Kathryn L. Pothoven

Dendritic cells with TGF-β1 differentiate naïve CD4+CD25− T cells into islet-protective Foxp3+ regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that β-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4+CD25+Foxp3+ T cells from naïve islet-specific CD4+CD25− T cells in the presence of TGF-β1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-β1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4+CD25+Foxp3+ T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.

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.

Cutting Edge: TGF-β-Induced Expression of Foxp3 in T cells Is Mediated through Inactivation of ERK

The peripheral induction of T regulatory cells can be accomplished by TGF-β through an epigenetic regulation leading to the expression of Foxp3. However, the exact mechanism of such a TGF-β-mediated action remains unclear. In the current study, we found that TGF-β treatment of CD4+CD25− T cells during T cell activation led to a transient inhibition of the phosphorylation of ERK followed by the induction of Foxp3 expression in these cells. Direct treatment with a specific ERK inhibitor, UO126, during CD4+CD25− T cell activation also induced Foxp3 expression and conferred a suppressive function to the induced Foxp3+ T cells. Furthermore, treatment of T cells with either TGF-β or UO126 significantly down-regulated the expression of DNMTs, a reaction normally elicited by demethylation agents, such as 5-Aza-2′-deoxycytidine. These results indicate that the epigenetic regulation of TGF-β-induced expression of Foxp3 may be mediated through the inactivation of ERK.

Ethylenecarbodiimide-Fixed Donor Splenocyte Infusions Differentially Target Direct and Indirect Pathways of Allorecognition for Induction of Transplant Tolerance

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c+ dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c+ dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4+CD25+Foxp3+ T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4+ regulatory T cells play a critical role in tolerance induction in transplantation. CD8+ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8+ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8+Foxp3+ T cells can be induced and significantly expanded by stimulating naïve CD8+ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8+Foxp3+ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8+Foxp3+ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8+Foxp3+ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4+Foxp3+ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8+Foxp3+ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4+CD25+Foxp3+ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4+CD25+Foxp3+ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4+CD25+Foxp3+ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4+CD25+Foxp3+ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG−/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4+CD25+Foxp3+ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4+CD25+Foxp3+ iTregs were further able to induce endogenous naïve T cells to convert to CD4+CD25+Foxp3+ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4+CD25+Foxp3+ iTregs. Such in vitro‐generated donor‐specific CD4+CD25+Foxp3+ iTregs are able to effectively control allogeneic islet graft rejection.

Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease

Background We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. Objective We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. Methods Paraffin‐embedded NP sections were stained with fluorescence‐labeled specific antibodies against OSM, GM‐CSF, and hematopoietic cell‐specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM‐CSF and follistatin‐like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. Results OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3‐5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45+ cells were OSM+, and of the OSM+ cells, 56% ± 7% were CD16+Siglec‐8−, indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45+ events from matched blood samples (n = 5) were OSM+, suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM‐CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05). Conclusions Neutrophils are a major source of OSM‐producing cells in patients with CRS and severe asthma.

The barrier hypothesis and Oncostatin M: Restoration of epithelial barrier function as a novel therapeutic strategy for the treatment of type 2 inflammatory disease

ABSTRACT Mucosal epithelium maintains tissue homeostasis through many processes, including epithelial barrier function, which separates the environment from the tissue. The barrier hypothesis of type 2 inflammatory disease postulates that epithelial and epidermal barrier dysfunction, which cause inappropriate exposure to the environment, can result in allergic sensitization and development of type 2 inflammatory disease. The restoration of barrier dysfunction once its lost, or the prevention of barrier dysfunction, have the potential to be exciting new therapeutic strategies for the treatment of type 2 inflammatory disease. Neutrophil-derived Oncostatin M has been shown to be a potent disrupter of epithelial barrier function through the induction of epithelial-mesenchymal transition (EMT). This review will discuss these events and outline several points along this axis at which therapeutic intervention could be beneficial for the treatment of type 2 inflammatory diseases.