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J Am Soc Nephrol 13:1517-1526, 2002
© 2002 American Society of Nephrology

Renal Tubular Epithelial Expression of the Costimulatory Molecule B7RP-1 (Inducible Costimulator Ligand)

Patricia Wahl*, Roland Schoop*, Grozdana Bilic*, Jörg Neuweiler{dagger}, Michel Le Hir{ddagger}, Steven K. Yoshinaga§ and Rudolf P. Wüthrich*

*Division of Nephrology and {dagger}Institute of Pathology, Kantonsspital, St. Gallen, Switzerland; {ddagger}Institute of Anatomy, University of Zurich, Zurich, Switzerland; and §Amgen Inc., Thousand Oaks, California.

Correspondence to Dr. Rudolf P. Wüthrich, Division of Nephrology, Cantonal Hospital, Rorschacherstrasse 95, 9007 St. Gallen, Switzerland. Phone: +41-71-494-10-32; Fax: +41-71-494-28-77; E-mail: rpw{at}kssg.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. MHC class II-expressing renal tubular epithelial cells (TEC) are able to present foreign peptide antigens to T cells. The costimulatory signals that are required for effective T cell activation upon antigen presentation by TEC have not been characterized. Various cultured TEC lines were examined for expression of the recently described costimulatory molecule B7RP-1 (B7h), a ligand of the T cell molecule inducible costimulator (ICOS), and expression was compared with that of B7.1, B7.2, and CD40. B7RP-1 and CD40 were abundantly expressed by cultured murine and human TEC, whereas B7.1 and B7.2 could not be detected. Stimulation with lipopolysaccharide or tumor necrosis factor-{alpha} did not induce B7.1 or B7.2 expression and did not alter B7RP-1 expression. Interestingly, interleukin-2 production by T cell hybridomas after antigen presentation by TEC was enhanced by blocking antibodies to B7RP-1 and ICOS. In contrast, blocking antibodies to B7RP-1 or ICOS exerted inhibitory effects on anti-CD3-activated murine splenocyte proliferation. Immunohistochemical staining of normal human kidneys demonstrated strong constitutive B7RP-1 expression in distal tubules, collecting ducts, and urothelium. In human kidneys with allograft rejection or interstitial nephritis, distinct B7RP-1 staining was also detected in proximal tubules, in areas of mononuclear infiltration. In conclusion, the B7RP-1/ICOS pathway negatively regulates T cell activation upon MHC class II-restricted antigen presentation by TEC. Because B7RP-1 is also expressed by tubules in vivo, it can be speculated that the B7RP-1/ICOS pathway could play an inhibitory role in TEC-mediated immune activation in the kidney.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Renal proximal tubular epithelial cells (TEC) play an important role in the pathogenesis of immune renal injury, both as initiators of T cell responses and as targets of T cells. Various studies, including our own, have demonstrated that TEC express MHC class II antigens, particularly in response to interferon-{gamma} (IFN-{gamma}) (1–3). TEC also have the ability to ingest and process antigen in the lysosomal compartment, then presenting it in the form of small antigenic peptides, in the context of MHC class II molecules, to CD4+ T cells (3–5). Specific CD4+ T cells may recognize antigen via T cell receptors. Various signaling pathways are then activated, transmitting the so-called signal 1 to the interior of the T cells (6).

Typical antigen-presenting cells initiate a second signal (signal 2), which is needed to fully activate the T cells. This second signal is usually received by T cells through the interaction of CD28 with B7 (7). Several studies have demonstrated, however, that cultured TEC lack the classic B7 molecules, including B7.1 and B7.2. Furthermore, B7.1 and B7.2 are not observed on tubules in vivo, in animal models of renal injury (8–10). This has raised many questions regarding the nature and importance of signal 2 in the interaction of T cells with TEC.

Ligation of T cell receptors in the absence of signal 2 may result in clonal anergy (11). This has also been demonstrated for TEC; the absence of B7 may induce antigen-specific anergy in CD4+ T cells in certain instances (12,13). However, because T cell activation by TEC occurs when foreign antigen is presented in the context of MHC class II antigens, it has been speculated that other costimulatory molecules could be involved in T cell activation by TEC. CD40 and intercellular adhesion molecule-1, for example, are expressed by TEC. The interactions of these molecules with their specific counter-receptors, i.e., CD40L (CD154) and leukocyte function-associated antigen-1, respectively, might be at least partly responsible for T cell activation by TEC (14–16). Vascular cell adhesion molecule-1 on TEC, interacting with the very late antigen-4 counter-receptor, could also play a role (17).

A novel member of the B7 family, namely B7RP-1 [also termed B7h, inducible costimulator (ICOS) ligand, or GL50], was recently discovered (18–21). B7RP-1 is the ligand of a novel T cell activation molecule termed ICOS. Whether B7RP-1 could be involved in the interaction of T cells with TEC has not been investigated. The purpose of this investigation was to characterize TEC expression of B7RP-1 at the cellular and molecular levels and to compare it with the expression of B7.1, B7.2, and CD40. Furthermore, we investigated the role of B7RP-1 on antigen presentation by TEC. The expression of B7RP-1 was also investigated in vivo, in normal kidneys and in kidneys with T cell-mediated renal injury, including allograft rejection and tubulointerstitial nephritis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Tissue culture reagents were obtained from Life Technologies (Gaithersburg, MD), and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Affinity-purified rabbit polyclonal antibodies against murine and human B7RP-1 were prepared after immunization of rabbits with a B7RP-1-Fc fusion protein. A polyclonal anti-murine ICOS antibody was prepared in a similar manner. A monoclonal antibody (mAb) against murine B7RP-1 (clone 1B7) was prepared by immunizing rats with B7RP-1-transfected CHO cells. Anti-murine B7.1 (clone RMMP-1, rat IgG2a), anti-murine CD40 (clone 3/23, rat IgG2a), anti-human B7.1 (clone DAL-1, mouse IgG1), anti-human B7.2 (clone BU63, mouse IgG1), and anti-human CD40 (clone LOB7, mouse IgG1) mAb were from Serotec (Oxford, UK). An anti-murine B7.2 mAb (clone GL-1, rat IgG2a) was from Pharmingen (San Diego, CA). An anti-murine CD3{epsilon} mAb (clone 145-2C11, Armenian hamster IgG) was from R&D Systems (Oxford, UK). Lipopolysaccharide (LPS) (derived from Escherichia coli; Sigma), recombinant tumor necrosis factor-{alpha} (TNF-{alpha}) (Sigma), and IFN-{gamma} (R&D Systems) were used to stimulate cells.

Cell Lines
The SV40-transformed TEC lines MCT and C1.1 and the HPV-16-transformed human kidney cortical tubular cell line HK-2 were used to study B7RP-1, B7.1, B7.2, and CD40 expression (3,22,23). Primary cultures of murine renal TEC were prepared from normal C57BL/6J mice as described (3). The murine monocyte/macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (Rockville, MD) and was used as a control cell line. MCT and RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s medium with Glutamax-1 (DMEM; Life Technologies, Gaithersburg, MD) containing 10% fetal bovine serum (FBS), 10 mM Hepes, 100 U/ml penicillin, and 100 µg/ml streptomycin. C1.1 cells, HK-2 cells, and primary cultures were grown in modified K1 medium, on collagen-coated plates, as described (3). Confluent MCT cell, C1.1 cell, HK-2 cell, and primary cultures were passaged with light trypsinization (0.5 g/L trypsin, 0.2 g/L ethylenediaminetetraacetate). All cell lines were grown at 37°C in 5% CO2.

Cell Stimulation Protocol
After TEC had grown to confluence, the medium was changed to DMEM with 1% FBS overnight, to bring the cells to rest. HK-2 cells were allowed to grow to 80% confluence and primary cultures were completely confluent before the medium was changed to modified K1 medium containing 1% FBS. Cells were then stimulated overnight with LPS (100 ng/ml) or TNF-{alpha} (50 ng/ml) and were examined for costimulatory molecule expression.

Flow Cytometric Analyses of Costimulatory Molecule Expression
Cultured TEC were trypsinized lightly, washed with Hanks’ balanced salt solution, and blocked with phosphate-buffered saline (PBS) containing 2% FBS. Cells were incubated with primary mAb (10 µg/ml) for 1 h at 4°C and were washed again. Cells were then incubated with the appropriate FITC-conjugated secondary antibody (diluted 1:100 in PBS/2% FBS) for 1 h at 4°C. After repeated washings, cells were analyzed by using a Becton Dickinson flow cytometer and Cell Quest software (Becton Dickinson, San Jose, CA).

Protein Extraction and Western Blotting for B7RP-1
After 24-h stimulation with LPS (200 ng/ml), the MCT, HK-2, and RAW 264.7 cells were washed twice with PBS and lysed with 600 µl of M-PER protein extraction reagent (Pierce, Rockford, IL). After gentle shaking for 5 min, the extracts were centrifuged at 13,000 rpm for 8 min at room temperature. The supernatants were mixed with 4x reducing sample buffer (0.38 M Tris base, 8% sodium dodecyl sulfate, 8% {beta}-mercaptoethanol, 4 mM ethylenediaminetetraacetate, 40% glycerol, 0.05% bromphenol blue, pH 6.8) and heated for 5 min. Equal amounts of protein extracts were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes (0.45-µm pore size; Bio-Rad, Hercules, CA). B7RP-1 was detected with a chemiluminescence detection system (Tropix, Bedford, MA), using affinity-purified polyclonal rabbit anti-murine and anti-human B7RP-1 antibodies.

RNA Extraction and Reverse Transcription-PCR
Total RNA was extracted from cultured cells with a RNeasy Mini kit (Qiagen, Valencia, CA). Cells were homogenized by passage of cell lysates through a shredder column and were processed according to the instructions provided by the manufacturer. All RNA samples were quantitated by measurement of the OD at 260 nm and were then analyzed for B7RP-1, B7.1, B7.2, CD40, or cytokine expression by semiquantitative reverse transcription (RT)-PCR, using a OneStep PCR kit (Qiagen). The primer sequences for murine and human B7RP-1, B7.1, B7.2, and CD40 were determined by using Primer 3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The rat housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was also examined, as described (24). One-microgram aliquots of total RNA were reverse-transcribed and amplified in 50-µl reaction mixtures, using a GeneAmp PCR System 9700 thermocycler (Perkin-Elmer, Foster City, CA). RT was performed at 50°C for 35 min. An initial PCR activation step was then performed at 95°C for 15 min. The general cycling parameters were as follows: denaturation at 94°C for 1 min, annealing at 54°C to 58°C for 1 min, and extension at 72°C for 1 min for 30 to 40 cycles, followed by a final extension step at 72°C for 10 min. RT-PCR products were resolved on 1% agarose gels and stained with ethidium bromide. Gels were then photographed with ultraviolet light.

Antigen Presentation by TEC and the Role of the B7RP-1/ICOS Costimulatory Pathway
Transformed TEC (C1.1 cells) or primary cultures of murine renal TEC were cultured for 3 d in the presence or absence of recombinant IFN-{gamma} (100 U/ml), for induction of MHC class II antigens. Antigen presentation was then determined by using the previously described T cell hybridoma C10 (3), which is Iak-restricted and specific for hen (chicken) egg white lysozyme (HEL) (Sigma). Varying numbers (104 to 105) of Iak-positive C1.1 cells or primary TEC were cocultured with 105 C10 T hybridoma cells, in 96-well plates with 500 µg/well HEL. Cells were also preincubated with anti-B7RP-1, anti-ICOS, anti-CD40, or anti-Iak antibodies, for examination of the effects of costimulation blockade on antigen presentation. After 48 h, supernatants were collected and assayed for interleukin-2 (IL-2) contents, using a sensitive specific enzyme-linked immunosorbent assay (Amersham, Dübendorf, Switzerland).

Splenocyte Proliferation Assays
Splenocytes were prepared from C57BL/6 mice by using a standard protocol. Cells were counted and dispersed into flasks (5 x 107 cells in 4 ml of DMEM). Cells were then activated with anti-CD3 mAb (final concentration, 5 µg/ml) and incubated at 37°C for 1 h. Blocking antibodies for B7RP-1 or ICOS were added (final concentration, 10 µg/ml), and cells were further incubated for a total of 3 d. Proliferation was then measured by using a CellTiter 96 Aqueous One Solution assay (Promega, Madison, WI), according to the instructions provided by the manufacturer.

Immunohistochemical Staining of Human Kidney Tissue Samples for B7RP-1
Paraffin-embedded tissue samples from normal human kidneys (tumor nephrectomy specimens, n = 5) and biopsy material from kidneys with acute allograft rejection (n = 4), tubulointerstitial nephritis (n = 4), or minimal-change glomerulonephritis (n = 4) were examined for the presence of B7RP-1 by immunohistochemical staining. Tissue sections were pretreated with proteinase K for 10 min and then microwaved twice for 10 min at 600 W. The primary antibody (polyclonal anti-human B7RP-1) was used at a dilution of 1:50 (8 µg/ml), with an incubation time of 25 min. Sections were further processed by using the diaminobenzidine-based ChemMate peroxidase detection kit (Dako, Glostrup, Denmark) and the Dako 500 Plus TechMate immunohistochemical staining system, according to the instructions provided by the manufacturer. Hematoxylin was used to provide a clear blue counterstain.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of B7RP-1 and Other Costimulatory Molecules in Murine and Human TEC
Using fluorescence-activated cell-sorting analysis, we examined murine and human TEC for cell surface expression of B7RP-1, in comparison with B7.1, B7.2, and CD40. The murine macrophage cell line RAW 264.7 was used as a positive control cell line (classic antigen-presenting cells expressing B7RP-1, B7.1, B7.2, and CD40). Figure 1 demonstrates that MCT and HK-2 cells constitutively expressed large amounts of B7RP-1. The intensity of expression was comparable to that of RAW 264.7 cells. After overnight stimulation with LPS (100 ng/ml), B7RP-1 expression increased only slightly in MCT cells and did not increase in HK-2 cells. A slight upregulation of B7RP-1 was also detected in RAW 264.7 cells after LPS stimulation. Similar results were observed with TNF-{alpha} stimulation in all three cell types (data not shown).



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  		Figure 1. Flow cytometric analysis of murine macrophages (RAW 264.7) and murine and human tubular epithelial cell (TEC) lines (MCT and HK-2) for B7.1, B7.2, CD40, and B7RP-1 expression. The gray curves in panels for unstimulated cells represent control staining with FITC-labeled secondary antibody alone. The gray curves in panels for lipopolysaccharide (LPS)-stimulated cell lines represent control staining with unstimulated positive cells. In all panels, the black curves represent cells stained with primary and secondary antibodies. LPS (100 ng/ml) stimulation was performed overnight. These results are representative of three experiments that yielded similar data.

 
Figure 1 also demonstrates that MCT and HK-2 cells did not express B7.1 or B7.2, whereas CD40 was markedly expressed by both cell lines. LPS stimulation increased CD40 expression in MCT cells, whereas CD40 expression could not be further stimulated in HK-2 cells. No upregulation was observed for B7.1 or B7.2 when MCT and HK-2 cells were stimulated overnight with LPS or TNF-{alpha} (data not shown).

To corroborate these findings, we also performed Western blot analyses of B7RP-1 expression in TEC. As observed in Figure 2, a 60-kD protein was detected in MCT and HK-2 cells and also in control RAW 264.7 cells. B7RP-1 expression was slightly enhanced after overnight stimulation with LPS in the control cell line RAW 264.7 but not in MCT or HK-2 cells.



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  		Figure 2. Western blot analysis of B7RP-1 protein expression in the murine cell lines RAW 264.7 and MCT and the human cell line HK-2. B7RP-1 protein expression was analyzed in response to LPS (200 ng/ml, overnight). These results are representative of three experiments that yielded similar results. Co, control.

 
We then examined B7RP-1, B7.1, B7.2, and CD40 expression in TEC at the mRNA level with RT-PCR. Consistent with the flow cytometric data, B7RP-1 and CD40 mRNA transcripts were readily detected in MCT and HK-2 cells (Figure 3). Transcripts for B7RP-1 and CD40 were also detected in C1.1 cells (a different TEC line; see below) and in primary cultures of murine TEC. Faint and inconsistent B7.1 but not B7.2 mRNA staining was detected in all TEC (data not shown). Again, LPS stimulation did not significantly enhance B7RP-1 mRNA expression, whereas CD40 mRNA expression was upregulated (Figure 3). Stimulation by LPS, TNF-{alpha}, or IFN-{gamma} also failed to upregulate B7.1 and B7.2 mRNA expression (data not shown). In summary, TEC display a unique profile of costimulatory molecules (B7RP-1+, CD40+, B7.1-, B7.2-), which is different from that of classic antigen-presenting cells such as macrophages (B7RP-1+, CD40+, B7.1+, B7.2+).



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  		Figure 3. Reverse transcription (RT)-PCR analysis of CD40 and B7RP-1 mRNA in TEC lines (MCT and HK-2) and macrophages (RAW 264.7). Stimulation with LPS (100 ng/ml) was performed overnight. These results are representative of four similar experiments. Co, control.

 
Functional Significance of B7RP-1 on TEC
To determine whether renal TEC B7RP-1 plays a costimulatory role, we examined T cell activation in an antigen presentation assay, using a HEL-specific T cell hybridoma. In a control assay, we tested the expected inhibitory effects of anti-B7RP-1 and anti-ICOS antibodies on anti-CD3-activated murine splenocytes.

The TEC line C1.1 was chosen for its ability to present antigen (HEL) to a HEL-specific, Iak-restricted, T cell hybridoma (C10 cells). C10 cells release IL-2 when cocultured with Iak-positive C1.1 cells in the presence of HEL. We first confirmed the presence of B7RP-1 in C1.1 cells (Figure 4). As in MCT cells, B7RP-1 mRNA and protein were present but were not upregulated by overnight stimulation with LPS (Figure 4, A and C). We also confirmed the presence of the B7RP-1 receptor ICOS in C10 cells, at the mRNA level (Figure 4B) and at the cell surface (Figure 4D).



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  		Figure 4. (A) Northern blot analysis (top) of B7RP-1 gene expression in antigen-presenting TEC (C1.1 cells), with or without LPS stimulation (100 ng/ml, overnight), and ethidium bromide staining of the gel (bottom). (B) RT-PCR analysis of inducible costimulator (ICOS) mRNA expression in C10 cells. (C) Flow cytometric analysis of B7RP-1 in C1.1 cells. (D) Flow cytometric analysis of ICOS in C10 cells. 2° Ab, secondary antibody (control).

 
For the antigen presentation assay, C1.1 cells were pretreated for 3 d with recombinant IFN-{gamma} (100 U/ml), to upregulate Iak. The C1.1 cells were then cocultured with C10 cells in the presence of HEL. IFN-{gamma}-induced, Iak-positive, C1.1 cells activated C10 cells effectively in the presence of HEL, as evidenced by a robust IL-2 response (Figure 5). In the absence of HEL, C10 cells were not activated by Iak-positive C1.1 cells. Furthermore, MHC class II-negative tubular cells (not stimulated with IFN-{gamma}) did not activate Iak-restricted C10 cells in the presence of HEL.



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  		Figure 5. Antigen presentation by C1.1 cells and the role of B7RP-1/ICOS inhibition. C10 cells [hen egg white lysozyme (HEL)-specific, Iak-restricted; 1 x 105] were cocultured in 96-well plates with varying numbers of unstimulated C1.1 cells or C1.1 cells that had been prestimulated for 3 d with 100 U/ml recombinant interferon-{gamma} (IFN-{gamma}). HEL was used at 500 µg/well, and cells were incubated for 48 h. Cells were also preincubated with blocking antibodies to Iak, CD40, B7RP-1, and ICOS. The interleukin-2 (IL-2) contents in the supernatants were assayed with an enzyme-linked immunosorbent assay. These results are representative of three experiments that yielded similar results.

 
C1.1 cells were then preincubated with antibodies targeting B7RP-1 and/or ICOS, for investigation of the role of this costimulatory pathway in antigen presentation. Interestingly, blocking with an anti-B7RP-1 mAb led to enhanced antigen-specific IL-2 production (Figure 5). The effect on IL-2 production was even more pronounced when C10 cells were blocked with an anti-ICOS antibody. A two- to threefold increase in the production of IL-2 by C10 cells was detected when anti-B7RP-1 and anti-ICOS antibodies were used in combination. Blocking with a CD40 antibody did not affect the amount of IL-2 produced, whereas blocking with an anti-Iak antibody markedly reduced the amount of IL-2 produced, as expected. When we repeated the experiments with primary cultures of TEC instead of C1.1 cells, we observed the same results. Blocking with the anti-Iak antibody decreased the amount of IL-2 produced, whereas blocking with anti-ICOS yielded a twofold increase in the amount of IL-2 produced (Figure 6).



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  		Figure 6. Antigen presentation by primary cultures of murine renal TEC and the role of B7RP-1/ICOS inhibition. C10 cells (HEL-specific, Iak-restricted; 1 x 105) were cocultured in 96-well plates with varying numbers of unstimulated primary culture cells or primary culture cells that had been prestimulated for 3 d with 100 U/ml recombinant IFN-{gamma}. HEL was used at 500 µg/well, and cells were incubated for 48 h. Cells were also preincubated with blocking antibodies to Iak and ICOS. The IL-2 contents in the supernatants were assayed with an enzyme-linked immunosorbent assay.

 
In a control experiment, we then tested the role of B7RP-1 in T cell activation in a splenocyte proliferation assay. Anti-CD3-activated murine splenocytes were incubated with B7RP-1/ICOS-specific blocking mAb for 3 d. Table 1 demonstrates that proliferation was inhibited by blocking with anti-B7RP-1 and anti-ICOS mAb. These data suggest that B7RP-1/ICOS blockade may have either inhibitory or stimulatory effects, depending on the experimental setting.


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  		Table 1. Effect of B7RP-1/ICOS blockade on anti-CD3-activated splenocytesa
 
Expression of B7RP-1 in Human Kidney Tissue Samples
We also examined the renal expression of B7RP-1 in human kidneys by immunohistochemical analysis, using renal cell carcinoma nephrectomy material and renal biopsy tissue samples. In normal kidneys, B7RP-1 was expressed in various tubular segments, with a predominance in the distal segments. B7RP-1 was strongly expressed in distal convoluted tubules (Figure 7, A and E), connecting segments, and collecting ducts (Figure 7, B and C). Apical and basolateral staining was observed. Some cytoplasmic staining was also detected. B7RP-1 was also expressed in the multilayered urothelium (Figure 7D). B7RP-1 was only weakly expressed by proximal tubules in normal kidneys, and B7RP-1 was not observed in glomeruli (Figure 7E). The vascular smooth muscle cells in arteries were also B7RP-1-positive. Cells in the tubulointerstitial space exhibited negative results. Table 2 summarizes these findings.



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  		Figure 7. Immunohistochemical analysis of B7RP-1 expression in normal human kidney. (A) Positive B7RP-1 staining in distal convoluted tubules. (B) Longitudinal view of collecting ducts. (C) Cross-sectional view of collecting ducts and thin segments of Henle’s loop. (D) Multilayered urothelium in the renal pelvis. (E) Negative B7RP-1 staining in glomeruli; adjacent distal convoluted tubules exhibit positive staining. Magnification: x100 in A, C, D, and E; x25 in B.

 

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  		Table 2. Expression of B7RP-1 in normal human kidneya
 
We also examined renal biopsies from patients with acute renal allograft rejection, tubulointerstitial nephritis, or minimal-change glomerulonephritis. In biopsies from patients with tubulitis resulting from acute allograft rejection, there was some proximal tubular staining in areas of tubulitis (Figure 8A). In biopsies from patients with tubulointerstitial nephritis, there was also some staining for B7RP-1 in proximal tubules (Figure 8B). Glomeruli remained negative in those biopsies. In kidneys with minimal-change glomerulonephritis, the expression of B7RP-1 was not altered, compared with that in normal kidneys (Figure 8C). In renal cell carcinoma specimens, there was heterogeneous expression of B7RP-1; most tumor cells were negative, but the cells occasionally exhibited weak staining for B7RP-1 (Figure 8D). Of interest, B7RP-1 was also expressed in the epithelium of a prostate gland with myoglandular hyperplasia (Figure 8E).



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  		Figure 8. Analysis of B7RP-1 expression in diseased human kidney. (A) Acute renal allograft rejection, demonstrating weakly positive proximal tubules adjacent to distal tubules, which are stained more intensely. (B) Tubulointerstitial nephritis, demonstrating focally positive proximal tubules adjacent to more intensely stained distal tubules. (C) Unchanged B7RP-1 expression in a kidney with minimal-change glomerulopathy. (D) Heterogeneous staining for B7RP-1 in a renal cell carcinoma (clear cell type). (E) Prostate epithelium with myoglandular hyperplasia, displaying strong epithelial B7RP-1 staining. Magnification: x100 in A, B, D, and E; x50 in C.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous studies, we and others demonstrated that renal parenchymal cells such as TEC have the capacity to present antigens to T cells (3–5). In general, the antigen-specific activation of T cells requires MHC class II molecule expression by antigen-presenting cells and a second signal via the B7/CD28 costimulatory pathway (7). In most studies, the classic costimulatory molecules B7.1 and B7.2 could not be detected in TEC in vitro or in vivo (8–10). Therefore, it was concluded that the B7/CD28 costimulatory pathway was not important for T cell activation by TEC. It was even postulated that the lack of B7 on TEC could confer a state of anergy to T cells (12,25). Controversy has arisen regarding whether TEC play a significant role in T cell activation and subsequent tubulointerstitial injury (13). It was thought that another costimulatory pathway could be involved (8) or that small amounts of B7.1, which were observed on cultured TEC after intense stimulation with IFN-{gamma} and LPS, could be responsible for the activation of T cells by TEC (26,27).

We now demonstrate that the B7-related cell surface molecule B7RP-1 (B7h) is constitutively expressed by cultured TEC. Large amounts of B7RP-1 could be detected in murine and human TEC lines, as well as in primary cultures of murine TEC. In addition, B7RP-1 could be detected in various nephron segments, including distal convoluted tubules and collecting ducts, in human kidneys. In immune injury, proximal tubular segments were also B7RP-1-positive in areas of mononuclear infiltration, suggesting that B7RP-1 expression is upregulated in vivo. This is the first time that the epithelial expression of B7RP-1 has been clearly demonstrated. The predominantly distal tubular expression of B7RP-1 in vivo contrasts somewhat with the expression on cultured TEC, which exhibits mostly proximal tubular characteristics. However, it is known that these cell lines also have some distal tubular characteristics, including, for example, vasopressin responsiveness (3).

In agreement with other studies (8–10), we did not observe significant B7.1 or B7.2 expression by TEC. CD40, a ligand for CD154 (CD40L) on T cells, was detected on all TEC tested, however. This is in agreement with findings from other studies, which also demonstrated CD40 on TEC in vitro and on proximal tubules in vivo (15,28,29). CD40/CD154 interactions may provide a costimulatory signal for T cells, although the importance of these interactions has not been examined for TEC.

We then tested the functional significance of B7RP-1 expression by TEC in an antigen presentation assay, using SV40-transformed TEC (C1.1 cells) and primary cultures of TEC. In previous studies, we observed that C1.1 cells and primary cultures of TEC could present HEL to Iak-restricted T cell hybridomas (3). We observed here that blocking antibodies to B7RP-1 and/or ICOS did not inhibit T cell activation, as assessed on the basis of IL-2 production, but rather increased IL-2 production by the T cell hybridoma. This suggests that the B7RP-1/ICOS pathway could inhibit T cell activation upon interaction with TEC. B7RP-1 blockade did not enhance IL-2 production as much as did ICOS blockade, but this could be attributable to differences in the blocking efficiencies of the various antibodies. In control experiments with anti-CD3-activated splenocytes, we observed that anti-B7RP-1/anti-ICOS had inhibitory effects on proliferation, as expected. These findings demonstrate that ICOS signaling is stimulatory in splenocytes activated by anti-CD3. Together, these results suggest that the B7RP-1/ICOS pathway could have dual effects on T cell activation, i.e., an activating effect when T cells are stimulated by classic antigen-presenting cells and a negative effect when T cells are stimulated by nontraditional antigen-presenting cells, such as TEC. With respect to the TEC/T cell system, the B7RP-1/ICOS pathway resembles the B7/CTLA-4 pathway, which also has known inhibitory effects on T cell activation (30).

In our experiments, we used T cell hybridomas rather than T cell clones for examination of the costimulatory role of B7RP-1/ICOS, because we observed in earlier studies that hybridomas display a proliferative response when activated by Iak-positive TEC, whereas T cell clones become anergic (3,9,12). The reason why T cell hybridomas are much easier to activate than T cell clones is not clear. Comparative studies using T cell clones are now underway, to determine whether anergy in T cell clones can be prevented by B7RP-1/ICOS blockade.

Because TEC lack B7 and because B7RP-1 on TEC mediates negative signaling in T cell hybridomas, we still need to identify the nature of the costimulatory signal that is required to activate T cell hybridomas. One possibility could be that CD40 plays a role in the costimulation provided by TEC. With anti-CD40 antibodies, however, we could not observe an inhibitory effect in IL-2 production by T cell hybridomas. Therefore, other TEC surface molecules must be examined, including some of the newer B7-related proteins, such as PD-L1 and PD-L2 (31,32).

The mechanisms of the inhibitory effect via B7RP-1 and ICOS must also be investigated. Others have demonstrated that IL-4 and IL-10 can be upregulated in T cells via the ICOS pathway (19,33). These inhibitory cytokines could explain immune deviation, whereby T cell activation could be reduced via ICOS signaling (34). Studies are also in progress to test the hypothesis that cytokine patterns change after engagement of ICOS on T cells by B7RP-1 on TEC.

Very recent data on murine experimental allergic encephalomyelitis (EAE), a model of autoimmune multiple sclerosis, are consistent with our data demonstrating an inhibitory role of B7RP-1/ICOS. In one study, ICOS-knockout mice demonstrated greatly enhanced susceptibility to EAE, indicating that ICOS has a protective role in this T cell-mediated autoimmune disease (35). In another study, ICOS blockade during antigen priming (1 to 10 d after immunization) exacerbated EAE, whereas blockade during the efferent immune response (9 to 20 d after immunization) abrogated disease (36). Excessive IFN-{gamma} production with priming blockade could be demonstrated in splenocytes from diseased mice, suggesting that an altered cytokine pattern, with predominant IFN-{gamma} production, could be the mechanism for accentuated autoimmunity. These data, together with our findings, suggest that ICOS could play an important physiologic role in negatively regulating the early afferent immune response.

In vivo, we observed upregulation of B7RP-1 by proximal tubular segments, in close association with an infiltrate. This suggests that TEC B7RP-1 could play an important regulatory role in T cell activation in vivo, preventing overactivation of the immune system in areas of T cell infiltration. It could even be speculated that B7RP-1/ICOS interactions between TEC and T cells could represent a mechanism for peripheral tolerance, whereby autoimmune processes are prevented.

In summary, we have demonstrated that the recently described ICOS ligand B7RP-1 is abundantly expressed by TEC in vitro and in vivo. Furthermore, we could demonstrate that the engagement of B7RP-1 with ICOS, in the context of TEC antigen presentation, had an inhibitory effect on T cell activation. The mechanisms of inhibition and the consequences for peripheral tolerance must be investigated further.


   Acknowledgments
 
This study was supported by the Swiss National Science Foundation (Grant 31-63551.00, to Dr. Wüthrich) and by a local grant from the Kantonsspital St. Gallen. We thank M. Hell for assistance with the immunohistochemical analyses and L. Bonetti for help with the illustrations. Part of this study was presented at the annual meeting of the American Society of Nephrology in San Francisco, October 10–17, 2001, and was published in abstract form (37).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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