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The Journal of Immunology, 2007, 179, 7466 -7477
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Autoimmune Diseases
*Kidney Diseases

Programmed Death 1 Ligand (PD-L) 1 and PD-L2 Limit Autoimmune Kidney Disease: Distinct Roles1

Julia Menke2,*, Julie A. Lucas2,*, Geraldine C. Zeller*, Mary E. Keir{dagger}, Xiao R. Huang{ddagger}, Naotake Tsuboi§, Tanya N. Mayadas§, Han Y. Lan{ddagger}, Arlene H. Sharpe{dagger} and Vicki R. Kelley3,*

* Laboratory of Molecular Autoimmune Disease, Renal Division, and {dagger} Department of Pathology, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA 02115; {ddagger} Department of Medicine, University of Hong Li Ka Shing Facility of Medicine, Hong Kong, China; and § Department of Pathology, Center of Excellence in Vascular Biology, Harvard Medical School, Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell- and macrophage (M{phi})-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1–/–), PD-L2 (PD-L2–/–), or both (PD-L1/L2–/–) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L–/– strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1–/– and PD-L2–/– mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68+ cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2–/– mice, but not PD-L1–/– mice. In contrast, we detected a rise in activated CD8+ T cells in PD-L1–/– mice, but not PD-L2–/– mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophage (M{phi})4 and T cells mediate injury and are prominent in nephrotoxic serum nephritis (NSN), an experimental model of autoimmune kidney disease (1, 2, 3). The interaction of M{phi} with self-reactive T cells and/or the interaction of kidney parenchymal cells with these invading leukocytes determine the extent of renal injury. Since the vast majority of humans do not develop kidney disease, there are undoubtedly layers of negative regulatory checkpoints that shield the kidney and preserve normal renal function.

One newer member of the B7:CD28 family, programmed death 1 (PD-1; also called CD279), which binds to two ligands, PD-1 ligand (PD-L1; also called B7-H1 and CD274) and PD-L2 (also called B7-DC and CD273), delivers negative regulatory signals that terminate immune responses (4). PD-1 is inducibly expressed on CD4+ T cells, CD8+ T cells, NK T cells, B cells, and monocytes (5, 6, 7). Based on their distinct patterns of expression, PD-L1 and PD-L2 may provide distinct functions that regulate immune responses. PD-L1 is more ubiquitously expressed than PD-L2. PD-L1 is constitutively expressed on T cells, B cells, M{phi}, dendritic cells (DC), and a variety of nonhemopoietic parenchymal cells, including heart (8, 9), lung (10), pancreas (11, 12, 13), colon (14), brain (15), and kidney tubular epithelial cells (TEC) (16), and is further up-regulated after activation (17). In contrast, PD-L2 expression is limited to activated monocytes/M{phi} and DC (17). Although a number of studies point to an important role for PD-L1 in regulating T cell tolerance (18, 19, 20), much less is known about the function of PD-L2 (12, 19, 20, 21).

The concept that the PD-1:PD-L pathway is a negative regulatory checkpoint in immune reactions was initially based on PD-1–/– mice, which spontaneously develop features of autoimmune disease. Importantly, genetic deletion of PD-1 in C57BL/6 (B6) mice resulted in a low incidence of mild glomerulonephritis (22). Although the incidence and severity of glomerular pathology increased in the B6-Faslpr strain, pathology in the interstitium (the area richest in invading M{phi} and T cells), tubules, and vasculature, as well as the mechanisms responsible for inducing glomerular disease were not explored. Since M{phi} and T cells do not normally infiltrate the kidney in the B6-Faslpr strain, we speculate that deleting PD-1 renders the kidney susceptible to invading M{phi} and T cells, as the PD-1-dependent protective barrier is lost. Thus, determining the intrarenal cells expressing PD-1, and its cognate ligands, and investigating the impact of each ligand on autoimmune kidney disease is central to understanding the role of the PD-1 pathway.

The expression of PD-L1 on parenchymal cells in peripheral tissues led to the hypothesis that PD-L1 may control T cells in nonlymphoid organs. Within this framework, increasing evidence suggests that expression of PD-L1 on parenchymal cells shields target tissues from invading leukocytes. For example, blocking PD-L1 (12) or eliminating PD-1 (23) in NOD mice results in enhanced T cell invasion of islets, increased islet destruction, and more severe diabetes. Bone marrow (BM) chimera studies demonstrated that PD-L1 on nonhemopoietic cells has a critical role in protecting the pancreas from self-reactive T cells (19). Since PD-L1 is expressed on a TEC line and is functionally significant, renal parenchymal cells may provide a checkpoint and protective barrier that thwarts immunoinflammatory reactions. Taken together, we hypothesize that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints in M{phi} and T cell-mediated autoimmune kidney disease.

To test this hypothesis, we induced NSN in mice lacking either PD-L1 (PD-L1–/–), PD-L2 (PD-L2–/–), or both (PD-L1/L2–/–). We now report that kidney pathology (glomerular and tubular/interstitial) and the loss of renal function increases in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice as compared with wild-type (WT) mice during NSN. Although the magnitude of renal disease in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice was similar, PD-L1 and PD-L2 regulate renal disease in distinct ways. Our studies provide the first in vivo evidence that M{phi} express PD-1, PD-L1, and PD-L2 and further demonstrate that PD-L1 on hemopoietic, rather than parenchymal, cells is largely responsible for the protective role of PD-L1 in suppressing kidney disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

We purchased B6 mice from The Jackson Laboratory. PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– (B6 background) mice were generated as previously described (18, 19). Mice were housed and bred in a pathogen-free animal facility. Use of mice in our study was reviewed and approved by the Standing Committee on Animals in the Harvard Medical School in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Nephrotoxic serum nephritis

We prepared nephrotoxic serum by immunizing sheep with a particulate fraction of mouse glomerular basement membrane from B6 mouse kidneys, as previously described (24). We primed PD-L1/L2–/–, PD-L1–/–, PD-L2–/–, and WT male mice (5–7 wk of age) s.c. in each flank with 0.375–0.5 mg of sheep IgG (0.75–1.0 mg total) in CFA (Sigma-Aldrich). We challenged these mice 7 days later by i.v. injection of sheep nephrotoxic serum (15 µl/g body weight). Mice were sacrificed and analyzed 14 days after challenge unless otherwise stated. We used nephrotoxic serum from rabbits to immunize PD-L1–/– and WT mice in one experiment (profiling expression of PD-L1 on parenchymal cells at days 4, 7, 14, and 28 following challenge) as previously described (25).

PD-1, PD-L1, and PD-L2 transcript expression

We analyzed the expression of PD-1, PD-L1, PD-L2 in the kidney and spleen by using real-time, two-step, quantitative PCR as previously described (26). The mRNA levels were normalized to those of GAPDH. The PCR primers used were as follows: GAPDH: sense, 5'-CAT GGC CTC CAA GGA GTA AG-3'; antisense, 5'-CCT AGG CCC CTC CTG TTA TT-3'; PD-1 sense, 5'-CCC TCA GTC AAG AGG AGC AT-3'; antisense, 5'-TCC CAG CTT GTG GTA AAC CT-3'; PD-L1 sense, 5'-TGG ACA AAC AGT GAC CAC CAA-3'; antisense, 5'-CCC CTC TGT CCG GGA AGT-3'; PD-L2 sense, 5'-GTA CCG TTG CCT GGT CAT CT-3'; and antisense, 5'-GCC AGG ACA CTT CTG CTA GG-3'.

Renal histopathology

After removing the kidneys, we fixed half of one kidney in 10% neutral-buffered formalin and embedded it in paraffin. We snap froze the other half in Tissue Tek OCT Compound (Sakura Finetek) for cryosectioning. We used the remaining kidney for flow cytometry and to prepare RNA. Paraffin sections (4 µm) were stained with periodic acid-Schiff reagent. We evaluated glomerular pathology by assessing 100 glomeruli/kidney cross-section and determining the percentage of obliterated glomeruli and glomeruli with crescents (defined as two or more cell layers within the Bowman’s space) or segmental lesions (exhibiting at least one of the following: necrosis, proliferation, hyalinosis) using coded slides. We assessed the percentage of damaged tubules (consisting of at least one of the following: dilatation, atrophy, necrosis) by scoring 400 renal cortical tubules/kidney cross-section in randomly selected high-power microscopic fields (x40).

Renal function

We measured blood urea nitrogen (BUN) levels using a colorimetric analysis kit (Infinity; Thermo Electron) according to the manufacturer’s instructions. Urinary albumin was measured using a Mouse Albumin ELISA Quantitation Kit (Bethyl Laboratories) according to the protocol provided. We measured creatinine levels in the urine using a creatinine reagent kit and a Creatinine Analyzer 2 (Beckman Coulter). To standardize urine albumin values, proteinuria was calculated as the ratio of urinary albumin per milligram of urinary creatinine (27).

Antibodies

We used the following purified primary Abs for immunostaining: rat anti-mouse CD4 (RM4-5) and rat anti-mouse CD8a (Ly-2; BD Pharmingen), rat anti-mouse CD68 (FA-11; Serotec), rat anti-mouse TNF-{alpha} (MP6-XT22), rat anti-mouse PD-L1 (MIH5) and rat anti-mouse PD-L2 (TY25; eBioscience), and rat anti-mouse PD-1 (29F.1A12; provided by G. J. Freeman, Boston MA) (28). The following isotype-specific Abs were used for controls: rat IgG2a (R35-95) and rat IgG2b (R35-38; eBioscience). The secondary Ab for immunostaining was biotin-conjugated rabbit anti-rat IgG (Vector Laboratories). We used the following Abs from eBioscience for FACS analysis: FITC-conjugated anti-CD4 (L3T4), anti-CD8 (Ly2), and anti-CD45.2 104; PE-conjugated anti-CD4 (L3T4), anti-CD45.1 (SJL), anti-CD69 (H1.2F3), anti-IL-12 (C17.8), anti-PD-1 (J43), anti-PD-L1 (MIH5), and anti-PD-L2 (TY25); PE-Cy5 conjugated anti-CD8 (Ly2) and anti-CD11c; and allophycocyanin-conjugated anti-CD4 (L3T4), anti-CD45.2 (SJL), and anti-F4/80 (BM8). We also used FITC-conjugated CD11b (M1/70) from BD Pharmingen and FITC- and allophycocyanin-conjugated anti-CD68 (FA11) from Serotec. Proximal tubules within the kidney were identified by fluorescein-conjugated lotus lectin (Vector Laboratories) (29, 30).

Immunohistochemistry

We stained frozen kidney sections for the presence of M{phi} using anti-CD68 Ab. However, although M{phi} represent the majority of intrarenal CD68+ cells during NSN, ~25% are DC (CD68+CD11c+; data not shown) (31). In light of this finding, we will refer to this population as CD68+ cells. We stained for the presence of T cells using anti-CD4 and anti-CD8 Ab as previously described (32). The number of cells bearing CD4, CD8, or CD68 determinants was assessed in 10 randomly selected high-power fields and within and surrounding 10 glomeruli.

To determine the number of activated CD68+ cells in the kidney during NSN, we fixed frozen kidney sections in paraformaldehyde, stained them with rat anti-mouse TNF-{alpha}-PE and CD68-FITC, and analyzed these sections using a fluorescence microscope. The frequency (percent) of activated M{phi} was assessed by enumerating the number of CD68+TNF-{alpha}+ cells within the total number of M{phi} in five high-power fields.

To detect the intrarenal PD-1, PD-L1, and PD-L2 expression, we fixed frozen kidney sections (4 µm) in ice-cold acetone for 10 min, washed these sections (0.1% BSA/1% FCS in PBS), and blocked endogenous peroxidase by incubating them in 0.6% H2O2/0.2% NaN3/PBS for 1 h. We blocked the nonspecific binding of avidin and biotin using an avidin/biotin blocking kit (Vector Laboratories) according to the manufacturer’s instructions. And we blocked the nonspecific binding of rabbit Abs by incubating with 10% normal rabbit serum in 10% BSA/1% FBS for 1 h. Sections were incubated with rat anti-mouse PD-1 (29F.1A12), PD-L1, or PD-L2 (1/100) Abs at 4°C overnight. We incubated these sections with biotinylated rabbit anti-rat Ab (1/100) for 1 h followed by peroxidase-conjugated ABC solution (Vector Laboratories) for 1 h and developed with 3',3-diaminobenzidine (Sigma-Aldrich). We counterstained sections with Mayer’s hematoxylin (Sigma-Aldrich).

Serum anti-sheep IgG isotypes

We measured the levels of mouse anti-sheep IgG Abs by ELISA using sera collected at day 14 during NSN as previously described (33). We detected the presence of bound mouse anti-sheep total IgG, IgG1, and IgG2a using peroxidase-conjugated rabbit anti-mouse IgG (1/5000; Southern Biotechnology Associates).

IgG deposits within renal glomeruli

We detected IgG deposits in the kidney as previously described (34). We assessed the incidence and severity of IgG deposits in glomeruli by titrating the fluorescein-conjugated goat anti-mouse IgG (MP Biomedicals) on serial sections using the following dilutions (1/3000 and 1/4500, 1/6000, and 1/7500). We scored 20 glomeruli/specimen as either positive or negative (incidence) and graded the amount (severity) of deposits in 20 positive glomeruli/specimen on a scale of 0–3 (26).

Flow cytometry

We prepared and stained single-cell suspensions from kidneys as previously described (35). We collected 0.5–1.0 x 106 total kidney cells using a FACSCalibur (BD Biosciences) and analyzed data using FlowJo software (Tree Star).

PD-L1 expression on primary TEC

We stimulated primary TEC from WT kidneys (2 x 105 cells/well), prepared as previously described (36), with IFN-{gamma} (100 and 500 IU/ml) for 24 h. These cells were treated with trypsin, stained for the presence of PD-L1, and analyzed by flow cytometry.

BM chimeras

We isolated BM from PD-L1–/– (CD45.2) and WT (CD45.1) donor animals as previously described (19). Briefly, PD-L1–/– and WT mice (4 wk of age) were lethally irradiated with 1200 rad in two doses and reconstituted with 5–7 x 106 WT or PD-L1–/– BM cells. We confirmed chimerism by evaluating CD45.1/CD45.2 expression on peripheral blood leukocytes. We induced NSN in BM chimeric mice 8–12 wk after reconstitution.

Statistical analysis

The data represent the mean ± SEM and were prepared using GraphPad Prism version 4.0. We used the nonparametric Mann-Whitney t test to evaluate p values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PD-1, PD-L1, and PD-L2 expression is increased in the kidney during NSN

To evaluate the relevance of the PD-1 pathway during kidney injury, our first goal was to measure PD-1, PD-L1, and PD-L2 expression during NSN. Note, in all studies throughout this report, we analyzed NSN at day 14 unless otherwise stated. Intrarenal PD-1, PD-L1, and PD-L2 transcripts increased dramatically during NSN as compared with untreated kidneys (Fig. 1). The increased expression of PD-1, PD-L1, and PD-L2 was specific to the kidney since expression of these molecules in the spleen did not increase significantly (Fig. 1B). Furthermore, we determined that PD-1 and PD-L1 transcripts were barely detectable in kidneys of untreated mice, whereas PD-L2 was constitutively expressed at low levels. Thus, intrarenal expression of PD-1 and its ligands increases in response to autoimmune-mediated renal inflammation.


Figure 1
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  		FIGURE 1. PD-1, PD-L1, and PD-L2 transcripts are up-regulated in the kidney cortex during NSN. We compared PD-L1 transcripts in the renal cortex (A) and spleen (B) from mice during NSN ({blacksquare}) with untreated WT mice (Figure 1) using quantitative real-time PCR. The average number of PD-1, PD-L1, and PD-L2 transcripts normalized to GAPDH values were determined for each sample. Data are presented as the mean ± SEM; **, p < 0.01, as determined by the Mann-Whitney t test.

 
PD-1, PD-L1, and PD-L2 are expressed on M{phi} and PD-1 and PD-L1 are expressed on T cells in the kidney during NSN

To begin to understand the roles of PD-1 and its ligands in kidney disease, we first examined their protein expression during NSN. Using immunostaining, we detected PD-1, PD-L1, and PD-L2 on the few resident hemopoietic cells (~0.7/3, 1.4/3, and 0.2/3 hematopoietic cells per high power field, respectively) and occasionally PD-L1 on TEC (<1%) in the kidney of untreated mice. After the induction of NSN, we detected an increase in intrarenal mononuclear cells expressing PD-1, PD-L1 (Fig. 2A, inset), and PD-L2 and TEC expressing PD-L1 (Fig. 2A). To identify the specific leukocytic populations that bear PD-1, PD-L1, and PD-L2, we analyzed their expression on T cells (CD4+ and CD8+) by flow cytometry (Fig. 2B). As expected, we detected PD-1 and PD-L1, but not PD-L2, expression on CD4+ and CD8+ T cell subsets in the kidney during NSN. About half of each T cell subset (CD4+ and CD8+) expressed PD-1 (CD4: 43 ± 2; CD8: 48 ± 5) on day 14 of NSN, while 75–80% of each T cell subset expressed PD-L1 (CD4: 76 ± 1; CD8: 74 ± 3). It should be noted that intrarenal T cells in untreated mice expressed PD-1 and PD-L1, although the frequency was much lower compared with mice during NSN (Fig. 2B2). Thus, resident and activated T cells in the kidney express PD-1 and PD-L1, but not PD-L2.


Figure 2
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  		FIGURE 2. PD-1, PD-L1, and PD-L2 are expressed on intrarenal CD68+ cells, PD-1 and PD-L1 are expressed on T cells, and PD-L1 is expressed on a small percentage of proximal TEC during NSN. A, Kidney sections from WT mice at day 14 during NSN (left panels) were stained with Abs to PD-1, PD-L1, or PD-L2. Negative controls (right panels) are kidneys from WT mice stained with an isotype control (top), PD-L1–/– mice stained with anti-PD-L1 (middle), and PD-L2–/– mice stained with anti-PD-L2 at day 14 during NSN. Thin arrows indicate mononuclear cell infiltrates. Arrowheads indicate TEC. We analyzed three to six WT mice per group in three separate experiments. B, Flow cytometric analysis of PD-1, PD-L1, and PD-L2 expression on CD4+, CD8+, CD68+, and F4/80+ cells within the kidney of WT mice at day 14 during NSN. 1, Representative FACS plots of expression in WT mice at day 14 during NSN. Plots are gated on live CD45.2+ cells. Note: PD-1, PD-L1, and PD-L2 expressing CD68+ and F4/80+ cells are indicated by asterisks. 2, Expression of PD-1, PD-L1, and PD-L2 is increased on hemopoietic cells in the kidney during NSN. Data are representative of three to eight mice in four separate experiments. Data were analyzed by the Mann-Whitney t test and are presented as mean ± SEM (*, p < 0.05; **, p < 0.01; and ***, p < 0.005; NE, not expressed. C, Primary TEC cells (1 x 105) were stimulated with 100 U/ml (Figure 2) or 500 U/ml ({blacksquare}) IFN-{gamma} for 24 h. PD-L1 expression was assessed by flow cytometry. The average of two separately stimulated samples is shown ± SEM. D, Flow cytometric analysis of PD-L1 expression on total live (left) and CD45.2– (right) gated kidney cells from untreated and WT or PD-L1–/– mice on day 14 during NSN. The PD-L1–/– kidneys were stained to verify specificity of the PD-L1 Ab. Numbers in box represent the total frequency of PD-L1+ parenchymal cells (CD45.2–). Note: Expression of PD-L1 on proximal TEC (lotus lectin+) is indicated by asterisk. Representative plots are shown (top panel). We analyzed three to four mice per group by the Mann-Whitney t test (bottom panel). Data are mean ± SEM (*, p < 0.05).

 
Next, we investigated the expression of PD-1 and its ligands on M{phi} in the kidney during NSN by flow cytometry. Previous experiments identifying PD-1, PD-L1, and PD-L2 expression on M{phi} were limited to ex vivo stimulation of primary human monocytes (8, 37) and murine peritoneal M{phi} (17). During NSN, we detected substantial numbers of intrarenal CD68+ and F4/80+ cells expressing PD-1 and PD-L1 (Fig. 2B). In fact, CD68+ cells are the dominant hemopoietic cell population expressing these proteins (20% or 26% of the total hemopoietic cells in the kidney are CD68+ that express PD-1 or PD-L1, respectively) (Fig. 2B1, last panel, *). When we specifically identify the expression of PD-1 and PD-L1 on CD68+ cells, we detected 39% ± 2 (n = 3) expressing PD-1 and 57% ± 7 (n = 3) expressing PD-L1 during NSN (Fig. 2B2). Similar results were noted using the M{phi} markers F4/80 (Fig. 2B) and CD11b (data not shown). Of note, PD-L1 expression was >2-fold higher on CD68+ cells than on either T cell population based on mean fluorescence intensity (mfi) (CD68+ PD-L1+ mfi: 123 ± 15, cf with CD4+ PD-L1+ mfi: 50 ± 2, CD8+ PD-L1+ mfi: 41 ± 3; data not shown). By comparison, the frequency of intrarenal PD-L2+ cells within the CD45.2+ population was lower (3–12%) and more variable than the frequency of PD-1+ and PD-L1+ cells. Interestingly, nearly every PD-L2+ cell in the kidney is CD68+ (Fig. 2B1). A subset of these cells are M{phi} as PD-L2 was detected on F4/80+ cells (Fig. 2B) and CD11b+ cells (data not shown). It is worth noting that we detected PD-1, PD-L1, and PD-L2 on CD68+ and F4/80+ cells in untreated kidneys, albeit on a lower frequency than during NSN (Fig. 2B2). Taken together, intrarenal M{phi} express PD-1, PD-L1, and PD-L2 during induced autoimmune renal disease.

PD-L1 is up-regulated by IFN-{gamma} on a subset of renal TEC in vitro, while only a small percentage of renal parenchymal and proximal TEC express PD-L1 during NSN

The constitutive and/or inducible expression of PD-L1 by parenchymal cells has been reported in many organs (8, 13). Although we detected PD-L1 on TEC by immunostaining, the vast majority of TEC did not express PD-L1 (Fig. 2A). To further investigate PD-L1 expression on TEC, we stimulated a proximal TEC cell line (C1.1) and confirmed that nearly every (>95%) cell expressed PD-L1 (data not shown) as has been reported previously (16). By comparison, primary TEC stimulated with IFN-{gamma} expressed PD-L1 (Fig. 2C), although to a lesser extent (35–40%) than the C1.1 cell line. Thus, a population of stimulated TEC is capable of expressing PD-L1.

To pinpoint the frequency of nonhemopoietic parenchymal cells expressing PD-L1 in the kidney during autoimmune disease, we used the hemopoietic cell marker CD45.2 to distinguish PD-L1 expression on parenchymal (CD45.2–) and hemopoietic (CD45.2+) cells at days 4, 7, 14, and 28 during NSN. We detected constitutive PD-L1 expression on a very small subset of parenchymal cells (0.4% ± 0.1, n = 5) in untreated WT mice. Of note, a subset of these parenchymal cells is proximal tubules since they express the proximal tubule cell marker lotus lectin (Fig. 2D). We verified the specificity of PD-L1 expression by staining for the presence of PD-L1 using kidney cells isolated from PD-L1–/– mice. The frequency of PD-L1 expression was similar to untreated mice at days 4 and 7 during NSN (n = 3/group; data not shown). By comparison, the frequency of PD-L1 expression was up-regulated on parenchymal cells on day 14 during NSN; 4.1% ± 2.3 (n = 3) of parenchymal cells expressed PD-L1 and the majority of these (2.3%) were proximal TEC (Fig. 2D). This up-regulated frequency returned to baseline by day 28 during NSN (n = 3; data not shown). Thus, PD-L1 is only expressed on a small population of proximal TEC and total renal parenchymal cells during NSN.

Renal disease is exacerbated in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice during NSN

To investigate the roles of PD-L1 and PD-L2 in the kidney during an autoimmune response, we first induced NSN in mice genetically deficient in both PD-L1 and PD-L2. We detected a notable increase in glomerular (sclerosis, crescents, obliteration) and tubular damage (dilatation, casts) in PD-L1/L2–/– mice as compared with WT mice during NSN (Fig. 3). These findings suggest that PD-L1 and/or PD-L2 provide a barrier to counter renal inflammation during NSN. To determine whether one or both of the PD-Ls are responsible for the protection of the kidney during NSN, we induced nephritis in mice deficient in either PD-L1 or PD-L2. We detected an increase in glomerular (Fig. 3) and tubular damage (Fig. 3, A and inset in B) in PD-L1–/– and PD-L2–/– mice as compared with WT mice that was similar to mice lacking both PD-L1 and PD-L2. Taken together, all three strains (PD-L1–/–, PD-L2–/–, and PD-L1/L2–/–) are similarly impaired in their ability to provide the normal regulatory checkpoint that counters renal inflammation during NSN.


Figure 3
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  		FIGURE 3. Renal histopathology is increased in PD-L1/L2–/–, PD-L1–/–, and PD-L2–/– mice during NSN. NSN was induced in PD-L1/L2–/–, PD-L1–/–, and PD-L2–/– mice along with WT controls for each experiment. A, We evaluated glomerular and tubular damage for each mouse at day 14 during NSN using paraffin sections stained with periodic acid-Schiff. Data were analyzed by the Mann-Whitney t test and are presented as mean ± SEM (*, p < 0.05; **, p < 0.01; and ***, p < 0.005). B, Representative glomeruli are shown from each group (original magnification, x40). Insets (left panel) are examples of tubular pathology.

 
To determine whether the increase in renal pathology in the absence of PD-L1 and/or PD-L2 during NSN was accompanied by a greater extent of impaired renal function, we compared BUN levels in the serum of PD-L1/L2–/–, PD-L1–/–, PD-L2–/–, and WT mice (Fig. 4). As expected, BUN levels rose above baseline (untreated mice) in WT mice during NSN. The BUN levels in PD-L1/L2–/–, PD-L1–/–, and PD-L2–/– mice rose even higher than in WT mice during NSN. In addition, although the PD-L2–/– mice had statistically significant higher albuminuria (urinary albumin/creatinine ratio) compared with WT mice, the PD-L1–/– and PD-L1/L2–/– mice did not reach significance (data not shown). Thus, mice deficient in one or both of the PD-Ls have poorer renal function (BUN) during NSN compared with WT mice that parallels the increase in renal pathology.


Figure 4
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  		FIGURE 4. BUN is higher in PD-L1/L2–/–, PD-L1–/–, and PD-L2–/– mice than in WT mice during NSN. BUN was analyzed in the serum of PD-L1/L2–/– (A), PD-L1–/– (B), and PD-L2–/– (C) mice and compared with WT mice on day 14 of NSN. For comparison, BUN was also measured in serum from untreated WT mice. Data are presented as mean ± SEM, *, p < 0.05; **, p < 0.01 (Mann-Whitney t test).

 
Intrarenal leukocytes are increased in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice during NSN

To determine whether PD-L1 and/or PD-L2 had an impact on specific leukocyte populations during NSN, we assessed and compared the number of intrarenal CD68+ and T cells (CD4+ and CD8+) in the PD-L1/L2–/–, PD-L1–/–, PD-L2–/–, and WT mice during NSN by immunostaining (Fig. 5). We detected an increase in the accumulation of multiple leukocyte populations (CD68+, CD4+, and CD8+ cells) in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice as compared with their WT counterparts. This increase in intrarenal hemopoietically derived cells was verified by analyzing the total percentage of intrarenal CD45.2+ cells in PD-L1–/–, PD-L2–/–, PD-L1/L2–/–, and WT mice using flow cytometry (Fig. 5B). The hierarchy of leukocytic populations (CD68+ cells > CD4+ T cells > CD8+ T cells) in the kidney during NSN was unchanged in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice compared with WT mice; however, in PD-L1–/– mice, we detected a substantial increase in the intrarenal CD8+ T cells (glomerular, 7-fold; interstitial, 4-fold, p < 0.01) as compared with WT mice (Fig. 5A2). In contrast, the number of intrarenal CD8+ cells did not rise in the PD-L2–/– mice during NSN (Fig. 5A3). The increase in the intrarenal CD8+ T cells (glomerular, 3-fold; interstitial, 2.5-fold) in PD-L1/L2–/– mice was less than in the PD-L1–/– mice, but greater than in the PD-L2–/– mice (glomerular, 1.5-fold; interstitial, 1.5-fold) (Fig. 5A1). We verified the specific increase in CD8+ T cells in PD-L1–/– mice, in comparison to PD-L2–/– and WT during NSN, by analyzing the frequency of CD8+ cells in the CD45.2+ population in the kidney by flow cytometry (Fig. 5B). Thus, PD-L1 and PD-L2 limit the intrarenal accumulation of CD4+ T cells and CD68+ cells during NSN, while only PD-L1 limits the expansion of CD8+ T cells in the kidney during this autoimmune kidney disease.


Figure 5
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  		FIGURE 5. T cell and CD68+ cell infiltrates are increased in the glomeruli and interstitium in PD-L1/L2–/–, and PD-L1–/–, and PD-L2–/– mice during NSN. A, We evaluated intrarenal T cells (CD4+ and CD8+) and CD68+ cells from 1) PD-L1/L2–/–, 2) PD-L1–/–, and 3) PD-L2–/– mice compared with WT mice on day 14 of NSN. B, Flow cytometric analysis of the frequency of CD8+ cells within the intrarenal CD45.2+ population of PD-L1–/–, PD-L2–/–, and WT mice at day 14 during NSN and untreated mice. Data are mean ± SEM, *, p < 0.05; **, p < 0.01; and ***, p < 0.005 (Mann-Whitney t test).

 
Intrarenal activated M{phi} are increased in PD-L2–/– mice during NSN

Activated M{phi} release mediators, including TNF-{alpha}, that are instrumental in inducing apoptosis of kidney parenchymal cells during inflammation (1). Since renal disease is exacerbated in PD-L1–/– and PD-L2–/– mice as compared with WT mice during NSN, we hypothesized that PD-1-PD-L interactions limit the number of intrarenal activated M{phi}. To evaluate M{phi} activation, we used dual immunostaining for the presence of CD68 (green) and TNF-{alpha} (red) and merged the images to identify double-positive cells, CD68+ cells producing TNF-{alpha} (yellow). Although a majority of the CD68+ cells are not TNF-{alpha}+ (green only), we determined that PD-L2–/– mice have a higher frequency of activated CD68+ cells compared with PD-L1–/– and WT mice (Fig. 6A, graph) as assessed for the presence of CD68+TNF-{alpha}+ cells (circled in photomicrographs) within the kidney during NSN (n = 3–4/group, day 7). In support of this finding, we detected an increase in the frequency of IL-12-expressing CD68+ cells isolated from PD-L2–/– kidneys as compared with PD-L1–/– and WT kidneys at day 7 during NSN (n = 3–4/group; data not shown). We point out that there are TNF-{alpha}-generating cells that are not CD68+ (red only), these are most likely TEC (36). Thus, the rise in intrarenal CD68+ cells in PD-L2–/– mice during NSN and the increased frequency of intrarenal activated CD68+ cells suggests that PD-L2 expression suppresses the intrarenal accumulation of activated CD68+ cells during NSN.


Figure 6
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  		FIGURE 6. Intrarenal activated CD68+ cells are increased in PD-L2–/– mice and intrarenal activated CD8+ T cells are increased in PD-L1–/– mice during NSN. A, We evaluated kidneys from PD-L1–/–, PD-L2–/–, and WT kidneys for the presence of activated CD68+ cells (green) by staining for TNF-{alpha} (red) at day 7 during NSN using immunofluorescence (n = 3–4 mice/group). PD-L2–/– kidneys have an increase in the frequency of activated CD68+ cells (TNF-{alpha}+, yellow) as compared with PD-L1–/– and WT kidneys during NSN. Graph equals the mean ± SEM. Representative photomicrographs illustrate CD68+ cells expressing TNF-{alpha}+ (circled areas) in the PD-L1-/, PD-L2–/–, and WT kidneys. B, We compared CD4+ and CD8+ populations for T cell activation (CD69+ and CD44+) in PD-L1–/–, PD-L2–/–, and WT mice during NSN using flow cytometry (n = 3–4/group). The graph depicts the average frequency of CD69+ or CD44high cells within CD45.2+CD4+ and CD45.2+CD8+ populations in PD-L1–/–, PD-L2–/–, and WT mice at day 14 during NSN. Data were analyzed by the Mann-Whitney t test and are presented as mean ± SEM (*, p < 0.05; **, p < 0.01).

 
Intrarenal activated CD8+ T cells are increased in PD-L1–/– mice during NSN

Because activated T cells regulate numerous immune mechanisms, we probed for activated CD4+ and CD8+ T cells in PD-L1–/– and PD-L2–/– as compared with WT kidneys during NSN. We did not detect a difference in the frequency of activated CD4+ T cells (CD69+, CD44+) in the PD-L1–/–, PD-L2–/–, or WT kidneys during NSN (Fig. 6B). By comparison, we detected an enhanced frequency of activated CD8+ T cells (CD69+, CD44+) in the PD-L1–/– kidneys as compared with the PD-L2–/– and WT kidneys during NSN (Fig. 6B). Thus, although PD-L1 and PD-L2 both limit renal inflammation during NSN, these ligands have a differential impact on activated T cell populations and, therefore, the mechanisms responsible for suppressing autoimmune attack are likely to be distinct.

Increased serum anti-sheep IgG isotypes and IgG glomerular deposits in PD-L2–/–, but not PD-L1–/– mice during NSN

To determine whether the increased severity of renal disease in the PD-L-deficient mice was accompanied by an increased humoral response, we measured serum titers of anti-sheep IgG isotypes in PD-L1/L2–/– and WT mice during NSN. We detected higher titers of serum IgG, IgG1, and IgG2b in PD-L1/L2–/– mice as compared with WT mice (Fig. 7A, top). To establish whether this enhanced anti-sheep Ig isotype expression was a reflection of the loss of PD-L1 and/or the PD-L2, we evaluated these isotypes in the individually deficient PD-L strains. Interestingly, we detected an increase in the anti-sheep Ig isotypes in the sera of PD-L2–/– mice, but not PD-L1–/– mice during NSN when compared with their WT counterparts (Fig. 7A, middle and bottom). Thus, the increase in serum anti-sheep Ig isotypes in the double-deficient PD-1/L2–/– mice is a reflection of the absence of PD-L2 and not PD-L1. Consistent with these findings, we determined that PD-L2–/–, but not PD-L1–/–, mice had an increased incidence and severity of glomerular IgG deposits as compared with WT mice during NSN (Fig. 7B). These data suggest that although the heightened severity of kidney disease in the PD-L1–/– mice is not driven by Ab-mediated mechanisms, the enhanced serum titers of anti-sheep IgG and glomerular IgG deposits in PD-L2–/– mice may be responsible, at least in part, for increasing the severity of renal disease during NSN. This interpretation is consistent with published findings suggesting that although generation of autologous murine IgG against the nephrotoxic Ab is not essential for disease initiation (38, 39), it may contribute to sustaining renal dysfunction (39).


Figure 7
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  		FIGURE 7. Anti-sheep specific Igs and IgG deposits in glomeruli are increased in PD-L2–/–, but not PD-L1–/– mice during NSN. A, The presence of anti-sheep IgG, IgG1, and IgG2b Abs was measured in serial serum dilutions from PD-L1/L2–/–, PD-L1–/–, and PD-L2–/– mice compared with WT mice at day 14 during NSN. B, We determined the 1) incidence (percent) of glomeruli with IgG deposits and 2) severity (intensity 0–3 scale) of IgG deposits in positive glomeruli by immunofluorescence in kidney sections from PD-L1–/– and PD-L2–/– mice on day 14 of NSN as compared with their WT counterparts. Representative photographs depict glomerular IgG deposits in each group (original magnification, x40). Data are mean ± SEM (*, p < 0.05; **, p < 0.01).

 
Expression of PD-L1 on hemopoietic cells limits intrarenal leukocytic infiltrates during NSN

To evaluate the role of PD-L1 on parenchymal vs hemopoietic cells, we generated chimeric mice that expressed PD-L1 exclusively on parenchymal cells by reconstituting lethally irradiated WT mice with PD-L1–/– (knockout, KO) BM (KO->WT). In addition, we generated chimeric mice that expressed PD-L1 exclusively on hemopoietic cells by reconstituting lethally irradiated PD-L1–/– mice with WT BM (WT->KO). WT mice reconstituted with WT BM (WT->WT) and PD-L1–/– mice reconstituted with PD-L1–/– BM (KO->KO) were used as controls. We detected an increase in CD68+, CD4+, and CD8+ infiltrates in glomeruli in the PD-L1–/– (KO->KO) chimeras as compared with the WT (WT->WT) chimeras during NSN (Fig. 8). Thus, the KO->KO and WT->WT chimeras mimicked our findings with PD-L1–/– and WT mice during NSN (Fig. 3). Interestingly, the increase in intrarenal CD4+ and CD68+ cells in KO->KO chimeras was strikingly similar to KO->WT chimeras, suggesting that PD-L1 on hemopoietic cells was primarily responsible for limiting the increase in these leukocytes during NSN. Furthermore, the increase in leukocytes in WT->KO mice was generally less than that of KO->WT, but higher than that of WT->WT (Fig. 8). Taken together, this suggests that PD-L1 primarily on hemopoietic cells and, to a far lesser extent, on parenchymal cells is instrumental in limiting inflammation during NSN.


Figure 8
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  		FIGURE 8. Expression of PD-L1 on hemopoietic cells is primarily responsible for limiting the number of leukocytic infiltrates during NSN. NSN was induced in four groups of BM chimeric mice. Interstitial and glomerular CD68+(A), CD4+(B), and CD8+ (C) infiltrates were assessed as in Fig. 5. Data are mean ± SEM (*, p < 0.05; **, p < 0.01. WT->WT = WT (CD45.1) BM cells into WT (CD45.2) hosts; KO->KO = PD-L1–/– (CD45.2) BM cells into PD-L1–/– (CD45.2) hosts; WT->KO = WT (CD45.1) BM cells into PD-L1–/– (CD45.2) hosts; KO->WT = PD-L1–/– (CD45.2) BM cells into WT (CD45.1) hosts.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report that signaling via the PD-1 pathway restricts the immune response during experimental autoimmune kidney disease. Our findings indicate that the magnitude of renal disease in PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice is similar, but the PD-L1- and PD-L2-dependent mechanisms responsible for countering renal disease differ. Our studies directly compare PD-L1–/–, PD-L2–/–, and PD-L1/L2–/– mice during autoimmune kidney disease, and identify distinct roles for PD-L1 and PD-L2 in regulating kidney disease. Taken together, our findings dissect the negative regulatory impact of the PD-1 pathway on dampening leukocyte-dependent autoimmune kidney disease.

M{phi} and T cells that accumulate in the kidney mediate renal injury during NSN (1, 40, 41, 42). In particular, activated M{phi} are a major culprit responsible for kidney cell damage, as we previously reported that activated M{phi} release mediators that induce apoptosis of intrarenal parenchymal cells during NSN (1). In contrast, activated T cells are pivotal in autoimmune kidney disease since they mediate inflammation (2). We now report that the numbers of intrarenal CD68+ cells and CD4+ T cells in PD-L1–/– and PD-L2–/– mice expand during NSN. Thus, both PD-1 ligands provide a barrier that suppresses the accumulation of CD68+ cells and CD4+ T cells in the kidney during autoimmune kidney disease. However, the increase in the frequency of resting and activated CD68+ cells was more pronounced in the PD-L2–/– as compared with PD-L1–/– mice, suggesting that PD-L2 plays a dominant role in controlling CD68+ cell activation. By comparison, the frequency of resting and activated CD8+ T cells was far greater in PD-L1–/– as compared with the PD-L2–/– mice, suggesting a specific role for PD-L1, and not PD-L2, in limiting CD8+ T cell activation. A specific role for PD-L1 in CD8+ T cell activation is consistent with the finding that PD-1-PD-L1 interactions are critical in inducing tolerance in CD8+ T cells (43). Hemopoietic and parenchymal cells express PD-L1 in the kidney; therefore, peripheral tolerance of PD-1+CD8+ T cells may result from engaging with PD-L1 on one or both of these cell types. In fact, PD-L1 expression on TEC inhibits ex vivo stimulation of OT-1+CD8+ T cells (44). However, our results using mixed BM chimeras suggest that PD-L1 expression on hemopoietic cells is primarily responsible for limiting the expansion of CD8+ T cells. Regardless of the exact mechanism, since CD8+ T cells promote NSN (45), an increase in this leukocytic population is likely to exacerbate renal pathology in PD-L1–/– mice during NSN. Thus, PD-L1 and PD-L2 regulate distinct leukocytic populations during autoimmune kidney disease.

The differential expression of PD-L1 and PD-L2 on intrarenal cells determines the impact of these ligands on the fate of the kidney during autoimmune attack. Intrarenal PD-L2 expression is far more restricted and less abundant than PD-L1 expression during NSN. Although M{phi} and DC are known to express PD-L2, the majority of CD68+ cells (75%) within the kidney during NSN are M{phi}, and not DC (31). Furthermore, we determined that a similar percentage of intrarenal leukocytes express other M{phi} markers (CD11b and F4/80). This suggests that the engagement of PD-L2 on M{phi} is central to limit renal inflammation. It is intriguing that kidney disease was not worse in the doubly deficient PD-L1/L2–/– mice, as compared with the single-deficient PD-L strains. One possible explanation is that the PD-L1 and PD-L2 distinct mechanisms partially negate each other. Thus, since PD-L1 suppresses CD8+ T cells while PD-L2 suppresses CD68+ cells, we speculate that these are partially counterregulatory.

Along with cell-mediated effectors, Ab-regulated immune reactions induce autoimmune kidney diseases (3). Therefore, we investigated whether enhanced kidney disease in the PD-L1–/– and PD-L2–/– mice during NSN was related to an increase in pathogenic Abs within the kidney. We detected an increase in total serum IgG and IgG isotypes to sheep IgG in PD-L2–/–, but not in PD-L1–/– mice as compared with WT mice during NSN. Thus, PD-L2, but not PD-L1, blunts Ab production, a finding consistent with the marked Leishmania mexicana Ab elevation in PD-L2–/–, but not PD-L1–/–, mice during cutaneous leishmaniasis infection (46). The rise in anti-sheep IgG in PD-L2–/– mice correlates with an enhanced deposition of IgG within the renal glomeruli. Thus, the exacerbation of renal pathology and the loss of renal function may, in part, result from pathogenic Ab effectors in PD-L2–/– mice during NSN. Therefore, it is intriguing to suggest that PD-L2 limits renal disease by reducing the production and deposition of pathogenic Abs.

Our findings suggest that hemopoietic, rather than parenchymal, cells, are primarily responsible for limiting autoimmune kidney disease. We did not anticipate these findings since previous in vitro studies established that IFN-{gamma}-stimulated TEC up-regulate PD-L1. In addition, it is well established that TEC express molecules that promote and thwart renal inflammation (47, 48). Thus, we hypothesized that PD-L1 expression on renal parenchymal cells provides a barrier to counter T- and M{phi}- mediated renal disease. However, using a BM chimera approach, we determined that PD-L1 on hemopoietic, rather than parenchymal, cells, is largely responsible for suppressing NSN. In contrast, PD-L1 expressed on the majority of β cells within the pancreas are primarily responsible for limiting the onset of autoimmune diabetes in an adoptive transfer model (19). The differing role of PD-L1 on parenchymal cells in these two distinct experimental models of autoimmune disease is likely to be related to the minimal PD-L1 expression on kidney parenchymal cells in the NSN model. Although an IFN-{gamma}-stimulated TEC cell line (<95%) expresses PD-L1 in vitro, a more limited population of primary IFN-{gamma}- stimulated TEC (35–40%) express PD-L1 and an even smaller population of proximal TEC (2.3%) express PD-L1 during NSN. One possible explanation for the discrepancy between these in vivo and in vitro findings is that the magnitude and locale of IFN-{gamma} generated during NSN induces PD-L1 expression on hemopoietic cells, but is insufficient to up-regulate PD-L1 on the vast majority of TEC. Further investigations are required to determine whether PD-L1 is more ubiquitously expressed by TEC during other immune-mediated kidney diseases. In conclusion, although PD-L1 on renal parenchymal cells are not a major barrier for inflammation during NSN, it remains to be determined whether PD-L1 on renal parenchymal cells are instrumental in suppressing other renal diseases.

Although PD-L1 and PD-L2 limit the accumulation of CD68+ cells in the kidney during autoimmune kidney disease, the exact mechanism is unclear. One possible explanation is that suppressing T cell proliferation and/or activation indirectly reduces CD68+ cell accumulation. Alternatively, since PD-1 is up-regulated on CD68+ cells during NSN, it is possible that PD-L1 and/or PD-L2 on adjacent M{phi} or PD-L1 on T cells engage with PD-1 on CD68+ cells and directly limit M{phi} expansion. In this regard, the frequency of PD-1+CD68+ cells did not rise in the absence of PD-L1 (data not shown), suggesting that PD-L1 is not the primary ligand for PD-1 on CD68+ cells. Alternatively, there may be cell intrinsic functions for PD-L1 or PD-L2 on M{phi}, as has been suggested for PD-L2 on DC (49, 50). Current studies are detailing the impact of the individual PD-1 ligands on M{phi} activation and proliferation, as well as M{phi} functions that regulate inflammation.

In conclusion, although PD-L1 and PD-L2 provide distinct checkpoints poised to protect the kidney during autoimmune attack, neither alone or together prevents disease. Thus, it will be intriguing to search for other negative regulatory pathways that together with PD-1 will confer total and enduring protection from autoimmune kidney disease.


   Acknowledgment
 
We thank Dr. Gordon J. Freeman for providing anti-PD-1 Ab.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported in part by National Institutes of Health Grants R01 DK 52369 (to V.R.K.) and PO1 AI 56299 (to A.H.S.) and grants from Genzyme Renal Innovations Program (to V.R.K.). J.M. and G.Z. received support from the Deutsche Forschungsgemeinschaft (Grants ME-3194/1-1 and ZE-711/1, respectively). J.L. was supported by Ruth L. Kirschstein National Research Service Award F32 DK078416-01. Back

2 J.M. and J.A.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Vicki Rubin Kelley, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: vkelley{at}rics.bwh.harvard.edu Back

4 Abbreviations used in this paper: PD-1, programmed-death 1; PD-L1, PD-1 ligand 1; PD-L2, PD-1 ligand 2; M{phi}, macrophage; NSN, nephrotoxic serum nephritis; WT, wild type; DC, dendritic cell; TEC, tubular epithelial cell; BUN, blood urea nitrogen; BM, bone marrow; mfi, mean fluorescence intensity. Back

Received for publication June 8, 2007. Accepted for publication September 21, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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