Significance The potential of proteasome inhibitors to prevent transplant rejection and to treat other immune disorders is hindered by mechanism-based toxicity from inhibition of constitutive proteasomes. Here, we demonstrate that briefly, reversibly, and selectively inhibiting the immunoproteasome prolonged the survival of transplanted hearts in mice and allowed long-term survival when combined with single-dose CTLA4-Ig. Immunoproteasome inhibition noncytotoxically reduced T-cell proliferation and the numbers of effector T cells in the allograft and draining nodes while increasing T-cell expression of exhaustion markers. The immunoproteasome thus appears to play a role in suppressing induction of T-cell exhaustion. Selective inhibition of the immunoproteasome may be a potential treatment option for the management of transplant rejection.

Abstract Constitutive proteasomes (c-20S) are ubiquitously expressed cellular proteases that degrade polyubiquitinated proteins and regulate cell functions. An isoform of proteasome, the immunoproteasome (i-20S), is highly expressed in human T cells, dendritic cells (DCs), and B cells, suggesting that it could be a potential target for inflammatory diseases, including those involving autoimmunity and alloimmunity. Here, we describe DPLG3, a rationally designed, noncovalent inhibitor of the immunoproteasome chymotryptic subunit β5i that has thousands-fold selectivity over constitutive β5c. DPLG3 suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and synergized with CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier. These findings demonstrate the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor toxic.

Allograft transplantation is an established, widespread intervention for organ failure. Unfortunately, complications often arise from prolonged administration of immunosuppressive agents to protect the graft from rejection. The drugs in current use cause broad immunosuppression. Some of them are toxic to cells outside the immune system, including to cells in the grafted organ. Because alloantibodies pose a great risk to allografts, one of the newest agents to be deployed in defense of the graft is bortezomib (1), a proteasome inhibitor that can kill plasma cells and was approved by the Food and Drug Administration (FDA) for the treatment of multiple myeloma (2). However, bortezomib inhibits the proteasomes in all cells, giving it a high potential for mechanism-based toxicity and requiring it to be used in transplantation medicine at subtherapeutic levels. Presumably, toxicity would be greatly diminished and efficacy improved if proteasomes could be inhibited selectively in immunocytes and especially in those that react to the alloantigens of the graft.

The possibility of inhibiting proteasomes selectively in certain types of cells arises from the existence of different isoforms of proteasome-associated proteases encoded by different genes whose expression is responsive both to cell lineage differentiation and to the cytokine milieu. Eukaryotic proteasomes have a barrel-shaped 20S core that contains two copies each of seven different α subunits and seven different β subunits arranged in four stacked rings in α 1–7 β 1–7 β 1–7 α 1–7 fashion (3). Three β subunits are proteolytic: β1 has caspase-like activity, whereas β2 is tryptic and β5 is chymotryptic. The isoform that is constitutively expressed in all cells (c-20S) controls diverse functions ranging from signal transduction to cell cycle, allowing cells to adapt to circumstances quickly both pretranscriptionally and posttranscriptionally through proteolytic degradation of temporarily dispensable proteins (4). Moreover, products of the proteasome are the major source of antigenic oligopeptides for major histocompatibility complex (MHC) class I antigen presentation (5). However, mononuclear phagocytes, dendritic cells (DCs), and lymphocytes (6, 7), as well as cells at sites of inflammatory and immune reactions exposed to cytokines such as IFN-γ, express variable proportions of the immunoproteasome (i-20S), in which some of the β1c, β2c, and β5c catalytic subunits of c-20S are replaced by β1i, β2i, and β5i (also called LMP7), respectively (8, 9).

Bortezomib and the other FDA-approved proteasome inhibitors (carfilzomib and ixazomib) comparably target both c-20S and i-20S. One compound, which binds the proteasome covalently and irreversibly, has relatively greater activity on i-20S than on c-20S and has shown efficacy in mouse models of inflammatory bowel disease, arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes in association with modulation of the function of Th1 and Th17 cells (10⇓⇓–13). However, to our knowledge, selective immunoproteasome inhibitors have not been tested for their role in promoting allograft acceptance, nor have effects been described on T-cell exhaustion and coinhibitory markers on DCs.

We hypothesized, first, that immunoproteasome inhibitors might contribute to allograft acceptance in association with pleiotropic effects on DCs and T cells, and second, that their efficacy and mechanism-based safety would be proportional to their isoform selectivity for i-20S over c-20S, and specifically for β5i over β5c. With regard to the second hypothesis, we reasoned that whatever degree of isoform selectivity was demonstrable in short-term assays, as long as a covalently reacting and irreversible β5i inhibitor reacted to some degree with β5c, it would show diminishing isoform selectivity with time. In contrast, a non-covalently reacting, reversible immunoproteasome inhibitor should maintain its selectivity during prolonged incubation with proteasomes. The recent discovery of a class of highly selective, non-covalently reacting immunoproteasome inhibitors based on an N,C-capped dipeptidomimetic scaffold (14) encouraged us to test both sets of hypotheses.

We report here that members of a distinct but related class of immunoproteasome inhibitors—N,C-capped dipeptides—are orders of magnitude more isoform selective than an irreversible inhibitor and increasingly so the longer the exposure to the target. Such compounds suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and, when given to mice for the first 14 d following transplant, synergized with a single dose of CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier.

Discussion Here, we describe a noncovalent N,C-capped dipeptide inhibitor of immunoproteasome subunit β5i (LMP7) with nanomolar potency whose selectivity for β5i over β5c rose from 7,200-fold to 99,000-fold as preincubation was extended from 0 to 24 h. The increase in selectivity with time reflected a time-dependent fall in IC 50 for β5i. We speculate that this may result from slow conformational changes favorable to binding of the inhibitor in the active site of β5i in response to noninhibitory occupancy of other β subunits. In any event, after 24 h of preincubation, the isoform selectivity of DPLG3 was four orders of magnitude greater than that of the peptide epoxyketone β5i inhibitor, ONX0914 (10), tested under the same conditions. A recent study reported a variant of ONX0914 with up to 600-fold isoform selectivity (21). Nonetheless, as another irreversibly acting peptide epoxyketone, the new agent is expected to inhibit β5c to a greater extent the longer that exposure continues, as well as to present a risk for inhibiting some unrelated targets (22). The exceptionally high, time-enhanced isoform selectivity of the reversibly acting N,C-capped dipeptide provided a biochemical rationale for expecting its mechanism-based effects to be limited to cells engaged in immunologic responses, as opposed to acting on nonimmunocytes. Moreover, increased expression of the target, β5i, in effector T cells reacting to antigen provided a biologic rationale for expecting the impact on immunocytes to be relatively restricted to those engaged in response to antigen at the time of treatment. Finally, the impact of an N,C-capped dipeptide immunoproteasome inhibitor on at least two classes of immunocytes—DCs and T cells—may contribute to the profound and prolonged impact on graft survival that resulted from transient treatment with the immunoproteasome inhibitor in combination with a single dose of CTLA4-Ig. Given the importance of alloantibody in chronic graft rejection, the impact of immunoproteasome inhibition on B cells and plasma cells warrants analysis as well, and will be the subject of future studies. The pleiotropic effects of selective immunoproteasome inhibition reflect both the protean biologic roles of the ubiquitin–proteasome system in all cells, and the variable degree to which c-20S subunits are replaced by i-20S subunits in some cells. Here, we show a selective up-regulation of β5i in T cells reacting to alloantigens, which suggests a specific role for this subunit in antigen-reactive effector T cells compared with other T cells. We are particularly intrigued that selective immunoproteasome inhibition led to the induction of exhaustion and coinhibitory markers on, and reduction in the proportions of, effector T cells in the allografts and recipients’ spleens. This suggests that a previously unappreciated biologic function of the immunoproteasome is to protect antigen-reacting effector T cells from exhaustion or inhibition. T-cell exhaustion is a state characterized by a progressive loss of production of certain cytokines such as IL-2 followed by a proliferation defect and apoptosis (23). Exhausted T cells express a variety of coinhibitory receptors, including PD1, TIM3, LAG3, CTLA4, CD160, and BLTA, that modulate the intracellular signaling responsible for this state (24). Although reversal of T-cell exhaustion may be useful in the treatment of chronic infections and cancers, induction of T-cell exhaustion may promote tolerance to autoantigens and alloantigens (24, 25). T-cell exhaustion has recently been shown to play a central role in determining clinical outcome in multiple autoimmune diseases (26), and it has been suggested that targeted induction of exhaustion could benefit patients with an aggressive course of disease. Nonselective proteasome inhibitors have shown benefit in small trials in patients with lupus nephritis, autoimmune hemolytic anemia, and autoantibody-associated mesangioproliferative glomerulonephritis (27), but it is unknown whether the benefit was related to induction of T-cell exhaustion, reduction in autoantibodies, and/or other mechanisms. Highly selective immunoproteasome β5i inhibitors such as DPLG3 represent powerful tools to dissect the role of the β5i subunit in various disease models and the impact of inhibition at different stages of the pathogenic process. Mice lacking β5i have limitations in this regard, both because absence of β5i precedes disease onset and because there is extensive compensation by β5c. Further study is required to identify the signaling, transcriptional, and posttranscriptional mechanisms by which immunoproteasome inhibition leads to changes in immunocyte activation, proliferation, surface marker expression, and secretion of cytokines. The present results call attention to the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor cytotoxic.

Materials and Methods Synthesis and Characterization of β5i Inhibitors. The synthetic route and methods of compound characterization were similar to those reported for N,C-capped dipeptidomimetics (15, 28), with modifications as given in the supplemental online material. All compounds were >95% pure. ONX0914 was obtained from BPBio. Human immunoproteasomes were purified from PBMCs and constitutive proteasomes from red blood cells. Suc-LLVY-AMC and Ac-ANW-AMC were purchased from Boston Biochem. Enzymatic assays were monitored on a Molecular Devices SpectraMax M5 plate reader. Cell viability was determined with ATP-lite assay kit (PerkinElmer). Cell-based Proteasome-Glo (G8660; Promega) was used to measure 20S chymotryptic-like β5 activity and its inhibition in cells. Biochemical Characterization of β5i Inhibitors. Assays with isolated proteasomes and in intact cells were performed as reported (29). Briefly, Karpas 1106P cells (80,000/well) were incubated with compound at indicated concentrations for 1 h at 37 °C. The plate was then spun at 188 × g for 1 min and the supernatant was removed. The overall β5 activity including β5i and β5c in each well was measured in situ with the Proteasome-Glo assay kit according to the manufacturer’s instructions. Luminescence was recorded on a SpectraMax M5 plate reader. Relative percentage of relative light units was used to calculate IC 50 values. Cell Cultures. We cultured Karpas1106P B lymphoma cell line (catalog no. 06072607; Aldrich) (15), mouse bone marrow-derived macrophages (18), PBMCs (17, 30), and HepG2 human hepatoma cells (31). Karpas cells were cultured in complete medium and the other cells in complete medium with 10% (vol/vol) FBS instead of 20% (vol/vol), all at 37 °C in a humidified air/5% (vol/vol) CO 2 . Cells in a 96-well plate were treated with compounds at indicated concentrations for 72 h at 37 °C in with 5% (vol/vol) CO 2 . Viable cells were counted using CellTiter-Glo assay kit. EC 50 values were calculated using PRISM. Human PBMC and PDC Assays. Buffy coats were obtained from the New York Blood Center (Long Island City, NY) with informed consent and used under a protocol approved by the institutional review board of the Hospital for Special Surgery. Total PBMCs were prepared using standard protocol (17) and cells (3 × 105 cells/well) were cultured for 24 h in 96-well plate. The viability of PBMCs was quantified using a colorimetric (MTT) assay (Millipore) according to manufacturer’s instructions. PDCs were isolated using positive selection using BDCA-4–conjugated beads (Miltenyi Biotec) as described (32). PDCs were 94–99% as determined by flow cytometry (BDCA2+ CD123+) and cells (2–5 × 104 cells per well) were cultured in U-bottom 96-well plate. Cells were activated with a phosphorothioate CpG-B or -C ODN at 0.3–1 μM (TLR9 agonist) as described (32), and their viability was assessed by flow cytometry using propidium iodide. Cells were cultured for 24 h, and human IFN-α and IP-10 production were assayed by ELISA with reagents from PBL Biomedical Laboratories and from Thermo Fisher, respectively. Monoclonal antibodies used for flow cytometry included the following: anti-CD3, anti-CD14, anti-CD19, anti-HLA-DR anti-CD83, anti-CD86 (BD Biosciences), anti-CD123, and anti-BDCA-2 (Miltenyi Biotec). Mice. Female C57BL/6 (H-2b), BALB/c (H-2d), FoxP3-GFP knock-in mice on a C57BL/6 background and Rag−/− mice on a BALB/c background were obtained from The Jackson Laboratory. Mice were 6–10 wk of age (20–25 g) at the start of the experiments. They were housed in accordance with institutional and NIH guidelines. The Harvard Medical School Animal Management Committee approved all animal experiments. Skin Transplantation. Full-thickness trunk skin grafts (1 cm2) harvested from BALB/c donors were transplanted onto the flank of Rag−/− C57BL/6 recipient mice, sutured with 6.0 silk, and secured with dry gauze and a bandage for 7 d. Cardiac Transplantation. Vascularized intraabdominal heterotopic transplantation of cardiac allografts was performed using microsurgical techniques (33). The survival of cardiac allografts was assessed by daily palpation. Rejection was defined as complete cessation of cardiac contractility as determined by direct visualization and confirmed by histology. Histological and Immunohistochemical Assessment. Five-micrometer-thick formalin-fixed paraffin-embedded sections were stained with standard hematoxylin and eosin (H&E) stain. Isolation of Lymphocytes from Hearts. Cardiac allografts were removed, perfused with PBS, minced finely with a razor blade, and digested at 37 °C with 1 mg/mL collagenase in 1 mL of complete RPMI medium 1640 for 1 h. Cells in the supernatant were washed twice, centrifuged at 620 × g using Percoll solutions at 33% (vol/vol) (cell suspension) and 66% (vol/vol). Lymphocytes were aspirated at the interface. Histology Score. Four sections stained with H&E were inspected from the allografts of each of three mice per group. To score lymphocyte infiltration, each section was divided into six sectors and lymphocyte infiltration in each sector was graded using a scale modified from the International Society for Heart and Lung Transplantation (0, no lymphocyte infiltration; 1, less than 25% lymphocyte infiltration; 2, 25–50% lymphocyte infiltration; 3, 50–75% lymphocyte infiltration; 4, more than 75% lymphocyte infiltration with hemorrhage and/or necrosis). The score for each sector was averaged, and the average from each of the four sections was used as the lymphocyte infiltration score for each heart. The severity of vasculopathy was determined by a combination of vascular occlusion score and perivascular lymphocyte infiltration score for each coronary vessel. Vascular occlusion was scored from grade 0–3 as follows: grade 0 (no or minimal, <10%), grade 1 (10–50% occlusion), grade 2 (50–75% occlusion), and grade 3 (more than 75% occlusion). The perivascular lymphocyte infiltration was scored as grade 0 (no lymphocyte infiltration), grade 1 (mild lymphocyte infiltration), 2 (moderate lymphocyte infiltration), and 3 (severe lymphocyte infiltration), and then added to the vascular occlusion score, for a final score that could range from 0 to 6. CD3/CD28 T-Cell Stimulation Assay and Mixed-Lymphocyte Reaction. Anti-CD3 Ab (100 μL) and soluble anti-CD28 Ab (1 μg/mL; BD Biosciences) were dispensed to wells of a 96-well flat-bottom plate containing C57BL/6 CD4 or CD8 T cells and the indicated concentrations of DPLG3 or the DMSO vehicle (final concentration of 1% DMSO). For the mixed-lymphocyte reaction, irradiated WT BALB/c splenocyte stimulators and C57BL/6 splenocyte responders from DPLG3-treated or vehicle-treated mice were added to each well in a 96-well round-bottom plate, then pulsed with 1 μCi of tritiated thymidine 9 h before the end of the 72-h assay. Incorporation efficiency was determined by scintillation counting. Flow Cytometry. Anti-mouse Abs against CD62L, CD44, CD4, CD25, CD8, FoxP3, GrB, PD1, TIM3, LAG3, LMP7, annexin, 7-amino-actinomycin D (7-AAD), and Ki67 were purchased from BD Biosciences. Cells recovered from spleens and peripheral lymphoid tissues were analyzed on a FACSCanto II flow cytometer (BD Biosciences) with FlowJo software, version 9.3.2 (Tree Star). RNA Sequencing. Total RNA was isolated from flow-sorted CD4 effector T cells (GFP−CD4+CD44high) and from spleens of DPLG3-treated mice using RNeasy Micro kit (QIAGEN) and subjected to library preparation using NEBNext Ultra RNA Library prep for Illumina kit (New England Biolabs). Library quality was evaluated by Bioanalyzer with High Sensitivity chips (Agilent Technologies). Sequencing was performed on a HiSeq 2000 (Illumina) by 2 × 50-bp paired-end reads at the Biopolymers Facility of Harvard Medical School. We used Bcbio_nextgen (https://github.com/chapmanb/bcbio-nextgen/) to process the RNA-seq data. Briefly, cutadapt (https://github.com/marcelm/cutadapt/) was used to trim adapters; trimmed reads were aligned to human reference genome (GRCh37) with tophat2; and read count for each gene was calculated by HT-seq. Genes with low expression (fragments per kilobase of transcript per million mapped reads < 1 across all samples) were filtered out. Statistical Analyses. Kaplan–Meier survival graphs were constructed and a log-rank comparison of the groups was used to calculate P values. The unpaired t test was used for comparison of experimental groups examined by Luminex assay, flow cytometry, and mixed-lymphocyte reactions. Differences were considered to be significant for P ≤ 0.05. Prism software was used for data analysis and graphing (GraphPad Software). Data represent means ± SEM.

Acknowledgments We thank Dr. J. David Warren at The Abby and Howard P. Milstein Synthetic Chemistry Core Facility at Weill Cornell Medicine for assistance and Drs. Jane Salmon and Xiaoping Qing at Hospital for Special Surgery for discussions. This work was supported by an American Heart Association grant (to J.A.), by the Milstein Program in Translational Medicine and Chemical Biology (C.F.N.), by the Alliance for Lupus Research (G.L.), and by the Daedalus Fund for Innovation at Weill Cornell Medicine (G.L.). The Department of Microbiology and Immunology at Weill Cornell Medicine is supported by the William Randolph Hearst Trust.

Footnotes Author contributions: C.F.N., G.L., and J.A. designed research; E.S.K., H.F., M.U., A.B.M., P.K.S., A.T.K., Z.S., T.L., S.R., J.P.A., R.W., G.S., L.S., G.L., and J.A. performed research; E.S.K., H.F., M.U., A.B.M., P.K.S., A.T.K., Z.S., L.V.R., I.G., T.L., S.R., J.P.A., R.W., L.S., F.J.B., C.F.N., G.L., and J.A. analyzed data; and E.S.K., F.J.B., C.F.N., G.L., and J.A. wrote the paper.

Reviewers: T.R.B., University of Pittsburgh Medical Center; F.G.L., University of Pittsburgh; and D.H.S., Massachusetts General Hospital.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1618548114/-/DCSupplemental.