Aaron Martin

Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis

Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.

Therapeutic Inflammatory Monocyte Modulation Using Immune-Modifying Microparticles

Negatively charged immune-modifying microparticles bind to the scavenger receptor MARCO on inflammatory monocytes, resulting in their apoptosis and reduced inflammatory damage in a range of diseases. A New Frontier in Immune Modulation Inflammatory monocytes markedly potentiate the immune pathology observed in many diseases, yet no therapy exists that specifically inhibits these cells. The therapeutic accessibility of monocytes in the bloodstream and their inherent propensity to engulf particulate material suggest that highly negatively charged microparticles might provide a readily translatable solution to this problem. These microparticles, referred to as immune-modifying microparticles (IMPs), may be derived from numerous compounds, including the biodegradable polymer poly(lactic-co-glycolic acid) (PLGA-IMP), already used in humans for inter alia dissolvable sutures. Getts et al. now show that upon infusion, IMPs bind to a receptor with a positive domain on inflammatory monocytes, resulting in monocyte sequestration in the spleen and apoptosis through a similar pathway observed for senescing leukocytes. This safe monocyte clearance pathway culminated in substantially reduced inflammatory tissue damage in mouse models of West Nile virus encephalitis, experimental autoimmune encephalomyelitis, peritonitis, colitis, and myocardial infarction. Together, the data suggest that IMPs could transform the treatment of acute inflammation. Indeed, phase 1/2 testing is planned to begin in 2014, with rapid translation supported by the availability of clinical-grade PLGA. Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3–mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate–induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

ECDI-fixed allogeneic splenocytes induce donor-specific tolerance for long-term survival of islet transplants via two distinct mechanisms

A major challenge for human allogeneic islet transplantation is the development of effective methods to induce donor-specific tolerance to obviate the need for life-long immunosuppression that is toxic to the insulin-producing β cells and detrimental to the host. We developed an efficient donor-specific tolerance therapy that utilizes infusions of ethylene carbodiimide (ECDI)-treated donor splenic antigen-presenting cells that results in indefinite survival of allogeneic islet grafts in the absence of immunosuppression. Furthermore, we show that induction of tolerance is critically dependent on synergistic effects between an intact programmed death 1 receptor–programmed death ligand 1 signaling pathway and CD4+CD25+Foxp3+ regulatory T cells. This highly efficient antigen-specific therapy with a complete avoidance of immunosuppression has significant therapeutic potential in human islet cell transplantation.

Tolerance Induced by Apoptotic Antigen-Coupled Leukocytes Is Induced by PD-L1+ and IL-10–Producing Splenic Macrophages and Maintained by T Regulatory Cells

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80+ macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1–dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.

Current landscape for T-cell targeting in autoimmunity and transplantation

In recent years, substantial advances in T-cell immunosuppressive strategies and their translation to routine clinical practice have revolutionized management and outcomes in autoimmune disease and solid organ transplantation. More than 80 diseases have been considered to have an autoimmune etiology, such that autoimmune-associated morbidity and mortality rank as third highest in developed countries, after cardiovascular diseases and cancer. Solid organ transplantation has become the therapy of choice for many end-stage organ diseases. Short-term outcomes such as patient and allograft survival at 1 year, acute rejection rates, as well as time course of disease progression and symptom control have steadily improved. However, despite the use of newer immunosuppressive drug combinations, improvements in long-term allograft survival and complete resolution of autoimmunity remain elusive. In addition, the chronic use of nonspecifically targeted immunosuppressive drugs is associated with significant adverse effects and increased morbidity and mortality. In this article, we discuss the current clinical tools for immune suppression and attempts to induce long-term T-cell tolerance induction as well as much-needed future approaches to produce more short-acting, antigen-specific agents, which may optimize outcomes in the clinic.

Ethylenecarbodiimide-Treated Splenocytes Carrying Male CD4 Epitopes Confer Histocompatability Y Chromosome Antigen Transplant Protection by Inhibiting CD154 Upregulation

In humans and certain strains of laboratory mice, male tissue is recognized as nonself and destroyed by the female immune system via recognition of histocompatibility Y chromosome Ag (Hya). Male tissue destruction is thought to be accomplished by CTLs in a helper-dependent manner. We show that graft protection induced with the immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SPs) with the chemical cross-linker ethylenecarbodiimide significantly, and often indefinitely, prolongs the survival of male skin graft transplants in an Ag-specific manner. In contrast, treatments with the Hya CD8 epitopes (Uty-/Smcy-SPs) failed to prolong graft survival. Dby-SP–tolerized CD4+ T cells fail to proliferate, secrete IFN-γ, or effectively prime a CD8 response in recipients of male grafts. Ag-coupled splenocyte treatment is associated with defective CD40–CD40L interactions as demonstrated by the observation that CD4 cells from treated animals exhibit a defect in CD40L upregulation following in vitro Ag challenge. Furthermore, treatment with an agonistic anti-CD40 Ab at the time of transplantation abrogates protection from graft rejection. Interestingly, anti-CD40 treatment completely restores the function of Dby-specific CD4 cells but not Uty- or Smcy-specific CD8 cells.

MicroRNA--managing the TH-17 inflammatory response.

The differentiation of interleukin 17–producing helper T cells is controlled by a complex network of cytokines, signaling pathways and transcription factors. Regulation by microRNA particles can now be added to this list.

Erratum: Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis (Nature Biotechnology (2012) 30 (1217-1224))

In the version of this article initially published, there were two errors in the discussion of epigenetic marks on page 854. In sentence 4, paragraph 2 of the section “Genetic and epigenetic changes predictive of malignancy,” dimethylated and trimethylated H3K9 were said incorrectly to be “polycomb” marks. “Polycomb” has been deleted from the sentence, and the following two sentences inserted for clarification: “In ES cells these genes are held in a ‘transcription ready’ state by two marks, a repressive H3K27me mark and an active mark, H3K4me64. Changes in the balance of repressive versus active marks can alter the activity of these genes, hypothetically keeping cells in a proliferative state.” Further down in the paragraph, DNMT3L was described incorrectly as catalytic. The original text, “... related to activation of the de novo methyltransferase DNMT3L68” has been revised to “...and maintain expression of the de novo methyltransferase–like protein DNMT3L68. Expression of DNMT3L appears to be a common feature in pluripotent cells including ES, EC and embryonic germ cells.” The errors have been corrected in the HTML and PDF versions of the article.

Targeting Th17 Cells for Therapy of Multiple Sclerosis

Multiple sclerosis is a demyelinating disease of the central nervous system mediated by autoreactive T lymphocytes. The Th17 lineage of effectors has been implicated in the inflammatory response against CNS autoantigens. Findings in experimental autoimmune encephalomyelitis (EAE, the animal model for multiple sclerosis) suggest that targeting the Th17 response may have a beneficial outcome for patients suffering from MS. Several existing and emerging therapeutic strategies will be discussed based on the manner in which they target Th17-mediated autoimmunity: lymphocyte depletion, prevention of Th17 development, and prevention of Th17 function. T cell-ablating agents are not Th17 specific and are associated with toxicity and opportunistic infections. The prevention of Th17 differentiation can be achieved experimentally by neutralizing cytokines specifically required for Th17 development and by the administration of cytokines or other drugs that interfere with differentiation; however, these strategies may also lead to enhanced rates of certain infectious diseases. Prevention of functional Th17 responses can be accomplished by inhibiting leukocyte trafficking or by neutralizing Th17 cytokines (IL-17 and/or GM-CSF). While several promising therapeutic candidates have been identified employing the EAE model, both the risks of immunomodulation and the efficacy of such candidates in human patients need to be completely characterized and carefully considered.

200. Generation of CAR-T Cells Lacking T Cell Receptor and Human Leukocyte Antigen Using Engineered Meganucleases

The manufacture of CAR-T cells depends on peripheral blood donations that contain T cells of sufficient quality and quantity. Currently, many CAR-T programs rely on autologous T cells, but several technical and commercial challenges hinder development. The majority of CAR-T trials have enrolled leukemia or lymphoma patients, many of which are unsuitable donors for CAR-T production due to their disease state or to previous treatments with lymphodepleting agents. In addition, a custom CAR-T production run for each patient is time consuming, lacks standardization and may present regulatory challenges. An alternative strategy is to source T cells from healthy donors and produce large batches of allogeneic CAR-T cells. Allogeneic T cells, however, will display mismatched human leukocyte antigens (HLA) that will be recognized by the recipients’ immune systems, contributing to immune rejection of engrafted CAR-T cells. Additionally, donor T cells will recognize the mismatched HLAs present in the recipient, contributing to graft-versus-host immune pathology. Both undesired immune responses are predicated on interactions between HLA and T cell receptors (TCR), and while the therapeutic effectiveness of CAR-T cells with targeted deletions in TCR genes has been reported by several groups, studies featuring both TCR and HLA deletion are limited. Here, we describe the use of meganucleases engineered to target regions of the TCR α chain constant region and β-2 microglobulin genes to generate TCR and HLA class I knockout primary human T cells. Both nucleases generate knockouts with approximately 75% efficiency and are well-tolerated by primary T cells from at least four separate donors. Purified double knockout cells do not demonstrate functional disadvantages in terms of proliferation or cytokine production, but do exhibit reduced allostimulatory potential toward HLA-mismatched T cells. Together, these findings demonstrate the feasibility of generating therapeutic quantities of CAR-T cells with reduced allo-reactive potential and collateral toxicity to normal tissues in recipients.