Diabetes with the Development of Autoimmune VTCN1 (B7-H4

Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune This information is current as Diabetes of October 1, 2021. Ilian A. Radichev, Lilia V. Maneva-Radicheva, Christina Amatya, Maryam Salehi, Camille Parker, Jacob Ellefson, Paul Burn and Alexei Y. Savinov

J Immunol published online 15 January 2016 Downloaded from http://www.jimmunol.org/content/early/2016/01/15/jimmun ol.1403251

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 15, 2016, doi:10.4049/jimmunol.1403251 The Journal of Immunology

Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes

Ilian A. Radichev,* Lilia V. Maneva-Radicheva,* Christina Amatya,* Maryam Salehi,* Camille Parker,* Jacob Ellefson,* Paul Burn,* and Alexei Y. Savinov*,†

Ag-speciﬁc activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set do-

main–containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice Downloaded from and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both a and b cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells’ VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the signiﬁcant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target http://www.jimmunol.org/ tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D. The Journal of Immunology, 2016, 196: 000–000.

ype 1 diabetes (T1D) is a life-threatening disease of an possess several protective mechanisms capable of downregulation autoimmune nature, for which the only currently available of autoimmune attack (3, 4). One of such mechanisms is at the treatment is continuous insulin administration. Clinical center of our investigation. T by guest on October 1, 2021 T1D arises as a consequence of the cytotoxic destruction of insulin- V-set domain–containing T cell activation inhibitor 1 (VTCN1), producing b cells by abnormally activated autoreactive T cells also known as B7-H4, B7S1, B7X, is a negative costimulatory specific for multiple islet cells’ Ags (1, 2). Accumulating evi- molecule, representing one of the newly discovered members of dence, however, suggests that islet cells do not merely play a role the B7 family (5–7). VTCN1 acts through a not yet identified of plain targets of autoimmune destruction, but, on the contrary, receptor on T cells, inhibiting T cell activation, proliferation, and cytokine production (5, 6, 8, 9). The persistence of autoreactive T cell responses during T1D prompted several experimental at- *The Sanford Project, Children’s Health Research Center, Sanford Research, Sioux Falls, SD 57104; and †Department of Pediatrics, University of South Dakota School tempts to alleviate diabetogenic autoimmunity via artificial en- of Medicine, Sioux Falls, SD 57105 richment of VTCN1-mediated coinhibition. Accordingly, matrix ORCIDs: 0000-0001-9585-8453 (I.A.R.); 0000-0003-4030-2399 (C.A.); 0000-0002- surface–bound VTCN1-Ig fusion protein suppressed the prolifer- 8493-8543 (C.P.); 0000-0002-7861-3355 (A.Y.S.). ation of islet-specific T cell clones derived from T1D patients. Received for publication January 5, 2015. Accepted for publication December 5, Furthermore, the treatment of diabetes-susceptible NOD mice 2015. with VTCN1-Ig protein significantly attenuated T1D (10). This work was supported by Sanford Research start-up funds, Juvenile Diabetes Unlike classical costimulatory molecules (B7-1 and B7-2), Research Foundation Grant 47-2013-522, and National Institutes of Health Subcontract UC4 DK104194 (all to A.Y.S.). Support was also received by Sanford Research’s Im- whose natural expression and action are strictly limited to APCs aging and Flow Cytometry core facilities funded by National Institutes of Health Centers (11, 12), VTCN1 is also expressed in several nonlymphoid organs of Biomedical Research Excellence Grants P20 GM103548 and P20 GM103620. and, most importantly, in pancreatic islets (6, 7, 9, 13–15). Con- Address correspondence and reprint requests to Dr. Alexei Y. Savinov, The Sanford sequently, VTCN1 has been hypothesized to not only inhibit Project, Children’s Health Research Center, Sanford Research, 2310 East 60th Street North, Sioux Falls, SD 57104. E-mail address: [email protected] classical T cell activation by APCs in the lymphoid compartment, The online version of this article contains supplemental material. but also to induce T cell tolerance within peripheral target tissues. Supporting this suggestion, upregulated VTCN1 expression was Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; BM, bone marrow; EdU, 5-ethynyl-29-deoxyuridine; Mf, macrophage; NRD1, N-arginine dibasic detected in multiple neoplasms (7, 13, 16–18), where it was as- convertase 1; PCSK1/2, proprotein convertase subtilisin/kexin type 1/2; PD-1, pro- sociated with tumor-protective downregulation of antitumor T cell grammed cell death-1 protein; PD-L1, programmed cell death–ligand 1; PLN, pancreatic lymph node; RA, rheumatoid arthritis; RT-qPCR, real-time quantitative PCR; responses (19). sVTCN1, soluble V set domain–containing T cell activation inhibitor 1; T1D, type 1 In the T1D setting, transfection of the VTCN1-Ig construct into diabetes; Treg, T regulatory cell; VTCN1, V-set domain–containing T cell activation human primary islet cells protected them from diabetogenic T inhibitor 1. cell clones isolated from T1D patients (14). Additionally, ex vivo Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 VTCN1 overexpression in mouse islets shielded them from T cell–

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403251 2 T1D IS ASSOCIATED WITH VTCN1 LOSS FROM PANCREATIC ISLETS induced damage in transplantation experiments (20), whereas In vivo bestatin treatments in vivo b cell–specific VTCN1 overexpression protected against + + Female NOD mice at 6 wk of age were injected daily (i.p.) with either diabetes induced by both CD4 and CD8 islet-specific clonal vehicle or 10 mg/kg bestatin (see Fig. 3A). Two groups (n = 7/group) were T cells (9, 21). Therefore, the distinctive combination of T cell treated for 4 wk, the other two (n = 12–14/group) were treated until dia- coinhibitory function with expression on islet cells uniquely po- betes development. To analyze the proliferation of insulitis-forming cells, sitions VTCN1 at the interface of pancreatic islets and the immune all animals in the 4 wk treatment groups received i.p. injections of 2.5 mg/kg 5-ethynyl-29-deoxyuridine (EdU) for the last four consecutive days of system. treatments. Mice were then euthanized and Mfs, pancreatic lymph nodes Despite the growing number of functional studies utilizing ge- (PLNs), spleens, and pancreata were collected for analysis. From 12 wk of netically manipulated VTCN1 (overexpression and/or deletion), age, all mice were monitored daily for urine glucose using Diastix strips. the state of natural VTCN1 on either APCs or islet cells in con- Mice with urine glucose $250 mg/dl on 2 consecutive days were confirmed diabetic by a blood glucose analysis and euthanized. Mfs were nection with T1D development is largely unknown. This is why we collected from diabetic mice. asked the question of whether a compromised function of endogenous VTCN1 can trigger enhanced vulnerability of islet tissue Adoptive transfers and generation of BM chimeras to diabetogenic autoimmunity. Recently, we unveiled an endoge- Adoptive transfers were performed by i.v. injection of 107 splenocytes nous pathway of functional VTCN1 inactivation in APCs (par- from diabetic NOD mice into irradiated (600 rad) B6g7 or nonirradiated ticularly in macrophages [Mfs] and dendritic cells) of NOD mice B6g7-Rag12/2 or NOD-scid recipients as previously described (26). Sham- and T1D patients. Specifically, a gradual loss of membrane- transferred mice were not irradiated and received PBS. Starting from day tethered VTCN1 due to a proteolytic cleavage mediated by 18 posttransfer, recipient mice were injected i.p. for 4 consecutive days with 2.5 mg/kg EdU per day. Mice were then sacrificed and pancreatic metalloproteinase nardilysin (N-arginine dibasic convertase 1

cryosections were prepared. Downloaded from [NRD1]) progressed alongside natural T1D development and To generate chimeric mice, lethally irradiated (950 rad) 6- to 7-wk-old triggered hyperproliferation of diabetogenic T cells (22). In the female recipients were injected i.v. with 1 3 107 BM cells, isolated from present study, we extend our previous findings and dissect a pat- tibias and femurs of donor mice as previously described (27). NOD-EGFP mice were used to distinguish between recipient and donor cells in the tern of VTCN1 expression and presentation on islet cells in con- chimeric animals. To confirm the chimerism, recipient PBMCs were an- nection with diabetogenesis. Subsequently, we define a general alyzed 3 and 10 wk after BM transfers for EGFP positivity by FACS. Mice mechanism of a progressive loss of VTCN1-mediated negative were euthanized 10 wk after BM transplantations. Peritoneal Mfs and costimulation, which occurs in multiple tissues/cells (islet endo- pancreata were collected for further analyses. http://www.jimmunol.org/ crine cells and APCs) due to the NRD1-dependent diminishment Isolation of macrophages of membrane VTCN1. This mechanism is linked to T1D sus- ceptibility and depends on two separate but synergistic processes. Thioglycollate-elicited mouse peritoneal Mfs (for the coculturing exper- g7 The first process is a result of an increased intracellular NRD1 iments and from B6 /NOD chimeras) or peritoneal cavity–residential Mfs (from bestatin-treated mice) were collected in PBS and plated for expression, ultimately leading to enhanced intracellular VTCN1 immunofluorescence (onto coverslips) or RNA isolation (in a six-well shedding. The second process includes a systemic upregulation of plate). Only the adherent cells were then used for analyses. NRD1 (an enzyme with both intra- and extracellular activities) (23, 24) in multiple tissues, which additionally potentiates VTCN1 Isolation and culture of mouse pancreatic islets by guest on October 1, 2021 proteolysis by extracellular NRD1. In summary, our findings point Islets were isolated by collagenase digestion as described (28). Handpicked toward VTCN1 stabilization along with systemic NRD1 inhibition islets (n . 50) were then either lysed in 1 ml TRIzol (Life Technologies) as future strategies for T1D treatment. for isolation of RNA, or dispersed for islet cell/T cell cocultures. Undis- persed islets were cultured to either conditioned medium or in vitro treatments. The effects of NRD1 inhibitors were evaluated after in- Materials and Methods culture treatments of freshly isolated whole islets for 24 h with either Chemicals bestatin (10 mM), phenanthroline (20 mM), or vehicle solution. The islets were then fixed with ice-cold methanol and VTCN1 levels were analyzed All chemicals were from Fisher Scientific (Suwanee, GA) unless stated by immunofluorescence. To prepare conditioned medium, islets (.20 per otherwise. 1,10-Phenanthroline, bestatin, collagenase P, and Histopaque well) were cultured in 48-well plates for 16 h in 200 ml serum-free RPMI were from Sigma-Aldrich (St. Louis, MO). 1640.

Human subjects T cell/islet cell cocultures Postmortem pancreatic tissue samples from diabetic and control donors For in vitro T cell activation assays, isolated islets were dispersed into were obtained from South Dakota Lions Eye and Tissue Bank. single-cell suspensions in cell-dissociation buffer (Life Technologies) for 15 min at 37˚C. Dispersed islet cells were then washed, depleted from plastic Mice and nylon wool–adherent APCs for 1 h at 37˚C, and incubated for 24 h in 96-well plates with RPMI 1640 medium containing 10% FBS in the NOD/ShiLtJ (NOD), NOD.CB17-Prkdcscid/J (NOD-scid), NOD.Cg-Prkdcscid presence or absence of 10 mM bestatin. Tg(CAG-EGFP)10sb/KupwJ (NOD-scid-EGFP), NOD.Cg-Rag1tm1Mom G9C8 or BDC 12-4.1 T cells were isolated from spleens of B6.G9C8 or Tg(TcraBDC12-4.1) 10Jos Tg(TcrbBDC12-4.1)82Gse/J (NOD.BDC12-4.1), NOD.BDC12-4.1 mice, respectively, via mechanical disruption of spleens B6.129S7-Rag1tm1Mom/J (B6-Rag12/2), and B6.NOD-(D17Mit21-D17Mit10)/ followed by the lysis of RBCs. In some experiments G9C8 T cells were LtJ (B6g7) mice were purchased from The Jackson Laboratory. B6.G9C8 mice, purified from bulk spleen population using the pan T cell purification kit II transgenic for TCR derived from insulin-specific CD8+ T cell clone G9C8, (Miltenyi Biotec). Isolated T cells were then labeled with CFSE (Life and H-2Kd MHC allele (25) were a gift of Dr. A. Chervonsky (University of Technologies) and 1 3 105 cells were added to the cultures of dispersed Chicago, Chicago, IL). B6.NOD-(D17Mit21-D17Mit10)-Rag1tm1Mom (B6g7- islets containing either bestatin or vehicle solution. In some experiments, Rag12/2) mice were generated by crossing B6-Rag12/2 with B6g7 mice, then 2 3 104 NOD peritoneal Mfs were incubated with disrupted islets for 24 h intercrossing F1 animals to produce the F2 population, which was typed by in the presence of either bestatin or vehicle solution before addition of FACS for presence of T cells in peripheral blood and by PCR for presence BDC12-4.1 T cells. After 5 d of cocultures, the nonadherent cells were of H-2g7 using 59-TGCACTTGCATAAGGAAAAC-39 and 59-GACTTTG- collected, washed, and analyzed by flow cytometry for CFSE dilution. On GGGCCTACTTATG-39 as forward and reverse primers, respectively. Only day 3, 100 ml conditioned medium was collected for IL-2 ELISA analysis, both H-2g7–positive and T cell–negative mice were used. To generate bone and then fresh medium with 5 U/ml rIL-2 (BioLegend) was added to the marrow (BM) chimeric mice, B6g7 or EGFP-positive (NOD 3 NOD-scid- cocultures. Nonspecific activation of G9C8 T cells with anti-CD3/anti- EGFP) F1 (NOD-EGFP) mice were used as either BM donors or recipients. All CD28–coated beads (Thermo Fisher Scientific) in the presence/absence of mice were maintained in the Sanford Research Animal Facility according to bestatin was carried out for 5 d in the same experimental conditions as the National Institutes of Health guidelines for animal use. cocultures. The Journal of Immunology 3

ELISA Immunoblot analysis IL-2 concentrations were analyzed using a mouse IL-2 Platinum ELISA kit Serum-free medium was conditioned by pancreatic islets for 24 h at 37˚C. (eBioscience, San Diego, CA) following the manufacturer’s recommen- Cell lysates were prepared in RIPA buffer as described before (22). The dations. samples (5 mg total protein) were subjected to electrophoresis through a 4– 12% NuPAGE Bis-Tris polyacrylamide gel. The membranes were blocked Immunofluorescence and histological analyses with 1% casein/0.1% Tween 20 in PBS and probed with goat anti-VTCN1 (LifeSpan BioSciences) or rabbit anti-NRD1 (Proteintech). HRP- Frozen in OCT compound, pancreata were cryosectioned into 5-mm-thick conjugated anti-goat or anti-rabbit (1:5000) (Jackson ImmunoResearch sections and placed on Superfrost glass slides. Slides were fixed in 100% Laboratories) were the secondary Abs followed by chemiluminescent acetone at 220˚C, air dried, and then stored at 280˚C before processing. substrate (SuperSignal West Dura substrate, Pierce). The membranes were Cryosections were stained with either goat anti-mouse VTCN1 (2 mg/ml; then scanned on a UVP BioSpectrum 500 imaging system. R&D Systems) or mouse anti-human VTCN1 (5 mg/ml; eBioscience) and then visualized with the respective secondary Alexa Fluor 488–conjugated Statistical analysis donkey anti-goat or donkey anti-mouse Abs (Jackson ImmunoResearch Laboratories). Pancreatic b and a cells were stained with guinea pig anti- Statistical analysis was performed using GraphPad Prism 6 (GraphPad insulin (1:100) and rabbit anti-glucagon (1:100), respectively (both from Software). Differences in T1D incidence rates were assessed using log-rank Abcam), followed by the corresponding secondary donkey Abs conjugated survival curve analysis. Differences in pairwise comparisons between either with Alexa Fluor 594 or Alexa Fluor 647 (Jackson ImmunoResearch groups were assessed using a two-tailed Student t test with a significance Laboratories). For staining of Mfs, rat anti-mouse F4/80 (5 mg/ml) threshold of p # 0.05. (eBioscience) was used. Peritoneal Mfs were fixed in 4% paraformalde- hyde and then stained with rat anti-mouse VTCN1 (5 mg/ml; R&D Sys- tems) followed by donkey anti-rat conjugated with Alexa Fluor 594 Results

(Jackson ImmunoResearch Laboratories). Pancreatic sections from ani- Proteolytic loss of endogenous VTCN1 from pancreatic islets Downloaded from mals injected with EdU were stained for insulin and CD3 and assessed for progresses alongside T1D development T cell proliferation via detection of EdU incorporation into the DNA of replicating cells, as previously described (29). CD3+ cells in the insulitis To investigate whether presentation of endogenous VTCN1 by islet + area were counted and the percentage of EdU cells was calculated. More cells can be tied to the development of diabetogenic autoimmunity, than 130 islets with .30,000 infiltrating cells per group were analyzed. To analyze insulitis, pancreatic sections were stained for insulin, and islet we compared the dynamics of VTCN1 levels in Langerhans islets g7 infiltration was graded in a blinded fashion from 0 (intact islets) to 4 in diabetes-susceptible NOD and diabetes-resistant B6 mice. (virtually no b cells within the islet infiltrate) and then divided into three Immunofluorescent staining of pancreatic sections revealed an http://www.jimmunol.org/ subcategories: 1) no insulitis, islets with grade 0; 2) mild insulitis, islets age-dependent progressive loss of VTCN1 protein from NOD is- with grades 1 and 2; and 3) severe insulitis, islets with grades 3 and 4. The lets (Fig. 1A, left panel). Such loss became noticeable at ∼9–10 percentage of islets falling into each subcategory was calculated for each experimental group. wk of age and led to widespread decline of VTCN1 by 15 wk of All images were acquired on a Nikon A1 confocal microscope. Imaging age, which is the average time of T1D onset in our NOD colony. conditions were kept constant for all samples within an experiment. Results Conversely, B6g7 animals had stable intraislet VTCN1 levels were expressed as relative fluorescent units of VTCN1 staining calculated throughout the observation time. The analysis of mean fluores- from at least 100 individual cells or .30 islets. cence intensities confirmed a statistically significant decrease of VTCN1 signal in NOD versus B6g7 islets (Fig. 1A, right panel). RNA isolation and cDNA generation by guest on October 1, 2021 VTCN1 mRNA, however, increased with age in islets of Total RNA was purified from Mfs or pancreatic islets using Direct-zol diabetes-prone NOD animals, whereas an opposite nonsignificant RNA MiniPrep kit (Zymo Research) from samples collected in TRIzol. Only RNAs with an RNA integrity number .8.0 were used for genera- trend toward age-dependent downregulation of VTCN1 expression g7 tion of cDNA and subsequent real-time quantitative PCR (RT-qPCR) anal- characterized the B6 islets (Fig. 1B). The augmentation of ysis. cDNA was generated using a GoScript reverse transcription system VTCN1 gene expression was even more evident and, importantly, (Promega). was paralleled by the significant increase in metalloproteinase NRD1 gene expression when islets from prediabetic NOD mice RT-qPCR were directly compared with the ones from age-matched B6g7 RT-qPCR analysis using primers specific for VTCN1, NRD1, proprotein animals (Fig. 1C). These findings echoed our recent report de- convertase subtilisin/kexin type 1 (PCSK1), or PCSK2 was performed as scribing a highly similar phenotype of progressive VTCN1 protein previously described (22). loss (coupled with compensatory VTCN1 mRNA upregulation) in Flow cytometry analysis (FACS) Mfs and DCs from T1D-succeptible subjects, which occurred due to proteolysis of cell-associated VTCN1 by the metalloproteinase All Abs were purchased from eBioscience unless stated otherwise. To NRD1 (22). Hence, we compared dynamics of islet NRD1 ex- analyze regulatory T cells (Tregs), single-cell suspensions from spleens and PLNs were blocked with 10 mg/ml anti-mouse CD16/CD32 and then in- pression and observed a significant age-dependent elevation of g7 cubated for 30 min with 2.5 mg/ml anti–CD4-FITC and 2 mg/ml anti– NRD1 mRNA in NOD, but not B6 , islets (Fig. 1D). Accordingly, CD25-allophycocyanin. The cells were then washed, incubated in fixation/ levels of NRD1 proteinase were also augmented in prediabetic permeabilization buffer, and stained for 30 min with 5 mg/ml PE- NOD islets in comparison with age-matched B6g7 (Fig. 1E). conjugated anti-Foxp3 Ab. Analysis of diabetogenic NRP-V7 mimotope–reactive CD8+ cells was Moreover, the amounts of intracellular NRD1 enzyme increased in performed in splenocytes and PLN cells that were consequently incubated NOD islets with age, paralleling T1D progression. To confirm with 10 mg/ml PE-conjugated H-2Kd-KYNKANVFL pentamer (Pro- that proteolytic shedding is indeed the mechanism liable for the Immune) and 2.5 mg/ml FITC-conjugated anti-CD8 for 10 and 30 min, observed VTCN1 decline, we cultured freshly isolated NOD respectively. Cells were then washed and stained with the TO-PRO-3 (Life and B6g7 islets and detected elevated levels of soluble VTCN1 Technologies) viability marker. The viability marker 7-aminoactinomycin D (7-AAD) (eBioscience) was used to detect the dead cells during analyses (sVTCN1) immunogenic fragments in medium conditioned by of in vitro–activated G9C8 or BDC12-4.1 T cells and in peripheral blood islets of prediabetic NOD mice (Fig. 1F). monocytes collected from chimeric mice. For analysis of cocultured To characterize VTCN1 expression in different types of pan- BDC12-4.1 T cell proliferation, the cells were also stained for 1 h with creatic endocrine cells and to examine whether VTCN1 loss ob- 5 mg/ml allophycocyanin-conjugated anti-CD69 (BioLegend). Samples were then recorded on an Accuri C6 cytometer (BD Biosciences) and served in islets of T1D-succeptible mice extends to human T1D analyzed with FlowJo software (Tree Star). All gating strategies are shown patients, we analyzed pancreatic sections immunostained for as supplemental material (see Supplemental Figs. 3, 4). VTCN1, insulin, and glucagon. In human subjects, both glucagon- 4 T1D IS ASSOCIATED WITH VTCN1 LOSS FROM PANCREATIC ISLETS Downloaded from http://www.jimmunol.org/

FIGURE 1. T1D development is accompanied by the proteolytic decrease of VTCN1 protein on islet cells. (A) Age-related decline of VTCN1 immunoreactivity in NOD islets. Representative images (left) and quantitative analysis (right) of pancreatic cryosections from NOD and B6g7 mice of indicated ages stained for VTCN1 (red) and insulin (green) are shown. Data are expressed as a percentage of relative fluorescence units when compared with islets from 5-wk-old B6g7 mice (n = 3–5 mice/group) 6 SEM. Relative fluorescence units were calculated for $20 individual islets per animal by sub- tracting mean control Ab fluorescence from mean test Ab fluorescence. Scale bars, 50 mm. **p , 0.01 (B) RT-qPCR analysis of VTCN1 mRNA (nor-

malized to GAPDH) isolated from pooled islets (n = 4–5 mice/group) from NOD mice at the indicated ages. Data are shown as mean arbitrary units (AU) 6 by guest on October 1, 2021 SEM. *p , 0.05. (C and D) RT-qPCR analysis of VTCN1 and NRD1 mRNAs in pancreatic islets from NOD and B6g7 mice. Data are shown as the mean 6 SEM (n = 4–5 mice/group). *p , 0.05, **p , 0.01, ***p , 0.001. (E) NRD1 immunoblot of lysates of islets isolated from B6g7 or NOD mice of indicated ages. (F) VTCN1 immunoblot of medium conditioned by islets isolated from NOD or B6g7 mice. Arrow indicates a possible degradation product of sVTCN1 observed in medium conditioned by NOD islets. expressing a cells and insulin-expressing b cells were VTCN1+ inhibitor (PCSK-1N) in islets isolated from NOD and B6g7 mice. (Supplemental Fig. 1A), although the VTCN1 signal was gener- A nonsignificant trend toward upregulation of all these molecules ally higher in a cells. Likewise, in murine islets both a and b cells was found in NOD islets (Supplemental Fig. 2). Therefore, to expressed VTCN1 (Supplemental Fig. 1B); however, contrary to ratify that the proteolytic clearance of VTCN1 from pancreatic humans, VTCN1 immunoreactivity in b cells was considerably islets is mediated by NRD1, we used two inhibitors known to stronger than in a cells. Evaluation of VTCN1 immunofluores- inhibit NRD1, but not PCSK1 or PCSK2: 1,10-phenanthroline and cence in islets from a small cohort of T1D patients revealed that, bestatin (30–32) for ex vivo in-culture treatments of nondispersed similarly to NOD mice, the islet remnants from a representative VTCN1-low islets freshly isolated from 13- to 15-wk-old NOD T1D donor displayed substantially reduced VTCN1 levels relative mice. Both inhibitors stabilized islet-associated VTCN1, as con- to islets from a representative healthy subject (Supplemental firmed by the increase of VTCN1 immunofluorescence (Fig. 2A), Fig. 1C). Interestingly, a small number of undersized islets in the thus implying that NRD1 is indeed the mediator of VTCN1 shedding T1D patient were strongly positive for both insulin and VTCN1 from islet cells. Similar ex vivo bestatin treatment of VTCN1-high (Supplemental Fig. 1D). islets from 6-wk-old either B6g7 or NOD mice did not change islet Taken together, these results suggest that proteolytic loss of VTCN1 levels in all animals analyzed (not shown). VTCN1 in multiple islet cells accompanies the course of T1D Next, we examined the functional impact of NRD1 inhibitor– development in both humans and mice. induced VTCN1 conservation for downregulation of anti-islet immune responses. For this study, dispersed into single-cell Inhibition of NRD1-mediated VTCN1 proteolysis in islets suspension VTCN1-low islets from 13-wk-old NOD mice were alters diabetogenic T cell responses pretreated with either bestatin or vehicle for 24 h and then cocul- Besides NRD1, pancreatic endocrine cells express two functional tured for 5 d (with inhibitor still present) with CFSE-labeled bulk proprotein convertases, PCSK1 and PCSK2, which by in silico spleen cells or purified T cells from B6.G9C8 mice. B6.G9C8 analysis using the Eukaryotic Linear Motif database (http://elm. animals express rearranged a- and b-chains of TCR from insulin eu.org/) are predicted to target VTCN1 cleavage. Hence, we com- B chain (aa 15–23)–specific CD8+ T cell clone G9C8, and also its pared mRNA expression of these enzymes as well as of PCSK1 MHC H-2Kd restriction allele (25). Therefore, these mice are an The Journal of Immunology 5

FIGURE 2. NRD1 inhibition improves surface localization and functionality of VTCN1. (A)Re- presentative images (left) and quantitative analysis (right) of VTCN1 immunostaining of isolated whole NOD islets cultured for 24 h with vehicle or with the indicated NRD1 inhibitor (n = 30–40 islets, three mice per group). Dots and the horizontal lines in each box indicate the mean and the median , values, respectively. Scale bars, 50 mm. **p Downloaded from 0.01, ***p , 0.001. (B) The histograms show ﬂow cytometry analysis of 7-AAD2 CFSE-labeled G9C8 cells cocultured for 5 d with dispersed NOD islets or anti-CD3/anti-CD28 beads in the presence or absence of 10 mM bestatin. The graph shows the percentage of G9C8 proliferating cells. *p , 0.05.

(C) Conditioned medium collected after 3 d of http://www.jimmunol.org/ G9C8 cells/islet cell cocultures [as in (B)] was analyzed by ELISA for IL-2 concentrations. (D) The histograms show ﬂow cytometry analysis of 7-AAD2 CFSE-labeled BDC12-4.1 CD4+ T cells cocultured for 5 d with dispersed NOD islets only (top panels) or dispersed islets and Mfs(lower panels) in the presence or absence of 10 mM bestatin. The graphs show percentage of BDC12-4.1 proliferating cells. *p , 0.05. For (B)–(D), n = 5–6. by guest on October 1, 2021 RFU, relative ﬂuorescence units.

excellent source of diabetogenic CD8+ T cells of a single G9C8 of bestatin in these control reactions did not affect the propagation clonotype, which are capable of the direct b cell recognition (33). of G9C8 cells (Fig. 2B), indicating that stabilization of endoge- Whereas purification decreased the absolute numbers of dividing nous VTCN1 in islet cells and, in particular, in b cells, via NRD1 G9C8 T cells, the overall proliferative behavior of purified T cells inhibition directly reduces proliferation of diabetogenic CD8+ in inhibitor-treated cocultures with islet cells (not shown) was T cells. very similar to that of the bulk G9C8 cells, which are illustrated in Previously we reported that a proteolysis-dependent decrease of Fig. 2B and 2C. Specifically, bestatin-treated islet cells prompted VTCN1 levels on APCs from diabetes-prone NOD mice potentiates a statistically significant reduction of proliferative capacity of in vitro hyperactivation and proliferation of autoreactive T cells cocultured G9C8 T cells when compared with vehicle-treated is- (22). To reveal whether such a mechanism contributes to the pe- lets (Fig. 2B). Moreover, the production of the proproliferative ripheral tolerance mediated by islet-resident APCs, presenting b cytokine IL-2 by G9C8 cells cocultured with bestatin-treated islets cell Ags to diabetogenic CD4+ T cells, we used CFSE-labeled was also decreased (Fig. 2C). To control for the possibility that splenocytes isolated from NOD.BDC12-4.1 mice in our cocul- the decline in G9C8 T cell propagation is due to a direct anti- ture experiments. NOD.BDC12-4.1 mice bear Rag1 recombinase proliferative effects of bestatin, rather than the consequence of deficiency coupled with the transgenic expression of both a- and bestatin-induced VTCN1 preservation, we used Ag-independent b-chains of TCRs from highly pathogenic I-Ag7–restricted insulin activation of G9C8 cells by anti-CD3/CD28 beads. The presence B chain (aa 9–23)–reactive CD4+ T cell clones (34). Analysis of 6 T1D IS ASSOCIATED WITH VTCN1 LOSS FROM PANCREATIC ISLETS

BDC12-4.1 cells cocultured, as described above, with bestatin- or Approaching the paradigm of connection between VTCN1 loss vehicle-treated suspensions of islet cells from 13-wk-old NOD and insulitic inflammation from an opposite angle, we analyzed mice revealed a significant bestatin-induced reduction in prolif- VTCN1 immunofluorescence in islets of NOD-scid mice, which erative rates of BDC12-4.1 T cells (Fig. 2D). The increase of APC have a diabetes-susceptible NOD genetic background but lack load (via addition of exogenous VTCN1-low peritoneal Mfs from autoimmunity due to the Prkdcscid mutation–caused absence of 17-wk-old NOD mice) in these conditions markedly augmented functional T and B lymphocytes (40). Similarly to NOD mice, proliferative responses of BDC12-4.1 T cells, whereas the sig- islets from NOD-scid animals displayed progressive age-dependent nificant bestatin-mediated reduction of BDC12-4.1 cell prolifera- loss of VTCN1 (Fig. 4B), suggesting that the diabetes-prone NOD tion was kept intact (Fig. 2D). background predisposes for such loss independently of lymphocyte Collectively, these results suggest that bestatin-induced NRD1 in- signaling. hibition stabilizes VTCN1 on multiple types of islet cells (in particular, These results raised the important functional question: What are islet-resident APCs and b cells), which cocontribute to the peripheral the islet-shielding effects of endogenous VTCN1 presented by islet tolerance via downregulation of anti-islet T cell responses. cells in vivo? Because by 15 wk of age B6g7 and NOD-scid mice display high and low VTCN1 levels on their islets, respectively Continuous in vivo administration of NRD1 inhibitor delays (Fig. 4C), we used 17- to 20-wk-old animals of these strains as diabetes in NOD mice recipients of adoptive transfers of NOD splenocytes. The VTCN1 To test whether the systemic NRD1 inhibition in vivo will alleviate levels on recipients’ islets were examined 3 wk after the transfers. VTCN1 loss on the islet cells and peripheral APCs and, conse- Formation of insulitic lesions did not influence VTCN1 levels, quently, alter the course of diabetes, 6-wk-old NOD female mice which remained stably high in B6g7 and stably low in NOD-scid were injected (i.p.) daily with 10 mg/kg bestatin (Fig. 3A). Ad- recipient mice (Fig. 4C). We then evaluated the T cell proliferation Downloaded from ministration of bestatin moderately but significantly delayed (by within the insulitis area of adoptively transferred mice. For these 3 wk, on average) the onset of disease (Fig. 3B, left) and produced experiments, 15-wk-old NOD-scid recipients were compared with significant stabilization of VTCN1 levels on peritoneal Mfs. In- B6g7-Rag12/2 mice, which, similarly to B6g7 animals, maintain terestingly, this effect was much more prominent early in the high VTCN1 levels on islets (not shown) and are lymphopenic treatment course (4 wk of administration, 10 wk of age) than at the equally to NOD-scid mice. The latter characteristic is important to latest time points (diabetic animals) (Fig. 3B, right). Similarly to eliminate the influence of the lymphopenic environment on ho- http://www.jimmunol.org/ the peritoneal Mfs, VTCN1 levels on the islets of mice treated for meostatic proliferation of transferred cells. The percentage of 4 wk with bestatin showed a trend toward stabilization, even dividing CD3+ T cells within peri-islet infiltrates was significantly though this effect did not reach the threshold of statistical signi- higher in NOD-scid recipient mice, correlating with their lower ficance (Fig 3C, left panel, p = 0.1). Such an incomplete islet islet-associated VTCN1 levels (Fig. 4D). Moreover, the percent- phenotype is most likely the consequence of the i.p. route of age of insulitis-free islets in B6g7-Rag12/2 mice was significantly bestatin delivery, which produced higher bestatin concentrations higher when compared with NOD-scid mice (Fig. 4D, right affecting peritoneal Mfs and lower intraislet concentrations. Al- panel). Therefore, VTCN1 on islet cells may function as an en- ternatively, even partial islet VTCN1 stabilization in bestatin- dogenous protective molecule, which attenuates proliferation of by guest on October 1, 2021 treated mice coincided with the significant decrease in numbers autoreactive islet-infiltrating T cells, shielding the pancreatic islets of proliferating cells within islet infiltrates, as well as with the from diabetogenic autoimmunity. significant delay in insulitis formation (Fig. 3C, middle and right panels), pointing toward partial activation of peripheral tolerance VTCN1 levels in multiple cell types are compromised on a within the islets. diabetes-prone NOD background by a general mechanism Finally, the analysis of T cell populations in spleens and PLNs combining cell-autonomous and systemic NRD1 upregulation of bestatin-treated (4 wk of treatment) animals revealed that Because mice of NOD background succumb to the progressive whereas inhibitor did not affect the relative sizes of both CD4+ NRD1-mediated loss of islet-protective VTCN1, we explored a and CD8+ T cell populations, it produced a significant elevation possibility to interfere with such loss. Accordingly, we asked of splenic Tregs, as well as a statistically significant decrease whether stable introduction of B6g7-originated myeloid cells, in numbers of highly diabetogenic NRP-V7 mimotope–specific which display low internal NRD1 and steady VTCN1 levels (22), CD8+ T cells (35) (Fig. 3D). These later observations mimic the will be able to prevent and/or delay loss of VTCN1 from NOD earlier reported effects of VTCN1-Ig treatments of autoimmune islets. For this, we constructed chimeric mice by transplanting BM mice (10, 36) and thus validate VTCN1-stabilizing, self-toler- from B6g7 donors into lethally irradiated NOD-EGFP recipients ance–promoting effects of systemic bestatin administration. (B6g7→NOD-EGFP). Reciprocal BM transfers from NOD-EGFP mice into B6g7 recipients (NOD-EGFP→B6g7) as well as control Endogenous VTCN1 shields pancreatic islets by alleviating B6g7→B6g7 and NOD-EGFP→NOD BM transplantations were proliferation of infiltrating T cells also performed. At the time of the transfers, all recipient mice As infiltration of multiple immune cells into peri-islet vicinity were 6–7 wk of age, which is before the start of noticeable (insulitis) is the major T1D trait in both mice and humans (37–39), VTCN1 loss. Although it is impossible to assess the effects of we assessed whether the loss of VTCN1 from islet cells is induced lethal irradiation on islet VTCN1 levels, sublethal irradiation by insulitis formation or, on the contrary, precedes and even po- doses used in our adoptive transfer experiments did not affect tentiates it. For this, we adoptively transferred splenocytes from either progressive loss of islet VTCN1 in NOD mice (not shown) acutely diabetic NOD mice into 15- to 20-wk-old B6g7 recipients, or the stable islet-tethered VTCN1 levels in B6g7 animals allowed 20 d for islet infiltrates to progress, and then evaluated (Fig. 4C). Ten weeks after the BM transplantations, .90% VTCN1 levels in recipients’ islets. Despite the formation of of PBMCs in chimeric mice were of the donor’s origin massive peri-islet infiltrates, recipient B6g7 mice maintained stable (Supplemental Fig. 3). Stable engraftment of B6g7 BM did not islet VTCN1 levels, which did not differ from the ones observed alleviate VTCN1 shedding in NOD islets, as VTCN1 immuno- in the islets of sham-transferred mice (Fig. 4A), implying that fluorescence in B6g7→NOD-EGFP chimeras was not significantly insulitis does not induce VTCN1 decline. different from NOD-EGFP→NOD animals (Fig. 5A). This indi- The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ FIGURE 3. Limited tolerance-promoting effects of systemic bestatin administration to NOD mice. (A) Schematic representation of treatment regimen. (B) Left, Spontaneous T1D incidence in NOD female mice continuously treated from 6 wk of age with daily i.p. injections of bestatin (n = 12, gray line) or vehicle (n = 14, black line). Difference in incidence (p , 0.05) was analyzed using log-rank survival curve analysis. Right, Peritoneal Mfs from mice treated for 4 wk (n = 7 mice/group) or until diabetes development (n = 12–14 mice/group) were fixed, stained for membrane VTCN1, and analyzed by immunofluorescence. The relative surface VTCN1 fluorescence of Mfs from bestatin-treated mice is plotted as a percentage from the vehicle-treated control animals. (C) Pancreatic sections from mice treated for 4 wk (n = 7 mice/group) were stained for insulin and VTCN1 (left) or EdU (middle). The relative fluorescence of VTCN1 in pancreatic islets was measured and plotted as a percentage from the vehicle-treated controls. Insulitis score (right) was evaluated by grading in a blinded fashion as described in Materials and Methods.(D) Percentage of CD4+, CD8+, Tregs, and NRP-V7+ cells in spleens and

PLNs isolated from bestatin- or vehicle-treated mice after 4 wk of treatment (n = 7 mice/group). Single-cell suspensions were stained for either CD4/CD25/ by guest on October 1, 2021 Foxp3 or CD8/NRP-V7 and then analyzed by FACS. *p , 0.05, **p , 0.01, ***p , 0.001. cated that VTCN1 loss is predominantly an islet cell–autonomous feedback mechanism activated in response to strong systemic in- process intrinsic for NOD islets. NOD-EGFP→B6g7 chimeric duction of VTCN1 loss. Finally, analysis of NRD1 expression, mice, however, exhibited islet VTCN1 levels significantly lower which was found to be elevated in Mfs from chimeric animals than for B6g7→B6g7 chimeras, but nevertheless, significantly harboring any NOD component (either hematopoietic or non- higher levels than for NOD-EGFP→NOD chimeric animals (Fig. hematopoietic; Fig. 5C, right panel), allowed us to conclude that: 5A). This suggests that a long-term introduction of NOD- 1) augmentation of NRD1 expression is the characteristic trait of originated hematopoietic cells can induce partial destabilization T1D-succeptible NOD background; 2) NRD1 is the most likely of islet VTCN1 in B6g7 animals through an extrinsic (islet non- candidate for the sought-after systemic VTCN1-destabilizing autonomous) mechanism. factor; and 3) the increased NRD1-mediated shedding is the ma- To investigate in more detail the nature of the systemic VTCN1- jor cause of VTCN1 decline observed in both Mfs and islet cells ablating stimuli produced by NOD BM-derived cells, we analyzed in conjunction with T1D development (Fig. 6). the phenotype of peritoneal Mfs isolated from the constructed chimeric animals. In agreement with our previous findings dem- Discussion onstrating massive VTCN1 shedding in APCs of diabetes-prone VTCN1 was identified as a negative costimulatory molecule with NOD mice (22), NOD BM-originated Mfs exhibited a significant primary function of downregulating immune responses by reducing VTCN1 decrease independently from the host’s environment that T cell proliferation and cytokine production (5–7). VTCN1 gene they developed and resided in (NOD-EGFP→B6g7 and NOD- expression is not limited to APCs, but it also occurs in several EGFP→NOD chimeras versus B6g7→B6g7 mice, Fig. 5B), con- peripheral nonlymphoid tissues. Therefore, in addition to backing firming the inherent ability of NOD Mfs to autonomously shed the anergic state of naive T cells populating lymphatic organs (41), VTCN1. Furthermore, the similar decline in VTCN1 levels was VTCN1-induced signaling can also negatively modulate already observed in B6g7-originated Mfs from B6g7→NOD-EGFP chi- stimulated T cells, contributing to induction of peripheral tolerance meras, indicating that nonhematopoietic cells of NOD mice in- (21, 42). Such a variety of expression patterns and functional ac- duce strong acceleration of VTCN1 loss from B6g7-derived cells. tivities suggests that VTCN1 plays a far more important role in the Interestingly, mRNA analysis showed increased VTCN1 expres- regulation of immune response than was previously appreciated. sion only in Mfs maturing in cellular environment of NOD, but Defective VTCN1 expression and/or presentation was coupled not of B6g7 BM recipients (Fig. 5C, left panel), pointing toward with exacerbation of several autoimmune diseases harboring upregulation of VTCN1 transcription as a likely compensatory hyperactivation of autoreactive T cells, namely T1D, rheumatoid 8 T1D IS ASSOCIATED WITH VTCN1 LOSS FROM PANCREATIC ISLETS Downloaded from http://www.jimmunol.org/

FIGURE 4. Autoimmunity is ameliorated by high levels of endogenous islet cell VTCN1. (A) Representative immunofluorescence images (left) and quantitative analysis (right) of pancreatic islets from B6g7 mice 20 d after reception of 107 splenocytes from acutely diabetic NOD mouse (Adoptive transfer) or vehicle solution (Vehicle) (n = 3–5 mice/group). Overlay of VTCN1 (red), insulin (green), and DNA (blue) is shown in the third column. Scale bars, 50 mm. (B) Representative images of pancreatic cryosections from NOD-scid mice of indicated ages stained for VTCN1 (red) and insulin (green). Scale bars, 50 mm. (C) Changes in the VTCN1 immunoreactivity in islets from vehicle-transferred (Sham) or splenocyte-transferred (Transferred) NOD- scid and irradiated B6g7 mice 3 wk after adoptive transfers of 107 splenocytes from acutely diabetic NOD mice (n = 3–4 mice/group). (D) Representative images (original magnification 3200) (left) and quantitative analysis (right) of EdU+ T cells within insulitic lesions of adoptively transferred [as in (C)] NOD-scid and B6g7-Rag12/2 mice, 3 wk after transfer (n = 3–4 mice/group). **p , 0.01, ***p , 0.001. by guest on October 1, 2021 arthritis (RA), and multiple sclerosis (8, 9, 21, 43). Moreover, the shedding, precedes T1D development (Fig. 1). Surprisingly, de- delineation of defective VTCN1 presentation in RA-associated spite the observed difference in the expression patterns between autoimmunity was directly related to the destruction of cell- mice and humans, both a and b cells were found to be VTCN1+ associated VTCN1 and release of sVTCN1 fragments into the (Supplemental Fig. 1). The drastic decrease of cell-associated periphery (8). Extending these studies into the context of experi- VTCN1 levels detected in islets of human T1D patients was not mental and natural T1D, we recently demonstrated that high blood limited to b cells, but, surprisingly, it affected several endocrine sVTCN1 concentrations in NOD mice and T1D patients are ac- cell types (Supplemental Fig. 1C), suggesting the presence of a companied by almost complete loss of VTCN1 from the APC general defect in VTCN1 presentation. Moreover, the fact that membranes (22). Based on these results, we suggested that VTCN1 loss in islets occurred despite elevated VTCN1 mRNA sVTCN1 may serve as an early marker of clinical T1D in pediatric expression (Fig. 1B, 1C) indicates the involvement of posttrans- patients. lational control mechanisms, such as protein degradation and/or In contrast to the autoimmunity, VTCN1 levels on tumor cells shedding, in regulation of this process. Importantly, similarly to and tumor-infiltrating APCs were found to be elevated in multiple the islets, elevated VTCN1 gene expression was also unable to neoplasms, providing for hypoactive antitumor T cell responses compensate VTCN1 protein loss in Mfs from T1D-prone animals (44–47). Interestingly, such augmentation of VTCN1 in the con- and human T1D patients (Fig. 5B, 5C) (22). Taken together, these text of cancer development was also combined with elevated observations suggest that a general proteolytic mechanism re- peripheral blood sVTCN1, which was proposed as a potential sponsible for defective VTCN1 presentation on multiple cell types prognostic marker for metastatic cancer spread and consequent is tied with the initiation and/or progression of diabetogenic au- poor outcome in cancer patients (19, 48). It seems, however, that toimmunity (Fig. 6). such a similar high sVTCN1 phenotype is a result of different Accordingly, we suggested the metalloproteinase NRD1 as mechanisms governing VTCN1 metabolism with opposite out- the common VTCN1-shedding enzyme responsible for impaired comes, that is, inhibited T cell responses in cancer or hyper- VTCN1 presentation in multiple tissues (Fig. 6). Stabilization of activated ones in autoimmunity. VTCN1 by NRD1 inhibitors in isolated islets (Fig. 2A), supple- Because VTCN1 was found to be expressed in the endocrine mented by the involvement of NRD1 proteinase in VTCN1 pro- pancreas (9, 15, 21), we started this study with an intent to ex- teolysis in Mfs during T1D progression (22), clearly validates this amine the functionality of endogenous VTCN1 in islet cells dur- suggestion. Moreover, application of the NRD1 inhibitor bestatin ing T1D development and to assess the natural VTCN1 capability ex vivo restored the functionality of endogenous VTCN1 on NOD to modulate autoimmune processes. We show that defective islets, as evidenced from the decreased proliferation and cytokine VTCN1 presentation from pancreatic islets, due to an increased production of G9C8 CD8+ T cells and BDC12-4.1 CD4+ cells The Journal of Immunology 9

FIGURE 5. Cell-autonomous and systemic augmentation of NRD1, characteristic for diabetes- prone NOD mice, determines defective VTCN1 presentation in multiple cell types. (A) Represen- tative images (left) and quantitative analysis (right) of pancreatic cryosections from NOD and B6g7 chimeric mice collected 10 wk after the BM transfer (n = 3–4 mice/group). Scale bars, 50 mm. (B) Representative images (left) and quantitative analysis (right) of peritoneal Mfs isolated from NOD and B6g7 chimeric mice. Scale bars, 10 mm. Downloaded from (A and B) Red dots and the horizontal lines in each box indicate the mean and the median values, respectively. (C) RT-qPCR analysis of VTCN1 (left) and NRD1 (right) mRNAs in peritoneal Mfsiso- lated from NOD and B6g7 chimeric mice. The generated chimeras were NOD-EGFP→NOD (NOD-EGFP http://www.jimmunol.org/ donor into NOD recipient), B6g7→NOD-EGFP (B6g7 donor into NOD-EGFP recipient), NOD- EGFP→B6g7 (NOD-EGFP donor into B6g7 recipient), and B6g7→B6g7 (B6g7 donor into B6g7 recipient). *p , 0.05, **p , 0.01, ***p , 0.001. RFU, relative ﬂuorescence units. by guest on October 1, 2021

(Fig. 2D) in coculturing experiments (Fig. 2B–D). These results consistent with the previous observations that VTCN1 over- evidently stressed the importance of endogenous VTCN1 pre- expression is protective against immune responses in two different sentation on both intraislet APCs and b cells for the local control systems: allograft survival of pancreatic islet transplants (20, 42) of autoimmunity. In agreement, several studies have also shown and in the AI4ab/B6.H2g7 severe diabetes mouse model (21). that ex vivo VTCN1 overexpression protects islet transplants/b Moreover, the inflammatory influence of massive multicell insu- cells from allograft rejection (20, 42, 49, 50). However, our results litis, which is widely accepted to be the source of cytokines, indicate that VTCN1 overexpression by itself will not address the growth factors, and other stimuli driving proliferation of diabe- shedding-dependent loss of functional VTCN1 protein. Therefore, togenic T cells within islet infiltrates, was trumped by VTCN1- development of better and more specific inhibitors of NRD1- mediated inhibitory signaling, as intrainfiltrate T cells proliferated directed VTCN1 proteolysis, as well as design of VTCN1- significantly better in low-VTCN1 presenting NOD islets in stabilizing agents of a different nature, should be an important comparison with high-VTCN1 B6g7 islets (Fig. 4D). Therefore, direction for future studies. This conclusion is in complete the intraislet coinhibition, delivered by proteolysis-uncompromised agreement with the observed partially protective antidiabetic ef- endogenous VTCN1, serves as a powerful local mechanism for fects of continuous bestatin administration in NOD mice, which self-protection against diabetogenicity. alleviated, to a certain extent, autoimmune responses via systemic Contrary to common expectations, our adoptive transfer ex- decrease of NRD1 activity (Fig. 3). periments demonstrated that stability of VTCN1 protein on islet Further extending our findings of endogenous VTCN1 down- cells was independent from accumulation of peri-islet lymphoid regulating anti-islet diabetogenic responses into an in vivo setting, infiltrates. VTCN1 levels were steadily high in heavily infiltrated we demonstrated that the proliferation of islet Ag-reactive T cells islets of B6g7 animals, but they displayed a gradual age-dependent within insulitic lesions was reversely correlated with the levels of decline in the infiltrate-free islets of NOD-scid mice (Fig. 4A–C). VTCN1 presented by the islet cells (Fig. 4C, 4D). This result is Hence, VTCN1 degradation originates at least in part within the 10 T1D IS ASSOCIATED WITH VTCN1 LOSS FROM PANCREATIC ISLETS Downloaded from http://www.jimmunol.org/

FIGURE 6. Schematic view of the impairment of VTCN1-dependent negative costimulation in the context of T1D development. (A) Cell-intrinsic and -extrinsic modes of VTCN1/NRD1 interactions. Cells with low internal levels of NRD1 display uninterrupted presentation of full-length VTCN1 tethered to the cell membrane (mVTCN1), whereas high intracellular NRD1 production results in VTCN1 proteolysis and generation of sVTCN1 fragments. NRD1, secreted from high-producing surrounding cells, complement VTCN1 proteolysis acting extracellularly. (B) Function of NRD1/VTCN1 axis in the development of T1D. Left, Outcome of Ag-specific activation of islet-reactive T cells by APCs in PLNs depends on NRD1/VTCN1 interplay. Low levels of by guest on October 1, 2021 intracellular and systemic NRD1 allow the continuous presentation of functional membrane-tethered VTCN1 by APCs, which provides negative costimulatory signal anergizing autoreactive T cells. When high NRD1 levels are present, VTCN1 is being cleaved, negative costimulation is abrogated, and hyperactivation of autoreactive T cells is induced. Right, Activated b cell–specific T cells migrate toward pancreatic islets and accumulate in islet infiltrates, where two potential scenarios, depending on NRD1 levels, occur: 1) low NRD1 content and consequent high mVTCN1 levels on islet cells are protective, as VTCN1 signaling reduces proliferation and cytokine production of accumulating T cells; or 2) absence of mVTCN1 on the islet cells due to an increased NRD1-mediated shedding results in hyperproliferation of autoreactive T cells and consequent b cell destruction. VTCN1R, putative VTCN1 receptor. islet cells of diabetes-prone animals, being therefore a cell- lation of NRD1 gene expression in islets of diabetes-prone NOD autonomous process characteristic for T1D-prone NOD back- mice (Fig. 1C), as well as in Mfs from BM-chimeric animals ground. harboring any NOD component (Fig. 5C). Moreover, elevation of A more detailed investigation into the balance of VTCN1 loss, NRD1 mRNA was accompanied by a gradual increase of NRD1 however, revealed that long-term engraftment of NOD BM cells protein secretion from NOD islets, which paralleled the progres- into B6g7 hosts jeopardized islet VTCN1 levels through an ex- sion of T1D (Fig. 1D). Therefore, NRD1 is the most likely con- trinsic, systemic mechanism (Fig. 5A). Additionally, the non- stituent to deliver both cell-intrinsic VTCN1 ablation (through its hematopoietic NOD cellular environment also induced accelerated intracellular activity) and systemic extrinsic VTCN1 loss (by its VTCN1 loss in B6g7 BM-originated Mfs (Fig. 5B). Taken to- proteolytic actions in an extracellular, secreted mode). In sum- gether, these data demonstrate that the persistent phenomenon of mary, our results open the door for new studies addressing an proteolytic impairment of functional VTCN1 presentation, de- important quest to identify the genetic and environmental factors tected in multiple cell types in conjunction with T1D suscepti- responsible for upregulation of NRD1 expression and enzymatic bility, is regulated in two synergistically acting manners: cell- activity with respect to autoimmunity in general and T1D in autonomous and systemic. The metalloproteinase NRD1, which particular. Our present and previous findings strongly indicate that has been found in several cell types, including pancreatic islets such factors are shared between autoimmunity-prone NOD mice (Fig. 1C, 1D) and immune cells (Fig. 5C), where its activity is and a substantial cluster of T1D patients. The elevated serum reported to be required for Ag processing and generation of cy- sVTCN1 levels (8), combined with an increase in PBMC NRD1 totoxic T lymphocyte epitopes (51), appears to fit both of these expression in RA patients (52), provide an initial insight into modes. NRD1 protein is mainly localized in the cytosol, but a this quest and hint that NRD1-dependent impairment of significant proportion of the active enzyme is secreted through an VTCN1-mediated negative costimulation is a general autoimmunity- unconventional secretory pathway and distributed on the cell associated pathway. In agreement, systemic NRD1 deficiency surface (23). In agreement, our experimental data show upregu- was recently reported to significantly reduce liver inflammation The Journal of Immunology 11 and protect mice from diet-induced nonalcoholic steatohepatitis 8. Azuma, T., G. Zhu, H. Xu, A. C. Rietz, C. G. Drake, E. L. Matteson, and L. Chen. 2009. 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It has previously been shown that NOD mice double- 3246–3255. deficient for PD-L1 and PD-L2 as well as PD-1–deficient animals 11. Schwartz, R. H. 1992. Costimulation of T lymphocytes: the role of CD28, developed accelerated diabetes with 100% penetrance in both CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 71: 1065–1068. male and female mice (4, 54). In light of these studies, the gen- 12. Lu, P., Y. L. Wang, and P. S. Linsley. 1997. Regulation of self-tolerance by eration of VTCN1-knockout mice on the NOD background would CD80/CD86 interactions. Curr. Opin. Immunol. 9: 858–862. 13. Choi, I. H., G. Zhu, G. L. Sica, S. E. Strome, J. C. Cheville, J. S. Lau, Y. Zhu, be an interesting future direction that would provide us with D. B. Flies, K. Tamada, and L. Chen. 2003. Genomic organization and ex- valuable information about the VTCN1 importance in diabetes pression analysis of B7-H4, an immune inhibitory molecule of the B7 family. J. development in comparison with the PD-1/PD-L1 pathway. Nev- Immunol. 171: 4650–4654. 14. Ou, D., X. Wang, D. L. Metzger, Z. Ao, P. Pozzilli, R. F. James, L. Chen, and ertheless, the existence of such redundancy in the negative co- G. L. Warnock. 2006. Suppression of human T-cell responses to beta-cells by stimulation signals on the periphery might be crucial for providing activation of B7-H4 pathway. Cell Transplant. 15: 399–410. multiple levels of defense of pancreatic islets against autoimmune 15. Cheung, S. S., D. Ou, D. L. Metzger, M. Meloche, Z. Ao, S. S. Ng, D. Owen, and G. L. Warnock. 2014. B7-H4 expression in normal and diseased human islet b destruction. cells. Pancreas 43: 128–134. Downloaded from Our present data have multiple implications for a variety of 16. Quandt, D., E. Fiedler, D. Boettcher, W. Ch. Marsch, and B. Seliger. 2011. B7-h4 disease conditions. For example, contrary to T1D, characterized by expression in human melanoma: its association with patients’ survival and antitumor immune response. Clin. Cancer Res. 17: 3100–3111. strong VTCN1 diminishment from the surface of multiple cell 17. Sun, Y., Y. Wang, J. Zhao, M. Gu, R. Giscombe, A. K. Lefvert, and X. Wang. types, in several cancers VTCN1 shedding is not associated 2006. B7-H3 and B7-H4 expression in non-small-cell lung cancer. Lung Cancer with loss, but rather with an increase of membrane-tethered 53: 143–151. 18. Zang, X., R. H. Thompson, H. A. Al-Ahmadie, A. M. Serio, V. E. Reuter, VTCN1. Possible explanations could include an intratumor J. A. Eastham, P. T. Scardino, P. Sharma, and J. P. Allison. 2007. B7-H3 and B7x loss/downregulation of NRD1 expression/activity or an over- are highly expressed in human prostate cancer and associated with disease http://www.jimmunol.org/ spread and poor outcome. Proc. Natl. Acad. Sci. USA 104: 19458–19463. compensatory excessive VTCN1 expression. Subsequently, the 19. He, C., H. Qiao, H. Jiang, and X. Sun. 2011. The inhibitory role of B7-H4 in control of NRD1 functionality appears to be a very attractive antitumor immunity: association with cancer progression and survival. Clin. Dev. target for future investigations aimed at the development of novel Immunol. 2011: 695834. 20. Wang, X., J. Hao, D. L. Metzger, A. Mui, Z. Ao, C. B. Verchere, L. Chen, D. Ou, therapeutics against either certain cancers or autoimmune condi- and G. L. Warnock. 2009. Local expression of B7-H4 by recombinant adeno- tions. Finally, our data show that disrupted control of costimula- virus transduction in mouse islets prolongs allograft survival. Transplantation tion, evidenced by VTCN1 loss in both APCs and pancreatic 87: 482–490. 21. Lee, J. S., L. Scandiuzzi, A. Ray, J. Wei, K. A. Hofmeyer, Y. M. Abadi, P. Loke, islets, ultimately results in altered balance of control of immune J. Lin, J. Yuan, D. V. Serreze, et al. 2012. B7x in the periphery abrogates responses. pancreas-specific damage mediated by self-reactive CD8 T cells. J. Immunol. 189: 4165–4174. by guest on October 1, 2021 22. Radichev, I. A., L. V. Maneva-Radicheva, C. Amatya, C. Parker, J. Ellefson, Acknowledgments C. Wasserfall, M. Atkinson, P. Burn, and A. Y. Savinov. 2014. Nardilysin- We thank Drs. A. Chervonsky and V. Varnasi (Department of Pathology, dependent proteolysis of cell-associated VTCN1 (B7-H4) marks type 1 diabetes development. Diabetes 63: 3470–3482. University of Chicago) for the transgenic B6.G9C8 mice. We are also 23. Hospital, V., V. Chesneau, A. Balogh, C. Joulie, N. G. Seidah, P. Cohen, and grateful to Brady Roby for critical editing of the manuscript and to A. Prat. 2000. N-arginine dibasic convertase (nardilysin) isoforms are soluble Drs. Satoshi Nagata and Tomoko Ise (Sanford Research) for helpful dis- dibasic-specific metalloendopeptidases that localize in the cytoplasm and at the cussions. We are deeply thankful to the staff of the South Dakota Lions cell surface. Biochem. J. 349: 587–597. 24. Seidah, N. G., and A. Prat. 2002. Precursor convertases in the secretory pathway, Eye and Tissue Bank for help with human tissue specimen collection. cytosol and extracellular milieu. Essays Biochem. 38: 79–94. 25. Varanasi, V., L. Avanesyan, D. M. Schumann, and A. V. Chervonsky. 2012. Cytotoxic mechanisms employed by mouse T cells to destroy pancreatic b-cells. Disclosures Diabetes 61: 2862–2870. The authors have no financial conflicts of interest. 26. Savinov, A. Y., F. S. Wong, and A. V. Chervonsky. 2001. IFN-g affects homing of diabetogenic T cells. J. Immunol. 167: 6637–6643. 27. Chen, Y. G., P. A. Silveira, M. A. Osborne, H. D. Chapman, and D. V. Serreze. 2007. 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