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The Journal of Immunology, 2001, 166: 3107-3113.
Copyright © 2001 by The American Association of Immunologists

Functional Expression of a Costimulatory B7.2 (CD86) Protein on Human Salivary Gland Epithelial Cells that Interacts with the CD28 Receptor, but Has Reduced Binding to CTLA41

Efstathia K. Kapsogeorgou, Haralampos M. Moutsopoulos and Menelaos N. Manoussakis2

Laboratory of Immunology, Department of Pathophysiology, School of Medicine, National University of Athens, Athens, Greece


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
B7 molecules expressed on classic APC play a critical role in the regulation of immune responses by providing activation or inhibitory signals to T cells, through the ligation with CD28 or CTLA4 receptors, respectively. We have recently described the expression of B7 molecules by the salivary gland epithelial cells (SGEC) of patients with Sjögren’s syndrome (also termed autoimmune epithelitis). The role of such expression needs to be clarified. Thus, in the present study, we sought to address the existence and function of B7.2 proteins on cultured nonneoplastic SGEC lines derived from Sjögren’s syndrome patients. The occurrence of B7.2 proteins on SGEC was verified by flow cytometry, immunocytochemistry, immunoprecipitation, and immunoblotting. The assessment of several cell lines in costimulation assays had revealed that the constitutive expression of B7.2 molecules is sufficient to provide costimulatory signals to anti-CD3-stimulated T cells. SGEC-derived costimulation induced IL-2-dependent proliferation of CD4+ T cells, which was associated with low production of IL-2, but probably also with the secretion of yet undefined autocrine T cell growth factor(s). B7.2 proteins expressed by SGEC were found to display distinctive binding properties denoted by the functional interaction with CD28 receptor and reduced binding to CTLA4. Finally, the detection of a functional soluble form of B7.2 protein in cell-free culture supernatants of both SGEC and EBV-transformed B cell lines is demonstrated. These findings imply a critical role for epithelial cells in the regulation of local immune responses in the salivary glands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The interaction of B7 proteins on classical APC, such as dendritic cells, macrophages, and B cells, with their counterreceptors CD28 and CTLA4 on T lymphocytes, induces critical regulatory signals for T cell activation (1, 2, 3). This represents the most extensively characterized pathway of costimulation with significant clinical implications in the pathogenesis and treatment of human disease (4, 5, 6, 7). The B7 family consists of at least two distinct but functionally interrelated glycoprotein molecules, namely the B7.1 (CD80) and the B7.2 (CD86) (8, 9, 10), whereas the existence of a third one (termed BB1/B7.3) has been elusive (11, 12, 13). Recently, an additional costimulatory homologue of B7.1 and B7.2 (termed B7H1) has been described, which apparently interacts with receptors distinct from CD28 or CTLA4 (14, 15). The B7.1 and B7.2 proteins appear to bind similarly to counterreceptors; however, their affinity for CD28 is reportedly lower than for CTLA4 (16, 17, 18). The engagement of CD28 receptor by the B7 proteins has been shown to elicit strong costimulatory signals for T cell activation, whereas their interaction with CTLA4 molecules is down-regulatory (10, 19, 20).

During the recent years, on the basis of reactivity with various anti-B7 mAbs, multiple cell types other than classical APC have been regarded as candidate populations for the delivery of costimulatory signals, such as activated T cells (21, 22), epithelial (23, 24), and endothelial cells (25, 26). In addition, the increased expression of B7 proteins by nonimmune tissues has been associated with the development of various inflammatory disorders, including autoimmune diseases (27, 28). More recent data, however, have cast doubt upon the actual occurrence of B7 proteins on the surface of these cells. The BB1 mAb was recently shown to recognize dually the CD74 (MHC II-associated invariant chain) and B7.1 proteins (12), a finding that probably dictates the reconsideration of several studies that applied the above mAb for the detection of B7.1. In addition, B7 protein expression by itself may not signify the capacity of nonclassic APC to deliver optimal costimulatory signals (22, 29, 30). Therefore, the presence and function of the native B7 proteins in such cells need to be elucidated.

Recently, using biopsy specimens and cultured nonneoplastic cell lines derived from patients with Sjögren’s syndrome (SS),3 we have presented evidence for the expression of B7.1 and B7.2 molecules by salivary gland epithelial cells (SGEC) at both the mRNA and the protein level (31). In fact, increased spontaneous expression of a BB1 mAb-reactive protein was demonstrated in SGEC lines. In subsequent experiments, however, the assessment of several SGEC lines by flow cytometry had failed to show reactivity with another anti-B7.1 mAb (L307.4), but revealed a low to moderate constitutive B7.2 protein expression in all cell lines. Thus, we aimed to further establish the presence of B7.2 molecules on cultured nontransformed SGEC lines and to determine their plausible costimulatory function. The study of B7.2 protein was facilitated by the availability of anti-B7.2 mAbs that were found suitable for protein detection by immunoprecipitation and immunoblotting as well as for the blocking of function in costimulation assays. By this approach, we presently demonstrate that cultured SGEC are capable to express functional T cell costimulatory B7.2 protein molecules that possess distinctive binding properties to CD28 and CTLA4 receptors, as indicated by its preferential binding to CD28. The costimulatory influence of epithelial B7.2 was found to lead to IL-2-dependent proliferation of CD4+ T cells. Such proliferation is probably associated with the production of an autocrine T cell growth factor distinct from IL-2, but which acts in synergy with this IL. In addition, this is the first study to demonstrate the production of a soluble form of B7.2 protein by cultured SGEC as well as EBV-transformed B cells. Our findings indicate the B7 expression capacity of SGEC and possibly attest to the potential of these cells to act as APC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents

The mAbs against human CD86/B7.2 (clones IT2.2 and FUN.1), CD28 (clone CD28.2), CD3 (clone HIT3a), CD14 (clone M5E2), and CD1a (clone MT102) were purchased from PharMingen (San Jose, CA). The mAbs against human CD80/B7.1 (clone L307.4), CD4 (clone SK3), CD8 (clone SK1), and to human cytokeratins 8 and 18 (clone CAM 5.2) were obtained from Becton Dickinson (San Jose, CA). The BB1 mAb against human CD80/B7.1 was a generous gift from Becton Dickinson Hellas (Athens, Greece). Anti-CD86/B7.2 mAb (clone BU63) was from Serotec (Oxford, U.K.), and neutralizing anti-IL-2 mAb (clone 5334.21) was from R&D Systems (Minneapolis, MN). The mAb to human dendritic reticulum cell (clone R4/23) was obtained from Dako (Glostrup, Denmark). The mAbs against human IL-2R{alpha} (CD25, clone 7G7B6), CD20 (clone 2H7), and the CD28-Ig fusion protein (human CD28 protein fused with mouse Ig) were purchased from Ancell (Bayport, MN). The CTLA4-Ig fusion protein (human CTLA4 protein fused with human Ig) was a kind gift from Repligen (Cambridge, MA). PE-conjugated F(ab')2 fragments of Abs against mouse and human IgG F(c) were from Rockland (Gilbertsville, PA). For use in immunoprecipitation and proliferation assays, mAbs were bound to sheep anti-mouse IgG-coated Dynabeads M450 (Dynal, Oslo, Norway) at a concentration of 1 µg mAb/107 beads, according to manufacturer’s instructions. Human rIFN-{gamma} was from Boehringer Ingelheim (Ingelheim, Germany).

Human EBV-transformed B cell and B7-transfected CHO cell lines

B7.1-, B7.2-, and mock-transfected Chinese hamster ovary (CHO) cell lines (CHO-B7.2, CHO-B7.1, and CHO-mock, respectively, kindly provided by V. Boussiotis, Dana-Farber Cancer Institute, Boston, MA) were maintained in complete RPMI 1640 medium supplemented with 400 µg/ml Geneticin (G418; Life Technologies, Grand Island, NY). The EBV-transformed B cell line JY, which expresses B7.1 and B7.2 (kindly provided by G. Thyphronitis, University of Athens, Greece), was cultured in RPMI 1640 (Life Technologies) supplemented with 10% FBS, 5 x 10-5 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Salivary gland epithelial cells

Labial minor salivary gland biopsies were obtained with informed consent from patients undergoing diagnostic evaluation for sicca symptoms indicative for SS. SS was diagnosed on the basis of the European SS classification criteria (32). Nonneoplastic long-term SGEC cultures were developed, characterized, and maintained, as previously described (31). Briefly, primary cultures were established by explant outgrowth technique in a medium consisting of a 3:1 mixture Ham’s F12 and DMEM (Life Technologies) supplemented with 2.5% FBS (Life Technologies), 0.4 µg/ml hydrocortisone (Upjohn, Kalamazoo, MI), 0.125 U/ml insulin (Novo, Bagsvaerd, Denmark), 10 ng/ml epidermal growth factor (Promega, Madison, WI), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. At 80% confluency, primary cultures were trypsinized and serially passaged in bovine collagen type I (Sigma, St. Louis, MO)-coated culture vessels in keratinocyte basal medium (KBM; Clonetics, Walkersville, MD) supplemented with 0.4 µg/ml hydrocortisone, 0.125 U/ml insulin, 10 ng/ml epidermal growth factor, and 25 µg/ml bovine pituitary extract (Sigma) at a density of 3 x 104 cells/cm2 (33). All cultures were incubated at 37°C, 5% CO2, and 99% humidity. The epithelial origin of SGEC lines was routinely verified immunocytochemically by the expression of epithelial-specific cell markers such as cytokeratins, as previously described (31). SGEC cultures were free of T and B cells, macrophages, and dendritic cells, as indicated by the absent expression of such cell type-specific markers (data not shown).

Preparation of SGEC-conditioned medium

Samples of culture supernatants were collected from subconfluent B7.2-expressing SGEC cultivated in serum-free KBM medium and immediately hypercentrifuged and filtered through a 0.2-µm filter (Millipore, Bedford, MA). Subsequently, supernatants were concentrated by ultrafiltration using a YM-30 membrane (Amicon, Danaverts, MA) and stored at -80°C until use. Before application in T cell proliferation assays, the concentrated CMs were appropriately diluted with the appropriate volume of complete RPMI medium to obtain 5-fold final concentration of the initial volume.

Flow cytometry

SGEC were harvested and resuspended in ice-cold PBS containing 2.5% FBS and 0.3% NaN3. About 5 x 104 cells were stained directly using FITC- or PE-conjugated mAbs to membranous B7 proteins, or isotype-matched control mAbs. Cell surface staining with CD28-Ig or CTLA4-Ig fusion proteins (50 µg/ml) was visualized by incubation with appropriate PE-conjugated IgG F(c)-specific secondary Ab. Analyses were performed using a FACSCalibur flow cytometer and CELLQuest software (Becton Dickinson), with appropriate forward and side scatter adjustments for epithelial cells. Mean fluorescence intensity (MFI) values obtained by staining with specific mAbs were corrected by the subtraction of background values (isotype control mAb).

Immunoprecipitation and immunoblotting

Cytoplasmic extracts of JY cells or SGEC were prepared by lysis in ice-cold buffer (2 x 106 cells/ml) containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 20 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, and 0.1 mM PMSF (all from Sigma). The nuclei and the insoluble cell debris were removed by centrifugation at 14,000 x g for 10 min at 4°C. Immunoprecipitation was performed using magnetic beads coated with anti-B7.2 FUN.1 mAb or the isotype control (2 x 107 beads/2 x 106 cells), according to the manufacturer’s instructions. Samples of total cytoplasmic cell extracts or immunoprecipitated proteins (corresponding to 2 x 106 cells/lane) were subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Immunoblotting was performed with anti-B7.2 (FUN.1 or BU63) or isotype control mAbs in 1% skim milk in TBS/0.1% Tween 20. Proteins were visualized by enhanced chemiluminescence using alkaline phosphatase-conjugated rabbit anti-mouse Igs (Dako) and CDP-star substrate (Boehringer Mannheim). For the detection of soluble B7.2 molecules, cell-free culture supernatants from subconfluent SGEC and JY cells were subjected to hypercentrifugation (100,000 x g for 1 h) for the removal of cellular elements and immediately analyzed by immunoprecipitation and immunoblotting, as above.

Immunocytochemistry

Cultured SGEC grown in multichamber slides were analyzed by immunocytochemistry using the avidin-biotin immunoperoxidase technique, as previously described (31).

B7 mRNA expression

The expression of B7.1 and B7.2 mRNA in cultured cell lines was detected by RT-PCR, as previously described (31). The integrity of all cDNA samples was tested by RT-PCR for {beta}-actin mRNA.

Isolation of CD4+ T cells

PBMC from healthy donors were separated by density-gradient centrifugation (lymphocyte separation medium; Life Technologies). The T cell population was enriched by sequential depletion of B cells by immunomagnetic anti-CD19-coated Dynabeads M450 (Dynal), and of adherent mononuclear cells by two successive rounds of incubation on plastic (for 2 and 24 h). Subsequently, CD4+ T cells were isolated from the remaining population by immunomagnetic positive selection using Dynabeads M450 coated with anti-CD4 (Dynal). The purity of CD4+ T cell population yielded by this procedure was routinely >99.3%, as assessed by flow cytometry. The purified CD4+ T cells were not expressing B7.2, were less than 5% positive for CD25 (IL-2R{alpha}), and were unresponsive to stimulation by anti-CD3 mAb only.

Costimulation assays

The capacity of B7.2-expressing cells to provide costimulatory signals to anti-CD3-activated allogeneic CD4+ T cells was assessed by a previously established assay (26).

For these studies, SGEC lines were selected on the basis of definitive constitutive expression of B7.2 (but not B7.1) mRNA and protein. In brief, 96-well microtiter plates (Costar, Cambridge, MA) were seeded with SGEC (at second passage, 2 x 104 cells/well in KBM), CHO transfectant cells (2 x 104 cells/well in G418 medium), or KBM medium alone. Upon confluency, the monolayers of epithelial cells were washed three times with PBS and fixed with 1% paraformaldehyde (Merck, Darmstadt, Germany) in PBS for 10 min at room temperature. Subsequently, wells were washed (seven times) with complete RPMI medium and received CD4+ T cells (2 x 104 cells/well, 200 µl/well in complete RPMI 1640) in the presence or the absence of anti-CD3-coated magnetic beads (three beads/CD4+ cell), anti-CD28 mAb (1 µg/ml), anti-IL-2-neutralizing mAb (1 µg/ml), anti-B7.2 mAb (IT2.2; 10 µg/ml), CTLA4-Ig (10 or 50 µg/ml), CD28-Ig (10 µg/ml), or control mAbs (10 µg/ml), in triplicates for each combination. The optimal conditions of the assay, including the concentration of added mAbs, were determined in preliminary experiments. All neutralizing reagents used in cultures (including mAbs and fusion proteins) were found nontoxic for T cells, SGEC, or CHO transfectants. The proliferative responses of CD4+ T cells were estimated after 5 days of culture by [3H]thymidine incorporation, except for CHO transfectant cell-costimulated T cell cultures, which were assayed at the third day (induction of maximal proliferation). Cells were pulsed with [3H]thymidine (1 µCi/well; ICN, Costa Mesa, CA) for the last 18 h of culture. Subsequently, cells were collected onto glass fiber filters (Skatron Instruments, Suffolk, U.K.), and radioactivity incorporation was measured in a liquid scintillation analyzer (Packard Instruments, Downers Grove, IL). The capacity of 5-fold concentrated SGEC-conditioned medium to costimulate the proliferation of anti-CD3-activated CD4+ T cells (105 cells/well) was also assessed, as above.

Assessment of cytokine production

Cell-free supernatants obtained from the cocultures of anti-CD3-activated CD4+ T cells with SGEC, CHO-B7.2, or CHO-neo transfectants in the presence or the absence of blocking mAbs were collected at different time points (12, 24, 48, and 72 h) and kept at -80°C until assayed. The occurrence of soluble T cell growth factor(s) was assessed by the induction of proliferation in anti-CD3 T cell blasts (see below). IL-2 production was measured in duplicates by an ELISA (Diaclone, Besancon, France), according to the manufacturer’s instructions.

Anti-CD3 blasts

Anti-CD3 blasts were generated by stimulation of purified CD4+ T cells with anti-CD3-coated magnetic beads (three beads/CD4+cell). Anti-CD3 blasts (5 x 104 cells/well) were cultured either in supernatants obtained by differentially costimulated CD4+ T cells, as described above in the presence or absence of a neutralizing anti-IL-2 mAb (1 µg/ml) for 5 days. Anti-CD3 blasts cultured with 2 ng/ml human rIL-2 (R&D Systems) were used as the positive control. In preliminary experiments, this concentration of human rIL-2 (rhIL-2) was found to induce submaximal proliferation of anti-CD3 T cell blasts under standard conditions. Proliferation was estimated in triplicate cultures by the measurement of [3H]thymidine incorporation.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The main scope of this study was to address the actual presence and function of B7 proteins on nonneoplastic SGEC. The expression of B7 proteins on SGEC was initially examined by flow cytometry analysis of several SGEC lines utilizing various mAbs to B7.1 and to B7.2 proteins. To date, our data cannot establish the expression of typical B7.1 protein molecules by SGEC. Despite reactivity to BB1 mAb and positivity for B7.1 mRNA in approximately one-third of cell lines, no positive reactions could be demonstrated with a well-established anti-B7.1 mAb (clone L307.4) by flow cytometry or immunocytochemistry (data not shown). Further studies are necessary to elucidate whether this discrepancy is due to novel BB1 mAb-reactive B7 molecules (13), or shared reactivity to non-B7 proteins (12). On the other hand, however, we were able to confirm the occurrence of low to moderate constitutive B7.2 protein expression in all cell lines (Fig. 1GoA), which was up-regulated by IFN-{gamma} (data not shown). Parallel experiments had demonstrated that B7.2 is significantly more sensitively detected on cultured SGEC by flow cytometry than immunocytochemistry (data not shown), which is in line with its more rare detection by the latter method (31). In fact, approximately one-fifth of SGEC lines (7 of 32 tested, 21.9%) had discernible amounts of surface B7.2 (mean MFI ± SD, 26 ± 3), albeit at significantly lower levels compared with EBV-transformed JY B cells (MFI, 125 ± 11) or CHO-B7.2 transfectant cells (MFI, 280 ± 18) (Fig. 1GoA). Taking in account the approximately 10-fold larger size of SGEC compared with JY B cells, it appears that the density of surface B7.2 expression is far lower on SGEC compared with B cells. B7.2 is a glycoprotein that runs with an apparent molecular mass of 65–100 kDa on SDS-PAGE. To verify its presence on epithelial cells, we utilized the well-characterized anti-B7.2 mAbs FUN.1 (Fig. 1GoB) and BU63 (data not shown) for detection by immunoprecipitation and immunoblotting. In this manner, B7.2 proteins were readily demonstrated in cytoplasmic extracts from both SGEC and JY B cells (Fig. 1GoB). In both cell types, immunoprecipitated B7.2 presented as multiple protein bands covering a relatively wide range of size, which is most likely consistent with the presence of variably glycosylated proteins. It is noteworthy that B7.2 proteins from SGEC run at a higher range of molecular mass size (75–110 kDa) compared with that from JY B cells (65–110 kDa; Fig. 1GoB), probably suggestive of differences in their glycosylation status. In addition, a soluble form of B7.2 of similar molecular mass size to cell-associated form was also found in culture supernatants derived from both SGEC and JY cells (Fig. 1GoB).



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  		FIGURE 1. Demonstration of constitutive B7.2 protein expression by SGEC. A, Flow cytometric analysis illustrating representative examples of constitutive B7 expression by cell lines utilized in the study, including human nonneoplastic SGEC (top panel), EBV-transformed B cells (JY, middle panel), and CHO-B7.2 transfectants (lower panel). SGEC lines studied were selected on the basis of definitive constitutive expression of B7.2 protein (FUN.1 mAb), but not B7.1, as demonstrated by lack of reactivity to anti-B7.1 mAbs (BB1 and L307.4). Filled and open curves represent the staining with the specific mAbs (indicated on top) and isotype control mAbs, respectively. B, Immunoprecipitation and immunoblotting analysis of cell-associated and soluble B7.2 proteins from SGEC and JY EBV-B cells. Cytoplasmic cell extracts (CE) and cell-free culture supernatants (Spn) derived from JY B cells (lanes 1 and 2) and SGEC (lanes 3–5) were subjected to immunoprecipitation and subsequently to immunoblotting using the anti-B7.2 mAb FUN.1 (lanes 1–4) or an isotype control mAb (lane 5; negative control). The 55-kDa bands correspond to Ig heavy chains, which are derived from the Abs used in immunoprecipitation and are recognized by the secondary Ab applied in immunoblotting. Lanes 6 and 7, Direct anti-B7.2 immunoblotting of JY and SGEC cytoplasmic cell extracts, respectively. Results are representative of four experiments with similar results.

 
CHO-B7.2 cells, but not CHO-mock transfectant cells, were fully capable of transmitting costimulatory signals to anti-CD3-stimulated CD4+ T cells (Fig. 2GoA) in a costimulation assay. To investigate the costimulatory function of B7.2 proteins expressed by SGEC, epithelial cell lines that displayed definitive constitutive expression of B7.2 mRNA and protein (but not B7.1/BB1) were similarly assessed, using anti-CD3-stimulated allogeneic CD4+ T cells (Fig. 2GoB). Accordingly, T cell proliferative responses as well as cytokine production were estimated in the presence or absence of B7.2-blocking reagents, such as neutralizing anti-B7.2 mAb (IT2.2), CTLA4-Ig or CD28-Ig fusion proteins, or control Abs. B7.2-expressing SGEC were found capable of providing significant costimulation for CD4+ T cell proliferation (Fig. 2GoB). The SGEC-assisted T cell proliferation was specifically inhibited by the addition of anti-B7.2 mAb (mean percentage inhibition ± SD of four experiments, 94.3 ± 5.6), but not by the isotype control mAb (percentage of inhibition, 0.8 ± 0.7; Fig. 3GoB). Surprisingly, the addition of CTLA4-Ig, even in high amounts (50 µg/ml), had limited inhibitory influence for SGEC-derived costimulation (percentage of inhibition, 21.9 ± 1), while it blocked completely the T cell proliferation costimulated by CHO-B7.2 cells (percentage of inhibition, 98.1 ± 0.3; Fig. 3GoB, lower panel). In contrast, the addition of CD28-Ig fusion protein in cocultures of SGEC and CD4+ T cells effectively blocked T cell proliferation assisted by SGEC (percentage of inhibition, 92.4 ± 3.9; Fig. 3GoB, upper panel), as well as by CHO-B7.2 (data not shown). The results from the above inhibition experiments were directly consistent with the binding profile of CD28-Ig and CTLA4-Ig to SGEC and CHO-transfectant cells, as assessed by flow cytometry. CD28-Ig, but not CTLA4-Ig, manifested binding to constitutively B7.2-expressing SGEC (Fig. 3GoA). In contrast to SGEC, both these fusion proteins reacted with CHO-B7.2 transfectants, and in absolute agreement with the differential affinity of these receptor proteins for B7.2 (16), the binding of CTLA4-Ig was stronger than CD28-Ig (Fig. 3GoA). Our data suggest that B7.2 protein expressed by SGEC can transmit costimulatory signals to T cells and is able to bind to CD28, but has reduced binding for CTLA4, the negative regulator of T cell activation.



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  		FIGURE 2. Induction of proliferative responses in purified CD4+ T cells by costimulation with anti-CD3 and CHO transfectants (CHO-tx, in A) or B7.2-expressing SGEC (in B). Triplicate cultures of 2 x 105 CD4+ T cells activated by anti-CD3-coated magnetic beads (three beads/T cell) and 1% paraformaldehyde-fixed CHO transfectants (CHO-mock or CHO-B7.2) or SGEC were set up in the presence or absence of blocking mAbs (10 µg/ml). Maximal proliferation of CD4+ T cells was measured by [3H]thymidine incorporation. Note the different scale of proliferation of CD4+ T cells costimulated by CHO-B7.2 and SGEC. Results involving the costimulation of CD4+ T cells by CHO transfectants or SGEC are representative of 4 and 10 experiments, respectively.

 


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  		FIGURE 3. The B7.2 protein expressed on SGEC binds functionally to CD28 receptor, but has reduced binding for CTLA4. A, The flow cytometry assessment of B7.2-expressing (but BB1/B7.1-negative) SGEC reveals the surface staining of cells by CD28-Ig fusion protein, but not by CTLA4-Ig. In contrast, CHO-B7.2 transfectant cells display reactivity to both these fusion proteins. Filled curves represent staining with anti-B7.2 mAb (FUN.1) or the indicated fusion protein. Control staining (open curves) was performed by isotype control mAb or control Igs. Results are representative of five experiments. B, The SGEC-assisted proliferation of anti-CD3-activated CD4+ T cells is completely blocked by anti-B7.2 mAb or CD28-Ig fusion protein, but not by CTLA4-Ig (10 µg/ml, upper panel). In contrast, CTLA4-Ig is capable of blocking CHO-B7.2-mediated costimulation, whereas the poor inhibitory effect of CTLA4-Ig for SGEC-costimulated T cell proliferation was evident even when high amounts (50 µg/ml) were used (lower panel). Results represent the mean percentage of inhibition (±SD) of four different experiments.

 
The nature and physiological significance of the differential binding of SGEC-derived B7.2 to its receptors are unclear. In this context, the increased molecular mass of B7.2 molecules that we found in SGEC is intriguing. We are currently investigating whether these features owe to distinctive biochemical characteristics of B7.2 expressed on our cultured epithelial cells. Alternatively spliced B7.2 mRNA isoforms have been described in mouse (34). On the other hand, posttranslational modification(s) of B7.2 proteins (e.g., in the glycosylation status) may have a regulatory effect in the function of B7.2 by favoring its preferential association with one of two receptors. Interestingly, the occurrence of a hypoglycosylated form of B7.2 on human T cells has been previously associated with inability to bind to CD28 receptor and to provide costimulatory signals (22).

To examine whether the SGEC-derived costimulatory function results in the production of a soluble growth factor, a T cell blast proliferation assay was employed. Thus, T cell blasts were exposed to cell-free supernatants obtained from costimulation assays, and the induction of proliferation was examined. In this manner, the supernatants obtained from the costimulation of CD4+ T cells by either SGEC or CHO-B7.2 (but not CHO-mock) were found to induce comparable proliferation (Fig. 4GoA), which confirms the production of a soluble T cell growth factor. This factor is presumably of T cell origin, as indicated by the lack of T cell blast proliferation by culture supernatants derived from fixed epithelial cells cultivated alone or together with unstimulated T cells (data not shown). In addition, consistent with the results from the costimulation assays, the production of this T cell growth factor was found dependent on B7.2, since the proliferative activity was diminished in supernatants obtained from cultures in which anti-B7.2 mAb was added (Fig. 4GoA).



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  		FIGURE 4. The proliferation of CD4+ T cells costimulated by B7.2-expressing SGEC is dependent on the presence of IL-2. A, The proliferation of anti-CD3 blasts that is induced by culture supernatants derived from SGEC-assisted costimulation assays ({blacksquare}) is blocked by a neutralizing anti-IL-2 mAb (). Bars (±SD) represent the proliferative response of T cell blasts exposed to medium only (no addition), or culture supernatants derived from the 48-h stimulation of CD4+ T cells by anti-CD3 alone (anti-CD3), or the costimulation of CD4+ T cells by anti-CD3 and CHO-B7.2 cells (designated as CHO-B7.2), or anti-CD3 and B7.2-expressing SGEC, in the presence or absence of anti-B7.2-blocking mAb. Anti-CD3 blasts cultured with rhIL-2 were used as the positive control. Results are representative of three experiments. B, IL-2 measurements by ELISA in culture supernatants obtained from costimulation assays (48 h) of CD4+ T cells in the presence or absence of anti-CD3, the indicated epithelial cell types, and/or blocking anti-B7.2 mAb. Although SGEC-assisted costimulation drives an IL-2-dependent proliferation (as shown in A), IL-2 is undetectable by ELISA (detection limit, 30 pg/ml) in culture supernatants at various time points (from 24 to 72 h). In contrast, costimulation by CHO-B7.2 cells is associated with the production of significant amounts of IL-2. Results are representative of nine experiments with similar results. For incomprehensible reasons, significant IL-2 production (624 pg/ml) was detected in one additional SGEC-assisted costimulation assay, which is not included in analysis.

 
Signaling and costimulation through CD28 have been shown to lead to the induction and secretion of various cytokines (35). Although the role of IL-2-independent pathways has been considered (36, 37, 38), the increased secretion of IL-2 appears critically involved in CD28-mediated T cell proliferation (39, 40, 41). We therefore examined the involvement of IL-2 production in the proliferative responses of CD4+ T cells costimulated by B7.2-expressing SGEC. In such costimulation assays, T cell proliferation was found largely dependent on IL-2, as demonstrated by its almost complete block by the addition of a neutralizing mAb against human IL-2 (mean percentage of inhibition ± SD of four experiments, 89.9 ± 2.4), but not the control mAb (0.7 ± 0.6). Similarly, the proliferation-inducing activity of culture supernatants derived from the above costimulation assays was also found to be IL-2 dependent, as illustrated by the abrogation of T cell blast growth following the addition of anti-IL-2 mAb (Fig. 4GoA). These findings strongly indicate the occurrence and importance of IL-2 production in the course of CD4+ T cell costimulation by B7.2-expressing SGEC. Using an ELISA assay, we failed to reveal detectable amounts of IL-2 in samples of culture supernatants obtained from SGEC-assisted costimulation assays (in 9 of 10 experiments performed), possibly due to low production and/or consumption of the cytokine to levels below the detection limit of the assay (30 pg/ml). In contrast, costimulation of CD4+ T cells by anti-CD28 mAb or CHO-B7.2 cells always resulted in significant amounts of IL-2 (167.5 ± 8.5 and 3.4 ± 0.2 ng/ml, respectively, and Fig. 4GoB). Despite the apparently low IL-2 concentration in the culture supernatants from SGEC-supported costimulation assays, these were able to support comparable proliferation of T cell blasts to 2 ng/ml rhIL-2 and to supernatants collected from CHO-B7.2-costimulated CD4+ cells (which as mentioned above contained approximately 3.4 ng/ml IL-2, as judged by ELISA) (Fig. 4GoB). From these data, we can conclude that SGEC-derived costimulatory signals lead to the production of relatively low amounts of IL-2, which, however, appear quite essential for the induction of T cell proliferation. This low IL-2 production seems unlikely to support T cell proliferation on its own. Thus, we speculate that SGEC-assisted proliferation owes largely to the co-secretion of additional T cell growth factor(s) distinct from IL-2, but whose action is virtually dependent on the presence of this IL. At present, the nature of such T cell growth factor is obscure. Certain cytokines, such as IL-4, have been previously shown to act in an IL-2-dependent fashion (42). In fact, the secretion of IL-4 has been associated with signaling through B7.2-CD28 molecules (43). However, this T cell growth cytokine is unlikely to correspond to the factor in question, as it was not detected by ELISA assays in culture supernatants of CD4+ T cells costimulated by SGEC (data not shown).

As demonstrated above, we were able to immunoprecipitate a native soluble form of B7.2 protein in cell-free culture supernatants derived from JY EBV-transformed B cells and B7.2-expressing SGEC (Fig. 1GoB). Although the existence of native human and porcine soluble B7.1 molecules (44, 45) as well as soluble porcine B7.2-like mediators of CD28 activation (26) has been previously described, to our knowledge, the occurrence of native soluble B7.2 protein has not yet been reported. The precise nature of this protein, including whether it represents shed or secreted forms of original molecules or an alternatively spliced product of B7.2, needs to be clarified. Soluble molecules generated by such mechanisms would be expected to have reduced size compared with the cell-bound protein, owing to the lack of the transmembrane or of both the transmembrane and cytoplasmic domains. In this context, the observed molecular mass similarity of the two B7.2 forms is noticeable, and may be attributed to posttranslational modification(s) of the soluble protein, such as increased glycosylation. Soluble B7.2 molecules may have agonistic or inhibitory (45) function for T cell costimulation, by acting as substitutes or competitors of cell-bound B7 molecules for their ligands. To examine whether soluble B7.2 proteins are capable of providing costimulatory signals to T cells, samples of 5-fold concentrated culture supernatants obtained from B7.2-expressing SGEC were assessed in costimulation assays, as above. Evidence of consistently low but definite costimulation of CD4+ T cell proliferation was thus obtained. The costimulatory activity in these supernatants was B7.2 mediated, as illustrated by blocking with anti-B7.2 mAb (mean percentage of inhibition ± SD of two experiments, 69.7 ± 3.1), but not an isotype control mAb (4.3 ± 0.9) (Fig. 5Go). Therefore, the soluble B7.2 protein molecules appear to retain some costimulatory activity, and thus, they may have a role in the modulation of immune responses.



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  		FIGURE 5. Soluble B7.2 molecules appear capable of providing costimulatory signals to CD4+ T cells. The costimulatory activity of serum- and cell-free culture supernatants of B7.2-expressing SGEC (SGEC-SPN) was assessed in CD4+ T cell costimulation assays, as described in Materials and Methods. Prior to assaying, the occurrence of soluble B7.2 proteins in SGEC-SPN was verified by immunoprecipitation. The results are representative of two experiments.

 
In conclusion, the data presented in this study support the notion that SGEC hold a role in the regulation of T cell responses. On the basis of expression of MHC molecules and functional B7.2 costimulatory proteins, these epithelial cells appear suitably equipped for the presentation of antigenic peptides and the transmittance of activation signals to T cells. The capacity of SGEC to act as nonclassical APC most likely indicates the implication of these cells in the pathogenesis of various disorders of the salivary glands, and particularly in the induction and/or maintenance of autoimmune epithelitis or SS (46, 47). Most probably, SGEC are less competent providers of T cell activation signals compared with dendritic cells, monocytes, or B cells. Nevertheless, the functional expression of B7.2 molecules by SGEC may be critical for the regulation of local immune responses in salivary glands. This is probably also implied by the characteristic binding features of epithelial B7.2 to its receptors on T cells. Although their biologic significance needs to be further assessed, our findings appear to unfold an important immunoregulatory potential of SGEC, and probably other types of epithelial cells.

Note added in proof.

Since the submission of the present work, the existence of functional soluble CD86 molecules produced by resting monocytes was described (48).


   Footnotes
 
1 This work was supported by grants from the Hellenic Secretariat for Research and Technology, the Lilian Voudouri Foundation, and the Hellenic Association of Immunology (to M.N.M.). Back

2 Address correspondence and reprint requests to Dr. Menelaus N. Manoussakis, Department of Pathophysiology, School of Medicine, National University of Athens, Athens 115 27, Greece. Back

3 Abbreviations used in this paper: SS, Sjögren’s syndrome; CHO, Chinese hamster ovary; KBM, keratinocytes basal medium; MFI, mean fluorescence intensity; rhIL-2, human rIL-2; SGEC, salivary gland epithelial cell. Back

Received for publication September 21, 2000. Accepted for publication December 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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