PD-L1 Monoclonal Antibody Treats Ischemic Stroke by Controlling Central Nervous System Inflammation
Background and Purpose—Both pathogenic and regulatory immune processes are involved in the middle cerebral artery occlusion (MCAO) model of experimental stroke, including interactions involving the programmed death 1 (PD-1) receptor and its 2 ligands, PD-L1 and PD-L2. Although PD-1 reduced stroke severity, PD-L1 and PD-L2 appeared to play pathogenic roles, suggesting the use of anti-PD-L monoclonal antibody therapy for MCAO.
Methods—Male C57BL/6 mice were treated with a single dose of anti-PD-L1 monoclonal antibody 4 hours after MCAO and evaluated for clinical, histological and immunologic changes after 96 hours of reperfusion.
Results—Blockade of the PD-L1 checkpoint using a single injection of 200 μg anti-PD-L1 monoclonal antibody given intravenously 4 hours after occlusion significantly reduced MCAO infarct volumes and improved neurological outcomes after 96 hours of reperfusion. Treatment partially reversed splenic atrophy and decreased central nervous system infiltrating immune cells concomitant with enhanced appearance of CD8+ regulatory T cells in the lesioned central nervous system hemisphere.
Conclusions—This study demonstrates for the first time the beneficial therapeutic effects of PD-L1 checkpoint blockade on MCAO, thus validating proposed mechanisms obtained in our previous studies using PD-1- and PD-L-deficient mice. These results provide strong support for the use of available humanized anti-PD-L1 antibodies for treatment of human stroke subjects.
Ischemic stroke, characterized by the rapid development of an infarct on disruption of cerebral blood flow,1,2 is a leading cause of death and disability worldwide. Treatment options currently are limited and mainly involve restoration of blood flow (reperfusion) by intravenous administration of tissue-type plasminogen activator within 4.5 hours after stroke onset. Although necessary, reperfusion can also enhance the inflammatory response and cause additional injury to adjacent brain tissue,3 in large part through the rapid transmigration of both innate (neutrophils and monocytes) and adaptive (T and B cells) immune cells from the periphery to the growing central nervous system (CNS) infarct.4–9 Clearly, there is an urgent unmet need for new therapeutic approaches that can prevent or reverse this process.
Previous research from our and other laboratories has firmly established both pathogenic and regulatory immune processes involved in the middle cerebral artery occlusion (MCAO) model of experimental stroke in mice. Of particular interest are immune interactions involving the programmed death 1 (PD-1) receptor and its 2 ligands, PD-L1 and PD-L2 that regulate the function of inflammatory immune cells. Initially, we demonstrated that PD-1 played a protective role in stroke because cortical, striatal, and hemispheric infarct volumes were significantly larger in PD-1−/− mice, with a marked recruitment of inflammatory cells from the periphery into the CNS.10 Studies were then extended to investigate the role of PD-L1 and PD-L2 in modulating severity of ischemic brain injury and associated CNS inflammation. Contrary to our expectations, PD-L1-deficient (PD-L1−/−) and PD-L2-deficient (PD-L2−/−) mice that were similarly subjected to 60 minutes of MCAO followed by 96 hours of reperfusion demonstrated smaller total infarct volumes compared with wild-type (WT) mice,11 suggesting a pathogenic rather than a regulatory role for both PD ligands. The opposing roles of PD-1 and PD-L in stroke suggested alternative ligand-/receptor-binding partners for PD-L1 and PD-L2 besides PD-1. We thus assessed the contribution of several other costimulatory molecules that could promote or inhibit T-cell proliferation in combination with PD-L1 or PD-L2 as well as verifying the PD-L-expressing cell types responsible for enhancing MCAO. We found that PD-L1 and PD-L2 have overlapping or distinct biological functions12 that derive in part from the broad expression of PD-L1 in lymphoid and nonlymphoid organs versus the more restricted but overlapping expression of PD-L2 on dendritic cells and macrophages.13 Our studies demonstrated that PD-L1 played a dominant role in promoting CD8+ and CD4+ T-cell proliferation that contributed to increased infarct volumes in WT mice subjected to MCAO, whereas in the absence of PD-L1, inhibitory CTLA-4/CD80 interactions became prevalent and PD-1/PD-L2 interactions emerged to control CD4+ T cell, antigen presenting cells and Breg-cell responses.
These results indicated that PD-L1 and PD-L2 differentially control induction of T- and Breg-cell responses after MCAO and suggested that selective targeting of PD-L1 and PD-L2 might represent a valuable therapeutic strategy in stroke. Thus, in this study, we evaluated for the first time the effects of anti-PD-L1 monoclonal antibody (mAb) therapy on stroke severity in male WT mice treated 4 hours after 60-minute occlusion and 96-hour reperfusion. Our results clearly demonstrate a significant reduction in cortical, striatal, and hemispheric infarct volumes and neurological deficit scores as well as reduced infiltration of inflammatory cells but concurrent enhancement of CD8+CD122+ Treg cells but not Breg cells in the lesioned brain hemisphere. Of critical importance, the effects of anti-PD-L1 mAb antibody treatment mirrored results obtained in PD-L1-deficient mice,11,12 thus indicating the potential for direct translation to the clinic using mAb blockade of the PD-L1 checkpoint. It is noteworthy that a human IgG1κ anti-PD-L1 mAb (MEDI4736) is currently being evaluated in 25 ongoing or planned clinical studies in multiple tumor types, with encouraging results in >800 treated patients.14 This antibody blocks PD-L1 binding to its receptors, has high affinity and selectivity for PD-L1 and sustained drug exposure for ≤1 year of dosing, is engineered to prevent collateral inflammatory damage and has no reported immunogenicity impacting its bioactivity, thus making it highly suitable for immediate testing as a novel treatment for stroke subjects.
Materials and Methods
Eight- to 12-week-old male WT C57BL/6J mice, weighing 20 to 25 g, were obtained from The Jackson Laboratory (Sacramento, CA). Naive WT male mice were housed in the Animal Resource Facility at the Portland Veterans Affairs Medical Center in accordance with institutional guidelines. Animals were randomized to treatment groups and induction of transient focal cerebral ischemia. All WT male mice were housed in a climate-controlled room on a 12-hour light/dark cycle. Food and water were provided ad libitum. All experiments were performed in accordance with National Institutes of Health guidelines for the use of experimental animals and the protocols were approved by the VA Portland Healthcare System and Oregon Health and Science University Animal Care and Use Committees.
All surgeries were conducted under aseptic conditions by a surgeon. Transient focal cerebral ischemia was induced in male mice for 1 hour by reversible MCAO in the right brain hemisphere under isoflurane anesthesia followed by 96 hours of reperfusion as described previously.15 Body temperature was controlled at 36.5±1.0°C throughout MCAO surgery with warm water pads and a heating lamp. Occlusion and reperfusion were verified in each animal by laser Doppler flowmetry (Model DRT4, Moor Instruments, Inc, Wilmington, DE). The common carotid artery was exposed and the external carotid artery was ligated and cauterized. Unilateral MCAO was accomplished by inserting a 6-0 nylon monofilament surgical suture (ETHICON, Inc, Somerville, NJ) with a heat-rounded and silicone-coated (Xantopren comfort light, Heraeus, Germany) tip into the internal carotid artery via the external carotid artery stump. Adequacy of MCAO was confirmed by monitoring cortical blood flow at the onset of the occlusion with a laser Doppler flowmetry probe affixed to the skull. Animals were excluded if mean intraischemic laser Doppler flowmetry was >30% preischemic baseline.15 At 1 hour of occlusion, the occluding filament was withdrawn to allow for reperfusion and the incision was closed with 6-0 surgical sutures (ETHICON, Inc). One-half milliliter of prewarmed normal saline was given subcutaneously to each mouse after surgery. Mice were then allowed to recover from anesthesia and were survived for 96 hours after initiation of reperfusion. The surgeon was blinded to treatment groups.
Mice were given 200 μg of either monoclonal anti-PD-L1 antibody (clone 10F.9G2, Biolegend, San Diego, CA) or an isotype-matched control (anti-Keyhole Limpet Hemocyanin [KLH], clone LTF-2, BioXcell, West Lebanon, NH) in 200-μL phosphate-buffered saline, 4 hours after MCAO. Intravenous injection via the lateral tail vein and intraperitoneal injection were both used for administration of antibodies with similar results in PD-L1 depletion and reduced infarct volumes. Hence, data presented in this study may derive from mice treated by either route of injection. The researcher delivering treatment was not blinded to treatment groups. Depletion of PD-L1 on peripheral leukocytes was evaluated 96 hours after reperfusion by flow cytometry.
Neurological Deficit Score
Neurological deficit scores (NDS) were determined at 4, 24, 48, 72, and 96 hours post MCAO to confirm ischemia and the presence of ischemic injury using a 0- to 4-point scale as follows: 0, no neurological dysfunction; 1, failure to extend left forelimb fully when lifted by tail; 2, circling to the contralateral side; 3, falling to the left; and 4, no spontaneous movement or in a comatose state.16 Mice without a deficit after 1 hour of reperfusion were excluded from the study.
Infarct Volume Analysis
The individual performing the infarct volume analysis was blinded to treatment group. Mice were euthanized, and brains were collected at 96 hours of reperfusion for 2,3,5-triphenyltetrazolium chloride histology (Sigma, St. Louis, MO), as described previously.17 The 2-mm brain sections were incubated in 1.2% 2,3,5-triphenyltetrazolium chloride for 15 minutes at 37°C, and then fixed in 10% formalin for 24 hours. Infarction volume was measured using digital imaging and images were analyzed using Sigma Scan Pro 5.0 Software (Systat, Inc, Point Richmond, CA). To control for edema, infarct volume (cortex, striatum, and hemisphere) was determined by subtraction of the ipsilateral noninfarcted regional volume from the contralateral regional volume. This value was then divided by the contralateral regional volume and multiplied by 100 to yield regional infarction volume as a percent of the contralateral region.
Isolation of Leukocytes From Spleen and Brain
Spleens from individual control and B-cell recipient WT mice were removed and a single-cell suspension was prepared by passing the tissue through a 100-μm nylon mesh (Fisher Scientific, Pittsburg, PA). The cells were washed using RPMI 1640. Red cells were lysed using 1× red cell lysis buffer (eBioscience, Inc, San Diego, CA) and incubated for 3 minutes. Cells were then washed twice with RPMI 1640, counted and resuspended in stimulation medium (RPMI containing 2% fetal bovine serum [GE Healthcare, Pittsburg, PA], 1% sodium pyruvate [Life Technologies, Carlsbad, CA], 1% l-glutamine [Life Technologies], and 0.4% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO]).
The brain was divided into the ischemic (right) and nonischemic (left) hemispheres, dissociated enzymatically in RPMI supplemented with 3 U/mL recombinant DNase I (Roche, Indianapolis, IN) and 1 mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich), resuspended in 80% Percoll (GE Healthcare) overlaid with 40% Percoll and subjected to density gradient centrifugation for 30 minutes at 1600 rpm (400 g) according to a previously described method.18 Inflammatory cells were removed from the interphase for further analysis. Cells were then washed twice with RPMI 1640, counted and resuspended in stimulation medium. Cells from individual brain hemispheres were evaluated by flow cytometry.
Analysis of Cell Populations by Flow Cytometry
All antibodies were purchased from BD Biosciences (San Jose, CA) or eBioscience, Inc (San Diego, CA) unless indicated otherwise. Four-color (FITC, PE, APC, and 7-aminoactinomycin D /PerCP/PECy7) fluorescence flow cytometry analyses were performed to determine the phenotype and cytokine production of splenocytes and brain leukocytes as previously published.19 Single-cell suspensions were washed with staining medium (phosphate-buffered saline containing 0.1% NaN3 and 0.5% bovine serum albumin; Sigma, St Louis, MO) and incubated with combinations of the following mAbs for extracellular stains: CD4 (clone GK1.5), CD8a (clone 53–6.7), CD11b (clone M1/70), CD19 (clone 1D3), CD45 (clone Ly-5), CD122 (clone TM-β1 BD), PD-L1 (clone MIH5), CD80 (clone 16-10A1), and CD11c (clone HL3) for 20 minutes at 4°C before washing the cells; 7-aminoactinomycin D (BD Biosciences) was added to identify dead cells whenever only 3 channels on the flow cytometer were used for detection of fluorescent antibody staining. FACS data acquisition was performed using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) and data were analyzed using fluorescence activated cell sorter express software (De Novo Software, Los Angeles, CA).
Intracellular staining was visualized using a published immunofluorescence protocol.16 Briefly, isolated leukocytes were resuspended (2×106 cells/mL) in complete medium and cultured with LPS (10 μg/mL) in addition to phorbol 12-myristate 13-acetate (50 ng/mL), ionomycin (500 ng/mL; all three from Sigma-Aldrich), and GolgiPlug (BD Biosciences) protein transport inhibitor for 4 hours. Fc receptors were blocked with anti-FcR mAb (2.3G2, BD Biosciences) before cell surface staining and cells were fixed and permeabilized with fixation/permeabilization buffer (BD Biosciences) according to the manufacturer’s instructions. Permeabilized cells were washed with ×1 permeabilization buffer (BD Biosciences) and stained with antibodies specific for the following intracellular targets: tumor necrosis factor-α (clone MP6-XT22), interleukin (IL)-10 (clone JES5-16E3), PD-1 (clone J43), and FoxP3 (clone FJK-16s), then resuspended in staining buffer for acquisition. Isotype-matched mAb served as negative controls.
RNA Isolation and Real-Time Polymerase Chain Reaction
Total RNA was isolated from the ischemic hemisphere from treated mice using the RNeasy mini kit protocol (Qiagen, Valencia, CA) and converted into cDNA using oligo-dT primers and Superscript RT II (both Life Technologies). Quantitative real-time polymerase chain reaction was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) using the following TaqMan Gene Expression Assays in Taqman Universal Master Mix (all Applied Biosystems): Mmp9, Il4, and Il10. ΔCt was calculated against the expression of the endogenous control gapdh. Fold change in targeted transcript expression was determined using the formula 2−ΔΔCt.
All values are reported as mean±SEM. The infarct volume data were analyzed using the GraphPad Prism 6 software (version 6.00; GraphPad Software, San Diego, CA). Infarct volume data are presented as mean+SEM. Differences in regional infarct volumes were determined with Student t test. Functional outcomes for neurological deficit scores were analyzed by Mann–Whitney Rank Sum test. For flow data analysis and representation of ≥3 groups, the 1-way ANOVA followed by post hoc Tukey test was applied. For real-time polymerase chain reaction, t tests with Welch correction were used to compare anti-PD-L1 mAb conditions to isotype mAb-treated controls. Statistical analyses were performed using GraphPad PRISM software version 5 (La Jolla, CA). For all tests, P≤0.05 were considered statistically significant.
Single Dose of Anti-PD-L1 mAb Depletes PD-L1 Expression Without Affecting Cell Composition in Naive Male WT Mice
To test our central hypothesis that the use of anti-PD-L1 mAb in experimental stroke will ameliorate functional outcome and stroke-induced neuroinflammation, we first evaluated the effects of anti-PD-L1 mAb treatment on PD-L1 expression in naive mice. Thus, naive WT male mice were injected intraperitoneally with either 200-μg anti-PD-L1 mAb or isotype control mAb to KLH dissolved in 200-μL sterile phosphate-buffered saline and administered once (ie, on D0) and evaluated 4 days later for PD-L1 expression. The results demonstrated that a single dose of anti-PD-L1 mAb was sufficient to deplete the expression of PD-L1 on different splenocyte subpopulations compared with the isotype control mAb (Figure 1A) without affecting their frequency (Figure 1B). Hence, for further stroke-related studies, a single dose of 200 μg/200 μL of mAb was used.
Treatment With Anti-PD-L1 mAb, 4 Hours After MCAO, Reduces Infarct Volumes and Improves Neurological Deficit Scores in Male WT Mice
On the basis of our previous work demonstrating smaller infarct volumes in PD-L1−/− mice,11 we reasoned that treatment of MCAO with anti-PD-L1 mAb might improve stroke outcome. Indeed, WT male mice treated intraperitoneally with anti-PD-L1 versus isotype control mAb 4 hours after 60-minute MCAO followed by 96 hours of reperfusion exhibited significantly reduced cortical (P=0.0013), striatal (P=0.0479), and total hemispheric (P=0.0013) infarct volumes (Figure 2A and 2B) and a significant improvement in median neurological deficit scores (P=0.0326; Table). These results demonstrate for the first time that post-MCAO treatment with anti-PD-L1 mAb can successfully ameliorate stroke-induced damage in the ischemic brain hemisphere.
Mortality and Exclusions
Overall mortality for all MCAO surgeries for infarct analysis and immunology studies was 11 mice out of a total of 133 mice, with mortality ranging from 4 to 7 mice within the experimental groups. Overall number of mice excluded because of intra ischemic laser Doppler flowmetry >30% preischemic baseline was 15 mice out of a total of 133 mice, with exclusions ranging from 7 to 8 mice within the experimental groups. Overall number of mice excluded because of failure in filament advancement was 2 mice out of a total of 133 mice, with exclusions ranging from 0 to 2 mice within the experimental groups. Overall, 5 mice were excluded because of severe hemorrhage after reperfusion in the anti-PD-L1 mAb-treated group of 73 mice. The 5 mice were excluded after 72 to 96 hours of reperfusion. One mouse died after 72 hours of reperfusion and 4 mice survived 96 hours of reperfusion but had high NDS (2–4). Severe hemorrhage of these 5 mice (6.8%) was detected when the brains were isolated after euthanization, suggesting that these 5 mice had delayed hemorrhagic transformation in the brain parenchyma.
Treatment With Anti-PD-L1 mAb, 4 Hours After MCAO, Reduces Inflammatory Responses but Enhances Accumulation of CD8+ Tregs in the Ischemic Brain Hemisphere
Leukocytes are major effectors of inflammatory damage after MCAO. To determine if PD-L1 blockade with anti-PD-L1 mAb altered leukocyte composition in brain after MCAO, absolute numbers of total viable leukocytes were enumerated. As shown in Figure 3A, the ischemic (ipsilateral) hemisphere in mice treated with either anti-PD-L1 or isotype control mAb had a significant increase (P=0.017 and P≤0.001, respectively) in the total number of viable leukocytes compared with the unaffected (contralateral) hemisphere, whereas no differences in total cell numbers were observed between treatment groups in either hemisphere (Figure 3A). However, treatment of MCAO mice with anti-PD-L1 versus control mAb significantly reduced the percentage of activated tumor necrosis factor-α+ CD11b+ cells, likely microglia/monocytes (Figure 3B and 3C), but enhanced the percentages of total CD8+ and CD8+CD122+ Treg cells (Figure 3B and 3D) within the ischemic hemisphere, with a corresponding nominal increase in IL-10 production. No effects of anti-PD-L1 mAb treatment of MCAO mice were observed on CNS infiltrating CD4+ T cells, although a nominal reduction in CD5+CD1dhi CD19+ Breg cells was observed (not shown).
To further assess the treatment effects of the anti-PD-L1 mAb on expression of pro- and anti-inflammatory cytokine/chemokine genes in the MCAO-affected hemispheres, real-time polymerase chain reaction was performed. The expression level of the matrix metalloproteinase Mmp-9 gene implicated in cerebral ischemia20,21 was significantly decreased (P=0.016; Figure 4A) in the affected hemisphere of the PD-L1 versus control mAb-treated MCAO mice. Moreover, the expression level of Il-10 in the ischemic hemispheres of the mAb-treated mice was significantly higher (P=0.038; Figure 4B), with an accompanying ≈3-fold, but not statistically significant, change in the expression level of Il-4 in the mAb-treated mice.
Treatment With Anti-PD-L1 mAb, 4 Hours After MCAO, Rescues Splenic Atrophy, Increases the Expression of Regulatory Molecules on T cells and Decreases the Expression of CD80 on APCs in Spleens, Post-MCAO
Effects of anti-PD-L1 mAb treatment were also evaluated on peripheral immunity (splenocytes) of MCAO-induced mice. As anticipated from results in naive WT mice, treatment with anti-PD-L1 versus control mAb 4 hours after MCAO strongly inhibited expression of PD-L1 on CD19+ B cells, CD4+ and CD8+ T cells, CD11b+ monocytes/macrophages, and CD11c+ DCs in the spleens of treated mice assessed after 96 hours of reperfusion (Figure 5A). This modulation of PD-L1 was apparently not cytotoxic because treatment with the anti-PD-L1 mAb significantly enhanced spleen cell numbers (Figure 5B), indicating partial rescue from MCAO-induced atrophy. We further evaluated possible regulatory cell types and found an increased frequency of CD8+CD122+ Tregs (P=0.016; Figure 5C) in anti–PD-L1-treated MCAO mice. However, no difference in the frequency of Foxp3+CD4+ Tregs was observed after treatment with anti-PD-L1 mAb (data not shown), suggesting the lack of involvement in our experiments of the PD-1/PD-L pathway in the generation of this Treg subpopulation.17,22
Because our previous work10,11 implicated PD-1 signaling as a key component in limiting CNS inflammation in MCAO, we next sought to assess the expression of this coinhibitory receptor on splenic T cells. Although there were nominal increases in PD-1 expression in CD4+ and CD8+ T cells in anti-PD-L1 mAb-treated mice, the differences were not significant when compared with the isotype control mAb-treated mice post MCAO (Figure 5C), indicating little effect of PD-L1 blockade on PD-1 expression and its possible regulatory effects after MCAO. Because PD-L1 can also bind to CD80 on APC to deliver inhibitory signals in T cells,23,24 it is possible that blockade of PD-L1 might obviate PD-L1/CD80 interactions and promote CNS inflammation through other costimulatory pathways (eg, PD-1/PD-L2). Consistent with this hypothesis, analysis of peripheral APCs demonstrated a significant reduction in CD80 expression on both CD11c+ dendritic cells (P=0.014) and CD11b+ monocytes (P=0.028) in PD-L1 mAb-treated mice when compared with the isotype control mice after MCAO (Figure 5D), suggesting reduced CD80 costimulatory potential after anti-PD-L1 mAb treatment.
The results presented above demonstrate that blockade of the PD-L1 checkpoint using a single injection of anti-PD-L1 mAb given 4 hours after occlusion can significantly reduce MCAO infarct volumes, partially reverse splenic atrophy, improve neurological outcome, and enhance levels of CD8+CD122+ Tregs after 96 hours of reperfusion. The current results using mAb therapy to directly neutralize PD-L1 in vivo after MCAO validated results from our previous studies in genetically deficient PD-L1−/− mice11,12 that also had reduced infarct volumes, a significant reduction in brain infiltrating proinflammatory cells, partial reversal of splenic atrophy, and increased levels of IL-10–producing CD8+CD122+ T-suppressor cells in the ischemic brain hemisphere compared with WT MCAO mice. Our current study is the first to demonstrate the therapeutic potential of anti-PD-L1 therapy for treatment of MCAO and supports potential use of humanized anti-PD-L1 mAb for treatment of human stroke subjects.
Five mice were excluded because of severe hemorrhage after reperfusion in the anti-PD-L1 mAb-treated group of 73 mice. These hemorrhages corresponded were visually apparent intracerebral hemorrhages occurring in a small percentage of mice, but only in the anti-PD-L1 group. We also considered the possibility that this treatment could favor small hemorrhages. Micro-hemorrhages accompanying anti-PD-L1 mAb treatment would be difficult to rule out using the 2,3,5-triphenyltetrazolium chloride–stained slices that were analyzed. Although there was no apparent RBC presence outside of noticeable hemorrhagic transformations, without having examined BBB permeability specifically, we can only say that using the current techniques, we could not detect any disruptions in BBB integrity large enough to allow substantial amounts of RBCs to enter the parenchyma. Studies examining the question of PD-L1 involvement in BBB permeability using endothelial cells derived from human brain tissue indicate that blockade of PD-L1 in tandem with PD-L2 results in increased susceptibility of endothelial cells to invasion by activated CD8+ T cells in vitro.25 In this model, blockade of PD-L1 alone did not produce statistically significant changes in the number of CD4+/8+ T cells or albumin able to translocate across an artificial BBB composed of human brain endothelial cells. Additional experiments would be necessary to conclusively determine tight junction integrity and permeability.
Results obtained by direct mAb blockade of PD-L1 function provide considerable support for proposed mechanisms of action in MCAO for PD-1, PD-L1, PD-L2, and various other costimulatory molecules belonging to the B7 family.12 This study used clone 10F.9G2 anti-PD-L1 antibody to treat the mice. Literature suggests that this clone blocks both PD1:PD-L1 and B7-1:PD-L1 interactions.26 Although we cannot rule out cell surface downregulation of PD-L1, the presence of a blocking Ab at PD-L1 on the cell could mean that the lack of binding of the detection Ab is because of occupation of PD-L1-binding site with the treatment Ab. Clinical studies have demonstrated high target occupancy for PD-L1 antibody on CD3+ T cells with a half-life of 15 days after injection.27 We, therefore, think that a 4-day effect of the PD-L1 Ab is likely. As previously discussed,28 the affinity of the CD80 costimulatory molecule for CTLA-4, PD-L1, and CD28 are kd=0.4, 1.4, and 4.0 μmol/L, respectively. Thus, at homeostatic levels in WT mice, PD-L1 competes with CTLA-4 for CD80 binding, thus providing in concert with MHC molecules a nonactivating signal that promotes naive T-cell survival, while simultaneously suppressing T-cell responses by outcompeting strong proinflammatory signaling through CD80/CD28. When stroke is induced in WT or PD-L2−/− mice that express PD-L1, the level of CD80 expression is significantly increased, thereby enhancing T-cell signaling through CD28 and further promoting T-cell activation that worsen stroke outcomes. However, after anti-PD-L1 blockade or in PD-L1-deficient mice with low CD80 expression and without PD-L1 to compete with CTLA-4, depressed T-cell signaling results through CD80/CTLA-4 interactions as well as inhibitory PD-1/PD-L2 interactions.
An important additional feature of PD-L1 blockade that likely contributed to better MCAO outcomes is the induction of IL-10–secreting CD8+CD122+ T-regulatory cells29 that were significantly increased in both the spleen (Figure 5C) and the lesioned brain hemisphere (Figure 3D) after anti-PD-L1 therapy. The contribution of this CD8+ Treg population could account for the increased expression of Il-10 in the ischemic brain hemisphere (Figure 4B) and may be crucial for successful treatment of MCAO in the absence of increased levels of other protective anti-inflammatory cell populations, including CD4+FoxP3+ Tregs17,22 and IL-10+ Bregs16,30 that seem to be downregulated by PD-L2 in the absence of PD-L1.11
Our understanding of the roles of PD-1, PD-L1, and PD-L2 in stroke has progressed concurrently with intense development of anti-PD-1 and anti-PD-L1 mAb for treatment of advanced solid tumors.14 PD-L1 is expressed on many cancer cell types as well as on APCs and prevents tumor cell killing by blocking the function of tumor-specific cytotoxic T cells. Treatment with anti-PD-L1 mAb releases this blocking effect and promotes T-cell killing of tumor cells. Currently, a human IgG1κ anti-PD-L1 mAb (MEDI4736) is being evaluated in 25 ongoing or planned clinical studies in multiple tumor types, with encouraging results in >800 treated patients.14 This antibody blocks PD-L1 binding to its receptors, PD-1 and CD80, has high affinity and selectivity for PD-L1 but not PD-L2, produces sustained drug exposure for ≤1 year of dosing, is engineered to prevent antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity that cause off-target inflammatory side effects and has no reported immunogenicity impacting its pharmacokinetics/pharmacodynamics. This new and powerful clinical reagent is thus highly suitable for immediate testing as a novel treatment approach for stroke subjects.
This study demonstrates for the first time the beneficial therapeutic effects of PD-L1 checkpoint blockade on MCAO, thus validating proposed mechanisms obtained in our previous studies using PD-1- and PD-L-deficient mice. Treatment of MCAO with anti-PD-L1 mAb 4 hours after occlusion significantly reduced infarct volumes and improved neurological outcome after 96 hours of reperfusion, providing strong support for the use of available humanized anti-PD-L1 antibodies for treatment of human stroke subjects.
Dr Bodhankar designed and performed the immunology experiments, carried out statistical analyses, prepared graphics, and wrote part of the article. Drs Chen and Wang performed the MCAO, carried out statistical analyses, and prepared the graphics for the histology procedure. A. Lapato assisted in tissue preparations and acquisition of immunologic data. Dr Dotson assisted in performing the immunology experiments, carried out statistical analyses, and prepared graphs and final Figure 1. Dr Vandenbark critiqued and edited the article. Dr Saugstad supervised the MCAO set-up and statistical analyses, processed the MCAO-related infarct volume, neurological deficit scores and mortality and morbidity data and edited the article. H. Offner directed the overall study and wrote the article. We read and approved the final version of the article. We wish to thank Gail Kent for assistance with article submission.
Sources of Funding
This work was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke 1RO1 NS075887 and 1RO1 NS047661. This material is based on work supported, in part, by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.
- Received June 26, 2015.
- Revision received July 24, 2015.
- Accepted August 5, 2015.
- © 2015 American Heart Association, Inc.
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