Cellular Immunology 258 (2009) 161–171

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Cellular Immunology

journal homepage: www.elsevier .com/locate /yc imm

Altered availability of PD-1/PD ligands is associated with the failure to controlautoimmunity in NOD mice q

Deepak Yadav a,1, Natasha Hill a,2, Hideo Yagita b, Miyuki Azuma c, Nora Sarvetnick a,3,*

a Department of Immunology, The Scripps Research Institute, 10446 North Torrey Pines Road, Mail Drop IMM-23, La Jolla, CA 92037, United Statesb Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113-8421, Japanc Department of Molecular Immunology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 April 2008Accepted 25 April 2009Available online 6 May 2009

Keywords:CostimulationDiabetesAutoimmunityB7-H1B7-DCPD-1

0008-8749/$ - see front matter � 2009 Published bydoi:10.1016/j.cellimm.2009.04.006

q This work was supported by grants from NIH (# DNora Sarvetnick. This is manuscript # 18759 from theThe Scripps Research Institute.

* Corresponding author. Fax: +402 559 6732.E-mail addresses: dyadav@ucsd.edu (D. Yadav), N

hyagita@med.juntendo.ac.jp (H. Yagita), miyuki.mnoras@unmc.edu (N. Sarvetnick).

1 Present address: Department of Pathology, Unive9500 Gilman Drive #0612, La Jolla, CA 92093, United S

2 Present address: School of Life Sciences, KingsKingston Upon Thames, Surrey, KT1 2EE, UK.

3 Present address: Lied Transplant Center, Nebraska68198-7690, United States.

Costimulation via the PD-1 and B7-H1/B7-DC pathway regulates immunity. We investigated whether thePD-1/PD-L pathway is impaired in autoimmune diabetes. A progressive increase in the expression of PD-1and B7-H1/B7-DC on T cells and APC, respectively, was observed in the pancreatic lymph nodes of femalenon-obese diabetic (NOD) mice as they developed diabetes. A significantly decreased expression of PD-1and B7-H1/B7-DC on T cells and APC, respectively, was observed in the periphery of prediabetic NODmice versus non-diabetic C57BL/6 strain. NOD islets also displayed a reduced capacity to upregulateB7-H1 following exposure to inflammatory cytokines. In vivo blocking studies in NOD/B7-2KONOD micerevealed that B7-H1 and B7-DC positively costimulate autoreactive CD4 and CD8 T cells and may co-oper-ate with B7-2 to augment priming and expansion of naïve autoreactive T cells. In summary, these datasuggest that diabetes susceptibility in NOD mice is associated with altered PD-1/PD-L availability.

� 2009 Published by Elsevier Inc.

1. Introduction

The programmed death (PD)-1/PD-1 ligand (PD-L) costimula-tory pathway, is a member of the B7/CD28 family, and consistsof the PD-1 receptor and its ligands B7-H1 (PD-L1, CD274) andB7-DC (PD-L2, CD273). PD-1 is expressed on CD4 T cells, CD8 Tcells, B cells, NKT cells and monocytes upon activation [1]. In thethymus, PD-1 is primarily expressed in the early double negative(DN) stage of thymocyte maturation, and PD-1low cells in the DNpopulation represent ab T cells [2,3]. B7-H1 is expressed in bothlymphoid and non-lymphoid organs. It is upregulated on T cells,B cells, dendritic cells (DCs) and macrophages after activation, aswell as being expressed by pancreatic b cells, placenta, lung, heartand brain [4]. In contrast, B7-DC expression is restricted, primarilyto activated macrophages and DCs [5]. The different expression

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K054063 and # AI51973) toDepartment of Immunology,

.Hill@kingston.ac.uk (N. Hill),im@tmd.ac.jp (M. Azuma),

rsity of California, San Diego,tates.ton University, Penrhyn Rd,

Medical Center, Omaha, NE

patterns of B7-H1 and B7-DC suggest that they have distinct bio-logical functions.

Engagement of PD-1 by its ligands inhibits T cell responses [6].For example, PD-1 deficient mice on the C57BL/6 (B6) backgrounddevelop a lupus-like glomerulonephritis, whereas BALB/c PD-1�/�

mice exhibit fatal dilated cardiomyopathy [7]. In non obese dia-betic (NOD) mice, PD-1 deficiency accelerates the onset and fre-quency of type 1 diabetes [8]. Thus PD-1 deficiency consistentlypotentiates autoimmunity, and genetic background determinesdisease specificity. PD-1 may play a negative regulatory role inthe turnover of homeostatically proliferating T cells [9], and abnor-mal homeostatic control plays a role in the development of auto-immunity in the NOD mouse [10].

The effects of B7-H1 and B7-DC on autoreactive T cell responsesare, however, not clear and there is evidence that B7-H1 can haveboth positive and negative effects on inflammation and autoimmu-nity. For example, B7-H1 deficiency enhances T cell responses andincreases susceptibility to EAE and diabetes [11,12]. B7-DC can alsoinhibit T cell responses [13]. In NOD mice B7-H1 is thought to playthe more dominant role in restricting autoimmunity [12,14]. How-ever, there is evidence that B7-H1 and B7-DC can costimulate T cellactivation and promote inflammatory disease [15–17]. Even tissue-specific expression of B7-H1 can have distinct outcomes dependingon the genetic strain. For example, on the C57BL/6 background B7-H1 over expression in islets promotes diabetes [18], but NOD miceare protected from diabetes by islet B7-H1 over expression [12,19].PD-1/PD-L1 interactions are also involved in positive selection of

162 D. Yadav et al. / Cellular Immunology 258 (2009) 161–171

thymocytes [20], although whether aberrant thymic PD-l/PD-L1signaling affects autoimmunity is not yet known.

To try to reconcile these opposing results, it has been suggestedthat the pro-inflammatory effects of B7-H1 may be mediated byinteraction with a receptor other than PD-1 [15,21,22]. B7-H1has recently been shown to bind to B7-1, demonstrating an unex-pected cross-talk within members of the B7 family [23]. However,this interaction also results in the inhibition of T cell proliferationand cytokine production, and therefore does not yet explain thepro-inflammatory effects of B7-H1. Reverse signaling throughB7-H1 may also occur, and could contribute to the dichotomous ef-fects of the PD-1–PD-L pathway [24]. Regulation of the PD-1/PD-Lpathway is clearly complex, and it will be important to understandhow the net balance of the interactions between different costimu-latory molecules determines the pathogenicity of T cells.

Significantly lower PD-1 expression levels on peripheral CD4+ Tcells have been described in human patients with type 1 diabetes(T1D) [25] and multiple sclerosis (MS) [26] compared to healthycontrol subjects. Furthermore, polymorphisms in the PD-1 geneare associated with SLE susceptibility [27]. Defects in the expres-sion or function of PD-1 may therefore contribute to susceptibilityto autoimmune disease in human patients.

In the NOD mouse islet B7-H1 expression inhibits pathogenic Tcell responses [12]. NOD islets are known to express B7-H1 expres-sion [4], yet still they are destroyed. This suggests that the B7-H1 ex-pressed in NOD islets is not sufficiently protective. We hypothesizedthat the PD-1/PD-L pathway is impaired in NOD mice and does notexhibit its full immunoregulatory potential in controlling pancreaticautoimmunity. We therefore examined the expression kinetics ofPD-1/PD ligands during the transition to diabetes in female NODmice with age, and also compared their expression between NODand B6 mice. We also addressed the unresolved question regardingthe role of specific PD ligands in the activation and expansion ofautoreactive T cells by blocking B7-H1 and B7-DC following adop-tive transfer of BDC2.5 and 8.3Scid islet-specific T cells, and alsocompared these responses in NOD and B7-2KONOD recipients toexamine the interplay between B7 molecules and PD ligands.

Our results show an increase in the frequency of PD-1+ T cellsand PD-L+ APCs in NOD mice during diabetes progression, and adecrease in the percentage of CD8+PD-1+ T cells in the spleen.Immunophenotypic comparison of non-diabetic NOD mice withnon-autoimmune C57BL/6 (B6) mice demonstrated decreasedexpression of PD-1/PD ligands in the spleen and PLN3 of NOD mice,and the ability of NOD islets to upregulate B7-H1 in response toinflammatory cytokines was also defective. Furthermore, in vivoblocking studies suggest differential requirements for B7-H1 andB7-DC costimulation by autoreactive CD4 and CD8 T cells. Our stud-ies demonstrate that susceptibility to diabetes in NOD mice is asso-ciated with distinct changes in the availability of PD-1/PD ligands.

2. Materials and methods

2.1. Mice

Female NODShi, C57BL/6 (B6), TCR transgenic BDC2.5NOD and8.3NODScid mice were obtained from The Scripps Research Insti-tute animal facility. NOD.B7-2KO mice were a kind gift from Dr.J. Bluestone (Univ. California, San Francisco, CA). Mice with twosuccessive blood glucose levels >250 mg/dl were considered dia-betic. Animals were maintained in the institute’s animal facilityin pathogen free conditions. All experiments were conducted inaccordance with institutional guidelines for animal care and use.

3 Abbreviations used: PLN, pancreatic lymph nodes; KO, knock out; WT, wild type.

2.2. Antibodies, reagents and flow cytometric analyses

Single cell suspensions from spleen and pancreatic lymph nodes(PLN) were prepared; RBCs were lysed with ammonium chloridelysis buffer and washed with PBS. Cells (0.5–1.5 � 106/sample)were incubated with appropriate amounts of monoclonal antibod-ies (1 lg/million cells) and stained for indicated cell surface mark-ers. Antibodies to CD4, CD8, CD11b, CD11c, CD44, Vb4, Vb8.18.2,PD-1, B7-H1, and Annexin V, were obtained from BD Pharmingen(La Jolla, CA). Anti-mouse B7-DC was purchased from eBioscience(San Diego, CA). Blocking anti-mouse B7-H1 (MIH6, rat-IgG2a)and anti-mouse B7-DC mAb (TY25, rat-IgG2a) have been describedbefore [5,28]. For flow cytometric analyses, both CellQuest (BD Bio-sciences) and FlowJo (Tree Star Inc.) software programs were usedand the dead cells were gated out on the basis of forward and sidescatter. Appropriate isotype controls were used to determine thebackground staining. All mean values quoted in the text are ±SEM.

2.3. Preparation of islet tissue and cytokine treatment

Pancreatic islets were isolated using the collagenase P pancreasinflation method described previously [29]. Islets from 5 week oldNOD and B6 mice were treated with 100 U/ml IFN-c and1000 U/ml TNF-a (BD Pharmingen) for 48 h, or with medium alone(RPMI supplemented with 10% FCS, 2 mM L-glutamine and 0.2%(v/v), penicillin/streptomycin) in 10% CO2. Islets were thendispersed into single cells using 0.25% trypsin/EDTA, washedthoroughly, and allowed to recover for 1 h at 37 oC. Cells wereresuspended in FACS buffer and stained with anti-B7-H1, or therelevant isotype control, and 7AAD was used to exclude dead cellsfrom the analysis. Islet b cells were identified by their high flavinadenine dinucleotide autofluorescence detectable on the FL1channel as previously described [30].

2.4. Staining of pancreatic tissue

Sections (4 lm) were cut from the pancreas of 12 week old fe-male NOD mice fixed in 10% NBF and embedded in paraffin. Sec-tions were subjected to standard citrate buffer antigen retrieval,stained with goat anti-mouse B7-H1 antibody (AF1019 from R&DSystems; used at 4 lg/ml), and counterstained with hematoxylin.Original magnification 40�.

2.5. In vivo antibody blockade, CFSE labeling, adoptive transfers andannexin staining

Four to eight week old NOD and B7-2KO recipients were in-jected intraperitoneally (i.p.) with anti-B7-H1, anti-B7-DC oranti-B7-H1 + anti-B7-DC (250 lg each/animal) on day-0 and day-2. The control group of animals was treated with equal amountsof rat-IgG (Sigma). As a positive control, NODScid mice transferredwith enriched T cells from 18 week old NOD mice were treatedwith blocking anti-B7-H1 and anti-B7-DC antibodies and diabetesincidence was compared with rat-IgG treated controls. As previ-ously described [14], blocking anti-B7-H1 treated animals exhib-ited significantly earlier onset of diabetes compared to rat-IgGtreated controls whereas blocking anti-B7-DC did not exhibit asignificant difference, confirming the functionality of the blockingantibodies (not shown). Twelve to twenty million CFSE labeled[31] splenocytes from either BDC2.5NOD (5–6 week old) or 8.3Scidmice (5–7 week old) were also injected intravenously (200 ll insterile PBS) on day 0 into each NOD and B7-2KO recipient. Onday 4 post injection of cells, PLNs from treated animals wereanalyzed for expansion (CFSE dilution), activation (CD44hi) andsurvival (Annexin) and CFSE+ cells were analyzed by flowcytometry.

D. Yadav et al. / Cellular Immunology 258 (2009) 161–171 163

2.6. Statistical analyses

The Student t test (unpaired) was used to determine the level ofsignificance of the data using Statview software (Abacus Concepts,Berkely, CA). A value of p < 0.05 was considered significant.

3. Results

3.1. The expression of PD-1/PD ligands in NOD mice changes with age

We hypothesized that alterations in the availability of PD-1/PDligands may correlate with diabetes progression. Therefore, theexpression of PD-1 ligands B7-H1 and B7-DC was measured inDC (CD11b–CD11c+) and macrophage (CD11b+CD11c�) compart-ments and the expression of PD-1 was measured on T cells (CD4and CD8). Spleen and pancreatic lymph nodes were analyzed usingfemale NOD mice of varying ages. Three groups of female NODmice were designated as non-diabetic (2–3 weeks old), pre-dia-betic (10–12 weeks old) and diabetic (20–23 weeks old).

Interestingly, splenic APCs exhibited a progressive increase inthe proportion of B7-H1+ cells within the DC and macrophagecompartments as NOD mice aged. A similar increase in the propor-tion of B7-DC+ cells within the DC population was also observed inspleen with age (Fig. 1A–C). In the PLNs, an increase in the propor-tion of B7-H1+ cells was found within the DC and macrophagecompartments in aged NOD mice. A similar increase in the propor-tion of B7-DC+ cells within the DC population with age was alsoobserved (Fig. 1A–C). The level of B7-DC+ signal in both the splenicand PLN macrophages was found to be undetectable. Thus thesedata show that there is a general increase in the availability ofPD-L+ immune cells in the periphery of NOD mice with age.

In peripheral T cells, a significantly higher percentage of PD-1+cells within the CD8+ T lymphocyte population was observed inthe spleen of non-diabetic mice, compared to both pre-diabetic(p = 0.043) and diabetic mice (p = 0.030). In contrast, a progressiveincrease in the proportion of PD-1+ cells within the CD8+ T lym-phocyte population was observed in the PLN of NOD mice withage (Fig. 1D and E). No significant change in the expression ofPD-1+ on splenic CD4 T cells was observed in NOD mice with age(Fig. 1D and E). In the PLNs, however, the percentage of PD-1+ cellswithin the CD4+ T lymphocyte population was significantly lowerin non-diabetic mice (p = 0.001) compared to pre-diabetic and dia-betic mice. Thus NOD mice exhibit distinct changes in the levels ofPD-1/PD ligands.

3.2. PD-1 and PD ligands are differentially expressed in the immunesystem of NOD and B6 mice

The data described above indicate that diabetes in the NODmouse progresses despite increased expression of PD-1/PD ligandsin peripheral lymphoid organs, suggesting that the PD-1/PD-Lpathway in NOD mice may not be fully effective in regulating auto-immunity. We therefore tested whether the levels of PD-1 and PD-L differ in NOD mice compared to the diabetes resistant strain. Wemeasured the expression of PD-1/PD ligands on APC and T cells in11–13 week old NOD and diabetes resistant C57BL/6 (B6) mice inthe peripheral lymphoid organs.

In peripheral T cells, comparable expression of PD-1 in bothCD4+ and CD8+ T cell was observed in the spleen of NOD and B6mice. However, in PLN B6 mice showed a significantly higher per-centage of PD-1+ cells both within CD4+ T cells (p = 0.031) andCD8+ T cells (p = 0.016) compared to NOD mice (Fig. 2A and C).

The expression of B7-H1 was significantly higher in the splenicDC population of B6 mice compared to NOD (p < 0.0001). In addi-tion, there was a modest but significant increase in B7-H1+ cells

in the macrophages of B6 mice in the spleen (p = 0.012), but notin the PLN. However, DCs in the PLN of B6 mice displayed a signif-icant increase in the proportions of both B7-H1+ cells (p = 0.003) aswell as B7-DC+ cells (p = 0.005) compared to NOD mice (Fig. 2B andD). These observations suggest a quantitative difference betweenNOD mice and diabetes resistant B6 strain regarding the availabil-ity of PD-1/PD ligands.

3.3. NOD islet cells have an impaired capacity to upregulate B7-H1expression

B7-H1 is induced by inflammatory cytokines [5,32] and isknown to be expressed in NOD islets during insulitis [4]. We con-firmed this observation, and were able to demonstrate B7-H1staining at the surface of inflamed NOD islet cells (Fig. 3A). DespiteB7-H1 expression, however, NOD islets still succumb to autoim-munity. We therefore tested in vitro whether NOD islets exhibitinherent defect in their ability to regulate B7-H1 expression ascompared to islets from diabetes resistant B6 strain. We used isletsfrom very young (5 week old) NOD mice in order to examine theregulation of B7-H1 expression in islets largely unaffected byendogenous inflammation. We treated islets with TNF-a (1000 U/ml) and IFN-c (100 U/ml) and using flow cytometry examinedthe ability of NOD and B6 islets to upregulate B7-H1. Cytokinetreatment was found to be a powerful inducer of B7-H1 expressionin islet b cells, increasing the MFI of B7-H1 staining in B6 islets byover 50-fold. However, B7-H1 expression in cytokine-treated NODislets was significantly lower compared to cytokine-treated B6 is-lets (average MFI of 2884 ± 352 in NOD versus 4328 ± 275 in B6 is-lets, p = 0.0006, n = 4 each strain) (Fig. 3B and C). This suggests thatNOD islets have an inherent defect in their ability to upregulate theimmunoregulatory molecule B7-H1 in response to inflammatorycytokines.

3.4. Distinct role of B7-H1 and B7-DC in autoreactive T cell activation,expansion and survival in vivo

PD ligands can deliver costimulatory or inhibitory signalsdepending on the type of immune cells involved, the activation sta-tus of the T cells, nature of the disease, and the genetic backgroundof the mice [6,18]. In addition, PD ligands have been shown toregulate the apoptosis of activated T cells [33]. We asked whetherB7-H1, alone or in combination with B7-DC, delivers positive ornegative signals to naïve anti islet T cells. Costimulation throughB7-2 is key in augmenting the pathogenicity of immune repertoirein pancreatic autoimmunity [31,34–36], and B7-DC and B7-1/B7-2molecules have been shown to work in concert to stimulate T cells[21]. We therefore tested the activation, expansion and survivalstatus of anti islet CD4 and CD8 T cells under B7-H1 and B7-DCblockade in NOD and B7-2KO mice. For this, we adoptively trans-ferred naïve (>85% CD44low, data not shown) CFSE labeled spleno-cytes from BDC2.5/8.3Scid mice, which express a b-cell-reactiveCD4+ and CD8+ T cell clones, respectively [37,38], into NOD andB7-2KO mice treated with anti-B7-H1 and anti-B7-DC blockingantibodies, alone or in combination. Four days post transfer, theexpansion (CFSE dilution), activation (CD44hi expression) and sur-vival (annexin) of adoptively transferred cells were monitored byflow cytometry. Interestingly, in NOD mice, combined B7-H1 andB7-DC blockade led to a significant reduction (p < 0.0001) in thepercentage of both proliferating (CD4+Vb4+CFSElow) auto reactiveCD4 T cells as well as activated (CD4+Vb4+CFSE+CD44hi) CD4 Tcells compared to rat-IgG treated mice (Fig. 4A). Anti-B7-H1 trea-ted mice also exhibited a significant reduction in the percentageof both proliferating (p = 0.016) and activated (p = 0.035) CD4 Tcells compared to rat-IgG treated controls. However, anti-B7-DCtreated mice did not exhibit a significant difference in the

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Fig. 1. Expression kinetics of PD-1/B7-H1/B7-DC in female NOD mice with age. The numbers in the dot plots depict the mean percentage ± SEM. (A) and (B) represent theproportion of B7-H1 and B7-DC positive cells in the dendritic cell (CD11c+) and macrophage (CD11b+) gated compartments respectively as also represented by the ‘‘logicle/bi-exponential” display histograms in (C). The black lines in histograms represent the isotype staining, whereas the red, blue and green lines represent the staining pattern innon-diabetic, pre-diabetic and diabetic animals respectively. (D) shows the proportion of PD-1+ T cells in the spleen and PLN as also represented by the dot plots in (E). Dataare pooled from two independent experiments. The error bars represent SEM. ND means not detected. NS means not significant.

164 D. Yadav et al. / Cellular Immunology 258 (2009) 161–171

percentage of either proliferating or activated CD4 T cells. Further-more, no significant change in the proportion of CD4+Vb4+CFSE+cells undergoing apoptosis was observed upon combined or singleB7-H1 or B7-DC blockade (Fig. 4A).

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recipients. However, a modest increase (p = 0.051) in the prolifera-tion of autoreactive CD8 T cells was observed upon B7-H1 blockade(Fig. 4B). On the other hand, combined B7-H1 and B7-DC blockadecaused significant reduction (p = 0.019) in the percentage of acti-vated (CD8+Vb8.1/8.2+CFSE+CD44hi) cells compared to rat-IgGtreated controls. No significant change in the proportion of acti-vated autoreactive CD8 T cells was observed upon B7-DC blockade,whereas a modest increase (p = 0.067) was observed upon B7-H1

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D. Yadav et al. / Cellular Immunology 258 (2009) 161–171 165

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Fig. 2. The differences in PD-1/PD-L expression in NOD mice vs. B6 mice. (A) and (B) represent the proportion of PD-1+ cells and B7-H1/B7-DC+ cells in T cell and APCcompartments respectively in NOD vs. B6 mice. (C) and (D) are the ‘‘logicle/bi-exponential” dot plot and histogram depiction of data described in (A) and (B), respectively. Theblack line in histograms represents the isotype staining, whereas the red line and blue line represent the staining pattern in NOD mice and B6 mice respectively. Data arepooled from six animals in each group (11–13 weeks old). The error bars represent SEM. ND means not detected. NS means not significant.

166 D. Yadav et al. / Cellular Immunology 258 (2009) 161–171

blockade (Fig. 4B). Interestingly, a significantly lower proportion ofCD8+Vb8.1/8.2+CFSE cells undergoing apoptosis was observedupon combined B7-H1 and B7-DC blockade (p = 0.007) as well asupon anti-B7-H1 treatment (p = 0.032) in NOD recipients (Fig. 4B).

Combined PD ligand blockade in B7-2KO mice also displayed asignificant reduction in the proportion of both proliferating(p = 0.011) and activated (p = 0.002) autoreactive CD4 T cells com-pared to rat-IgG treated controls. Anti-B7-DC treated B7-2KO mice

showed a modest reduction (p = 0.048) in CD4 T cell proliferationbut the difference between the percentages of activated cells inthe anti B7-DC treated group was not significantly different. Treat-ment of B7-2KO mice with anti-B7-H1 caused a significant reduc-tion (p = 0.027) in the activation status of autoreactive CD4 T cellsbut B7-H1 blockade alone did not further inhibit proliferation inthe absence of B7-2 (Fig. 4C). Also, no significant change in the pro-portion of apoptotic CD4+Vb4+CFSE+ cells was observed upon

(A)5000

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4500 NOD B6)

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B6

MFI=69+/-2 MFI=2,884+/-352

NOD

B7-H1B7 H1

Fig. 3. Reduced capacity of NOD islets to upregulate B7-H1 expression. (A) represents pancreas sections from 12 week old NOD stained with anti-PD-L1 antibody. Thestaining with secondary alone antibody showed negligible background staining. Islets from 5 week old NOD and B6 mice (pooled from 2 donors for each strain) treated with100 U/ml IFN-c+ 1000 U/ml TNF-a for 48 h, or with medium alone, then dispersed and stained with anti-B7-H1 or the relevant isotype control and 7AAD (to exclude deadcells from analysis). Average mean fluorescence intensity (MFI) of B7-H1 staining ± SEM (n = 4 for each treatment; gated on live FL1+b cells) is shown in (B), andrepresentative histograms of B7-H1 expression (solid line) and isotype control staining (dotted line) are shown in (C). The data are representative of two independentexperiments.

D. Yadav et al. / Cellular Immunology 258 (2009) 161–171 167

combined or single B7-H1 or B7-DC blockade in B7-2KO mice(Fig. 4C).

The adoptively transferred anti islet CD8 T cells exhibited a sig-nificant reduction in their proliferation (p = 0.013), activation(p = 0.032) and susceptibility to apoptosis (p = 0.019) upon com-bined B7-H1 and B7-DC blockade compared to the rat-IgG treatedgroup in B7-2KO recipients. However, no significant change in theproportion of proliferating or activated anti islet CD8 T cells wasobserved upon B7-H1 blockade (Fig. 4D). These results indicatethat in NOD mice, PD ligands and B7-H1 in particular can providepositive signals for autoreactive CD4 T cell priming and expansionwithout having a detectable effect on cell survival. Also, B7-H1/B7-DC blockade in NOD mice reduced the activation of anti islet CD8 Tcells while enhancing their survival. The blocking data regardingB7-2KONOD mice suggests that B7-2 provides a strong positivesignal for naïve anti islet T cell activation and expansion, but thatcombined blockade of PD ligands can further inhibit T cell activa-tion and proliferation in B7-2 deficient mice. B7-H1/B7-DC block-ade enhanced anti islet CD8 T cell survival in B7-2KO recipientswithout affecting CD4 T cells.

4. Discussion

There remain many unanswered questions concerning theimmunobiology of the PD-1/PD-L pathway. Whether the availability

of PD ligands B7-H1 and B7-DC correlated with progression ofautoimmune diabetes is not known. Also, the nature of costimula-tion (positive or negative) provided by the PD ligands to T cells isstill under debate. While the inhibitory effect of B7-H1 on effectorT cell responses and tissue damage has been shown in many stud-ies [6–8,12] there is growing evidence to suggest that B7-H1 canalso promote the activation and proliferation of naïve T cells[18,22,39]. It has been suggested that the stimulatory effect ofB7-H1 may occur via interaction with an as yet unknown ligandon T cells that is distinct from PD-1 [40]. This hypothesis stemsfrom the contrast between the largely unambiguous inhibitory ef-fects of PD-l ligation on T cell activation, as shown by the develop-ment of autoimmunity in PD-1 deficient mice [8,41,42], comparedto the more mixed effects of B7-H1 modulation on T cell activationand autoimmunity.

Our results clearly show that in NOD mice blocking B7-H1 andB7-DC inhibits the proliferation and activation of naïve autoreac-tive CD4 T cells in the draining lymph node. While the effect ofblocking B7-H1 was more pronounced than blocking B7-DC, block-ing both receptors had a greater effect than single treatment alone.This data therefore provides further support for the ability of B7-H1/B7-DC to stimulate naïve CD4 T cell responses. In contrast,blocking B7-H1/B7-DC did not significantly affect the proliferationof naïve autoreactive CD8 T cells, although a marginal inhibition ofCD8 T cell activation and apoptosis by the combination of blocking

Fig. 4. B7-H1 and B7-DC modulate anti islet CD4 T cell and CD8 T cell priming, expansion and survival. CFSE-labeled splenocytes (1.5–2 � 107cells) from 5 to 6 week oldBDC2.5 NOD mice were administered i.v. into 4–6 week old NOD (A) and B7-2KO (C) recipient mice, also intraperitoneally injected (250 lg each � 2/animal) with anti-B7-H1,anti-B7-DC, anti-B7-H1 + B7-DC or rat-IgG as controls (as described in materials and methods). In order to study the anti islet CD8 T cell response, 12–20 million cells from 5–7 weeks old 8.3Scid mice were administered i.v. into 6–8 weeks old NOD (B) and B7-2KO (D) recipients, which were also injected with blocking B7-H1/B7-DC antibodies asdescribed above. Four days post injection of cells; the PLN cells were analyzed by flow cytometry to assess the extent of proliferation (% of CD4+Vb4+cells or CD8+Vb8.1/8.2+cells that are CFSElow), activation (% of CD4+Vb4+CFSE+ cells or CD8+Vb8.1/8.2+CFSE+ cells that are CD44hi) and survival (% of CD4+Vb4+CFSE+ cells or CD8+Vb8.1/8.2+CFSE+cells that are Annexin+). Analysis of BDC2.5/8.3Scid cells before transfer indicated >85% of them were naive (i.e. CD44low). The data are pooled from 2–3 independentexperiments. p values in histograms represent the statistical significance with respect to corresponding rat-IgG treated control group. Numbers in parenthesis indicate thenumber of animals in that group. NS means not significant. ND means not done. Values represent mean percentage ± SEM.

168 D. Yadav et al. / Cellular Immunology 258 (2009) 161–171

B7-H1 and B7-DC was observed. The costimulatory action of B7-H1/B7-DC may therefore affect primarily naïve CD4 T cells. PD-1is not expressed on naïve T cells but is upregulated upon activa-tion; its expression peaks at day 3 and remains on the cell surface[6]. Thus the costimulatory effect of B7-H1/B7-DC may dependupon the relative expression of PD-1, and potentially also the puta-tive pro-inflammatory B7-H1/B7-DC receptor on T cells. A recent

study by Guleria et al. [43] addressed similar questions. They ob-served a comparable trend towards increased 8.3 T cell expansionduring B7-H1 blockade, although they did not observe the de-creased expansion of BDC2.5 T cells during B7-H1 blockade thatwe observed. The reasons for this discrepancy are not clear, butmay be related to differences in the purification or activation stateof BDC2.5 T cells transferred. It will be of interest in future studies

Fig. 4 (continued)

D. Yadav et al. / Cellular Immunology 258 (2009) 161–171 169

to address these questions, and such investigations may help us tounderstand the conditions under which B7-H1 promotes or inhib-its T cell activation.

The expression of multiple co-stimulatory molecules on the APCsurface is dynamically controlled and determines T cell activation,as well as regulating the strength and duration of the response.Whether co-operation exists between B7 molecules and PD ligandsis not yet clear. B7-2KONOD mice have severe defects in anti islet Tcell priming and expansion [31]. Our current studies show thatblocking B7-H1 and B7-DC further impairs the activation of naiveautoreactive T cells in these mice, and affects both CD4 and CD8T cell subsets. These observations suggest that PD ligands, particu-larly B7-H1, cooperate with B7-2 to promote initial T cell priming.

A similar co-operation between B7-DC and B7 molecules has alsobeen proposed [21].

The important immune regulatory role of PD-1/PD-L signalingsuggests the possibility that genetic variants affecting this pathwaymay influence the development of autoimmune disease [6]. Ourdata show that the islets of NOD mice have an intrinsic defect intheir ability to upregulate B7-H1 in response to cytokines. Isletslacking B7-H1 are more rapidly destroyed by an autoreactive re-sponse than wild type islets, demonstrating that B7-H1 on isletcells is an important negative regulator of tissue destruction [12].It is possible that the 50% reduction in B7-H1 expression that weobserve in NOD islets is sufficient to impact regulation of the effec-tor T cell response in the target tissue, although further studies

170 D. Yadav et al. / Cellular Immunology 258 (2009) 161–171

using genetically similar strains such as NOR mice will be requiredto test this hypothesis.

We also found that NOD mice have significantly reducedexpression of B7-H1 and B7-DC on APC from lymphoid organscompared to B6 mice, and show decreased expression of PD-1 onboth CD4 and CD8 T cells in the PLN. This suggests that there is alsoreduced signaling through the PD-1/PD-L pathway in the second-ary lymphoid organs of NOD mice compared to B6. However, inNOD mice the expression of both B7-H1 and B7-DC increases inmultiple APC subsets during diabetes progression. The expressionof PD-1 on CD8 T cells in the PLNs of NOD mice also increases asthey age and develop diabetes. Therefore, immunophenotypicanalysis of NOD mice indicates that progression to diabetes doesnot fully correlate with PD-1/PDL availability in lymphoid tissues.The increase in PD-1 expression may reflect an accumulation ofactivated T cells during disease progression, since PD-1 is ex-pressed by activated but not naïve T cells [1]. Moreover, increasedexpression of B7-H1 is associated with chronic inflammation in arange of disorders [40]. Whether this increased availability is con-tributing to the emergence of autoreactivity is not clear since theexact nature of the expression of positive costimulatory non PD-1 ligand is not yet described. It is possible that PD-1 and nonPD-1 receptors compete with each other to influence the TCR acti-vation threshold [22]. Thus, the different threshold levels of regu-lation through the PD-1/B7-H1 and nonPD-1/B7-H1 may lead tothe divergent results in terms of co-inhibition or co-stimulation.Furthermore, an elegant study by Keir et al. [12] suggests thatB7-H1 and B7-DC may have more subtle roles during the activationof self-reactive T cells in lymphoid organs, but the unique expres-sion of PD-L1 in the pancreas may lead to the predominant role forPD-L1 during autoimmune diabetes. It is possible that suboptimalavailability of PD-1/PD ligands may result in inadequate protectionagainst autoimmunity. Indeed a continuous PD-1 engagement bothon APCs and peripheral tissues has been suggested to be essentialfor the maintenance of peripheral tolerance [44]. It has also beenreported that NOD T cells are sub optimally activated due to re-duced availability of the costimulatory molecule B7–2, which inturn leads to insufficient induction of tolerogenic signals viaCTLA-4 [45]. Hence, diabetes susceptibility may be due to broadimbalances in T cell costimulation.

Thus our data support a model in which co-stimulatory and co-inhibitory roles of PD ligands, especially B7-H1, depend on thestate of inflammation and stage of T cell priming. During the initi-ation of autoimmunity, PD ligands and particularly B7-H1 mayprovide positive signals to naïve autoreactive T cells in the draininglymph nodes, possibly via a non PD-1 receptor expressed on naïveT cells. After migration into the infected tissue, activated effector Tcells and other immune cells release inflammatory cytokines suchas IFN-c that upregulate B7-H1 on islets. The interaction of isletB7-H1 with PD-1 on activated T cells then inhibits the pathogenicautoreactive response within the target tissue. Future immuno-therapies that enhance the availability of negative costimulatorysignals may represent an attractive approach for inhibiting effectorresponses in the target tissue, or for combating the emergence ofthe autoreactive T cell repertoire.

Acknowledgments

The authors also wish to thank Jack Bui, Malin Flodstrom-Tull-berg, Sandrine Dabernat and members of the Sarvetnick lab fortheir helpful comments and Mary Cleary, Cody Fine and, PatrickSecrest for their technical help with flow cytometric staining andanimal handling. Deepak Yadav is the recipient of postdoctoral fel-lowship awards from Myasthenia Gravis Foundation of America(MGFA, 2003) and Juvenile Diabetes Research Foundation Interna-tional (JDRFI, 2005).

References

[1] Y. Agata, A. Kawasaki, H. Nishimura, Y. Ishida, T. Tsubata, H. Yagita, T. Honjo,Expression of the PD-1 antigen on the surface of stimulated mouse T and Blymphocytes, Int. Immunol. 8 (1996) 765–772.

[2] H. Nishimura, T. Honjo, N. Minato, Facilitation of beta selection andmodification of positive selection in the thymus of PD-1-deficient mice, J.Exp. Med. 191 (2000) 891–898.

[3] H. Nishimura, Y. Agata, A. Kawasaki, M. Sato, S. Imamura, N. Minato, H. Yagita,T. Nakano, T. Honjo, Developmentally regulated expression of the PD-1 proteinon the surface of double-negative (CD4-CD8-) thymocytes, Int. Immunol. 8(1996) 773–780.

[4] S.C. Liang, Y.E. Latchman, J.E. Buhlmann, M.F. Tomczak, B.H. Horwitz, G.J.Freeman, A.H. Sharpe, Regulation of PD-1, PD-L1, and PD-L2 expression duringnormal and autoimmune responses, Eur. J. Immunol. 33 (2003) 2706–2716.

[5] T. Yamazaki, H. Akiba, H. Iwai, H. Matsuda, M. Aoki, Y. Tanno, T. Shin, H.Tsuchiya, D.M. Pardoll, K. Okumura, M. Azuma, H. Yagita, Expression ofprogrammed death 1 ligands by murine T cells and APC, J. Immunol. 169(2002) 5538–5545.

[6] S.J. Khoury, M.H. Sayegh, The roles of the new negative T cell costimulatorypathways in regulating autoimmunity, Immunity 20 (2004) 529–538.

[7] T. Okazaki, T. Honjo, The PD-1–PD-L pathway in immunological tolerance,Trends Immunol. 27 (2006) 195–201.

[8] J. Wang, T. Yoshida, F. Nakaki, H. Hiai, T. Okazaki, T. Honjo, Establishment ofNOD-Pdcd1�/� mice as an efficient animal model of type I diabetes, Proc. Natl.Acad. Sci. USA 102 (2005) 11823–11828.

[9] S.J. Lin, C.D. Peacock, K. Bahl, R.M. Welsh, Programmed death-1 (PD-1) definesa transient and dysfunctional oligoclonal T cell population in acutehomeostatic proliferation, J. Exp. Med. 204 (2007) 2321–2333.

[10] C. King, A. Ilic, K. Koelsch, N. Sarvetnick, Homeostatic expansion of T cellsduring immune insufficiency generates autoimmunity, Cell 117 (2004) 265–277.

[11] Y.E. Latchman, S.C. Liang, Y. Wu, T. Chernova, R.A. Sobel, M. Klemm, V.K.Kuchroo, G.J. Freeman, A.H. Sharpe, PD-L1-deficient mice show that PD-L1 on Tcells, antigen-presenting cells, and host tissues negatively regulates T cells,Proc. Natl. Acad. Sci. USA 101 (2004) 10691–10696.

[12] M.E. Keir, S.C. Liang, I. Guleria, Y.E. Latchman, A. Qipo, L.A. Albacker, M.Koulmanda, G.J. Freeman, M.H. Sayegh, A.H. Sharpe, Tissue expression of PD-L1mediates peripheral T cell tolerance, J. Exp. Med. 203 (2006) 883–895.

[13] Y. Latchman, C.R. Wood, T. Chernova, D. Chaudhary, M. Borde, I. Chernova, Y.Iwai, A.J. Long, J.A. Brown, R. Nunes, E.A. Greenfield, K. Bourque, V.A. Boussiotis,L.L. Carter, B.M. Carreno, N. Malenkovich, H. Nishimura, T. Okazaki, T. Honjo,A.H. Sharpe, G.J. Freeman, PD-L2 is a second ligand for PD-1 and inhibits T cellactivation, Nat. Immunol. 2 (2001) 261–268.

[14] M.J. Ansari, A.D. Salama, T. Chitnis, R.N. Smith, H. Yagita, H. Akiba, T. Yamazaki,M. Azuma, H. Iwai, S.J. Khoury, H. Auchincloss Jr., M.H. Sayegh, Theprogrammed death-1 (PD-1) pathway regulates autoimmune diabetes innonobese diabetic (NOD) mice, J. Exp. Med. 198 (2003) 63–69.

[15] H. Dong, G. Zhu, K. Tamada, L. Chen, B7-H1, a third member of the B7 family,co-stimulates T-cell proliferation and interleukin-10 secretion, Nat. Med. 5(1999) 1365–1369.

[16] S.Y. Tseng, M. Otsuji, K. Gorski, X. Huang, J.E. Slansky, S.I. Pai, A. Shalabi, T. Shin,D.M. Pardoll, H. Tsuchiya, B7-DC, a new dendritic cell molecule with potentcostimulatory properties for T cells, J. Exp. Med. 193 (2001) 839–846.

[17] T. Kanai, T. Totsuka, K. Uraushihara, S. Makita, T. Nakamura, K. Koganei, T.Fukushima, H. Akiba, H. Yagita, K. Okumura, U. Machida, H. Iwai, M. Azuma, L.Chen, M. Watanabe, Blockade of B7-H1 suppresses the development of chronicintestinal inflammation, J. Immunol. 171 (2003) 4156–4163.

[18] S.K. Subudhi, P. Zhou, L.M. Yerian, R.K. Chin, J.C. Lo, R.A. Anders, Y. Sun, L. Chen, Y.Wang, M.L. Alegre, Y.X. Fu, Local expression of B7-H1 promotes organ-specificautoimmunity and transplant rejection, J. Clin. Invest. 113 (2004) 694–700.

[19] C.J. Wang, F.C. Chou, C.H. Chu, J.C. Wu, S.H. Lin, D.M. Chang, H.K. Sytwu,Protective role of programmed death 1 ligand 1 (PD-L1) in nonobese diabeticmice: the paradox in transgenic models, Diabetes 57 (2008) 1861–1869.

[20] M.E. Keir, Y.E. Latchman, G.J. Freeman, A.H. Sharpe, Programmed death-1 (PD-1): PD-ligand 1 interactions inhibit TCR-mediated positive selection ofthymocytes, J. Immunol. 175 (2005) 7372–7379.

[21] T. Shin, G. Kennedy, K. Gorski, H. Tsuchiya, H. Koseki, M. Azuma, H. Yagita, L.Chen, J. Powell, D. Pardoll, F. Housseau, Cooperative B7–1/2 (CD80/CD86) andB7-DC costimulation of CD4+ T cells independent of the PD-1 receptor, J. Exp.Med. 198 (2003) 31–38.

[22] S. Wang, J. Bajorath, D.B. Flies, H. Dong, T. Honjo, L. Chen, Molecular modelingand functional mapping of B7-H1 and B7-DC uncouple costimulatory functionfrom PD-1 interaction, J. Exp. Med. 197 (2003) 1083–1091.

[23] M.J. Butte, M.E. Keir, T.B. Phamduy, A.H. Sharpe, G.J. Freeman, Programmeddeath-1 ligand 1 interacts specifically with the B7–1 costimulatory moleculeto inhibit T cell responses, Immunity 27 (2007) 111–122.

[24] M.E. Keir, L.M. Francisco, A.H. Sharpe, PD-1 and its ligands in T-cell immunity,Curr. Opin. Immunol. 19 (2007) 309–314.

[25] Y. Tsutsumi, X. Jie, K. Ihara, A. Nomura, S. Kanemitsu, H. Takada, T. Hara,Phenotypic and genetic analyses of T-cell-mediated immunoregulation inpatients with Type 1 diabetes, Diabet. Med. 23 (2006) 1145–1150.

[26] M. Krakauer, P.S. Sorensen, F. Sellebjerg, CD4(+) memory T cells with highCD26 surface expression are enriched for Th1 markers and correlate withclinical severity of multiple sclerosis, J. Neuroimmunol. 181 (2006) 157–164.

D. Yadav et al. / Cellular Immunology 258 (2009) 161–171 171

[27] L. Prokunina, C. Castillejo-Lopez, F. Oberg, I. Gunnarsson, L. Berg, V.Magnusson, A.J. Brookes, D. Tentler, H. Kristjansdottir, G. Grondal, A.I.Bolstad, E. Svenungsson, I. Lundberg, G. Sturfelt, A. Jonssen, L. Truedsson, G.Lima, J. Alcocer-Varela, R. Jonsson, U.B. Gyllensten, J.B. Harley, D. Alarcon-Segovia, K. Steinsson, M.E. Alarcon-Riquelme, A regulatory polymorphism inPDCD1 is associated with susceptibility to systemic lupus erythematosus inhumans, Nat. Genet. 32 (2002) 666–669.

[28] F. Tsushima, H. Iwai, N. Otsuki, M. Abe, S. Hirose, T. Yamazaki, H. Akiba, H. Yagita,Y. Takahashi, K. Omura, K. Okumura, M. Azuma, Preferential contribution of B7–H1 to programmed death-1-mediated regulation of hapten-specific allergicinflammatory responses, Eur. J. Immunol. 33 (2003) 2773–2782.

[29] M. Solomon, M. Flodstrom-Tullberg, N. Sarvetnick, Differences in suppressor ofcytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normalBALB/c and spontaneously-diabetic NOD recipient mice, Transplantation 79(2005) 1104–1109.

[30] D.G. Pipeleers, P.A. In’t veld, M. Van de Winkel, E. Maes, F.C. Schuit, W. Gepts, Anew in vitro model for the study of pancreatic A and B cells, Endocrinology 117(1985) 806–816.

[31] D. Yadav, V. Judkowski, M. Flodstrom-Tullberg, L. Sterling, W.L. Redmond, L.Sherman, N. Sarvetnick, B7-2 (CD86) controls the priming of autoreactive CD4T cell response against pancreatic islets, J. Immunol. 173 (2004) 3631–3639.

[32] P. Loke, J.P. Allison, PD-L1 and PD-L2 are differentially regulated by Th1 andTh2 cells, Proc. Natl. Acad. Sci. USA 100 (2003) 5336–5341.

[33] S.E. Sandner, M.R. Clarkson, A.D. Salama, A. Sanchez-Fueyo, C. Domenig, A.Habicht, N. Najafian, H. Yagita, M. Azuma, L.A. Turka, M.H. Sayegh, Role of theprogrammed death-1 pathway in regulation of alloimmune responses in vivo,J. Immunol. 174 (2005) 3408–3415.

[34] H. Bour-Jordan, B.L. Salomon, H.L. Thompson, G.L. Szot, M.R. Bernhard, J.A.Bluestone, Costimulation controls diabetes by altering the balance ofpathogenic and regulatory T cells, J. Clin. Invest. 114 (2004) 979–987.

[35] D. Yadav, N. Sarvetnick, Immunomodulation of the anti-islet CD8 T cellresponse by B7-2, J. Clin. Immunol. 27 (2007) 221–226.

[36] D. Yadav, N. Sarvetnick, B7-2 regulates survival, phenotype, and function ofAPCs, J. Immunol. 178 (2007) 6236–6241.

[37] J.D. Katz, B. Wang, K. Haskins, C. Benoist, D. Mathis, Following a diabetogenic Tcell from genesis through pathogenesis, Cell 74 (1993) 1089–1100.

[38] J. Verdaguer, D. Schmidt, A. Amrani, B. Anderson, N. Averill, P. Santamaria,Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabeticmice, J. Exp. Med. 186 (1997) 1663–1676.

[39] N. Martin-Orozco, Y.H. Wang, H. Yagita, C. Dong, Cutting edge: programmeddeath (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance totissue antigens, J. Immunol. 177 (2006) 8291–8295.

[40] H. Dong, X. Chen, Immunoregulatory role of B7-H1 in chronicity ofinflammatory responses, Cell Mol. Immunol. 3 (2006) 179–187.

[41] H. Nishimura, M. Nose, H. Hiai, N. Minato, T. Honjo, Development of lupus-likeautoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor, Immunity 11 (1999) 141–151.

[42] H. Nishimura, T. Okazaki, Y. Tanaka, K. Nakatani, M. Hara, A. Matsumori, S.Sasayama, A. Mizoguchi, H. Hiai, N. Minato, T. Honjo, Autoimmune dilatedcardiomyopathy in PD-1 receptor-deficient mice, Science 291 (2001) 319–322.

[43] I. Guleria, M. Gubbels Bupp, S. Dada, B. Fife, Q. Tang, M.J. Ansari, S.Trikudanathan, N. Vadivel, P. Fiorina, H. Yagita, M. Azuma, M. Atkinson, J.A.Bluestone, M.H. Sayegh, Mechanisms of PDL1-mediated regulation ofautoimmune diabetes, Clin. Immunol. 125 (2007) 16–25.

[44] M.E. Keir, G.J. Freeman, A.H. Sharpe, PD-1 regulates self-reactive CD8+ T cellresponses to antigen in lymph nodes and tissues, J. Immunol. 179 (2007)5064–5070.

[45] E. Dahlen, G. Hedlund, K. Dawe, Low CD86 expression in the nonobese diabeticmouse results in the impairment of both T cell activation and CTLA-4 up-regulation, J. Immunol. 164 (2000) 2444–2456.