©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Novel Costimulatory Molecules
A PROTEIN OF 38-42 kDa FROM B CELL SURFACE IS CONCERNED WITH T CELL ACTIVATION AND DIFFERENTIATION (*)

(Received for publication, January 11, 1995; and in revised form, June 13, 1995)

Dass S. Vinay Manoj Raje Rakesh K. Verma Gyan C. Mishra (§)

From the Institute of Microbial Technology, Sector 39-A, Chandigarh 160014, India

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Optimal activation of T cells often requires signals delivered by the ligation of T cell receptor (TcR) and those resulting from costimulatory interaction between certain T cell surface accessory molecules and their respective counter receptors on antigen presenting cells. The molecular events underlying the co-stimulatory activity are still not understood fully. Here we describe a 38-42-kDa (B3) protein, present on the surface of lipopolysaccharide-activated B cells, which can provide costimulation to resting T cells leading to a predominant release of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2 and interferon-. Binding assay and electron microscopic autoradiography data suggest that this molecule binds T cells, and the same can be competed by unlabeled B3. Characterization experiments point out that B3 shows up as a single prominent peak on reverse phase-high performance liquid chromatography, runs as a single spot in reducing two-dimensional gel electrophoresis, and is a phosphoglycoprotein. The Western analysis indicate that it does not cross-react with antibodies directed against murine ICAM-1, LFA-1alpha, VCAM-1, HSA, and B7 suggesting the novelty of the protein. The internal amino acid sequence of this molecule suggests that it does not belong to a known category of murine B cell surface molecules.


INTRODUCTION

T helper cell activation is accomplished by recognition of antigen-Ia complex, expressed by antigen presenting cell (APC), (^1)via a clonally restricted heterodimeric receptor (TcR)(1, 2, 3) . The precise mechanism by which APCs activate T cells is quite complex and not fully understood. The current dogma is that at least two signals are required. The first signal is provided by the occupancy of TcR, which is major histocompatibility complex restricted(1) , and the second non-major histocompatibility complex restricted signal (costimulatory signal) is delivered by certain molecules present on the surface of APCs(4, 5, 6) . The participation of costimulatory signal in T cell activation is of paramount importance as it results in two potential outcomes, activation or clonal anergy(7, 8) . The two different outcomes of antigen recognition, by T cells, is first explained by the dual signal model of T cell activation by Bretscher and Cohn (9) and updated recently by Jenkins and Schwartz(10) . Since then, efforts of numerous researchers have culminated in the identification of various molecules capable of providing costimulatory signal(11) . The list of these second signal generating molecules, however, is still far from complete as reports are rapidly appearing in the literature regarding the possible existence of certain hitherto unknown molecules with costimulatory properties(12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) .

To identify additional cell surface-associated molecules that provide costimulatory signals to T cells, we have isolated proteins from lipopolysaccharide (LPS)-activated B cell membrane. When reconstituted into lipid bilayer, at least three proteins (B1, B2, and B3) gave differential levels of costimulatory help to primary T cell activation. The present document presents the results obtained with one of the above proteins with a molecular mass range of 38-42 kDa (B3) (the data of B1 and B2 have been communicated elsewhere) and its relation to primary T cell activation and differentiation.


EXPERIMENTAL PROCEDURES

Animals

Female inbred BALB/C mice, 8-10 weeks old, were obtained from the National Institute of Nutrition, Hyderabad, India, and from our conventional conditions and were allowed free access to food and water.

Cell Lines and Hybridomas Used

All the cell lines and hybridomas used in this study, viz. anti-Thy 1 (TIB 99), anti-L3T4 (TIB 207), anti-CD8 (TIB 150), anti-Mac2 (TIB 166), anti-Mac3 (TIB 168), 33D1 (anti-dendritic cell Ab; TIB 227), anti-IL-2 R (CRL 1698), HT-2 (CRL 1841), anti-Ia^d (HB3), anti-IFN- (HB 170), anti-IL-2 (HB 8794), anti-IL-4 (HB 188), anti-LFA-1 (TIB 217), and anti-ICAM-1 (CRL 1878) were procured from the American Type Culture Collection (ATCC), Rockville, MD. Anti-CD3 (145.2C11) was a kind gift from Dr. Charles A. Janeway Jr., Howard Hughes Medical Institute, New Haven, CT. WEHI-279 (CRL 1704), A20 (TIB 208), and anti-HSA (TIB 183) were kind gifts from Dr. Satyajit Rath, National Institute of Immunology, New Delhi, India.

Primary T Cells

CD4 T cells were prepared from mice spleens as follows. Briefly, a single cell suspension of spleens was prepared in balanced salt solution (pH 7.2). Red cells were lysed using hemolytic Gey's solution. Non-adherent cells, collected by allowing cells to adhere to plastic Petri plates (Nunc, Denmark) at 37 °C with 7% CO(2) for 2 h, were treated sequentially with a mixture of anti-Mac2 and anti-Mac3 (45 min on ice), 33D1 (45 min on ice), and anti-Ia^d (45 min on ice). The cells were then washed and incubated with two rounds of anti-Lyt-2.2 (Cedarlane, Ontario, Canada) with 45 min each on ice followed by treatment with low toxicity baby rabbit complement. CD4 T cells were enriched by passing through nylon wool column. The cells were collected after five to six washes with prewarmed RPMI, 10% FCS (37 °C) and plated on Petri plates, previously coated with goat anti-mouse IgM, for 1 h at 37 °C. The non-adherent cells were used as a source of CD4 T cells and the purity of such cell population routinely exceeded 98% as estimated by FACScan (Beckton Dickinson) in preparations stained with anti-L3T4.

Preparation of Resting B Lymphocytes

Resting B cells from mice spleens were prepared as follows. Briefly, a single cell suspension of spleens was prepared in balanced salt solution (pH 7.2). The red blood cells were removed by treatment with Gey's solution. After removing the macrophages by allowing them to adhere twice to plastic surface (1 h each at 37 °C and 7% CO(2)), the cells were treated twice (45 min each on ice) with a mixture of anti-Mac2, anti-Mac3, and 33D1 and a mixture of anti-Thy1, anti-L3T4, and anti-CD8 followed by labeling (30 min at 37 °C) with low toxicity baby rabbit complement. The cells obtained were then loaded on a discontinuous Percoll gradient (100, 70, and 50%) and centrifuged at 1600 times g for 30 min at 4 °C. The cells collected from 100-70% interface layer were considered as resting B cells.

Preparation of LPS-activated B Lymphocytes

Activated B cells were prepared as follows. Briefly, a single cell suspension of mice spleens was prepared in balanced salt solution (pH 7.2). The red blood cell were depleted by treatment with hemeolytic Gey's solution. The cells were then plated on plastic Petri plates (Nunc, Denmark) for 2 h at 37 °C and 7% CO(2). The non-adherent cells were treated sequentially on ice for 45 min each with a mixture of anti-Mac2 and anti-Mac3, and a mixture containing anti-Thy1, anti-L3T4, and anti-CD8 antibodies followed by complement mediated killing. The cells were then incubated at a concentration of 4 times 10^7/10 ml/pertiplate with 10 µg/ml LPS (from Salmonella typhosa; Sigma) for 72 h at 37 °C and 7% CO(2). The purity of such cells was over 98% as analyzed by FACscan (Beckton Dickinson).

Isolation of LPS-activated B Cell Surface Proteins

The LPS-activated B cells were harvested and washed three times with PBS (pH. 7.2) and frozen overnight at -70 °C. The cells were thawed and homogenized in the presence of 0.25 M sucrose, 20 mM Tris-HCl (pH 7.4), and 1 mM EDTA along with a protease inhibitor mixture (leupeptin 10 µg/ml, aprotinin 10 µg/ml, iodoacetamide 10 mM, antipain 10 µg/ml, pepstatin 10 µg/ml, chymostatin 10 µg/ml, and phenylmethylsulfonyl fluoride (1 mM). The nuclear fraction was removed by centrifugation for 10 min at 700 times g at 4 °C. The supernatant (S1) was collected and kept aside. The pellet was rehomogenized and spun as above. The supernatant (S2) was collected, and the pellet was discarded. S1 and S2 were mixed and subjected to centrifugation at 1,10,000 times g for 2 h at 4 °C. The supernatant was discarded, and the pellet was solubilized in 1% Triton X-100, 20% glycerol, and 20 mM Tris-HCl (pH 7.5) and protease inhibitor mixture (composition as mentioned above) and agitated overnight at 4 °C followed by centrifugation at 100,000 times g for 1 h at 4 °C. The proteins, from the supernatant, were separated using 10% preparative polyacrylamide gel electrophoresis according to Laemmli(15) . After electrophoresis, the protein bands were located by staining a strip of the gel with Coomassie Blue, and the appropriate unstained regions were crushed and eluted with 1% SDS, 100 mM NH(4)HCO(3),50 mM Tris-HCl, 0.1 mM EDTA, and 0.15 M NaCl (pH 8.0) at 37 °C for 48 h. After filtration and centrifugation to remove polyacrylamide particles, the solution was dialyzed against 0.1% SDS, 10 mM NH(4)HCO(3), 10 mM Tris-HCl, 0.01 mM EDTA, 50 mM NaCl (pH 8.0) for 24 h at 4 °C.

SDS Removal, Estimation, and Partial Protein Renaturation

To the protein solution was added Lubrol Px (a neutral detergent) to effect a final 10% of the detergent concentration, and it was kept at 37 °C for 6 h to enable a micelle mixture of SDS-Lubrol Px. This was followed by dialysis against 10 mM Tris-HCl (pH 8.0), 1% Lubrol Px, 0.01 mM EDTA, 50 mM NaCl at 4 °C for 96 h. The dialysis buffer was replaced by a fresh one after every 8 h. The extent of SDS removal, from the protein in the dialyzing bag, was estimated with a basic fuschin method(16) .

Reconstitution of Proteins into Liposomes

Preparation of Liposomes

These were prepared by a reverse phase evaporation method using DL-alpha-phosphatidylcholine, dipalmitoyl, Sigma) in 1:1 chloroform/methanol ratio. The lipid film was left in vacuum for 2 h at room temperature. The film was then hydrated in 10 mM Tris-HCl (pH 8.0), 60 mM NaCl and sonicated in a bath type sonicator (Bransonic, model B 2200 E4, Danbury, CT) to clarity at 4 °C for 30 min. The liposomes were sequentially sized through 0.4 and 0.2 µm polycarbonate membranes before use.

Protein-Liposome Coupling

A 1 to 500 ratio of SDS-depleted and partially renatured protein sample to liposomes was placed in a dialysis bag and dialyzed for 96 h at 4 °C against 10 mM Tris (pH 8.0), 0.01 mM EDTA, and 10 mM NaCl with at least 12 changes of dialysis buffer. The contents of the dialyzing bag were spun down for 2 h at 4 °C at 178,000 times g. The supernatant was discarded, and the pellet was dissolved in physiological saline and later passed through a Sephadex G-50 minicolumn according to Fry et al.(17) to remove unliposomized protein.

Density Gradient Centrifugation

2 ml of the above sample was layered on top of a discontinuous gradient of 5-40% sucrose (w/v) in 10 mM Tris-HCl (6.8), 0.15 M NaCl, and 0.1 mM EDTA. The sample was centrifuged overnight at 98,000 times g in a Beckman SW28 rotor at 4 °C. 2-ml samples were collected from the 5 and 10% interface, washed in 5 volumes of physiological saline by centrifuging at 178,000 times g for 2 h at 4 °C. The pellet was dissolved in physiological saline and stored sterile for not more than 3 months at -20 °C. The protein content, coupled to liposomes, was estimated after lysing with 1% SDS by the BCA method(18) . The lipid phosphorous was estimated as per the method of Ames and Dubin (19) .

T Cell Proliferation Assay

CD4 T cells were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with penicillin (70 µg/ml), streptomycin (100 µg/ml), glutamine (4 mM), 2-mercaptoethanol (50 µM) sodium pyruvate (1 mM), HEPES (20 µM), and 10% heat-inactivated FCS (Sera Laboratories, Crawley Down, Sussex, United Kingdom). Affinity purified anti-CD3 (145.2C11) (10 µg/ml), diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6), was immobilized on the surface of 96-well flat-bottomed (Costar, Cambridge, MA) culture plates by overnight incubation at 4 °C. The wells were washed three times with PBS (pH 7.2) before adding the cells along with varying concentrations of experimental, reconstituted, and control proteins. Phorbol myristate acetate (PMA) (Sigma) was used at a concentration of 10 ng/ml. The proliferation was assessed after 72 h of culture with 1 µCi [^3H]thymidine added during the last 16 h of culture. Cells were harvested on a multiple sample harvestor (Skatron, Norway), and the incorportated radioactivity was assessed in a scintillation counter.

Lymphokine Bioassay

CD4 T cells were cultured in 24-well plates at a density of 0.25 times 10^6/well with different stimuli. The culture supernatants were collected after 22 h for lymphokines assay.

Interleukin-2 and -4 Assay

Interleukin-2 and interleukin-4, in culture supernatants, were determined by the induction of HT-2 proliferation as described by Fernandez-Bortan et al.(2) . Briefly, HT-2 cells (1 times 10^4/well) were cultured in 96-well flat bottomed plates, containing RPMI 10% FCS and various concentrations of culture supernatants from control and experimental wells. Since HT-2 cells are responsive to both these lymphokines, therefore, while measuring IL-2 level, the activity of IL-4 was neutralized using anti-murine IL-4 antibody (600 ng/ml). Likewise, for IL-4 assay, the activity of IL-2 was inhibited by adding anti-IL-2 and anti-IL-2 receptor antibodies. The cultures were incubated for 24 h at 37 °C/7% CO(2) followed by pulsing with 1 µCi [^3H]thymidine during the last 6 h of culture. The cells were harvested, and the incorporated radioactivity was measured by liquid scintillation counting. The lymphokines activity, in terms of units, was derived by extrapolating the standard curve values obtained by using recombinant interleukin-2 and -4 (Genzyme).

Interferon- Assay

Interferon- was assayed by its ability to inhibit the proliferation of WEHI-279 cells. Cells were cultured in 96-well flat bottomed plates with different concentrations of culture supernatants from control as well as experimental wells. The cultures were pulsed with [^3H]thymidine and the incorporated radioactivity was measured as above. Recombinant IFN- served as standard to extrapolate the lymphokine activity in terms of units.

Interleukin-5 Assay

The activity of IL-5 was assayed by the induction of proliferation of mouse splenic resting B cells using dextran sulfate as a comitogen as described by Swain(21) . Resting B cells were obtained from splenocyte suspension as mentioned above. After washing, 1 times 10^5 cells/well were added into individual wells of 96-well flat bottomed microtiter plate. 100-µl aliquots of a range of dilutions of the culture supernatants under test were added in triplicate wells along with 50 µg/ml dextran sulfate. Murine recombinant IL-5 added in different dilutions to obtain a standard curve whose specificity was cross-checked with anti-IL-5 (500 ng/ml). The cultures were pulsed with 1 µCi of [^3H]thymidine after 72 h and harvested 16 h later. The incorporated radioactivity was determined by liquid scintillation counting. IL-5 activity, expressed in terms of units/ml, was obtained from the standard values.

Northern Blotting of mRNA

CD4 T cells (5 times 10^6/ml) were cultured in 24-well plate (Costar, MA) for 8 h at 37 °C, 7% CO(2) in the presence of previously immobilized anti-CD3 (10 µg/ml), PMA (10 ng/ml), and B3 (0.01 µg/ml) in separate sets of experiments. Thereafter, the cells were harvested and washed repeatedly in cold PBS (pH 7.2) and stored at -70 °C in pellets until RNA extraction was performed. Total cytoplasmic RNA was prepared according to White and Bancroft (22) and blotted over to Immobilon-N (Millipore, MA) and cross-linked to the membranes by Stratalinker 1800 (Stratagene, La Jolla, CA). Membranes were washed with 1 times SSC and 0.1% SDS for 1 h at 65 °C and then prehybridized in a solution containing 50% formamide, 5 times SSPE (20 times SSPE = 3 M NaCl, 0.2 M Na(2)HPO(4), and 20 mM EDTA) and 5 times Denhardt's reagent (50 times Denhardt's = 1% each of Ficoll, BSA fraction V, polyvinylpyrrolidone, 0.1% SDS) overnight at 42 °C. Specific RNA was detected by probing the membranes with [-P]ATP-labeled cDNA probes for murine IL-2, IL-4, IL-5, and IFN- (Amgen Biological, Thousand Oaks, CA) in fresh prehybridization buffer for 24 h at 42 °C. After washing, the membranes were exposed to x-ray film with an intensifying screen at -70 °C for 48 h.

Gel Electrophoretic and Lectin Gel Binding Analysis

SDS-PAGE was performed in 0.5-mm thick slab gels containing 4% acrylamide in stacking gel and a 10% acrylamide in separating gel, with a buffer system according to Laemmli(15) . The lane containing B3 was transferred on to Immobilon-P (Millipore, CA), washed, and visualized by staining with 0.2% aqueous Ponceau S, destained, and its ability to bind I-ConA was tested in conjunction with autoradiography on x-ray film.

Phosphorylation Procedure

The assay mixture (final volume, 0.2 ml) contained 50 mM MOPS/KOH (pH 6.0), [-P]ATP (0.2 mCi/ml), 0.3 M MgCl(2), and approximately 75 µg protein of LPS-activated B cell membrane lysate. The reaction carried out at room temperature was started by addition of the proteins and stopped by adding 50 µl of 50% trichloroacetic acid, 0.3 M unlabeled ATP, and 0.3 M MgCl(2). All the following operations were carried out at 4 °C. The proteins were washed four times. The pellet, extracted by 1 ml of diethylether, was further processed and electrophoresed according to Amory et al.(23) After electrophoresis, the gel was stained with Coomassie Blue, destained, and B3 was located, excised, dried, and exposed to x-ray film.

Iodination and Competitive Binding Assay

I-Labeling of B3 was performed using the IODO-bead method with PD-10 column (Pharmacia, Sweden). Iodinated samples were trichloroacetic acid- precipitated and an equivalent fraction containing 2 ng of protein was allowed to bind the CD4 T cells preactivated with anti-CD3 (10 µg/ml) or PMA (10 ng/ml) and/or both as the case may be. The reaction was carried out for 30 min at 37 °C in RPMI containing 10% FCS, 0.2% sodium azide, 20 mM HEPES (pH 7.0). For competition binding, a range of (5-100 times the protein concentration) non-radiolabeled dilutions of B3 was used. The reaction mixture was incubated at 4 °C for 2 h on an orbital shaker and mixed with a vortex mixer at 15-min intervals. After extensive washing, the cell pellets were subjected to gamma counting (Beckmann).

Electron Microscopy

Negative Staining of Liposomes

A 20-µl droplet of aqueous suspension of liposomes was placed on a fresh piece of parafilm, and a sample droplet was picked up by touching a carbon-coated grid to it. After allowing the excess liquid to drain off, the grid was gently dipped in 1% phosphotungstic acid (pH 7.0) and dried by blotting on a filter paper. Grids were observed in a transmission electron microscope (JEOL 1200 EXII, Japan) and representative fields were photographed.

Electron Microscopic Autoradiography

The B3bulletliposome complex as well as goat anti-mouse IgM (Sigma) were I-labeled by IODO-bead method essentially as described in the previous section. The radiolabeled samples were incubated with CD4 T cells (previously activated at 37 °C for 30 min with 10 µg/ml anti-CD3) for 30 min at 37 °C. After incubation, the cells were washed thrice with PBS to remove unbound material by centrifuging for 10 min at 500 times g at 4 °C. To the pellet was added an equal volume of low melting agarose (Sigma) and allowed to gel, and the latter was cut into 1-mm^3 pieces. Trapped cells were fixed in 1% paraformaldehyde, 1% glutaraldehyde in PBS at 4 °C for 1 h. After washing with PBS, the cells were post-fixed in 1% osmium tetraoxide for 90 min at 4 °C in the dark. After washing with PBS and passing through graded acetone series, the samples were embedded in epoxy resin (Bio-Rad). Ultrathin sections, cut on Ultracut S (Richert-Jung, Austria), were coated in dark with photographic emulsion (Ilford nuclear L4 emulsion) and incubated in the dark for 2-3 weeks in a dessicator at 4 °C. The autoradiographs were developed and stained with aqueous uranyl acetate and lead citrate and observed in a transmission electron microsope (JEOL 1200 EXII, Japan).

Western Blotting

Western immunoblots were made from SDS-PAGE(24) . Samples were electrophoresed for 90 min with constant amperage of 14 mA in a minigel apparatus (Atto, Japan). The proteins were transferred to Immobilon-P polyvinylidine difluoride membranes (Millipore, MA) using a Bio-Rad electrophoretic transfer unit with 10 mM CAPS buffer (pH 11.0). After washing in PBS, pH. 7.2, for 15 min, the membranes were blocked with 3% nonfat dry milk and 0.2% Tween-20 in PBS for 2 h at 37 °C with gentle agitation. After washing in PBS, the membranes were incubated overnight at 4 °C with appropriate antibody in PBS-Tween, washed, and incubated with anti-rat horseradish peroxidase-conjugated IgG for 2 h at room temperature on an orbital shaker. Bound antibody was detected with metal ion enhanced diaminobenzidine.

Reverse Phase-HPLC

B3 was located, isolated, and eluted as mentioned earlier in this section. The protein sample was then diluted with 0.5% trifluoroacetic acid and loaded on to a microbore HPLC column (C8) (Aquapore RP 300, Brownlee columns, ABI) and monitored.

Protein Sequencing

B3 isolated from activated B cell membranes, as described above, was purified by 10% SDS-PAGE(15) . After the electrophoresis, B3 was blotted on to ``ProtBlot'' (Applied Biosystems) in CAPS buffer (pH 11.0) followed by staining in 0.2% Ponceau S, 1% acetic acid and washed with PBS. The protein was then digested with trypsin as its N terminus was found to be blocked and a sample equivalent of 2 pmol, from one of the digested fractions, was subjected to internal sequencing up to 15 residues on Applied Biosystem (model 492 A) Procise Sequencer (at The Protein Sequencing Facility, Worcester Foundation for Experimental Biology, Shrewsbury, MA). The repetitive yields of the first 15 amino acids sequenced were found to be at least 90%.

Two-dimensional Electrophoresis

The reagents for this purpose were prepared essentially according to Amory et al.(23) and performed as advocated by Penin et al.(25) .


RESULTS

Identification and Partial Characterization of B3

The membranes of LPS-activated murine splenic B cells, when subjected to SDS-PAGE analysis, revealed about 15 major protein bands when stained with Coomassie Brilliant Blue (Fig. 1a). B3 was localized, crushed, and eluted as described under ``Experimental Procedures.'' The accuracy with which B3 was isolated was checked by rerunning the protein on SDS-PAGE. When stained with Coomassie Brilliant Blue, it demonstrated a single band both under non-reducing (Fig. 1b), reducing (Fig. 1c), and as a single spot in two-dimensional gel electrophoretic (Fig. 1e) conditions. When B3 was subjected to lectin gel binding (Fig. 1f) and phosphorylation (Fig. 1g) assays it was noticed that B3 binds I-ConA and incorporates radiolabeled phosphate indicating the possibility that it is a phosphoglycoprotein. The results of our attempts to localize B3 on the surface of resting B cells demonstrated that it is hardly detectable even when probed by silver stain of SDS-gel possibly indicating that its high expression is induced when pretreated with LPS (Fig. 1d).


Figure 1: SDS-PAGE analysis of LPS-activated B cell membrane, stained with Coomassie blue, revealing protein bands. a, the samples were analyzed on a 10% gel under non-reducing conditions. The arrowhead denotes the position of B3. b and c, one-dimensional profile of B3 under non-reducing (b) and reducing (c) conditions (see arrowheads). d, SDS-PAGE analysis of resting B cell membrane. The sample was run on 10% gel under non-reducing conditions and probed with silver stain. The arrowhead denotes the possible position of B3. e, two-dimensional non-reducing (SDS-PAGE)/reducing (TDAB-PAGE) electrophoretic pattern of B3. Gel electrophoresis was performed on 10% gels in both the dimensions and stained with Coomassie Blue. e, lectin gel binding analysis of B3. This was performed with I-ConA as outlined under ``Experimental Procedures.'' The arrowhead indicates the position of B3. f, phosphorylation assay of B3. The B cell membrane proteins were subjected to phosphorylation analysis with [-P]ATP. B3 was located, isolated by PAGE, and analyzed by autoradiography.



B3 Binds to T Cell Surface

B3, before checking for its costimulatory ability, was reconstituted into lipid bilayers. Prior to reconstituting the protein, it was ensured that a fairly homogeneous preparation of liposomes was obtained. Fig. 2a reveals the negative staining of liposomes indicating a near homogeneous preparation of lipid vesicles. In order to demonstrate the protein reconstitution in lipid vesicles, the aid of electron microscopic (EM) autoradiography was employed. Since B3 is a membrane protein, presumably with a hydrophobic stretch, it was expected to be inserted into the lipid bilayer. In order to illustrate the liposome-protein coupling, the reconstituted protein was iodinated and processed for EM autoradiography. Fig. 2b (arrows) shows the predominant presence of iodinated protein on the vesicle surface suggesting by that each of the liposome has a fairly even distribution of B3. Such I-B3-bearing liposomes when incubated with anti-CD3 activated T cells, and it was observed that B3 was distributed uniformly all along their surface (arrows, Fig. 2c).


Figure 2: Binding and distribution of B3 on T cell surface. B3 coupled to liposomes was labeled with I and incubated with anti-CD3-activated CD4 T cells and EM autoradiography was performed as mentioned under ``Experimental Procedures.'' Cells were examined by transmission electron microscopy. a, negative staining of liposomes by phosphotungstic acid (times10,000, bar = 200 nm). b, I-labeled B3 coupled to liposomes. Arrows show the liposomes frequently bearing the iodinated B3 (times20,000, bar = 200 nm) c, CD4 T cells labeled with iodinated B3 (arrows; times20,000; bar = 200 nm). Note the distribution of B3 only on the T cell surface.



That the binding of B3 to T cell surface was specific was demonstrated in experiments with nonspecific control like murine anti-IgM. The data obtained clearly shows that there was no binding of liposome-coupled I-anti-IgM on the T cell surface presumably because T cells do not possess receptors for IgM (Fig. 3a). On the other hand, when liposomized I-anti-IgM was incubated with A20 cells, as a positive control, the radiolabeled antibody was seen to be evenly distributed on the plasma membrane (PM, arrows, Fig. 3b) and also localized in the interior of the cell (arrows, Fig. 3c).


Figure 3: Murine anti-IgM coupled to liposomes and I-labeled iodinated was incubated with T and A20 cells. EM autoradiography was performed as described under ``Experimental Procedures.'' The cells were examined by transmission electron microscopy. PM, plasma membrane; Cyt, cytoplasm; Nuc, nucleus; EV, endocytotic vesicle. a, anti-CD3-activated T cells incubated with liposomized and I-labeled murine anti-IgM (times25,000, bar = 200 nm); b, A20 cells incubated with liposomized and I-anti-IgM (times40,000, bar = 200 nm). Note the absence of labeled anti-IgM binding on the T cell membrane. Also note the distribution of labeled anti-IgM in and on the surface of A20 cells.



To further confirm the specificity of binding of B3 to T cells, competitive binding assay was performed. When unreconstituted I-B3 was incubated with T cells in the absence of anti-CD3, very less binding (1,250 ± 252 cpm) was noticed. However, the number of receptors for B3 appeared to be up-regulated when the T cells were preactivated with anti-CD3 (10,509 ± 2,562 cpm). Further, the binding specificity of B3 to T cells was tested by competing I-B3 with unreconstituted and unlabeled B3 to bind the anti-CD3-activated T cells. The data clearly show that the binding capacity of I-B3 is diminished by unlabeled B3 (Fig. 4, a-g).


Figure 4: Competitive binding assay with B3. CD4 T cells (1 times 10^6/ml) were incubated with or without anti-CD3 or unlabeled B3 30 min at 37 °C. After washing, I-labeled B3 (2 ng) was added to final a volume of 200 µl and incubated for 2 h at 4 °C with gentle agitation. After extensive washing, the incorporated radioactivity in cell pellets was monitored on a gamma counter. a, cells + I-B3; b, cells + anti-CD3 + I-B3; c, cells + anti-CD3 + 100:1 (B3:I-B3); d, cells + anti-CD3 + 75:1 (B3:I-B3); e, cells + anti-CD3 + 50:1 (B3:I-B3); f, cells + anti-CD3 + 25:1 (B3:I-B3); g, cells + anti-CD3 + 5:1 (B3:I-B3). The range of dilutions of unlabeled B3 used was in terms of protein content.



Antibodies against Murine LFA-1alpha, ICAM-1, HSA-1, B7, and VCAM-1 Do Not Cross-react with B3

It is known that a majority of costimulatory molecules identified thus far are adhesive in nature. In our efforts to rule out the possibility of B3 being a known costimulatory molecule, a Western analysis was performed. The data obtained are depicted in Fig. 5which demonstrates that B3 did not cross-react with any of the antibodies against murine LFA-1-alpha, ICAM-1, HSA, B7, and VCAM-1. LPS-activated B cell membrane lysate was run in appropriate lanes as a positive control for these adhesive molecules.


Figure 5: Western blotting. Proteins were run on SDS-PAGE (10%) and transferred onto Immobilon-P membrane and stained in 0.2% aqueous Ponceau S to ensure that all lanes contained the transferred protein. After washing and blocking, the membrane was probed with antibodies against ICAM-1, LFA-1alpha, HSA, B7, and VCAM-1. As a positive control, LPS-activated B cell membrane sample was run in appropriate lanes, and the bound antibody was detected by diaminobenzidine.



B3 Costimulates Primary T Cells to Proliferate

Fig. 6a shows that the addition of B3 to the cultures led to an increase in [^3H]TdR incorporation of T cells in a dose-dependent manner. A maximum statistically significant (p < 0.05) proliferation (39,426 ± 4,214 cpm) was noticed at a B3 concentration of 1 µg/ml as against the basal value (955 ± 268 cpm) obtained with cells plus anti-CD3 only. When the concentration of B3 was increased, no further amplification in T cell proliferation was observed. Thus, in all the subsequent experiments, only the half-maximal concentration (0.01 µg/ml) of B3, as determined by dose-response pattern, was used. Further, when the cultures were stimulated with controls like liposomes, SDS, B3 alone, gel eluate, and LPS, no statistically significant (p < 0.05) proliferation (<2,000 cpm) of T cells was noticed (Fig. 6b) pointing out thereby that the activity elicited by B3 was indeed specific. On the other hand, when PMA was added to anti-CD3-activated T cells, as a positive control, a maximum incorporation of [^3H]TdR (10,871 ± 2,827 cpm) was observed.


Figure 6: Effect of various concentrations of B3 on the proliferation of CD4 T cells. 1 times 10^5 T cells/well were cultured in 0.2 ml of RPMI, 10% FCS for 72 h with various concentrations of liposomized B3. The cells were pulsed with 1 µCi [^3H]thymidine during the last 16 h, and the incorporated radioactivity was determined by liquid scintillation counting. All the values represent the mean cpm ± S.D. of at least three experiments. a demonstrates the dose-response profile of B3, and b represents the behavior of various controls like anti-CD3 (10 µg/ml), PMA (10 ng/ml), B3 (0.01 µg/ml), liposomes (2.326 nmol inorganic phosphorous content), SDS (0.02 nmol), gel eluate (10 µl/well), and LPS (1 µg/ml). Asterisk denotes statistically significant (p < 0.05) over its respective control (i.e. cells + anti-CD3-treated group).



Induction of Secretion of IL-4 and IL-5 by B3

After confirming that B3 was able to enhance the proliferation of primary T helper cells, we next determined whether or not this protein could elicit the secretion of any lymphokine. To verify this, T cells were cultured with anti-CD3 and/or B3, and the supernatants collected after 22 h were tested for IL-2, IL-4, IL-5, and IFN- levels. The data show that only the cultures stimulated with anti-CD3 and B3 produced significant levels of IL-4 and IL-5 and a very poor level of IL-2 and IFN- (Fig. 7). As seen in the proliferative studies, cultures containing T cells and B3 did not bring about any significant secretion of the above lymphokines. The trend obtained with bioassay of lymphokines was further confirmed by Northern analysis, the details of which are highlighted in Fig. 7(see inset).


Figure 7: Induction of lymphokines by B3. Cells were cultured as described in the legend to Fig. 6. The supernatants from the control and experimental wells were collected after 22 h and tested for IL-2 and IL-4 using HT-2 cell line (1 times 10^4 cells/well), IFN- on WEHI-279 cell line (2 times 10^4 cells/well), and IL-5 on resting splenic B cells (1 times 10^5 cells/well). The cells were incubated in RPMI, 10% FCS (100 µl/well in the case of IL-2, IL-4, and IFN- and 200 µl/well in the case of IL-5). The cells were pulsed with 1 µCi of [^3H]thymidine during the last 6 h (for IL-2, IL-4, and IFN-gamma) and the last 16 h (in the case of IL-5) of culture. The radioactivity incorporated was monitored by liquid scintillation counting. The values expressed in terms of units/milliliter were derived from the standard curve. The Northern analysis data (see inset) also coincide with the pattern obtained with the lymphokine bioassays. Student's t test was performed to calculate the degree of significance. Asterisk denotes the statistically significant values (p < 0.05) over its respective control (i.e. cells + anti-CD3-treated group).



Reverse Phase-HPLC and Protein Sequence Analysis

B3, when subjected to reverse phase-HPLC, showed a single prominent peak (at 18.21 min) in the chromatogram (Fig. 8a) demonstrating thereby that this protein is homogeneous in its present form of isolation coinciding with our two-dimensional data. This protein was subjected to internal amino acid sequence upon tryptic digestion (because of its blocked N terminus) up to 15 residues, and the details are depicted in Fig. 8b.


Figure 8: Reverse phase-HPLC and internal sequence of B3. a, reverse phase-HPLC profile of B3. This was performed as described under ``Experimental Procedures,'' the summary of internal sequence of B3. The upper case letters represent the three-letter code of each of the amino acid identified while the lower case numbers indicate its position. Unknown positions are indicated by the letter X.




DISCUSSION

Optimal T cell activation depends not only on the occupancy of TcR, but also on accessory molecules, provided by the APCs, that function in cell-cell adhesion and/or signal transduction(26) . During the recent past, several new costimulatory molecules have been identified(12, 13, 14) , and the list still appears to be incomplete as the evidence for existence of additional cell surface proteins involved in signal transduction is being raised by several workers(27, 28) . It may be mentioned here that on the basis of their ability to secrete specific lymphokines, T helper cells have been divided into Th1 and Th2. Th1 cells produce IL-2 and IFN-, lymphotoxin, etc., and primarily participate in stimulting the cell-mediated immunity(29, 30) , while Th2 cells secrete IL-4, IL-5, IL-6, etc., and are involved mainly in the induction of humoral immunity(29, 31, 32, 33, 34) . It has been postulated that these two T helper subsets are not only functionally different but also need qualitatively and quantitatively distinct requirements for costimulation(35) . However, to date and to the best of our knowledge, no costimulatory molecules are reported which exclusively activate Th2 cells.

In the present study, we describe the biochemical and functional analysis of a novel LPS-activated murine splenic B lymphocyte cell surface-associated costimulatory molecule, provisionally termed B3, that chiefly activates Th2-like cells. Our data suggest that B3 molecule is specifically involved in the costimulation of resting T helper cells upon cross-linking TcRbulletCD3 complex with anti-CD3 monoclonal antibody resulting in predominant secretion of IL-4 and IL-5 and very poor levels of IL-2 and IFN-. Our rationale for choosing B cell surface molecules to provide costimulatory signal to T cells lies in the observation that B lymphocytes are major APCs for the clonal expansion of normal murine CD4 T cells(36) . Further, our selection of LPS-activated B cells for identifying the costimulatory molecules is based on the premise that resting B cells are poor APCs (37) and do not constitute costimulatory activity(38, 39) ; only upon treatment with either LPS or IL-1 or immunoglobulin or IFN-, or cross-linking surface major histocompatibility complex class-II molecules or neuraminidase etc. (36, 40, 41, 42) do the B cells acquire enhanced ability to stimulate T cells. Moreover, cytokine secretion is induced from naive T cells only when activated B cells are used as APCs (43) . It may also be stressed here that the molecule, described in the present study, is bearly detectable on the membranes of resting B cells even when loaded five times the concentration of LPS-activated B cell membrane lysate probed with silver strain.

In biochemical experiments, we have characterized B3 as a single molecule with an approximate molecular mass of 38-42 kDa when analyzed by SDS-PAGE. Upon reducing, this molecule was recovered as a single sharp band. The reverse phase-HPLC approach to purify this protein clearly showed a solitary and distinct peak (at 18.21 min) in the chromatogram. This information, in conjunction with the SDS-PAGE analysis, conclusively proves that this protein is homogeneous in its present form of isolation. In addition to these aproaches, the two-dimensional profile of B3 always consistently yielded a single pattern (in about 12 repetitions) which also reiterates the absence of any other contaminants sticking either specifically or nonspecifically to the said protein. As assessed by its ability to bind I-ConA, B3 appears to be glycosylated a fact which was further substantiated by partial digestion of this protein by N-glycosidase F that resulted in two distinct fragments of 22 and 18 kDa (data not shown). The phosphorylation assay, on the other hand, revealed that this molecule is capable of incorporating radiolabeled phosphate. These results indicate that B3 is a phosphoglycoprotein.

For an effective signal transduction, a costimulatory molecule is expected to bind its counter ligand on the target cell. Studies were undertaken to explore this possibility, and the results obtained indicate that B3 molecule binds to T cells and this binding can be diminished by competing with unlabeled B3. This fact is further strengthened by the results obtained with electron microscopic autoradiographic studies which show that I-labeled reconstituted B3 molecule when incubated with anti-CD3-activated T cells, this protein is found associated with the surface of the T cell. It is interesting to note that the receptors for this molecule, on T cells, appear to be significantly up-regulated when prior activated with anti-CD3. In the light of these observations, it is safer to assume that T cells bear ligands for B3 molecule.

On the basis of its molecular mass, B3 is clearly distinct from the most characterized costimulatory molecules such as ICAM-1 (90-114 kDa) (44) . VCAM-1 (100-110 kDa)(45) , B7 (45-60 kDa)(46) , HSA (35-60 kDa)(47) , and human LFA-3 (60-80 kDa)(48) . Our Western analysis data strengthens this view by the fact that the antibodies directed against the above molecules failed to cross-react with B3. As regards to LFA-3, it still has not been reported in the murine system.

The internal amino acid sequence was obtained by tryptic digestion (as its N-terminal was found to be blocked) of the protein blotted onto the polyvinylidene difluoride membrane. This yielded a single sequence, and at present we only have a partial sequence of 15 amino acids. A data base (non-redundant amalgamation data base of SwissProt, PDB, SP update, PIR, GP update) search of this yielded somewhat surprising results, as it showed a high homology (95%) with pyruvate kinase and is not a part of any known surface proteins. The pyruvate kinase is a well known cytosolic enzyme(49) . In a rare event of misidentifying pyruvate kinase as the present protein, is not possible due to the following reasons: 1) this protein is isolated using the well known procedures for isolation of membranes and was washed extensively to remove any nonspecific contaminants; 2) the present protein is found to be heavily glycosylated from the data shown as under ``Results,'' and it is very uncertain for cytosolic protein be heavily glycosylated; and 3) the molecular mass of pyruvate kinase is about 56-60 kDa (50) while the protein described in the present study has an approximate molecular mass of 38-42 kDa. These points strongly disfavor the argument that B3 is pyruvate kinase. The fact that B3 showed high homology with pyruvate kinase (PK), prompted us to investigate as to whether B3 possesses any PK-like activity. When B3 was allowed to react with phosphoenol pyruvate, substrate for pyruvate kinase, it did not show any enzymatic activity thereby further demonstrating that B3 and PK are unrelated entities (data not shown).

The only known costimulatory molecule closest to B3, in terms of molecular weight, is B7-2. Like B7 (now called CD80), B7-2 (a 34-kDa protein) is a counterreceptor for CD28 and CTLA-4 T cell surface molecules and induces the predominant secretion of IL-2(51) . In contrast, the molecule described in the present study activates CD4 Th cells to secrete IL-4 and IL-5 but a very little IL-2 and IFN-. These observations lend support to the view that B3 molecule is not a counter ligand for either CD28 or CTLA-4. Also, when anti-CD3-activated T cells incubated with anti-CD28 and were allowed to interact with I-labeled B3, there was no substantial change in the binding capacity of labeled B3 to T cells. Further on, our experiments to identify the receptor for B3 on T cells, using the homobifunctional cross-linker disuccinimidyl suberate, clearly demonstrated B3 binds a 60-kDa protein on T cell surface (data not shown). It may be mentioned here that B7 (CD80) binds a 44-kDa glycoprotein (CD28) on T cell surface.

Thus, all the generated evidence favors the conclusion that B3 is a novel costimulatory molecule that activates Th2-like cells. Using a similar approach, we have recently demonstrated the presence of a 150-kDa protein (M150) from the membranes of thioglycollate-elicited murine peritoneal macrophages that selectively activate Th1 type of cells leading to the secretion of significant levels of IL-2 and IFN- but negligible amounts of IL-4(52) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 0091-172-690127; Fax: 0091-172-690585/690632.

(^1)
The abbreviations used are: APC, antigen presenting cell; LPS, lipopolysaccharide; IFN, interferon; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; FCS, fetal calf serum; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; MOPS, 4-morpholinepropanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HPLC, high performance liquid chromatography; cpm, counts/minute.


ACKNOWLEDGEMENTS

We are grateful to Dr. C. M. Gupta for consistent advice and encouragement especially in connection with the reconstitution of proteins into lipid bilayers. We also thank Drs. N. K. Ganguly and Harpreet Vohra, Post Graduate Institute of Medical and Research, Chandigarh, India for helping in FACS analysis. The technical assistance of R. Das and C. S. Gill is greatly appreciated. Our thanks are also due to Vijay Pal for excellent secretarial assistance.


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