2/]i induced by mAb 6E3.C4 (data not shown). These results suggest that the release of Ca2/ is in part mediated by IP3 as a result of phosphatidyl inositol metabolism by phospholipase activity and is not linked to protein kinase or phosphatase activity. This release of calcium from intracellular stores into the cytoplasm of the cell may be a factor in mediating the observed inhibition of T cell activation. Physical Description of Antigen Bound by mAb 6E3.C4 The mAb 6E3.C4 bound to a 52-kDa molecule from cell lines from all mammalian species tested, including human, simian, rat, and hamster as determined by Western blot analysis both under reducing and nonreducing conditions (Fig. 4a). This molecule had a pI of 5.6–5.8 as demonstrated by 2D Western blot analysis (Fig. 4b). Enzymatic digestion of cell lysates indicated that the antigen bound by mAb 6E3.C4 consisted of protein (Fig. 4c) and little or no carbohydrate (Fig. 4d). Examination of freshly isolated mouse tissues showed reactivity of this antibody by Western blot analysis

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with brain but not liver, kidney, muscle, or heart, demonstrating a restricted expression pattern for this molecule. Freshly isolated thymus, spleen, and lymph node cells were weakly positive for mAb 6E3.C4 reactivity by Western blot analysis (data not shown). Protein Isolation and Amino Acid Sequence Analysis To obtain positive identification of the antigen bound by mAb 6E3.C4, purification and sequencing of the antigen was undertaken. Molt-4 cellular membranes were isolated and proteins were separated by ion-exchange chromatography (Fig. 5A) and size exclusion gel electrophoresis. The separated protein was subjected to a final round of SDS–PAGE and the excised mAb 6E3.C4-reactive band was submitted for internal sequence analysis. Peptide fragments were generated from the protein in the excised band and two internal fragments were chosen for analysis. These two peptide fragments were found to be identical to sequences of a-tubulin (Fig. 5B). a-Tubulin also corresponds to the molecule recognized by mAb 6E3.C4 by virtue of its molecular weight (52 kDa) and charge (pI 5.6–5.8) (Figs. 4a and 4b). Furthermore, mAb 6E3.C4 was found by Western blot analysis to react with purified a-tubulin (Sigma Chemical Co.) as indicated by the arrow in Fig. 5c. These analyses definitively demonstrate that mAb 6E3.C4 binds an epitope associated with a-tubulin or a molecule expressing a cross-reactive epitope. DISCUSSION

In our laboratory, we have generated several murine mAb’s that appear to react with self antigens following

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FIG. 3. Induction of calcium release from intracellular stores of KgV cells by monoclonal antibody (6E3.C4). KgV cells were labeled with the calcium-sensitive dye Fura-2 as described under Materials and Methods. Fluorescence intensity is displayed as a ratio of the fluorescence intensity detected at 510 nm for excitation wavelengths of 340 and 380 nM. (a) KgV cells were incubated at 377C and ascites were added every 300 sec at the indicated final concentrations. As a control for the effect of ascites, mAb HB36 was added in a similar manner. (b) To assess the effect of a phospholipase inhibitor on the calcium flux induced by anti-a-tubulin monoclonal antibody (6E3.C4), KgV cells were preincubated with 10 mM manoalide at 377C for 15 min prior to the addition of 0.5% ascites of mAb 6E3.C4. Changes in the cytoplasmic free calcium were determined as described above.

infection with the retrovirus E-55/ MuLV. In this study, we have presented evidence that one of these antibodies, mAb 6E3.C4, binds a molecule on the surface of activated lymphocytes as well as other dividing cells that, therefore, may be classified as an activation antigen. Furthermore, binding of this cell surface molecule by mAb 6E3.C4 early in the activation process

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leads to an inhibition of T cell proliferation associated with the release of intracellular calcium stores. Subsequent studies determined that mAb 6E3.C4 bound atubulin. Thus, the inhibitory effect of this antibody on cell proliferation appears to be the result of its binding to a cell surface form of a-tubulin or an unidentified cross-reactive molecule.

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FIG. 4. Physical characterization of the molecule bound by mAb 6E3.C4. (a) Western blot analysis of 25 mg of protein from cell lysates of various mammalian species under reducing (lanes 1–6) and nonreducing (lanes 7–12) conditions is shown. Lanes 1 and 7 are molecular weight standards; lanes 2 and 8, 3 and 9, 4 and 10, 5 and 11, and 6 and 12 are cell lysates from mouse (KgV), human (Molt-4), cat (PG4), hamster (CHO), and monkey (Cos-7) cell lines, respectively. (b) Western blot analysis of two-dimensional gels of 25 mg of Molt-4 cell lysates shows this molecule has a pI of 5.6–5.8 (as indicated by the arrowhead). (c) Protease sensitivity of epitope recognized by mAb 6E3.C4 on 25 mg of KgV cell lysate. Western blot analysis demonstrates in lane 2 the untreated 52-kDa band recognized by the antibody, while lane 3 shows the effect of 4 hr of incubation with Glu-C endoprotease as described under Materials and Methods (lane 1 is the molecular weight standards). (d) The glycosidase insensitivity of the epitope was determined by treatment of KgV cell lysates (25 mg) with combinations of 2.0 mU neuraminidase with either 0.4 units of recombinant N-glycosidase F alone (lanes 3 and 7) or together with 2.5 mU O-glycosidase (lanes 4 and 8) as described under Materials and Methods. Western blot analyses were performed and immunostained with either mAb 6E3.C4 (lanes 1–4) or polyclonal goat anti-Rausher gp70 (lanes 5–8) as a control. The arrow indicates the deglycosylated form of gp70.

Sporadic reports concerning the cell surface expression of tubulin have been published in the past 20 years (26–28). In one compelling article, Quillen et al. reported that tubulin was readily iodinated on the surface of viable cells and that this molecule was removed from the surface by treatment with trypsin. However,

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the functional significance of this surface tubulin expression was not addressed in these previous reports. More recently, the cytoplasmic portions of several cell surface molecules such as CD2, have been shown to associate with membrane tubulin and it has been suggested that this interaction may play a role in the T cell

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FIG. 5. Amino acid sequence analysis of an isolated molecule bound by mAb 6E3.C4 matches with a-tubulin. Molt-4 membranes were prepared and subjected to ion-exchange separation (A) using a Pharmacia FPLC system as described under Materials and Methods. Fractions were tested by Western blot analysis for reactivity, and positive fractions were pooled for further purification. Proteins in the pooled fractions were separated by preparative size exclusion gel electrophoresis and electroblotting to PVDF membranes. The immunoreactive band was excised from the blot and the proteins eluted in preparation for internal sequence analysis as performed by Dr. Spiecher’s laboratory at the Wistar Institute. (B) A search of GeneBank revealed that the sequences obtained matched with previously published sequences of a-tubulin. The most dominant sequences (17) obtained and the least prevelent sequences (27) obtained are shown for both peptide fragments (x, unknown amino acid). (C) Western

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activation process mediated via these receptors (18). It has been demonstrated that CD2 and a-tubulin dissociate from each other upon T cell activation. An activation-dependent dissociation from CD2 or another relevant cell surface molecule (e.g., CD3, CD4, or Fas) could result in exposure of a unique epitope of a-tubulin on the cell surface allowing an interaction with mAb 6E3.C4. The dissociated cell surface a-tubulin may then direct the organization of cytoplasmic microtubules via a tubulin–tubulin interaction. The binding of mAb 6E3.C4 may interfere with this process perhaps by mimicking the nondissociated state of a-tubulin. Indeed, Quillen et al. hypothesized that a specific tubulin binding site on the surface of certain tumor cells and activated lymphocytes may exist which associates with a unique form of tubulin (27). Perhaps this ‘‘tubulin binding site’’ is one of these aforementioned immunologically relevant cell surface molecules. However, the possibility that a-tubulin exists as a cell surface molecule is complicated by the fact that atubulin does not have a hydrophobic membrane spanning domain. The lack of such a domain suggests the possibility that mAb 6E3.C4 does not bind cell surface a-tubulin, but rather binds an as yet unidentified molecule that expresses a cross-reactive epitope. This crossreactivity may occur as the result of binding to an epitope formed by the primary amino acid sequence or the conformation of the molecule. In this case, binding of mAb 6E3.C4 may inhibit T cell activation inhibiting cytokine:receptor binding or send a negative signal in a manner similar to certain anti-CD4 antibodies that inhibit T cell activation (29). We hypothesize that a cell surface form of a-tubulin or a molecule that expresses a cross-reactive epitope plays a role in the T cell activation process as evidenced here by the inhibitory effect of mAb 6E3.C4 on mitogen stimulation of T cells. Further support for our results demonstrating the expression and function of cell surface a-tubulin comes from the observations of Rubin et al. suggesting that chemotherapeutic agents such as vinblastine, an anti-microtubule agent, might act directly on the cell membrane through the binding of cell surface a-tubulin since the therapeutic dosage for these agents is far below the concentrations which affect microtubules (28). The exact mechanism of mAb 6E3.C4mediated inhibition as well as the effect of other mAb’s that bind a-tubulin is currently being investigated by studying the various events which occur in T cells following stimulation including phosphorylation of proteins, ion fluxes, generation of second messengers, and transcription of early activation genes.

blot analysis of 2 mg of a-tubulin purified from brain tissue (Sigma). Western blot analyses were performed and immunostained with either monoclonal anti-a-tubulin (Sigma) (lane 1) or mAb 6E3.C4 (lane 2) as described under Materials and Methods.

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Received August 28, 1996; accepted with revision November 18, 1996

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