Allograft Survival Programmed Death-Ligand 1 in Corneal The

The Function of Donor versus Recipient Programmed Death-Ligand 1 in Corneal Allograft Survival

This information is current as Linling Shen, Yiping Jin, Gordon J. Freeman, Arlene H. of September 26, 2021. Sharpe and M. Reza Dana J Immunol 2007; 179:3672-3679; ; doi: 10.4049/jimmunol.179.6.3672 http://www.jimmunol.org/content/179/6/3672 Downloaded from

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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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Function of Donor versus Recipient Programmed Death-Ligand 1 in Corneal Allograft Survival1

Linling Shen,* Yiping Jin,* Gordon J. Freeman,† Arlene H. Sharpe,‡ and M. Reza Dana2*

Programmed death-ligand (PD-L)1 and PD-L2, newer B7 superfamily members, are implicated in the negative regulation of immune responses and peripheral tolerance. To examine their function in alloimmunity, we used the murine model of orthotopic corneal transplantation. We demonstrate that PD-L1, but not PD-L2, is constitutively expressed at high levels by the corneal epithelial cells, and at low levels by corneal CD45؉ cells in the stroma, whereas it is undetectable on stromal fibroblasts and corneal endothelial cells. Inflammation induces PD-L1 up-regulation by corneal epithelial cells, and infiltration of significant numbers of PD-L1؉CD45؉CD11b؉ cells. Blockade with anti-PD-L1 mAb dramatically enhances rejection of C57BL/6 corneal allografts by BALB/c recipients. To examine the selective contribution of donor vs host PD-L1 in modulating allorejection, we used PD-L1؊/؊ ؊/؊ mice as hosts or donors of combined MHC and minor H-mismatched corneal grafts. BALB/c grafts placed in PD-L1 C57BL/6 Downloaded from hosts resulted in pronounced T cell priming in the draining lymph nodes, and universally underwent rapid rejection. Allografts from PD-L1؊/؊ C57BL/6 donors were also significantly more susceptible to rejection than wild-type C57BL/6 grafts placed into BALB/c hosts, primarily as a result of increased T cell infiltration rather than enhanced priming. Taken together, our results identify differential roles for recipient vs donor PD-L1 in regulating induction vs effector of alloimmunity in corneal grafts, the most common form of tissue transplantation, and highlight the importance of peripheral tissue-derived PD-L1 in down-regulating

local immune responses. The Journal of Immunology, 2007, 179: 3672–3679. http://www.jimmunol.org/

here has been considerable recent interest in the roles of protects against autoimmune diabetes by inhibiting autoreactive T programmed death-ligand (PD-L)31 (B7-H1) and PD-L2 cells. T (B7-DC), members of the expanding B7 family, in regu- The PD-L1/PD-1 pathway has also been linked to allograft sur- lating T cell-mediated immunity (1–4). Although PD-L1 is not vival. It has been shown that PD-L1.Ig, in combination with cy- expressed on the surface of most resting cells at high levels, its closporin A, significantly prolongs cardiac allograft survival (15). surface expression is inducible on both hemopoietic and parenchy- Similarly, concurrent use of PD-L1.Ig and anti-CD154 is able to mal cells (2, 5–7). PD-L2 expression, however, is restricted to prevent rejection of allogeneic islet cells (16). Moreover, a more macrophages and dendritic cells (3, 4). A large body of in vitro recent study by Watson et al. (17) demonstrated that treatment with by guest on September 26, 2021 evidence has demonstrated that ligation of PD-Ls with their re- PD-L1.Ig alone substantially prolongs corneal allograft survival. ceptor PD-1 on activated T cells down-regulates TCR-mediated T However, all of these manipulations have involved systemic im- cell proliferation and cytokine production (2, 4, 6–9). However, munomodulatory strategies, leaving open the specific role of tis- other studies have shown that PD-Ls can stimulate the T cell re- sue-expressed PD-Ls in regulating the alloimmune response. sponse, suggesting the presence of another receptor, different from The cornea is known as an immune-privileged tissue whose PD-1, that may also regulate T cell immunity (1, 3, 10). Studies transparency is a prerequisite for vision. Corneal transplants enjoy with blocking Abs and knockout mice have provided important a higher acceptance rate compared with other solid tissue trans- evidence for the inhibitory functions of PD-L1 in vivo in autoim- plants (18). In this study, we test the hypothesis that PD-L1 may munity (11–13). As an example, Keir et al. (14) recently have serve as an immunoinhibitory factor protecting the cornea graft provided in vivo evidence that tissue-specific PD-L1 expression against T cell-mediated alloimmune responses. In a murine model of orthotopic corneal transplantation involving no immunosup- pression or pharmacological intervention, nearly one-half of C57BL/6 corneal grafts survive indefinitely in BALB/c recipients *Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary, Depart- (19–23), whereas 15–20% of BALB/c corneal grafts survive in ment of Ophthalmology, Harvard Medical School, Boston, MA 02114; and †De- partment of Medical Oncology, Dana-Farber Cancer Institute, and ‡Departments C57BL/6 hosts (24). The indefinite survival of a significant portion of Pathology, Harvard Medical School and Brigham and Women’s Hospital, of corneal allografts allows us to readily investigate constitutive Boston, MA 02115 negative immunoregulatory factors in allograft rejection. Addition- Received for publication August 16, 2006. Accepted for publication June 20, 2007. ally, this model has a number of other distinguishing features, as The costs of publication of this article were defrayed in part by the payment of page follows: first, the direct pathway of sensitization has a minimal role charges. This article must therefore be hereby marked advertisement in accordance ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. in graft rejection due to the low numbers of MHC class II APC in the cornea; in contrast, the indirect pathway is the principal 1 This work was supported by National Institutes of Health Grants R01-EY12963, P20 RR20753, AI56299, and AI39671. route of T cell sensitization mediated by host APC (18, 20, 21). ϩ 2 Address correspondence and reprint requests to Dr. M. Reza Dana, Schepens Eye Second, CD4 Th1 cells are the primary effectors of rejection in Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, this model, whereas B cells and CTLs play no essential function in MA 02114. E-mail address: [email protected] acute corneal transplant rejection (19–23). Corneal transplantation 3 Abbreviations used in this paper: PD-L, programmed death-ligand; BM, bone is, therefore, a very useful model to evaluate the specific role of marrow. PD-L1 in modulating CD4ϩ T cell-mediated alloimmune re- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 sponses in vivo. www.jimmunol.org The Journal of Immunology 3673

In the present study, we demonstrate that PD-L1 is constitu- Orthotopic corneal transplantation and PD-L1 blockade tively expressed on corneal epithelial cells, as well as on infiltrat- Orthotopic penetrating keratoplasty was performed, as described previ- ing bone marrow (BM)-derived cells. Abrogating PD-L1-mediated ously, in one (right) eye of each mouse (16). Briefly, donor center corneas signaling via systemic PD-L1 blockade significantly enhances cor- (2 mm diameter) were excised from wild-type C57BL/6, PD-L1Ϫ/Ϫ neal allograft rejection. Furthermore, using PD-L1-deficient mice, C57BL/6, or wild-type BALB/c mice using Vannas scissors (Storz Instru- we provide evidence that corneal allograft survival relies not only ments) and placed in chilled PBS. The recipient graft bed was prepared by excising a 1.5-mm site in the central cornea of BALB/c, C57BL/6, or on the suppression of the induction of alloreactive T cells by re- PD-L1Ϫ/Ϫ C57BL/6 mice. The donor button was then placed onto the cipient PD-L1, but also significantly on attenuated allospecific T recipient bed and secured with eight interrupted 11-0 nylon sutures, fol- effector responses in the target tissue by the graft PD-L1, delin- lowed by application of antibiotic ointment. The corneal sutures were eating distinct functions for host-vs-graft-derived PD-L1. We thus removed 7 days after surgery. To block PD-L1-mediated signaling, al- lografted mice were treated from the day of transplantation with anti- conclude that PD-L1 expression in donor corneal tissue, as well as murine PD-L1 (10F.9G2, rat IgG2b, 150 ␮g/mouse i.p.) (7, 9) or control rat in the recipient hemopoietic compartment, serves critical functions IgG (MP Biomedicals) three times per week for 8 wk. All grafts were in maintaining the relative immune-privileged status of corneal evaluated using slit-lamp biomicroscopy weekly over 8 wk. Grafts were grafts, the most common form of transplantation. defined as rejected when they became opaque and the iris details could not be recognized clearly using a standardized opacity-grading (ranges, 0–5) scheme (19). Materials and Methods RT-PCR Mice Total RNA was isolated from full-thickness corneas, cornea epithelial

Eight- to 12-wk-old C57BL/6 and BALB/c mice were obtained from Tac- Downloaded from Ϫ/Ϫ sheets, and the combined sheets of stroma and endothelium using RNeasy onic Farms. PD-L1 C57BL/6 mice were generated, as previously de- Micro Kit (Qiagen). Fixed amounts of RNA were reverse transcribed into scribed (12). They were housed in a specific pathogen-free environment at cDNA with SensiScript reverse transcriptase (Qiagen). For PCR detection the Schepens Eye Research Institute animal facility. All procedures were of PD-L1 and PD-L2, the following primers were used: PD-L1 forward, approved by the Institutional Animal Care and Use Committee. 5Ј-dTGCGGACTACAAGCGAATCA-3Ј and PD-L1 reverse, 5Ј-dGCTTG TATCTTCAACGCCAC-3Ј; PD-L2 forward, 5Ј-GTGCGATTTTGACCGC Cauterization of corneal surface AGAG-3Ј and PD-L2 reverse, 5Ј-CTAGGGATGTGGAACAAAGCC-3Ј; GAPDH forward, 5Ј-GAAGGGCATCTTGGGCTACAC-3Ј and GAPDH Application of electrocautery to the cornea is a standard experimental http://www.jimmunol.org/ reverse, 5Ј-GCAGCGAACTTTATTGATGGTATT-3Ј. The PCR condi- method of inducing corneal inflammation (25). Briefly, mice were anes- tions consisted of 35 cycles at 95°C for 30 s, 58°C for 30 s, and 72°C for thetized and placed under the operating microscope. Using the tip of a 1 min. PCR products were analyzed by agarose gel electrophoresis. handheld thermal cautery (Aaron Medical Industries), five light burns were applied to the central 50% of the cornea. Immediately after the procedure, Real-time PCR antibiotic ointment was applied. Corneas were excised 7 days after cautery application and used for cell preparation and RNA extraction. Frozen corneal buttons were homogenized, and RNA was isolated with RNeasy miniprep columns (Qiagen) and treated on-column with DNase I, Immunofluorescence staining according to the manufacturer’s protocol. RNA (300 ng) was reversed transcribed using Superscript III and random hexamers (Invitrogen Life Tech- Eyes were frozen in OCT compound (Miles), and 6-␮m-thick sections nologies) for 50 min at 50°C. Quantitative PCR was performed with Taq- were made. The sections were blocked with 2% BSA and anti-FcR mAb man Universal PCR Mastermix and preformulated primers for IFN-␥ by guest on September 26, 2021 (BD Pharmingen), followed by biotin-avidin block (Vector Laboratories). (assay ID Mm00801778_ml), TNF-␣ (assay ID Mm99999068_ml), and The sections were then stained with biotin-labeled primary Abs to PD-L1 GAPDH (assay ID Mm99999915_gl) (Applied Biosystems), according to (MIH5) or PD-L2 (TY25) (eBioscience) in combination with anti-CD45 the manufacturer’s recommendation. PCR conditions were 2 min at 50°C FITC (BD Pharmingen) for 2 h, followed by streptavidin-rhodamine (Jack- and 10 min at 95°C, followed by 35 cycles of 15 s at 95°C and 60°C for son ImmunoResearch Laboratories) for1hasasecondary reagent. Isotype- 1 min using an ABI PRISM 7900 HT (Applied Biosystems). The results of matched biotin-labeled rat IgG2a (eBioscience) and FITC rat IgG2b (BD real-time PCR were analyzed by the comparative threshold cycle method Pharmingen) were also incubated with tissue sections. All staining proce- and normalized by GAPDH as an internal control. dures were performed at room temperature; each staining step was followed by three thorough washings in PBS. Finally, the sections were cov- ELISPOT assay ered with mounting medium (Vector Laboratories) and examined by the The ELISPOT assay was performed to delineate the contribution of direct eclipse E800 epifluorescent microscope (Nikon). and indirect allosensitization to graft immunity, as described previously (27). Briefly, 96-well ELISPOT plates (Polyfiltronics) were coated with 4 Isolation of corneal cells ␮g/ml primary anti-IFN-␥ mAb (BD Pharmingen) in sterile PBS overnight. The plates were then washed three times with PBS and blocked for 1.5 h Single-cell suspensions were prepared from the corneal samples by colla- with PBS containing 1% BSA. Next, cells were harvested from draining genase digestion, as previously described (26). Briefly, corneal buttons lymph nodes ipsilateral to the grafted eyes (n ϭ 6). Cells harvested from were aseptically removed. To separate the epithelial layer from underlying lymph nodes of ungrafted animals served as controls. Responder T cells stroma, they were incubated with PBS-EDTA for 1 h. The full-thickness were purified using anti-CD90 magnetic beads and an autoMACS cell sep- corneal buttons, the epithelial sheets, or the combined sheets of stroma and arator (Miltenyi Biotec) and added to wells previously loaded with irradi- endothelium were cut into small fragments and incubated with 2 mg/ml ated donor splenocytes as APC (direct response), or irradiated syngeneic collagenase type IV (Sigma-Aldrich) and 0.05 mg/ml DNase I (Roche) for splenocytes as APC together with donor sonicates (indirect response) in a 1 h at 37°C in a humidified atmosphere of 5% CO . Thereafter, the frag- 2 final volume of 200 ␮l of AIM-V medium, as previously described (27). ments were triturated through 21-gauge needle, followed by passing Cells were incubated for 48 h. The plates were washed three times with through 70-␮m cell strainer to create a single-cell suspension. Cells were PBS, then four times with PBS containing 0.025% Tween 20. Biotinylated counted with trypan blue exclusion with high viability. anti-IFN-␥ detection mAbs were added at 2 ␮g/ml (BD Pharmingen) and incubated for2hatroom temperature. The washing steps were repeated, Flow cytometry and after1hofincubation with avidin-HRP, the plates were washed again The single-cell suspensions obtained from corneal samples were blocked three times with PBS/0.025% Tween 20 and then three times with PBS alone. The spots were developed by the addition of the aminoethylcarba- with anti-FcR mAb for 30 min at 4°C in 1% BSA/0.02% NaN3/PBS. Cells were then stained with the following Abs for 45 min at 4°C. Anti-CD45 zole staining solution (Sigma-Aldrich). The resulting spots were counted FITC, anti-CD11b FITC, anti-CD3 PE, and their isotype-matched rat and analyzed on a computer-assisted ELISPOT image analyzer (C.T.L.). IgG2a FITC and hamster IgG FITC were purchased from BD Pharmingen. Statistics Anti-PD-L1-PE (MIH5), anti-PD-L2-PE (TY25), anti-PD-1-FITC (J43), and their isotype-matched rat IgG2b PE and hamster IgG FITC were pur- Kaplan-Meier analysis was adopted to construct survival curves, and the chased from eBioscience. Finally, cells were washed and analyzed on an log-rank test was used to compare the rates of corneal graft survival in EPICS XL flow cytometer (Beckman Coulter). different settings. Student’s t test was used for comparison of statistical 3674 CORNEAL PD-L1 AND ALLOIMMUNITY

FIGURE 2. RT-PCR analysis of PD-L1 and PD-L2 transcripts in the normal cornea. Total RNA from full-thickness corneas (lane 1), corneal epithelial layers (lane 2), combined corneal stroma/endothelium (lane 3), and lymph nodes (lane 4) were used to generate cDNA, and PD-L1 and PD-L2 expression was analyzed by RT-PCR. Upper panel, PD-L1 transcripts; middle panel, PD-L2 transcripts; lower panel, GAPDH transcripts used as housekeeping internal control.

vested. To delineate PD-L1 expression in the epithelium, corneas

were separated into epithelial layers and the subjacent layer of Downloaded from stroma and endothelium for flow cytometric analysis. Comparing the inflamed to the normal corneas, PD-L1 expression was only slightly elevated on CD45Ϫ epithelial cells and the few BM-derived CD45ϩ cells (previously shown by us to be comprised of Langerhans cells) (Fig. 3, A and C) (25). In contrast, corneal in- FIGURE 1. Expression of PD-L1 in the normal uninflamed cornea. ϩ

flammation led to a dramatic increase in the number of PD-L1 http://www.jimmunol.org/ Cross-sections of normal corneas were dual stained with anti-CD45 FITC cells (Ͼ10-fold) in the stroma/endothelium, with nearly all being and biotinylated anti-PD-L1 (B), or with isotype-matched control Abs (A), ϩ followed by streptavidin-rhodamine. Normal corneas were incubated with CD45 , suggestive of infiltrating BM-derived cells (Fig. 3, B and 2 mg/ml collagenase IV for1hat37°C, and the cells were harvested and D). Staining with anti-CD11b further identified the majority of ϩ stained with either anti-PD-L1-PE (D) or anti-PD-L2-PE (F), in combina- CD45 cell infiltrates as monocytic cells (Fig. 3, E and F), com- tion with anti-CD45 FITC, and analyzed by flow cytometry. The appro- patible with the ingress of host APC into the graft, as previously priate control Abs were also used (C and E). Data shown are representative demonstrated (28). of three experiments. Ep, Epithelial layer; St, stromal layer; En, endothelial layer. by guest on September 26, 2021 significance between groups; p values of Ͻ0.05 were considered significant. Results The cornea constitutively expresses PD-L1 To investigate the protein expression of PD-L1 and PD-L2 in normal corneas, we performed immunofluorescence microscopy on corneal cross-sections and flow cytometry of collagenase-digested corneal cells. Strong PD-L1 staining was detected primarily in the corneal epithelium with lower intensity staining in the stroma; there was no expression in the corneal endothelium (Fig. 1). Con- sistent with the immunohistochemistry results, flow cytometry revealed that PD-L1 was almost exclusively expressed by corneal parenchymal (CD45Ϫ) rather than CD45ϩ BM-derived cells (Fig. 1D). In marked contrast, PD-L2 staining was negligible in the cornea (Fig. 1F). To confirm PD-L1 expression on the corneal epithelium, RT-PCR was performed; PD-L1 transcripts were readily detected in the epithelial layer, but barely identifiable in the combined layer of stroma/endothelium; no PD-L2 transcripts were detected in the cornea (Fig. 2). FIGURE 3. Flow cytometric analysis of PD-L1 expression in the normal uninflamed (A and B) vs inflamed (C–F) corneas. Corneas received ϩ PD-L1 is expressed on infiltrating CD45 cells of the inflamed electrocautery to induce inflammation and were excised after 7 days. After cornea treatment with 20 mM EDTA, normal and inflamed corneas were separated into epithelial sheets (A and C) and combined sheets of stroma/endothelium To determine whether the expression of PD-L1 is altered in the (B and D). The cells were collected from collagenase digestion of cornea inflamed cornea, we induced corneal inflammation by application epithelial sheets and the combined sheets of stroma and endothelium, and of electrocautery, a commonly used laboratory method for the dual stained with anti-PD-L1-PE and anti-CD45 FITC, or their control study of corneal inflammation (25). Profound corneal inflamma- Abs. Cells derived from inflamed full-thickness corneas were stained with tion was evident, with attendant influx of monocytes 1 wk post- anti-PD-L1-PE, in combination with either anti-CD45 FITC or anti-CD11b cauterization. Mice were euthanized, and their corneas were har- FITC (E and F). Data shown are representative of three experiments. The Journal of Immunology 3675

FIGURE 4. Fate of C57BL/6 corneal allografts in eyes of BALB/c mice treated with anti-PD-L1 mAb or control rat IgG. BALB/c mice received orthotopically corneal transplants from C57BL/6 mice and were random- ized to receive anti-PD-L1 mAb (n ϭ 14) or control rat IgG (n ϭ 12). FIGURE 5. Ϫ/Ϫ Transplants were assessed biomicroscopically, and graft survival data are Fate of corneal allografts in PD-L1 and wild-type presented as Kaplan-Meier survival curves. Survival of graft in anti-PD- C57BL/6 recipients. BALB/c corneal buttons were grafted orthotopically Ϫ/Ϫ n ϭ n ϭ L1-treated group was significantly lower than the survival in the rat IgG- onto either PD-L1 ( 12) or wild-type C57BL/6 recipients ( 14) treated group (p ϭ 0.019). and scored for 8 wk. As isograft controls, corneas from C57BL/6 mice were grafted onto either PD-L1Ϫ/Ϫ (n ϭ 10) or wild-type C57BL/6 mice Downloaded from (n ϭ 10). Data are reported as Kaplan-Meier survival curves. Survival of allografts in PD-L1-deficient recipients was significantly lower than in PD-L1 blockade enhances T cell-mediated corneal allograft wild-type recipients (p ϭ 0.028). Isografts retained clarity with no sign of rejection failure, even when placed in PD-L1-deficient hosts. To examine the role of corneal PD-L1 in regulating the alloim-

mune response in vivo, we used PD-L1 blockade in an orthotopic http://www.jimmunol.org/ model of murine corneal transplantation. Corneal grafts were pre- 0.028). Interestingly, however, when wild-type BALB/c mice were pared from eyes of C57BL/6 mice, and then transplanted ortho- used as recipients of allogeneic PD-L1Ϫ/Ϫ C57BL/6 corneal frag- topically to BALB/c mice. To abolish the PD-L1/PD-1-mediated ments, the rejection of PD-L1-deficient grafts was also enhanced signaling, a blocking anti-murine PD-L1 mAb (10F.9G2) was ad- compared with wild-type allografts by the end of 8 wk (20 vs 53%, ministered systemically into BALB/c recipients three times per p ϭ 0.047; Fig. 6). These findings (Figs. 5 and 6), in conjunction week from the time of engraftment for up to 8 wk, a time point at with the fate of corneal grafts in anti-PD-L1-treated recipients which tolerance to corneal grafts is well established (19–23). At weekly intervals thereafter, graft survival was assessed clinically by slit-lamp biomicroscopy. The results of this experiment, con- by guest on September 26, 2021 ducted over an 8-wk interval, are shown in Fig. 4. Administration of neutralizing anti-PD-L1 mAb to allograft recipients led to a cumulative survival of only 7% at 8 wk, a rate significantly lower than the 50% survival observed among the recipients treated with control rat IgG ( p ϭ 0.019). Corneal allografts placed in anti- PD-L1 mAb-treated recipients exhibited an accelerated rejection, beginning as early as 2 wk. Furthermore, the severity of the rejection, as measured by the mean opacity score of the grafts, was greater for the grafts in the anti-PD-L1 mAb-treated BALB/c recipients (mean opacity ϭ 3.9 Ϯ 0.24) as compared with the control Ab-treated group (mean opacity ϭ 3 Ϯ 0.52).

Corneal allograft survival relies not only on recipient PD-L1 expression, but also significantly on donor PD-L1 expression The data above clearly demonstrate that PD-L1 blockade via systemic administration of anti-PD-L1 mAb accelerates the onset, and increases the severity, of corneal allograft rejection. However, because both recipient APC infiltrating the grafts (Fig. 3, D–F), as well as the donor tissue (Fig. 1) can express surface PD-L1, it remained unclear at which sites the blockade of PD-L1 affected FIGURE 6. Fate of PD-L1Ϫ/Ϫ or wild-type C57BL/6 corneal allografts enhanced allorejection. To investigate whether the protection of placed in eyes of BALB/c mice. BALB/c mice were engrafted orthotopi- Ϫ Ϫ corneal allograft is directly achieved through PD-L1 on donor cor- cally with corneal allografts from either PD-L1 / (n ϭ 15) or wild-type neal tissue or through PD-L1 in the recipient hemopoietic com- (n ϭ 19) C57BL/6 mice and scored clinically for 8 wk. As isograft con- Ϫ/Ϫ partment, we used PD-L1Ϫ/Ϫ C57BL/6 mice as either corneal al- trols, PD-L1 C57BL/6 corneas were transplanted onto syngeneic wild- type mice (n ϭ 10). Data are reported as Kaplan-Meier survival curves (A). lograft donors or recipients. When PD-L1-deficient C57BL/6 mice Survival of PD-L1-deficient allografts was significantly lower than wild- were used as recipients of allogeneic BALB/c corneal fragments, type allografts (p ϭ 0.047); isografts remained healthy and clear. Slit-lamp the rejection of the corneal grafts was enhanced compared with photographs of representative grafts from wild-type (B) and PD-L1Ϫ/Ϫ (C) that seen in wild-type recipients (Fig. 5). Indeed, all corneal allo- donors. B, Demonstrates a clear accepted allograft at 8 wk; C, a highly grafts were rejected in these recipients by 4 wk postengraftment, vascularized and opacified rejected allograft at 8 wk (vessels in the pe- whereas 21% survived in wild-type recipients after 8 wk ( p ϭ riphery are iris vessels easily visible in all BALB/c hosts). 3676 CORNEAL PD-L1 AND ALLOIMMUNITY

FIGURE 7. The indirect pathway of allorecognition in PD-L1Ϫ/Ϫ and wild-type C57BL/6 recipients grafted with BALB/c corneas. Responder T cells were isolated from lymph nodes of PD-L1Ϫ/Ϫ or wild-type C57BL/6 recipients (n ϭ 6) 7 days after transplantation, or from ungrafted mice, and stimulated with irradiated syngeneic APC (wild-type or PD-L1Ϫ/Ϫ C57BL/6; ratio 1:3) and BALB/c donor sonicates. The frequency of indirectly activated T cells in grafted mice was measured by the IFN-␥-ELISPOT assay and compared with their respective background signals from ungrafted mice. Signiﬁcantly stronger T cell priming was Downloaded from p Ͻ 0.01). The results are depicted as the mean number of spots per million responder T cells loaded Ϯ SEM and represent ,ءء ;p Ͻ 0.05 ,ء) evident in PD-L1Ϫ/Ϫ recipients one of three independent experiments.

(Fig. 4), strongly suggest that both donor and recipient PD-L1 http://www.jimmunol.org/ contribute significantly to corneal allograft survival. So as to ensure that PD-L1 depletion does not lead to graft failure through mechanisms other than allorejection, corneal transplantation was also performed from PD-L1Ϫ/Ϫ C57BL/6 to wild- type C57BL/6, and wild-type C57BL/6 to PD-L1Ϫ/Ϫ C57BL/6 mice, to exclude the possibility that PD-L1 expression modifies inflammation, angiogenesis, healing, or other relevant biological events that can affect graft survival without allospecificity. No graft failure occurred in any of these isograft groups (Figs. 5A and by guest on September 26, 2021 6), suggesting that nonallospecific responses could not explain the enhanced rejection seen with PD-L1 KO donors or recipients. Upon sacrifice of the mice after 8 wk, these isografts showed negligible T cell infiltration or neovascularization (data not shown).

PD-L1 expression in the recipient, but not the donor, enhances corneal allograft survival by suppressing the induction of alloreactive T cells To gain further insight into the mechanisms by which recipient and donor PD-L1 exerts its protective effect on T cell-mediated graft rejection, we separately tested the effect of PD-L1 depletion in the hosts vs donors on the initial priming of T cells in lymph nodes using the IFN-␥-ELISPOT assay. Because the indirect pathway of sensitization represents the predominant mechanism of corneal graft rejection (18, 20, 21), indirect recognition of corneal alloan- FIGURE 8. Indirect and direct sensitization in hosts grafted with either tigen was measured by culturing responder T cells from cornea- PD-L1Ϫ/Ϫ or wild-type corneas. The frequency of alloreactive T cells ac- transplanted hosts with syngeneic APC and donor sonicates (as tivated through the indirect (A) and the direct (B) pathways was evaluated source of alloantigen). As shown in Fig. 7, a potent indirect re- by IFN-␥-ELISPOT analysis. A, Responder T cells, isolated from lymph Ϫ Ϫ sponse was triggered in the PD-L1 / C57BL/6 recipients 1 wk nodes of BALB/c recipients grafted with either wild-type or PD-L1Ϫ/Ϫ after surgery (a time point just preceding the manifestation of clin- C57BL/6 corneas (n ϭ 6), were incubated with irradiated syngeneic ical rejection), reflected by an 8- to 9-fold increase in the frequency BALB/c APC (ratio 1:3) and C57BL/6 donor or BALB/c (negative control) of IFN-␥-producing T cells over ungrafted controls in response to sonicates. B, Responder T cells isolated from lymph nodes of BALB/c Ϫ/Ϫ either wild-type or PD-L1Ϫ/Ϫ syngeneic stimulators. In contrast, recipients grafted with wild-type and PD-L1 C57BL/6 corneas were incubated with irradiated allogeneic C57BL/6 or syngeneic BALB/c stim- only a 2- to 3-fold increase in the frequency of IFN-␥-secreting T ulators (ratio 1:1). Ungrafted BALB/c mice served as controls. The fre- cells was observed in wild-type C57BL/6 allograft recipients as quencies of both the indirectly and directly primed IFN-␥-producing T compared with ungrafted controls similarly stimulated, suggesting cells were comparable in BALB/c mice transplanted with either PD-L1Ϫ/Ϫ that corneal transplantation elicited a significantly more vigorous T or wild-type C57BL/6 corneas. The results are depicted as the mean num- cell sensitization in PD-L1-deficient as compared with wild-type ber of spots per million responder T cells loaded Ϯ SEM, and are repre- recipients. sentative of one of three independent experiments. The Journal of Immunology 3677

tected in accepted wild-type allografts (Fig. 9A). Correspondingly, we observed an increase in T cell infiltration in PD-L1Ϫ/Ϫ allografts compared with wild-type allografts 4 wk posttransplantation (Fig. 9B). Because the normal cornea is naturally devoid of T cells (25), the T cells identified in these grafts were comprised of infiltrating alloreactive cells.

Discussion There is now mounting evidence that PD-L1 plays a crucial role in down-regulating immune responses and maintaining or promoting peripheral tolerance. A noteworthy feature of PD-L1 is its broad expression on both lymphoid and nonlymphoid tissues. The peripheral tissue-speciﬁc PD-L1 expression indicates that it may have a key role in regulating or terminating immune responses in inﬂamed tissues. Although a majority of in vitro studies to date show that PD-L1 up-regulation in nonlymphoid tissues can inhibit T cell activity as assessed by proliferation and cytokine secretion

(6, 7), the exact function of nonlymphoid tissue PD-L1 expression Downloaded from FIGURE 9. Influence of graft PD-L1 expression on T cell infiltration in vivo is still unfolding. The preponderance of in vivo evidence and IFN-␥ levels. Wild-type and PD-L1Ϫ/Ϫ corneas from C57BL/6 mice supports the role of parenchymal PD-L1 as a negative regulator of Ϫ Ϫ were transplanted onto BALB/c mice. A, Wild-type and PD-L1 / allo- T cell responses in tolerance and autoimmunity, whereas other grafts undergoing acute rejection were harvested and subjected for real- studies indicate its role as a positive regulator, suggesting the pres- ␥ time PCR analysis of IFN- mRNA levels. Accepted wild-type allografts ence of potentially another receptor, different from PD-1, that may were used as a negative control. The expression of IFN-␥ transcript was also regulate T cell-mediated immunity (10, 14). http://www.jimmunol.org/ significantly higher in PD-L1Ϫ/Ϫ rejected allografts than in wild-type re- The use of PD-L1Ϫ/Ϫ mice as both donors and recipients in the p Ͻ 0.05), but not detectable in accepted wild-type ,ء) jected allografts allografts. B, Flow cytometric analysis of infiltrating CD3ϩ T cells in the orthotopic model of corneal transplantation provides a highly use- wild-type and PD-L1Ϫ/Ϫ allografts 4 wk posttransplantation. Numbers in- ful means to dissect the in vivo functional significance of PD-L1 dicate the percentage of the stained cells relative to the total cells yielded expression on peripheral tissues vs the lymphoid compartment. We from collagenase digestion of six corneal allografts. Negligible staining show in this study that in the cornea, PD-L1 is constitutively ex- (Ͻ1%) was observed with isotype control Ab (data not shown). Data are pressed on epithelial cells at high levels, and is further up-regu- representative of two similar experiments. lated during inflammation (Fig. 3). Differing significantly from other tissues, MHC class IIϩ APC are absent in the central portions of the cornea (18, 25, 28); hence, donor APC play no appreciable by guest on September 26, 2021 We then assessed the function of graft-derived PD-L1 in regu- role in priming T cells unless the host graft bed is significantly Ϫ Ϫ lating host T cell priming by using PD-L1 / or wild-type inflamed (29), in which case the highly immature APC in the cor- C57BL/6 mice as donors. Compared with ungrafted controls, a neal graft can express adequate MHC class II and accessory mol- higher frequency of IFN-␥-producing T cells was observed in re- ecule expression for T cell priming. For this reason, the protective sponse to donor alloantigen stimulation in BALB/c hosts harboring role of corneal tissue PD-L1 against potentially alloreactive effec- Ϫ Ϫ either wild-type or PD-L1 / C57BL/6 allografts (Fig. 8A). How- tor T cells can be specifically tested using PD-L1-deficient mice as ever, the number of these indirectly primed T cells was comparable donors. As shown in Fig. 6, PD-L1Ϫ/Ϫ corneal allografts under- in these two donor groups, suggesting that graft PD-L1 has min- went significantly more rapid rejection, concurrent with increased imal effect on indirect allorecognition. Finally, donor APC-de- IFN-␥ levels and enhanced recruitment of effector T cells in the pendent direct sensitization was measured to explore the pos- grafts (Fig. 9). The priming of T cells, however, was not affected sibility of enhanced T cell priming as a result of PD-L1 by the absence of PD-L1 expression in the grafted tissue. Both deficiency on graft-derived APC. Once again, no significant dif- PD-L1Ϫ/Ϫ and wild-type corneal allografts produced a comparable ference in the magnitude of the direct response was noted in increase in the frequency of indirectly primed T cells in the re- Ϫ Ϫ BALB/c mice grafted with either wild-type or PD-L1 / gional lymph nodes (Fig. 8A). Furthermore, the lack of PD-L1 on C57BL/6 corneas (Fig. 8B). graft-borne APC did not render them any more capable of mounting a direct alloresponse than wild-type controls (Fig. 8B). This Donor PD-L1 promotes corneal allograft survival via finding offers additional evidence that donor passenger leukocytes attenuating allospecific effector T cell responses do not play a significant role in corneal alloantigen presentation. The increased susceptibility of PD-L1Ϫ/Ϫ corneal grafts to rejec- Finally, PD-L1Ϫ/Ϫ isografts remained clear indefinitely, further tion and the minimal involvement of graft PD-L1 in T cell priming validating that the rapid graft rejection seen with PD-L1 depletion raised the question as to whether corneal graft-expressed PD-L1 is in the host or donor tissue was allospecific and not due to non- immunoprotective by directly suppressing the effector phase of specific modulation of tissue inflammation by PD-L1. Taken to- alloimmunity, which in corneal transplantation is orchestrated al- gether, these data strongly indicate that the increased vulnera- most exclusively by Th1 effectors (17, 18, 20, 21, 28). We there- bility of PD-L1Ϫ/Ϫ grafts to allorejection is not due to the fore examined the Th1 cytokine IFN-␥ levels in rapidly rejected priming phase, and provide strong in vivo evidence that PD-L1 wild-type and PD-L1Ϫ/Ϫ corneal allografts in addition to the fre- expression on corneal parenchymal cells is an important immu- quency of allograft-infiltrating T cells. Of note, during the time of noinhibitory factor in limiting the allospecific T effector re- acute rejection, significantly higher IFN-␥ mRNA levels were de- sponse in the target tissue. tected in PD-L1Ϫ/Ϫ allografts as compared with rejecting wild- Given the primacy of the indirect route of allosensitization in type allografts. By contrast, the expression of IFN-␥ was not de- corneal transplantation (22, 23), it is likely that PD-L1 expression 3678 CORNEAL PD-L1 AND ALLOIMMUNITY by recipient APC serves to inhibit recipient T cell priming, pro- ripheral tissues, possibly through tissue-specific overexpression, to viding an explanation for enhanced rejection of grafts in PD- improve the success of tissue grafts and to promote immune L1Ϫ/Ϫ hosts. Our data clearly suggest that systemic PD-L1 block- quiescence. ade with anti-PD-L1 produces a significantly more rapid and higher rate of graft rejection than can be explained by loss of Acknowledgments PD-L1 expression in the graft alone. Therefore, to examine any We thank Dr. Mohamed Sayegh and Dr. Indira Guleria, Brigham and protection exerted by recipient PD-L1, we performed corneal Women’s Hospital, Harvard Medical School, for helpful discussions re- transplantation in recipient mice lacking PD-L1 expression, and garding our research. noted a significant augmentation in allograft rejection as compared with wild-type recipients (Fig. 5). ELISPOT studies revealed a Disclosures greater expansion of indirectly primed alloreactive Th1 cells in the The authors have no financial conflict of interest. draining lymph nodes in PD-L1-deficient recipients as compared with wild-type recipient controls (Fig. 7). Hence, our results indi- References cate that PD-L1 signaling within the recipient also plays a critical 1. Dong, H., G. Zhu, K. Tamada, and L. Chen. 1999. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. 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