Suppressed T-cell activation by IFN-γ-induced expression of PD-L1 on renal tubular epithelial cells
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(2) 2714. PD-1 on T cells, T-cell proliferation and cytokine production is inhibited [11]. Here, we demonstrate that PD-L1 is markedly upregulated in cultured TEC in response to IFN-g and that it exhibits an inhibitory function on TECinduced T-cell activation. PD-L1 is also upregulated on cortical tubules in mouse kidney transplants during rejection, suggesting that PD-L1 could influence T-cell activation in vivo.. Subjects and methods General reagents Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) and Sigma (St Louis, MO). Blocking monoclonal antibodies (mAbs) specific for murine PD-1 (clone J43, Armenian hamster IgG), PD-L1 (clone MIH5, rat IgG2a) and PD-L2 (clone TY25, rat IgG2a) were obtained from eBioscience (San Diego, CA). Monoclonal antibodies specific for murine I-Ak, I-Ek and FITC-labelled Armenian hamster antibodies were obtained from BD PharMingen (San Diego, CA). Anti-rat IgG antibodies (goat, affinity-purified) were purchased from Sigma. A rat IgG control antibody (BD PharMingen) was used as an irrelevant antibody in the antigen presentation assays. Recombinant mouse IFN-g was purchased from R&D Systems (Oxford, UK). Lipopolysaccharide derived from Escherichia coli and recombinant mouse tumour necrosis factor-a (TNF-a) was purchased from Sigma.. R. Schoop et al.. harvested, washed twice with Hanks’ balanced salt solution and blocked with 1 phosphate-buffered saline (PBS) containing 2% FBS and 0.1% azide. TEC were tested by flow cytometry for surface expression of PD-L1 and PD-L2 as described previously [14]. T-cell clones (A.E7 and D10.G4.1) were stained for PD-1 using a similar staining protocol.. Western blot analysis TEC were stimulated for 0, 4 or 8 h with IFN-g (500 U/ml). Cells were washed with 1 PBS and lysed with mammalian protein extraction reagent (M-PERTM ; Pierce, Rockford, IL). After shaking gently for 5 min, the lysates were centrifuged at 13 800 g for 10 min and the protein concentration of the supernatant was measured spectrometrically at 280 nm. Supernatants were mixed with 4 reducing sample buffer (0.38 M Tris base, 8% SDS, 8% 2-mercaptoethanol, 4 mM EDTA, 40% glycerol, 0.05% bromophenol blue) and were heated for 5 min at 95 C. Equal amounts of protein extracts were separated by 12.5% SDS–PAGE. Proteins were then transferred to nitrocellulose membranes (0.45 mm pore size; Bio-Rad, Hercules, CA). Quantitative protein transfer was checked by incubating blots in Ponceau S staining solution (0.1% w/v in 5% acetic acid). The gel was stained with Coomassie blue to ensure that protein transfer to the membrane was complete (data not shown). PD-L1 protein was detected by chemiluminescence (ChemiLucentTM western blot detection system; Chemicon International, Ternecula, CA). Emitted light was detected on X-ray film (X-OMAT; Kodak, Rochester, NY).. Cell lines. RNA extraction and RT–PCR analysis. The SV40-transformed murine renal TEC lines C1.1 and MCT were used to study the expression of PD-L1 and PD-L2 [1,12]. Primary cultures of murine renal TEC were prepared from AKR/J mice as described previously [1]. Primary cultures of TEC and C1.1 were cultured on collagen-coated plates in modified K1 medium with 5% fetal bovine serum (FBS). MCT cells were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax-1 and 10% FBS [1]. The C10 T-cell hybridoma (hen-egg lysozyme-specific, I-Ak-restricted) was grown in DMEM with 10% FBS [1]. A.E7, a CD4þ Th1 T-cell clone, and D10.G4.1, a CD4þ Th2 T-cell clone, were kindly provided by L.H. Glimcher (Boston, MA). Both clones were maintained as described previously [13]. Cells were cultured by repeated stimulation every 3–4 weeks with irradiated splenocytes from AKR/J mice and either 60 ng/ml pigeon cytochrome C (A.E7) or 100 mg/ml conalbumin (D10.G4.1) in RPMI-1640 supplemented with 10% fetal calf serum, 50 mM 2-mercaptoethanol and 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% (vol/vol) conditioned medium from cultures of concanavalin A (con A)-stimulated rat splenocytes (Rat T STIMTM without con A; Becton Dickinson, San Jose, CA). Tcell clones were used for antigen presentation experiments 5–10 days after stimulation. All cells were grown at 37 C with 5% CO2.. Total RNA was extracted from C1.1 and MCT cell lines (RNeasyÕ mini kit; Qiagen, Valencia, CA). Cells were homogenized by passaging cell lysates through a shredder column and were further processed according to the manufacturer’s protocol. All samples were quantitated by the measurement of the optical density at 260 nm and equal amounts were amplified by reverse transcription–polymerase chain reaction (RT–PCR) (QiagenÕ OneStep PCR kit; Qiagen). Specific primer sequences were determined using the Primer 3 software (http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi) for amplification of murine PD-1, PD-L1 and PD-L2. To ensure even amounts of template, the rat housekeeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH) was co-amplified as described previously [15]. Reverse transcription was performed at 50 C for 35 min. An initial PCR activation step was performed at 95 C for 15 min. The following 28 amplification cycles consisted of denaturation at 94 C, annealing at 58 C and extension at 72 C, each step running for 1 min, before the final extension took part at 72 C for 10 min. Reaction mixtures were separated on 1.5% agarose gels containing ethidium bromide and bands were detected under UV light. Gels were analysed using densitometry software (Scion Image; Scion Corp., Frederick, MA).. Antigen presentation assay Flow cytometric analysis TEC were stimulated with IFN-g (500 U/ml) for various time intervals (1–32 h). TEC were then lightly trypsinized,. Antigen presentation was performed as described previously [14]. Briefly, TEC were stimulated with IFN-g for 3 days to induce expression of I-Ak and I-Ek. Cells were then.
(3) PD-1 ligand on tubular epithelial cells. co-cultured with C10 T-cells and antigen (3.3 mg/ml hen-egg lysozyme) or Th-cell clones (A.E7 or D10.G4.1) and antigen (100 mg/ml pigeon cytochrome C for A.E7 and 60 ng/ml conalbumin for D10.G4.1). Blocking anti-murine PD-1 and/or anti-murine PD-L1 mAbs (10 mg/ml) were added to test the role of PD-1/PD-L1 in antigen presentation. Supernatant was collected after 24 h and detection of interleukin-2 (IL-2) (C10), IFN-g (A.E7) or IL-4 (D10.G4.1) production was performed using cytokine-specific enzymelinked immunosorbent assays (ELISAs) (OptEIATM mouse IFN-g/IL-4/IL-2 kits; PharMingen).. Mouse kidney transplantation Male BALB/c (H-2d) donor mice weighing 25–30 g were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) given intraperitoneally. The kidney graft harvesting was performed as described previously [16,17]. The kidney and its vascular supply with the ureter were removed en bloc and stored in lactated Ringer’s solution at 4 C. The recipient’s native left kidney was then removed and the donor kidney was transplanted in the right flank. After completing venous and arterial anastomoses, the recipient bladder was pierced with a 20-gauge needle allowing curved forceps to be inserted through the bladder to pull the ureter through. The periureteral tissue was stitched to the exterior wall of the bladder and the ureter was allowed to retract inside the bladder. The abdomen was then closed in a single layer with a continuous 5–0 silk suture. Mice were given 1.5 ml normal saline subcutaneously after surgery and were placed on a heat pad until recovery.. Immunofluorescence staining of mouse kidney tissue samples After 5 days, the transplanted kidneys were excised and shockfrozen in liquid nitrogen. Cryostat sections (5 mm thick) were air-dried and fixed in acetone at 0 C. After rehydration in 1 PBS, the sections were incubated overnight at 4 C with the primary antibody. Primary antibodies were diluted in 1 PBS supplemented with 1% bovine serum albumin. Anti-PD-L1 (clone MIH5) and anti-PD-L2 (clone TY25) antibodies were used for staining. After rinsing in 1 PBS, the sections were incubated for 1 h at room temperature with the Cy3-labelled (red fluorescence) secondary antibody in 1 PBS supplemented with phalloidin-FITC (Molecular Probes Europe BV, Leiden, The Netherlands) and 4,6-diamino-2-phenylindole dihydrochloride (DAPI; Boehringer, Mannheim, Germany). Phalloidin-FITC (green fluorescence) binds to filamentous actin and DAPI (blue fluorescence) binds to DNA. Together they provide a counterstain that ameliorates the identification of structures in the tissue. After rinsing, the sections were mounted using an aqueous mounting medium (Immu-mount; Shandon, Pittsburgh, PA). CD3 on infiltrating T cells was detected by immunofluorescence in cryosections using a rat mAb (clone KT3). The second antibody was Cy3-labelled mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).. Statistics Results for mean channel fluorescence of flow cytometry are expressed as means ± SEM of three separate experiments.. 2715. Results from cytokine ELISAs are expressed as means±SEM of triplicate determinations from one experiment representative of at least three similar experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA). Significance was accepted at P 0.05.. Results Expression and inducibility of PD-L1 and PD-L2 on murine TEC The cell surface expression of PD-L1 and PD-L2 was analysed by flow cytometry. Figure 1A and B show that PD-L1 was constitutively expressed on SV40-transformed TEC (C1.1) and by primary cultures of TEC (primary TEC). Induction of TEC with IFN-g (500 U/ml) showed a notable increase in the expression of PD-L1 in C1.1 and in primary TEC. Treatment with TNF-g did not upregulate PD-L1 (data not shown). In contrast to PD-L1, the expression of PD-L2 was negative on C1.1 and primary TEC, even after prolonged stimulation with IFN-g (Figure 1C and D). Figure 2A and B show a time course of IFN-ginduced upregulation of PD-L1 in an additional TEC line (MCT). Upon stimulation with 500 U/ml IFN-g there was a significant upregulation of PD-L1 after 4 h. Half-maximal stimulation was achieved at 8 h with a plateau being reached after 32 h (Figure 2B). To confirm that the detected inducibility of PD-L1 corresponds to an increased production of PD-L1 protein, we verified the flow cytometric data by western blot analysis. As shown in Figure 3A, the PD-L1 band at 50 kDa became more prominent after stimulating MCT cells with IFN-g for 4 and 8 h. We also examined expression of PD-L1 transcripts in MCT by RT–PCR analysis (Figure 3B). A significant increase in PD-L1 mRNA was observed after 1 h of stimulation with IFN-g. PD-L2 transcripts could not be found even after prolonged stimulation with IFN-g (data not shown). Amplification of the housekeeping gene GAPDH confirmed that approximately equal amounts of templates were used. Densitometrical quantification of detected bands showed that maximal transcription was reached after 6 h. Functional role of PD-1/PD-L1 interaction in T-cell activation Using antigen presentation assays, we then examined the functional relevance of PD-L1 in TEC-mediated T-cell activation. SV40-transformed TEC (C1.1) or primary TEC were incubated for 72 h with IFN-g to express MHC class II molecules (I-Ak and I-Ek) and were then loaded with specific foreign antigens. The T cells (C10, A.E7 or D10.G4.1) and TEC were co-cultured for 24 h and cytokines were subsequently assayed in the supernatants. Figure 4 demonstrates that the production of IL-2 by C10 T cells is enhanced in the presence but not the.
(4) 2716. R. Schoop et al.. Fig. 1. Flow cytometric analysis for PD-L1 and PD-L2 in TEC. C1.1 (A and C) and primary cultures of TEC (B and D). Constitutive expression of PD-L1 and its induction upon stimulation with IFN-g (500 U/ml for 24 h) is demonstrated for both cell lines (A and B). PDL2 surface expression was not detected on either C1.1 cells (C) or on primary cultures of TEC (D).. Fig. 2. Time course of IFN-g-induced cell surface expression of PD-L1 on MCT cells. Flow cytometric analysis after 8 and 22 h of stimulation with 500 U/ml IFN-g. The figure is representative of three separate experiments (A). Mean channel fluorescence values of three separate experiments after 4–32 h stimulation with 500 U/ml IFN-g (B).. Fig. 3. Western blot analysis of PD-L1 protein induction in response to stimulation with 500 U/ml IFN-g for 4 and 8 h (A). RT–PCR analysis of PD-L1 and GAPDH mRNA in response to 1–10 h stimulation with 500 U/ml IFN-g. Numbers between gels represent normalized densitometric ratios (PD-L1/GAPDH) (B)..
(5) PD-1 ligand on tubular epithelial cells. absence of antigen (hen-egg lysozyme). Addition of a mAb targeting I-Ak inhibited the increase in IL-2 completely, as expected. A slight increase of IL-2 was seen when PD-1 was blocked; however, a significant increase was detected when PD-L1 was blocked. Although blocking with anti-PD-1 did not lead to a significant increase in IL-2 production when used alone, it did have a significant effect when used in combination with anti-PD-L1. We presume that this could be due to different binding affinities of these antibodies. Another possibility could be that PD-L1 is capable of binding to another receptor on T cells.. 2717. Similar findings were obtained with the Th1 and Th2 clones and primary TEC. Figure 5A demonstrates a robust IFN-g production by A.E7 (I-Ek restricted Th1) T cells in the presence but not in the absence of antigen (pigeon cytochrome-c). When anti-PD-1 was used in combination with anti-PD-L1, the IFN-g response was markedly increased. Using anti-PD-L1 mAb or antiPD-1 alone resulted in a lesser increase in IFN-g production (data not shown). Complete inhibition of IFN-g production was seen when blocking with anti-I-Ek. Figure 5B shows data using the D10.G4.1 (I-Ak restricted Th2) T-cell clone. Again, a strong cytokine response (IL-4) was obtained in the presence but not in the absence of antigen (conalbumin A). A marked increase of IL-4 was detected when anti-PD-L1 was used in combination with anti-PD-1 mAbs. Blocking with anti-I-Ak inhibited IL-4 production, as expected. Together, these findings suggest that the PD-L1/PD-1 pathway negatively regulates both Th1 and Th2 cell responses when TEC are involved as antigen-presenting cells. Immunofluorescence staining of PD-1 ligands in mouse kidney tissue samples. Fig. 4. Antigen presentation assay with IFN-g-induced (500 U/ml, 3 days) TEC (C1.1 cells) and C10 T-cells. In the absence of antigen (hen-egg lysozyme), the production of IL-2 by C10 cells is marginal. In the presence of antigen, the anticipated increase of IL-2 production was inhibited with the anti-I-Ak mAb, whereas the IL-2 production was enhanced in the presence of blocking mAbs for PD-1 and PD-L1. *P<0.05 and **P<0.01 when compared with control (ANOVA).. We then performed immunofluorescence stainings of mouse kidneys to examine PD-L1 and PD-L2 expression in vivo. In the cortex of allotransplants with acute cellular rejection, PD-L1 was abundantly expressed in all proximal and distal tubules as well as in the glomerular tuft and Bowman’s capsule (Figure 6B). In the medulla the strongest expression was found in thick ascending limbs whereas the collecting ducts were weakly stained (data not shown). To prove that the upregulation of PD-L1 was due to allogenicity and not to the surgical intervention itself,. Fig. 5. Antigen presentation assay with IFN-g-induced (500 U/ml, 3 days) primary TEC and Th1 cells (A.E7) and Th2 cells (D10.G4.1). IFN-g was assayed for Th1 cells (A) and IL-4 for Th2 cells (B). Blocking mAbs for PD-1 and PD-L1 or an irrelevant mAb (co) were used at 10 mg/ml. Insets show the presence of PD-1 on A.E7 and D10.G4.1 (flow cytometric analysis). **P 0.01 when compared with control (ANOVA)..
(6) 2718. R. Schoop et al.. Fig. 6. Immunofluorescence detection of PD-L1 in mouse kidney transplants. Control sections in which the primary antibody was omitted are shown for allogenic (A) and syngenic (C) kidneys. PD-L1 was strongly expressed in all tubular profiles and in glomeruli (g) of rejected allogenic transplants (B), but undetectable in grafts from syngenic donors (D). Additionally, endothelial cells of arteries (E) and arterioles (F) were strongly labelled in rejected organs but not in syngenic grafts (G).. kidneys from syngenic strains were also stained for PD-L1. In syngenic transplants no PD-L1 was detected (Figure 6D). Staining for PD-L2 gave no relevant signals in any kidney tissue tested (data not shown). PD-L1 staining was also found on vascular endothelial cells of arteries (Figure 6E) and arterioles (Figure 6F). Apart from the PD ligands, we examined in transplanted kidneys the expression of further proteins that potentially play a role in the interaction of T cells and renal cells (data not shown). The interstitium of rejected grafts was dilated and contained large amounts. of T cells (CD3þ) and antigen-presenting cells (MHC class IIþ). The interstitium of isogenic grafts contained increased amounts of infiltrating cells as compared with normal kidneys, although much less than in rejected grafts. Figure 7 demonstrates the low incidence of T cells in isogenic grafts (Figure 7A) in contrast to the increased accumulation of T cells in rejected grafts (Figure 7B). Tubular expressions of MHC class I, MHC class II, VCAM-1 and ICAM-1 were diffusely and strongly upregulated in rejected kidneys. The co-stimulatory molecules CD80 and CD86 were not detected in tubules. In rejected grafts, CD80 was found.
(7) PD-1 ligand on tubular epithelial cells. Fig. 7. Distribution of T cells in the cortex of an isogenic graft and a heterologous graft using immunofluorescent staining. The presence of infiltrating T cells, as determined by a-CD3 antibody, is clearly seen in the cortex of an isogenic graft (A) and of a heterologous graft (B). T cells were detected by immunofluorescence in cryosections with a-CD3. Bar: 100 mm.. in the vasa recta and CD86 was present in a few interstitial cells.. Discussion PD-L1 and the related molecule PD-L2 have significant homologies with the B7 family of co-stimulatory molecules. Upon interaction with PD-1 on T cells, both molecules are capable of providing a negative signal for T-cell activation. This inhibitory effect has been shown in the setting of T-cell activation by dendritic cells [18] and by endothelial cells [19]. Thus far, the epithelial expression of PD-L1 and PD-L2 has not been investigated. Our observations clearly demonstrate that renal tubular epithelial cells express PD-L1 but not PD-L2 and that this expression is regulated by IFN-g. Co-stimulatory and co-inhibitory signals determine the strength of the T-cell activation process [3–6]. We, and others, have reported previously that the classical co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) are not expressed by TEC in vitro and in vivo. 2719. [2,14]. Other TEC accessory pathways, including CD40/CD154, ICAM-1/LFA-1, VCAM-1/VLA-4 or CD2/LFA-3, could mediate an interaction of TEC with T cells [20]. It is believed, however, that these pathways do not provide a true second signal but will rather help to sustain the response of activated T cells. In an attempt to identify other co-stimulatory pathways that might be operating in TEC-mediated T-cell activation, we recently examined the tubular expression of B7RP-1, a novel member of the B7 family. Contradictory to our expectations, we found that B7RP-1, through interaction with its ligand ICOS on T cells, also provides an inhibitory signal for T-cell activation in the setting of renal tubular antigen presentation [14]. Thus, a true co-stimulatory signal that is provided by TEC still remains to be identified. Expanding our research to the PD-L1/PD-1 pathway, we have now identified yet another inhibitory signal for T-cell activation in an assay where TEC present foreign protein antigen to CD4þ Th1 and to Th2 cells. These data, together with our previous work on TEC-mediated T-cell activation, demonstrate an intricate interaction scheme between TEC and T cells. The net effect of signalling through these different pathways between TEC and T cells is also complex. It is the balance of co-stimulatory and inhibitory signals that determines the final outcome of the immune response in the kidney. PD-1, the binding partner of PD-L1 and PD-L2, is an immunoinhibitory receptor expressed by T, B and myeloid cells [5]. Initially, it was felt that PD-1 plays a role in apoptosis, as it was originally identified in apoptotic T cell hybridomas [8]. However, it was found that the overexpression of PD-1 did not lead to apoptosis and that its expression correlated more with T-cell activation than with apoptosis [21]. Recent studies have shown that PD-1 downregulates immune responses and that its loss leads to a breakdown of peripheral tolerance. Thus, PD-1 knockout mice develop a lupus-like disease with glomerulonephritis and arthritis [9]. Furthermore, data from Ansari et al. [22] showed that PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in pre-diabetic female non-obese diabetic (NOD) mice. They also found that PD-L1 but not PD-L2 was expressed on inflammed islets of NOD mice. Recent data in the transplantation setting also exemplify the inhibitory nature of PD-L1/PD-1 interactions. For example, PD-L1/PD-1 blockade in conjunction with chemokine targeting leads to rejection and abrogates long-term survival of islet allografts [23]. Using skin allografts, it could be shown that blocking with anti-PD-L1 mAb leads to a significant increase in T-cell expansion and reduced apoptosis [24]. In the setting of graft-vs-host disease studies, it could be demonstrated that PD-1 blockade potently enhances T-cell alloresponses [25]. In certain instances, PD-L1/PD-1 interactions may also provide a stimulatory signal for T-cell activation. Thus, studies by Dong et al. [26] using resting T cells and PD-L1 Ig fusion protein document that PD-L1 provides a positive signal, stimulating T-cell proliferation.
(8) 2720. and inducing IL-10 production. One important difference is the use of resting cells in most of the studies that document a co-stimulatory rather than an inhibitory function for PD-1. However, some experiments with resting T cells also show inhibition. One possible explanation might be that there is a second receptor for the PD-1 ligands with the capacity to deliver a stimulatory signal, analogous to the CD28/CTLA-4 molecules. Another important aspect in determining the signalling function of PD-L1/PD-1 interaction is whether antibodies or fusion proteins are used. In summary, whether this ligand pair generates a co-inhibitory or a co-stimulatory signal may depend on additional factors that need to be investigated further. In summary, we have identified a novel co-inhibitory pathway of renal TEC-induced Th-cell activation, namely the PD-L1/PD-1 pathway. PD-L1 is effectively upregulated by IFN-g and provides a negative signal for foreign antigen-induced T-cell activation. As PD-L1 is also upregulated in vivo on cortical TEC, we speculate that it might negatively regulate T-cell responses and protect the renal epithelium from immune-mediated tubulointerstitial injury. The PD-L1/PD-1 pathway might, thus, be comparable to the B7/CTLA-4 pathway, which also provides an inhibitory signal in classical antigen presenting systems. Future work will need to address how PD-L1 acts in conjunction with the other co-stimulatory and co-inhibitory molecules and how it may be influenced therapeutically to prevent or moderate immune renal injury. Acknowledgements. Special thanks are due to Brandon Sullivan for his help with the T-cell clones. We would also like to thank L. Bonetti for help with the illustrations. Part of this study was presented at the annual meeting of the Swiss Society of Nephrology (SGN) in St Gallen, 2002. This study was supported by the Swiss National Science Foundation (Grant 31-63551.00 to R.P.W.) and by a local grant from the Kantonsspital St Gallen. Conflict of interest statement. None declared.. References 1. Wu¨thrich RP, Glimcher LH, Yui MA, Jevnikar AM, Dumas SE, Kelley VE. Generation of highly differentiated murine renal tubular epithelial cell lines: MHC class II regulation, antigen presentation and tumor necrosis factor production. Kidney Int 1990; 37: 783–792 2. Hagerty DT, Evavold BD, Allen PM. Regulation of the costimulator B7, not class II major histocompatibility complex, restricts the ability of murine kidney tubule cells to stimulate CD4þ T cells. J Clin Invest 1994; 93: 1208–1215 3. Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol 2002; 2: 116–126 4. Bugeon L, Dallman MJ. Costimulation of T cells. Am J Respir Crit Care Med 2000; 162: 164–168 5. Greenwald RJ, Latchman YE, Sharpe AH. Negative coreceptors on lymphocytes. 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J Exp Med 2000; 192: 1027–1034 12. Haverty TP, Kelly CJ, Hines WH et al. Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J Cell Biol 1988; 107: 1359–1368 13. Lederer JA, Liou JS, Todd MD, Glimcher LH, Lichtman AH. Regulation of cytokine gene expression in T helper cell subsets. J Immunol 1994; 152: 77–86 14. Wahl P, Schoop R, Bilic G et al. Renal tubular epithelial expression of the costimulatory molecule B7RP-1 (inducible costimulator ligand). J Am Soc Nephrol 2002; 13: 1517–1526 15. Benz N, Fan X, Wu¨thrich RP. Enhanced CD44 expression in MRL-lpr lupus nephritis. Kidney Int 1996; 50: 156–163 16. Fisher B, Lee S. Microvascular surgical techniques in research, with special reference to renal transplantation in the rat. Surgery 1965; 58: 904–914 17. Zhang Z, Schlachta C, Duff J, Stiller C, Grant D, Zhong R. Improved techniques for kidney transplantation in mice. Microsurgery 1995; 16: 103–109 18. Brown JA, Dorfman DM, Ma FR et al. Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 2003; 170: 1257–1266 19. Mazanet MM, Hughes CC. B7-H1 is expressed by human endothelial cells and suppresses T cell cytokine synthesis. J Immunol 2002; 169: 3581–3588 20. Rubin-Kelley VE, Wu¨thrich RP. Antigen presentation by renal parenchyma. In: Neilson EG and Couser WG, eds Immunologic Renal Diseases 2001; Lippincott-Raven Publishers, Philadelphia, 1997; 195–205 21. Vibhakar R, Juan G, Traganos F, Darzynkiewicz Z, Finger LR. Activation-induced expression of human programmed death-1 gene in T-lymphocytes. Exp Cell Res 1997; 232: 25–28 22. Ansari MJ, Salama AD, Chitnis T et al. The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 2003; 198: 63–69 23. Lee I, Wang L, Han R, Wells AD. Targeting the chemokine MCP-1/CCR2 chemokine pathway induces permanent survival of Islet allografts through a PD-L1-dependent mechanism. Am J Transplant 2003; 3 [Suppl 5]: 551 24. Sander SE, Salama AD, Kewalramani R, Yagita H. Functions of the regulatory T cell costimulatory pathways CTLA-4 and PD-1 in alloreactive CD4þ T cell responses in vivo. Am J Transplant 2003; 3 [Suppl 5]: 152 25. Blazar BR, Carreno BM, Panoskaltsis-Mortari A et al. Blockade of programmed death-1 engagement accelerates graft-versus-host disease lethality by an IFN-g-dependent mechanism. J Immunol 2003; 171: 1272–1277 26. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999; 5: 1365–1369 Received for publication: 5.11.03 Accepted in revised form: 23.6.04.
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