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Lee, Gap Ryol. "Phenotypic and Functional Properties of Tumor-Infiltrating Regulatory T Cells." Mediators of Inflammation 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/5458178.
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Regulatory T (Treg) cells maintain immune homeostasis by suppressing excessive immune responses. Treg cells induce tolerance against self- and foreign antigens, thus preventing autoimmunity, allergy, graft rejection, and fetus rejection during pregnancy. However, Treg cells also infiltrate into tumors and inhibit antitumor immune responses, thus inhibiting anticancer therapy. Depleting whole Treg cell populations in the body to enhance anticancer treatments will produce deleterious autoimmune diseases. Therefore, understanding the precise nature of tumor-infiltrating Treg cells is essential for effectively targeting Treg cells in tumors. This review summarizes recent results relating to Treg cells in the tumor microenvironment, with particular emphasis on their accumulation, phenotypic, and functional properties, and targeting to enhance the efficacy of anticancer treatment.
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Storb, Rainer F., Guido Lucarelli, Peter A. McSweeney, and Richard W. Childs. "Hematopoietic Cell Transplantation for Benign Hematological Disorders and Solid Tumors." Hematology 2003, no. 1 (January 1, 2003): 372–97. http://dx.doi.org/10.1182/asheducation.v2003.1.372.0010372.
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Allogeneic hematopoietic cell transplantation (HCT) has been successfully used as replacement therapy for patients with aplastic anemia and hemoglobinopathies. Both autologous and allogeneic HCT following high-dose chemotherapy can correct manifestations of autoimmune diseases. The impressive allogeneic graft-versus-tumor effects seen in patients given HCT for hematological malignancies have stimulated trials of allogeneic immunotherapy in patients with otherwise refractory metastatic solid tumors. This session will update the status of HCT in the treatment of benign hematological diseases and solid tumors. In Section I, Dr. Rainer Storb reviews the development of nonmyeloablative conditioning for patients with severe aplastic anemia who have HLA-matched family members. He also describes the results in patients with aplastic anemia given HCT from unrelated donors after failure of responding to immunosuppressive therapy. The importance of leuko-poor and in vitro irradiated blood product transfusions for avoiding graft rejection will be discussed. In Section II, Dr. Guido Lucarelli reviews the status of marrow transplantation for thalassemia major and updates results obtained in children with class I and class II severity of thalassemia. He also describes results of new protocols for class III patients and efforts to extend HCT to thalassemic patients without HLA-matched family members. In Section III, Dr. Peter McSweeney reviews the current status of HCT for severe autoimmune diseases. He summarizes the results of autologous HCT for systemic sclerosis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, and reviews the status of planned Phase III studies for autologous HCT for these diseases in North America and Europe. He also discusses a possible role of allogeneic HCT in the treatment of these diseases. In Section IV, Dr. Richard Childs discusses the development and application of nonmyeloablative HCT as allogeneic immunotherapy for treatment-refractory solid tumors. He reviews the results of pilot clinical trials demonstrating graft-versus-solid tumor effects in a variety of metastatic cancers and describes efforts to characterize the immune cell populations mediating these effects, as well as newer methods to target the donor immune system to the tumor.
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Storb, Rainer F., Guido Lucarelli, Peter A. McSweeney, and Richard W. Childs. "Hematopoietic Cell Transplantation for Benign Hematological Disorders and Solid Tumors." Hematology 2003, no. 1 (January 1, 2003): 372–97. http://dx.doi.org/10.1182/asheducation-2003.1.372.
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Abstract Allogeneic hematopoietic cell transplantation (HCT) has been successfully used as replacement therapy for patients with aplastic anemia and hemoglobinopathies. Both autologous and allogeneic HCT following high-dose chemotherapy can correct manifestations of autoimmune diseases. The impressive allogeneic graft-versus-tumor effects seen in patients given HCT for hematological malignancies have stimulated trials of allogeneic immunotherapy in patients with otherwise refractory metastatic solid tumors. This session will update the status of HCT in the treatment of benign hematological diseases and solid tumors. In Section I, Dr. Rainer Storb reviews the development of nonmyeloablative conditioning for patients with severe aplastic anemia who have HLA-matched family members. He also describes the results in patients with aplastic anemia given HCT from unrelated donors after failure of responding to immunosuppressive therapy. The importance of leuko-poor and in vitro irradiated blood product transfusions for avoiding graft rejection will be discussed. In Section II, Dr. Guido Lucarelli reviews the status of marrow transplantation for thalassemia major and updates results obtained in children with class I and class II severity of thalassemia. He also describes results of new protocols for class III patients and efforts to extend HCT to thalassemic patients without HLA-matched family members. In Section III, Dr. Peter McSweeney reviews the current status of HCT for severe autoimmune diseases. He summarizes the results of autologous HCT for systemic sclerosis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, and reviews the status of planned Phase III studies for autologous HCT for these diseases in North America and Europe. He also discusses a possible role of allogeneic HCT in the treatment of these diseases. In Section IV, Dr. Richard Childs discusses the development and application of nonmyeloablative HCT as allogeneic immunotherapy for treatment-refractory solid tumors. He reviews the results of pilot clinical trials demonstrating graft-versus-solid tumor effects in a variety of metastatic cancers and describes efforts to characterize the immune cell populations mediating these effects, as well as newer methods to target the donor immune system to the tumor.
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Im, Ho Joon, Kyung Nam Koh, Jin Kyung Suh, Eun Seok Choi, Seongsoo Jang, Chan-Jeoung Park, and Jong Jin Seo. "Haploidentical Hematopoietic Stem Cell Transplantation in Pediatric Patients: Comparison of Early Post-Transplant Outcome According to in Vitro Depletion Method." Blood 124, no. 21 (December 6, 2014): 1229. http://dx.doi.org/10.1182/blood.v124.21.1229.1229.
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Abstract Currently, haploidentical hematopoietic cell transplantation (HHCT) is considered an established option for patients who have diseases curable with HCT but who lack a suitable donor. We compared the early post-transplant outcomes of the two transplant groups using different in vitro depletion method. Between July 2008 and June 2014, 49 pediatric patients underwent in vitro T cell-depleted HHCT. Of 49 patients, 28 received CD3-depleted stem cells (CD3-HHCT) and 21 received TCRαβ-depleted grafts (TCRαβ-HHCT). Among 28 patients of CD3-HHCT, nine had hematologic malignancy [HM, one with ALL in non-CR, six with AML (3 CR1, 2 CR2, 1 non-CR), and two with MDS-RCMD], 18 had non-malignant disease (one with Fanconi anemia, 16 with acquired SAA, and one with CDA), and one had refractory neuroblastoma. Among 21 patients of TCRαβ-HHCT, 16 had HM [five with ALL (2 CR1, 2 CR2, 1 CR3), five with AML (2 CR1, 2 CR2, 1 non-CR), one with mixed lineage leukemia in non-CR, one with MDS-RCC, two with JMML, and two with NHL (1 CR2, 1 CR3)], two had SAA, and three had solid tumors [two with RMS (1 CR2, 1 refractory), and one with Ewing sarcoma in CR3]. Of 28 patients who received CD3-HHCT, two patients failed to achieve primary engraftment, and five patients experienced graft rejection (GR) within 28 days post-transplant. No patients of TCRαβ-HHCT experienced graft failure (GF). Early GF (primary GF and GR) was more common in CD3-HHCT compared to TCRαβ-HHCT (P=0.011). The cumulative incidence of acute GVHD ≥ grade II was comparable between two groups (33.3% for CD3-HHCT and 27.0% for TCRαβ-HHCT). Extensive chronic GVHD occurred in three patients from CD3-HHCT and one from TCRαβ-HHCT. T and T4 cell counts at 2 months post-transplant were higher in TCRαβ-HHCT than that of CD3-HHCT (P=0.007 for T and P=0.074 for T4). In CD3-HHCT, four patients died of non-relapse causes (two of CMV disease, one of encephalopathy, and one of autoimmune hemolytic anemia) and one died of leukemia. In contrast, five patients from TCRαβ-HHCT died of disease but, none died of non-relapse cause. Of 20 patients (18 from CD3-HHCT and two from TCRαβ-HHCT) with non-malignant disease, only one patient of CD3-HHCT died of TRM. As for 29 patients (10 from CD3-HHCT and 19 from TCRαβ-HHCT) with malignant disease, the EFS at 1 year for CD3-HHCT and TCRαβ-HHCT were 60.0% and 56.6%, respectively (P>0.05). In this study, TCRαβ-depleted HHCT showed a better early post-transplant outcome in terms of engraftment, immune recovery, and NRM, compared to CD3-HHCT. However, further study is warranted to evaluate the efficacy of TCRαβ-HHCT in preventing relapse in advanced malignancy. Disclosures No relevant conflicts of interest to declare.
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Emerson, Amy E., Emily M. Slaby, Shivani C. Hiremath, and Jessica D. Weaver. "Biomaterial-based approaches to engineering immune tolerance." Biomaterials Science 8, no. 24 (2020): 7014–32. http://dx.doi.org/10.1039/d0bm01171a.
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Bordron, A., A. Mankaï, B. Bendaoud, A. Saraux, V. Devauchelle, I. Ghedira, C. Jamin, J. C. Pers, C. Berthou, and P. Youinou. "B Cell-Ablative Therapy: Where are We Now?" International Journal of Immunopathology and Pharmacology 20, no. 4 (October 2007): 655–59. http://dx.doi.org/10.1177/039463200702000401.
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Based on their multifaceted functions, B cells participate in several pathological settings such as lymphoproliferative disorders, autoimmune diseases and graft rejection. B cell-ablative therapy has thus emerged as a mainstay in these diseases. A number of anti-B cell antibodies (Abs) have been generated, among which anti-CD20 Abs appear to be efficient. Rituximab (RTX) is one of these anti-CD20 monoclonal Abs. Originally approved for the treatment of non-Hodgkin lymphoma, RTX is now being administered in other malignant proliferations, applied to an increasing number of autoimmune diseases and required to prevent rejection of a graft. Although this medication is remarkably safe, a handful of laboratory tests have been proposed to monitor RTX-treated patients. The efficacy in different diseases, and the emergence of new anti-CD20 Abs raise many questions. Thus, their detailed understanding can lead to a better issue for inhibition of immune responses.
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Niemand, Claudia, Carina Conrads, Ramona Siemer, and Mario Assenmacher. "Rapid Clinical Scale Isolation of CD25hiCD4+ Regulatory T Cells." Blood 104, no. 11 (November 16, 2004): 4969. http://dx.doi.org/10.1182/blood.v104.11.4969.4969.
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Abstract Several publications during the last few years have reported CD25hiCD4+ regulatory T cells (Tregs) to prevent or to reverse disease in different mouse models of experimental autoimmune encephalomyelitis (EAE), colitis, graft rejection and graft-versus-host-disease (GvHD). As mouse and human Tregs share many phenotypical and functional characteristics, Tregs could provide a promising therapeutic approach for various human autoimmune diseases and pathological alloresponses. Here we have shown that Tregs can be isolated from leukapheresis harvests by CD25 enrichment using the CliniMACS technology (n=13). By this procedure we obtained 2.32x108 (± 1.12x108, range 0.71–4.42x108) cells out of 1010 mononuclear cells with a mean purity of 52.12% (± 12.11%, range 25.48–66.61%) for CD25hiCD4+ cells. Around 90% of enriched cells were CD25+CD4+. Among contaminating CD4− cells most cells were CD25+ which were further characterized by counterstaining to be mainly CD19+ B cells and a few CD8+, CD56+ or CD123+ cells. It is possible to deplete the CD19+ or CD8+ cells by using CD19 Microbeads or CD8 Microbeads respectively with the CliniMACS Instrument before CD25 enrichment. Combined depletion of different cells, e.g. CD19+ and CD8+ cells is conceivable. Isolated cells were phenotypically and functionally characterized. The majority of the CD25hiCD4+ T cells expressed glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L and CD45RO. In addition, isolated cells were able to suppress the proliferation and activation of cocultured conventional CD4+ cells after polyclonal stimulation with anti-CD3 antibody. We conclude that the large-scale isolation of CD25hiCD4+ regulatory T cells for clinical applications (e.g. therapy of autoimmune diseases, graft rejection or GvHD) is possible by using the CliniMACS CD25 Reagent and the CliniMACS Instrument.
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Jenh, Chung-Her, Mary Ann Cox, Long Cui, Eva-Pia Reich, Lee Sullivan, Shu-Cheng Chen, David Kinsley, et al. "A selective and potent CXCR3 antagonist SCH 546738 attenuates the development of autoimmune diseases and delays graft rejection." BMC Immunology 13, no. 1 (2012): 2. http://dx.doi.org/10.1186/1471-2172-13-2.
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Kaouther, Mnasria, and Oueslati Ridha. "Dendritic Cell-Based Graft Tolerance." ISRN Pharmacology 2011 (April 10, 2011): 1–4. http://dx.doi.org/10.5402/2011/347134.
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It has recently been demonstrated that mouse and human dendritic cells (DCs) can produce IL-2 after activation. However the role of the IL2/IL2R pathway in DC functions has not yet been fully elucidated. The results presented in this study provide several new insights into the role of this pathway in DCs. We report that stimulation of human monocyte-derived DCs with LPS strongly upregulated CD25 (α chain of the IL2R) expression. In additon, by using a humanized monoclonal antibody against CD25, we demonstrated that the IL2 signalling in DC upregulated both IL-12 and γIFN production but decreased IL10 synthesis. We also found that LPS-matured DCs produced IL2. Taken together, these results suggest that IL-2 actively contributes to the DC activation through an autocrine pathway. Furthermore, our results indicate that the IL2 pathway in DC is involved in the development of T-helper priming ability and in the upregulation of surface markers characteristic of a “mature” phenotype. This study therefore provide new molecular clues regarding the split between these two phenomena and unravel new mechanisms of action of anti-CD25 monoclonal antibodies that may contribute to their action in several human immunological disorders such as autoimmune diseases and acute allograft rejection.
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Zulpaite, Ruta, Povilas Miknevicius, Bettina Leber, Kestutis Strupas, Philipp Stiegler, and Peter Schemmer. "Tryptophan Metabolism via Kynurenine Pathway: Role in Solid Organ Transplantation." International Journal of Molecular Sciences 22, no. 4 (February 15, 2021): 1921. http://dx.doi.org/10.3390/ijms22041921.
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Solid organ transplantation is a gold standard treatment for patients suffering from an end-stage organ disease. Patient and graft survival have vastly improved during the last couple of decades; however, the field of transplantation still encounters several unique challenges, such as a shortage of transplantable organs and increasing pool of extended criteria donor (ECD) organs, which are extremely prone to ischemia-reperfusion injury (IRI), risk of graft rejection and challenges in immune regulation. Moreover, accurate and specific biomarkers, which can timely predict allograft dysfunction and/or rejection, are lacking. The essential amino acid tryptophan and, especially, its metabolites via the kynurenine pathway has been widely studied as a contributor and a therapeutic target in various diseases, such as neuropsychiatric, autoimmune disorders, allergies, infections and malignancies. The tryptophan-kynurenine pathway has also gained interest in solid organ transplantation and a variety of experimental studies investigating its role both in IRI and immune regulation after allograft implantation was first published. In this review, the current evidence regarding the role of tryptophan and its metabolites in solid organ transplantation is presented, giving insights into molecular mechanisms and into therapeutic and diagnostic/prognostic possibilities.
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Normanton, Marília, and Luciana Cavalheiro Marti. "Current data on IL-17 and Th17 cells and implications for graft versus host disease." Einstein (São Paulo) 11, no. 2 (June 2013): 237–46. http://dx.doi.org/10.1590/s1679-45082013000200019.
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Human interleukin 17 was first described in 1995 as a new cytokine produced primarily by activated T CD4+ cells that stimulate the secretion of IL-6 and IL-8 by human fibroblasts, besides increasing the expression of ICAM-1. Various authors have reported that IL-17A has a role in the protection of organisms against extracellular bacteria and fungi due to the capacity of IL-17A to recruit neutrophils to the areas of infection, evidencing a pathological role in various models of autoimmune diseases, such as experimental autoimmune encephalitis and arthritis. The participation of IL-17A has also been described in the acute rejection of organ transplants and graft versus host disease. However, the greatest revolution in research with IL-17 happened in 2000, when it was proposed that IL-17 cannot be classified as Th1 or Th2, but rather, simply as a new lineage of IL-17-producing T-cells. These findings modified the previously established Th1/Th2 paradigm, leading to the definition of the CD3+ CD4+ Th17 cellular subtype and establishment of a new model to explain the origin of various immune events, as well as its implication in the graft versus host disease that is discussed in depth in this article.
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Ostermann, D., N. Perico, O. Imberti, C. Barbui, M. Bontempelli, and G. Remuzzi. "Colchicine allows prolonged survival of highly reactive renal allograft in the rat." Journal of the American Society of Nephrology 4, no. 6 (December 1993): 1294–99. http://dx.doi.org/10.1681/asn.v461294.
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Colchicine, with its immunosuppressive properties, has been used with beneficial effects in autoimmune diseases. Whether colchicine, by virtue of the above properties, could attenuate the process of kidney allograft rejection in the rat is investigated in this report. Untreated Lewis rats (N = 6) given an incompatible kidney allograft from Brown-Norway rats rejected the graft within 12 days. Colchicine at a daily ip dose of 40 (N = 6) or 10 (N = 4) micrograms/kg promoted long-term survival (> 170 days) of major histocompatibility complex-incompatible kidney grafts. Animals (N = 4) given 4 micrograms of colchicine per kilogram had a graft failure within 10 days. Experiments have also been performed to evaluate the effect of colchicine withdrawal at different time intervals from transplantation on subsequent allograft survival. Colchicine (40 micrograms/kg per day ip) was given for 12, 6, or 1 mo or for 15 days to an additional four groups of six animals each without any other immunosuppressants. The withdrawal of colchicine did confer long-term inhibition of the immune system in animals treated for at least 1 mo, as documented by a graft survival of more than 80 days. By contrast, those animals who discontinued colchicine after only 15 days of treatment had graft rejection within the next 8 days. Mixed lymphocyte culture experiments showed a significant (P < 0.01) reduction of the proliferation of peripheral blood lymphocytes taken from all groups of animals 30 days after colchicine withdrawal when challenged with Brown-Norway lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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Jovanovic, Dijana. "Serum amyloid a in clinical practice." Srpski arhiv za celokupno lekarstvo 132, no. 7-8 (2004): 267–71. http://dx.doi.org/10.2298/sarh0408267j.
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Serum amyloid A (SAA) is an acute phase first class protein discovered a quarter of the century ago. Its concentration depends on clinical findings of the patient, illness activity and the therapy applied. SAA increases moderately to markedly (100-1000 mg/l) in bacterial and fungal infections, invasive malignant diseases, tissue injuries in the acute myocardial infarction and autoimmune diseases such as rheumatoid arthritis and vasculitis. Mild elevation (10-100 mg/l) is often seen in viral infections, systemic lupus erythematosus and localized inflammation or tissue injuries in cystitis and cerebral infarction. SAA as sensitive, non-invasive parameter is used in organ transplantation where early and correct diagnosis is needed as well as where prompt therapy is required. Besides acute kidney allograft rejection, SAA is used in the diagnosis of rejection after liver transplantation, simultaneous pancreas and kidney transplantation and also in bone marrow transplantation (acute ?graft vs. host disease"). Simultaneous determination of C-reactive protein (CRP) and SAA may point to acute kidney allograft rejection. Standard immunosuppressive therapy with cyclosporine A and prednisolone significantly suppresses the acute phase CRP reaction both in operation itself and acute rejection, but not in infection. On the other hand, SAA rejection in operation, acute allograft rejection and infection is present in spite of cyclosporine A and steroids therapy. Different reaction of SAA and CRP in transplant patients to cyclosporine A therapy helps in differentiation between the infection and rejection. Although CRP and SAA are sensitive and acute phase reactants, their serum concentrations cannot be valued as prognostic and diagnostic criteria without creatinine serum concentration and clinical findings. In addition, they offer important information for clinical diagnosis as well as the kind of therapy.
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Kappel, Lucy W., Gabrielle L. Goldberg, Christopher G. King, David Y. Suh, Odette M. Smith, Cassandra Ligh, Amanda M. Holland, et al. "IL-17 contributes to CD4-mediated graft-versus-host disease." Blood 113, no. 4 (January 22, 2009): 945–52. http://dx.doi.org/10.1182/blood-2008-08-172155.
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Abstract CD4+ interleukin-17 (IL-17)+ T cells (Th17 cells) have been implicated in allograft rejection of solid organs and several autoimmune diseases. However, the functional role of Th17 cells in the development of acute graft-versus-host disease (GVHD) has not been well-characterized. We detected significant numbers of alloreactive CD4+ donor T cells expressing IL-17, IL-17F, or IL-22 in the lymphoid organs of recipients of an allogeneic bone marrow transplant. We found no differences in GVHD mortality or graft-versus-tumor (GVT) activity between wild type (WT) and IL-17−/− T-cell recipients. However, upon transfer of murine IL-17−/− CD4+ T cells in an allogeneic BMT model, GVHD development was significantly delayed behind recipients of WT CD4+ T cells, yet overall GVHD mortality was unaffected. Moreover, recipients of IL-17−/− CD4+ T cells had significantly fewer Th1 cells during the early stages of GVHD. Furthermore, we observed a decrease in the number of IFN-γ–secreting macrophages and granulocytes and decreased production of proinflammatory cytokines (interferon [IFN]-γ, IL-4, and IL-6) in recipients of IL-17−/− CD4+ T cells. We conclude that IL-17 is dispensable for GVHD and GVT activity by whole T cells, but contributes to the early development of CD4-mediated GVHD by promoting production of proinflammatory cytokines.
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Baeke, Femke, Evelyne Van Etten, Lut Overbergh, and Chantal Mathieu. "Vitamin D3and the immune system: maintaining the balance in health and disease." Nutrition Research Reviews 20, no. 1 (June 2007): 106–18. http://dx.doi.org/10.1017/s0954422407742713.
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1,25-Dihydroxyvitamin D3(1,25(OH)2D3), the active form of vitamin D3, is a central player in Ca and bone metabolism. More recently, important immunomodulatory effects have been attributed to this hormone. By binding to its receptor, the vitamin D receptor, 1,25(OH)2D3regulates the expression of various genes and consequently affects the behaviour of different cell types within the immune system. 1,25(OH)2D3can potently inhibit pathogenic T cells and gives rise to elevated numbers of regulatory T cells via the induction of tolerogenic dendritic cells. These immunomodulatory activities of 1,25(OH)2D3have also been proven usefulin vivo: administration of 1,25(OH)2D3in several animal models can prevent or cure different autoimmune diseases and graft rejection. To overcome the dose-limiting side effects of 1,25(OH)2D3on Ca and bone, less calcaemic structural analogues (alone or in combination with synergistically acting drugs or bone-resorption inhibitors) have been successfully used in animal models. Furthermore, as 1,25(OH)2D3also contributes to host defence against infectious agents by the induction of antimicrobial responses, this molecule might provide a new strategy to deal with drug-resistant infections. According to the pleiotropic effects of 1,25(OH)2D3in the immune system, increasing epidemiological data underline the importance of adequate vitamin D intakes in reducing the risk of several autoimmune diseases and infections such as tuberculosis.
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Geraud, Arthur, Paul Gougis, Aurore Vozy, Celine Anquetil, Yves Allenbach, Emanuela Romano, Elisa Funck-Brentano, Javid J. Moslehi, Douglas B. Johnson, and Joe-Elie Salem. "Clinical Pharmacology and Interplay of Immune Checkpoint Agents: A Yin-Yang Balance." Annual Review of Pharmacology and Toxicology 61, no. 1 (January 6, 2021): 85–112. http://dx.doi.org/10.1146/annurev-pharmtox-022820-093805.
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T cells have a central role in immune system balance. When activated, they may lead to autoimmune diseases. When too anergic, they contribute to infection spread and cancer proliferation. Immune checkpoint proteins regulate T cell function, including cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) and its ligand (PD-L1). These nodes of self-tolerance may be exploited pharmacologically to downregulate (CTLA-4 agonists) and activate [CTLA-4 and PD-1/PD-L1 antagonists, also called immune checkpoint inhibitors (ICIs)] the immune system.CTLA-4 agonists are used to treat rheumatologic immune disorders and graft rejection. CTLA-4, PD-1, and PD-L1 antagonists are approved for multiple cancer types and are being investigated for chronic viral infections. Notably, ICIs may be associated with immune-related adverse events (irAEs), which can be highly morbid or fatal. CTLA-4 agonism has been a promising method to reverse such life-threatening irAEs. Herein, we review the clinical pharmacology of these immune checkpoint agents with a focus on their interplay in human diseases.
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Perseghin, Paolo. "Extracorporeal Photochemotherapy as a Challenging Treatment for Cutaneous T-Cell Lymphoma, Acute and Chronic Graft-versus-Host Disease, Organ Rejection and T-Lymphocyte-Mediated Autoimmune Diseases." Transfusion Medicine and Hemotherapy 35, no. 1 (December 21, 2007): 8–17. http://dx.doi.org/10.1159/000111755.
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Oliveira, Régis Linhares, Pedro Cesar Chagastelles, Patrícia Sesterheim, and Patricia Pranke. "In Vivo Immunogenic Response to Allogeneic Mesenchymal Stem Cells and the Role of Preactivated Mesenchymal Stem Cells Cotransplanted with Allogeneic Islets." Stem Cells International 2017 (2017): 1–12. http://dx.doi.org/10.1155/2017/9824698.
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Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into cells from the mesenchymal lineage. The hypoimmunogenic characteristic of MSCs has encouraged studies using allogeneic MSCs for the treatment of autoimmune diseases and inflammatory conditions. Promising preclinical results and the safety of allogeneic MSC transplantation have created the possibility of “off-the-shelf” clinical application of allogeneic cells. This study has aimed to evaluate the survival of untreated and IFN-γ- and TNF-α-treated (preactivated) allogeneic MSCs transplanted under the kidney capsule of immunocompetent mice together with the role of preactivated MSCs after cotransplantation with allogeneic islets. The preactivation of MSCs upregulated the gene expression of anti-inflammatory molecules and also enhanced their immunomodulatory capacity in vitro. In vivo, allogeneic MSCs provoked an immunogenic response, with the infiltration of inflammatory cells at the transplant site and full graft rejection in both the untreated and preactivated groups. Allogeneic islets cotransplanted with preactivated MSCs prolonged graft survival for about 6 days, compared with islet alone. The present results corroborate the hypothesis that allogeneic MSCs are not immune-privileged and that after playing their therapeutic role they are rejected. Strategies that reduce allogeneic MSC immunogenicity can potentially prolong their in vivo persistence and improve the therapeutic effects.
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Kizhakayil, Dhanya, Abbirami Sathappan, Giusy Gentilcore, Zoltan Pos, Nikolett Lupsa, Mohammed A. Al-Aghbar, Cristina Maccalli, Nicholas Van Panhuys, Jean-Charles Grivel, and Sara Deola. "B-T Cell Interactions in GRAFT-Versus-Host Disease." Blood 136, Supplement 1 (November 5, 2020): 38. http://dx.doi.org/10.1182/blood-2020-141277.
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Cytotoxic T cells (CTLs) and B cells engage distinct interactions in GVHD patients' blood and tissues, detectable in regular flow-cytometry screenings, by size and by double positive CD19-CD8 antibody markers (Deola, BMT 2017). B-CTL couplets are formed by alpha-betaTCR+ CD8+ CTLs preferentially targeting CD27+ CD19+ cells displaying an activated CD80 and CD86 phenotype. Interactions may last from 5 minutes to roughly 1 hour, and release a pattern of T cell attracting chemokines, as IP10, MIG, ITAC, which are also known GVHD biomarkers. To further unravel the mechanism of this cell interaction, we built an in-vitro model where human PBMCs cells are expanded with cognate peptides and IL2 for 1-2 weeks, then immune-selected for CD8 antigen by Miltenyi microbeads negative-selection and incubated (2-18 hours) with fresh autologous CD19-B cells, immune-selected with the same method. The interactions are studied under confocal microscope video-imaging (Zeiss LSM 880+Imaris 3D analysis software) and in flow-cytometry (SymphonyA5 BD) after deep phenotype antibody staining. The intensity of interaction, measured by fluorescence interference on cell membranes, revealed an active engagement of CD19 and CD8 antigens. CD19 antigen penetrates deeper in contacting T cells, than CD8 on B cells, and consistently with this finding, after the interactions there is an antigen exchange between cells with CD19 antigen actively transferred in CD8 cells (p value =<0.001), but not the contrary. We already proved that this type of B-T interaction is not antigen specific in CTL-to-B direction (Deola et a, JI 2008) but to exclude cross-presentation from B to CTLs and to unravel the role of CD8, we interfered by antibody blocking of MHC class I pathway on B cells and CD8 on CTLs. B-T cell interactions are not abolished after MHC-I or CD8 blocking, the intensity of coupling is unchanged after MHC-I block, and is higher after blocking CD8 (p value=<0.001). In particular, by blocking CD8 molecule, T cells target preferentially CD19+/CD27- cells rather than CD19+/27+ cells. Interestingly, B cell engagement follows 2 repetitive patterns of interaction: a high intensity interaction that visually corresponds to tight coupling cells with high CD19 penetration in T cells, and a low-intensity continuous interaction, visually measurable by cells "sniffing" each other. Both patterns correspond to diverse Calcium flux activation on T cells and B cells, suggesting functional different pathways triggered by the 2 type of interactions. Deep phenotype flow cytometry analyses after coupling reveals distinct programs triggered by the contact in both B cells and T cells. While after the interaction CTLs double their pool of perforin bearing effectors and their fraction of CD45RA-/CD27+ memory CTLs, CD19 preferentially undergo a deletion of IgD- CD27- (DN) cells (13,85%+/-1,1 and 22,95%+/-4,5 CD95/Fas+, respectively in B cells alone and B+CTLs, n=2) and a rescue of affinity mature CD27+ IgD- cells (39.8%+/-25,47 and 21,2%+/- 29% CD95/Fas+ in the same groups) CTLs are the ultimate line of "tissue attack" in GVHD and several diseases, as autoimmune diseases, cancer, viral diseases, sharing a common pathological program definable as "immune rejection". B cells are key players in immune rejection, but a link between these 2 types of cells is still unclear. Our findings enforce the hypothesis of a program of peripheral tolerance/activation triggered directly between B cells and activated CTLs in the context of inflammation and of GVHD. Disclosures No relevant conflicts of interest to declare.
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Gao, Wenda, Kenichiro Yamashita, Jennifer Sullivan, Abraham Scaria, Terry B. Strom, and Xian C. Li. "Adenovirus-Mediated PD-L1 Over-Expression Has Differential Effects on Allograft Survival in Murine Islet and Heart Transplant Models." Blood 104, no. 11 (November 16, 2004): 4960. http://dx.doi.org/10.1182/blood.v104.11.4960.4960.
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Abstract Program death-1 (PD-1) is a negative regulator of the immune system. Blocking PD-1-mediated negative signaling accelerates autoimmune diseases, while engaging PD-1 with recombinant PD-L1Ig fusion protein potentiates the efficacy of co-stimulation blockade in prolonging allograft survival. However, soluble PD-L1Ig itself showed no graft-protecting effect, in contrast to its strong inhibition of T and B cell activation in vitro when applied in a plate-bound form. In this study, we tested the hypothesis that membrane-bound PD-L1 should prolong allograft survival due to its increased ability to crosslink PD-1 receptor. An adenovirus (Ad.PD-L1) was constructed to encode the full-length mPD-L1, followed by green fluorescent protein (GFP) gene linked by an IRES sequence. A control adenovirus (Ad.Ctrl) was similarly constructed that carries only the GFP gene. In islet transplant model, B6AF1 (H-2b/a) islets were infected with the adenoviruses, and then transplanted into C57BL/6 (H-2b) mice induced diabetic by streptozotocin. In heart transplant model, DBA/2 (H-2d) hearts were perfused with adenoviruses, and then transplanted into C57BL/6 mice. PD-L1 over-expression in islet did not prolong graft survival, but accelerated islet rejection (Ad.PD-L1: 9.0+/−3.5 days; Ad.Ctrl: 13.3+/−2.2 days). In contrast, infection with Ad.PD-L1 prolonged heart allograft survival (15.4+/−5.7 days) in C57BL/6 mice, which promptly rejected DBA/2 hearts (7.0+/−2.3 days, p<0.02). Thus, over-expression of membrane-bound PD-L1 has beneficial effect in an organ/tissue specific manner. Strategies other than direct expression of PD-L1 in the islet b cells need to be devised in order to utilize this negative pathway to prevent rejection.
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Vassiliadis, S., A. Ranella, L. Papadimitriou, A. Makrygiannakis, and I. Athanassakis. "Serum levels of pro- and anti-inflammatory cytokines in non-pregnant women, during pregnancy, labour and abortion." Mediators of Inflammation 7, no. 2 (1998): 69–72. http://dx.doi.org/10.1080/09629359891199.
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Disturbance of the cytokine equilibrium has been accused for many pathological disorders. Microbial infections, autoimmune diseases, graft rejection have been correlated to over- or under-production of specific cytokines which are produced as responder molecules to the various immune stimuli. The sole naturally occurring immune reaction in the organism is developed during the gestational period where, despite the presence of a semi-allogeneic graft, maternal immunoreactivity is driven to support fetal growth. The successful embryo development has been attributed to the important intervention of cytokines where some have been characterized as indispensable and others deleterious to fetal growth. However, the physiological levels of many factors during the gestational process have not been determined. Thus, in the present study we have measured and established the values of IL-1α, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, GM-CSF, TNF- α and IFN-γ during all phases of human pregnancy (first, second and third trimester of pregnancy, labour, abortions of the first trimester) as well as in the non-pregnant control state. This is an attempt to assess serum protein concentrations and present the physiological levels of these cytokines at certain time intervals providing thus a diagnostic advantage in pregnancy cases where the mother cannot immunologically support the fetus. Exploitation of this knowledge and further research may be useful for therapeutic interventions in the future.
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Taylor, Andrew W., and Darren J. Lee. "The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells." Journal of Transplantation 2011 (2011): 1–7. http://dx.doi.org/10.1155/2011/246856.
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The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) has an important role in modulating immunity and homeostasis. The production of IFN-γby effector T cells is suppressed byα-MSH, while TGF-βproduction is promoted in the same cells. Suchα-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of theα-MSH-induced Treg cells showed that the cells areCD4+T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed theα-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential forα-MSH-induced Treg cells to be from the effector T-cell population. We found that theα-MSH-induced Treg cells areCD25+ CD4+T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, theα-MSH treatment augments FoxP3 message in the effector T cells, andα-MSH induction of regulatory activity was limited to the effectorCD25+T-cell population. Therefore,α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.
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BILOUS, V. L. "PRODUCTION AND APPLICATION OF ANGIOSTATINS FOR THE TREATMENT OF OCULAR NEOVASCULAR DISEASES." Biotechnologia Acta 14, no. 1 (February 2021): 5–24. http://dx.doi.org/10.15407/biotech14.01.005.
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Angiostatins comprise a group of kringle-containing proteolytically-derived fragments of plasminogen/plasmin, which act as potent inhibitory mediators of endothelial cells proliferation and migration. Angiostatins are involved in modulation of vessel growth in healthy tissues and various pathological conditions associated with aberrant neovascularization. The aim of the present paper was to summarize available information, including our own experimental data, on prospects of angiostatin application for treatment of ocular neovascular diseases (OND), focusing on retinal pathologies and corneal injury. In particular, literature data on prospective and retrospective studies, clinical trials and animal models relating to the pathophysiology, investigation and management of OND are described. Special emphasis was made on the laboratory approaches of production of different angiostatin isoforms, as well as comparison of antiangiogenic capacities of native and recombinant angiostatin polypeptides. Several studies reported that angiostatins may completely abolish pathologic angiogenesis in diabetic proliferative retinopathy without affecting normal retinal vessel development and without exhibiting adverse side effects. Angiostatins have been tested as a tool for corneal antiangiogenesis target therapy in order to manage diverse ocular surface pathological conditions induced by traumas, chemical burns, previous surgery, chronic contact lens wear, autoimmune diseases, keratitis and viral infections (herpes, COVID-19), corneal graft rejection, etc. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Application of adenoviral genetic construct encoding angiostatin K5 as a promising tool for OND treatment illustrates a vivid example of upcoming revolution in local gene therapy. Further comprehensive studies are necessary to elucidate the clinical potential and optimal regimes of angiostatinbased intervention modalities for treating ocular neovascularization.
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Feng, Xingmin, Elena E. Solomou, Keyvan Keyvanfar, Thomas Herndon, Jichun Chen, Sachiko Kajigaya, and Neal S. Young. "Rabbit ATG but Not Horse ATG Promotes Expansion of Functional CD4+CD25highFoxP3 Regulatory T Cells In Vitro." Blood 110, no. 11 (November 16, 2007): 2312. http://dx.doi.org/10.1182/blood.v110.11.2312.2312.
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Abstract CD4+CD25+ regulatory T cells (Treg) are believed to play important roles in suppressing immune responses and maintaining tolerance. Treg have the ability to prevent the development of autoimmune diseases, graft rejection and graft versus host disease (GVHD) in mice and perhaps also in humans. Immunosuppressive drugs such as rabbit ATG (rATG), horse ATG (hATG) and cyclosporine A (CsA) are widely used in conditioning for transplantation and for the treatment of autoimmune diseases and GVHD, but their effects on Treg remain to be fully elucidated. Lopez et al. (J Am Soc Nephrol. 2006;17:2844–2853.) first reported that in vitro culture of human peripheral blood mononuclear cells (PBMC) with rATG resulted in expansion of CD4+CD25+ T cells. In the current study, we show that in vitro culture of normal human PBMC with low dose rATG (10 μg/ml) resulted in marked expansion of CD4+CD25high T cells (rATG 8.00±0.95% versus untreated 0.99±0.11%, n=10, p<0.0001) and CD4+CD25high FoxP3 T cells (rATG 2.24±0.11% versus untreated 0.90±0.10%, n=10, p<0.0001). rATG exposure converted CD4+CD25− T cells into CD4+CD25+ T cells, which proliferated better in comparison to CD4+CD25− T cells. In immunoblots of protein extracted from PBMC treated with rATG, there was increased expression of FoxP3 and nuclear factor of activated T cells (NFAT1) in CD4+CD25− and CD4+CD25+ T cells; rATG-induced NFAT1 expression correlated with FoxP3 expression. Expanded Treg suppressed autologous T-cell proliferation after T-cell receptor (TCR) stimulation by 64% when cultured with autologous PBMC at a 1:1 ratio, consistent with functional activity. Culture supernatants of PBMC treated with rATG showed increased levels of IL-10, compared with supernatants of PBMC treated with hATG or CsA, but no differences in INF-γ, IL-2, and IL-4. Unexpectedly, hATG did not expand but rather decreased Treg [For CD4+CD25high T cells (n=10): hATG 0.67±0.10% versus untreated 0.99±0.11%, p=0.0386; For CD4+CD25high FoxP3 T cells (n=10): hATG 0.62±0.08% versus untreated 0.90±0.10%, p=0.0435]. Furthermore, rATG and hATG showed differences in binding to lymphocytes, they contained different amounts of CD3 and TCRαβ antibodies, and they induced different activation states (expression of glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4, and CD62L) for CD4+ T cells. In vitro, Treg expansion mediated by rATG occurred at submitogenic concentrations (< 50 μg/ml) rather than at lymphocyte depletion levels (50–100 μg/ml). Our findings suggest that rATG expanded Treg by converting CD4+CD25− T cells into CD4+CD25+ T cells, probably through a mechanism of transcription regulation, and enhanced NFAT1 expression, in turn conferring on CD4+CD25− T cells FoxP3 expression and regulatory activity. The therapeutic effects of rATG in the treatment of autoimmune diseases and GVHD may occur due to not only lymphocyte depletion but also enhanced Treg cell number and function. Our observation may provide a useful method for expansion of Treg in cellular treatment in transplantation and autoimmune diseases.
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Luo, Xiaofeng, Jing Li, Juan Chen, Jocelyn A. Schroeder, Jianda Hu, and Qizhen Shi. "Platelet-Targeted Gene Transfer Prevents Graft Rejection and Induces Immune Tolerance Even in a Primed Model." Blood 128, no. 22 (December 2, 2016): 2315. http://dx.doi.org/10.1182/blood.v128.22.2315.2315.
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Abstract Induction of antigen-specific immune tolerance is desirable in autoimmune diseases, transplantation, gene therapy, and some protein therapies in order to prevent or reverse adverse immune responses. Our previous studies have demonstrated that targeting FVIII or FIX expression to platelets under control of the platelet-specific aIIb promoter can restore hemostasis and induce immune tolerance in hemophilia mice. Here we explored if this approach can be applied to prevent graft rejection and induce immune tolerance to a non-coagulant protein even with pre-existing immunity. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). We designed another vector, 2bVpOVA, which includes the VWF propeptide (Vp) cassette to secure OVA storage in platelet granules. The third construct, 2bGFP, in which the GFP cassette is driven by the same promoter, was used as a control vector. We confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to hematopoietic stem cells (HSCs) can result in OVA expression with greater than 95% of OVA stored in platelet a-granules. Under a non-myeloablative conditioning 6.6Gy total body irradiation (TBI), platelet-OVA expression levels were 24.22±8.72 ng/108 platelets and 1.41±0.73 ng/108 platelets in 2bOVA and 2bVpOVA transduced wild type (WT) recipients, respectively. When recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in un-transduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. To explore whether platelet targeted gene transfer can be applied to prevent graft rejection, skin grafts from Act-mOVA transgenic mice, in which OVA is expressed on the cell surfaces of all organs, were transplanted onto transduced recipients. Full thickness tail skin successfully grafted onto OVA-immunized 2bOVA- or 2bVpOVA-transduced recipients and was sustained for the rest of the animals' lives or during the study course for up to 6 months. In contrast, skin grafts were rejected in OVA-immunized untransduced WT and 2bGFP-transduced animals within 6 weeks. To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP and transplanted into CD45.1/B6 WT recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (85.7±4.3%, 84.9±3.9%, and 86.5±2.9%, respectively), but donor-derived CD45.2+CD4+ T cells in the 2bOVA (0.2±0.1%, n=10) and 2bVPOVA (0.8±0.3%, n=11) groups were significantly lower than in the 2bGFP group (2.6±0.5%, n=11) in peripheral blood. Similarly, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy. To investigate whether platelet-targeted gene transfer can still induce the immune tolerance when immune system is primed, Sca-1+ cells from OTII mice were transduced with 2bOVA lentivirus and transplanted into OVA-primed CD45.1/B6 WT animals preconditioned with 6.6Gy TBI. We found that the platelet-OVA expression level in the OVA-primed group after 2bOVA gene transfer was not significantly different from the unprimed group (26.47±4.47 vs. 29.56±6.29 ng/108 platelets). The engraftments were comparable among the primed, the unprimed, and the primed untransduced control groups. Similar to the unprimed model, OVA-specific CD4+ T cells in 2bOVA-transduced OVA-primed recipients were significantly lower than in the untransduced controls (0.23±0.07% vs. 3.98±0.61%). Importantly, the anti-OVA total IgG declined with time after 2bOVA gene transfer, even when the animals were rechallenged with OVA. In summary, our data demonstrate that platelet-targeted gene transfer can prevent graft rejection and induce immune tolerance even when the immune system is primed, suggesting that platelet gene therapy can be a promising approach to induce immune tolerance and prevent undesired immune responses. Disclosures No relevant conflicts of interest to declare.
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Maruf, Abdullah Al, Luke Wan, and Peter J. O’Brien. "Evaluation of Azathioprine-Induced Cytotoxicity in anIn VitroRat Hepatocyte System." BioMed Research International 2014 (2014): 1–7. http://dx.doi.org/10.1155/2014/379748.
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Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using “Accelerated Cytotoxicity Mechanism Screening” techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 μM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition ofN-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition ofN-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N′-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.
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Ratnasingam, Sumita, Patricia A. Walker, Huy Tran, Zane Kaplan, James David McFadyen, Huyen Tran, Tse-Chieh Teh, et al. "Bortezomib Yields High Response Rates in Antibody-Mediated Autoimmune Hematological Diseases Refractory to Conventional Immunosuppression." Blood 126, no. 23 (December 3, 2015): 3457. http://dx.doi.org/10.1182/blood.v126.23.3457.3457.
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Abstract Introduction Certain patients with antibody-mediated autoimmune disease exhibit poor responses to conventional immunosuppression. Proteasome inhibitors (PI) have been prospectively evaluated in humoral graft rejection, where rapid alloantibody depletion occurs in the context of combined immunosuppression. In addition to killing antibody-producing cells, PIs deplete autoreactive T-cells, suppress inflammatory NFkB signaling and modulate MHC class I antigen presentation. The durability of rituximab-based B-cell depletion may be abrogated by promotion of long-lived autoreactive (CD20 neg) plasma cell development. Therefore there is a strong rational for utilising PIs in antibody mediated autoimmune disease. Based on our initial experience re-purposing bortezomib (BTZ) to deplete ADAMTS-13 antibodies in thrombotic thrombocytopenic purpura (TTP) (Shortt et al NEJM 2013; 69: 90-2.), we have utilized off-label BTZ in cases of severe refractory antibody-mediated hematological disease via a compassionate access program. We report the first case-series of BTZ use in this broader patient group. Patients & Methods Outcome data were collected for all patients treated between 2012 and 2015 with off-label BTZ salvage for autoantibody-mediated hematological disease across a combined health-care network in the Australian state of Victoria (catchment area >1.5 million patients; 3 major academic centers). Eligible patients were refractory to standard of care and at risk of severe morbidity/mortality from underlying disease or immunosuppression. All patients demonstrated on-going autoantibody production despite rituximab-based B-cell depletion. We sought to correlate biological responses (e.g. reductions in autoantibody titer) with clinical efficacy (e.g. remission rates and capacity to wean concurrent immunosuppression) and to document BTZ toxicities. Compassionate BTZ access was subject to institutional drug and therapeutic committee review and Australian Therapeutic Goods Administration notification. Routine antiviral prophylaxis was administered. Results: Treatment episodes (n=8) included TTP (n=3), warm autoimmune hemolytic anemia (AIHA) (n=3), cold AIHA (n=1) and an acquired FVIII inhibitor (n=1) in 7 patients (median age 53 years, range 34-80; M/F 4:3). Patients had received a median of 3.5 prior lines of therapy (range: 2-6) and all were rituximab exposed. BTZ was predominantly administered at 1.3mg/m2; D1, 4, 8, 11 q21d, with subsequent dose modification to weekly where repeated cycles were required. Initial IV therapy was preferred in TTP patients, to maximize exposure between exchanges; where recurrent cycles were administered all patients transitioned to subcutaneous administration to avoid neurotoxicity. With a median follow-up of 10 months (censored June 2015; range: 4-35 months), the overall response rate was 75%, including 50% durable complete remissions and 25% partial remission with reductions in transfusion requirements and baseline immunosuppression. Biological responses correlated with reductions in autoantibody titer. Most patients demonstrated an immediate response within 1 cycle (median cycles delivered 1.5; range: 1-6). BTZ was generally well tolerated with grade 4 hepatotoxicity observed in one patient in the context of high alcohol intake. No infective complications or neuropathy were observed. Table 1.CaseAgeM/FDiseasePrior Lines of TreatmentCyclesResponseDuration of response170MAIHA (warm)31NR02a53FTTP41CR7m2b54FTTP21CR25m364FAIHA (cold)56PR5m449FTTP21CR32m534MHemophilia A34CR4m647MAIHA (warm)52PR2m780MAIHA (warm)62NR0NR - no response; CR - complete response; PR - partial response (reduction in immunosuppression or disease activity) Conclusions: BTZ rapidly downregulates autoantibodies, correlating with a high response rate in relapsed/refractory autoimmune hematological disease with an acceptable/expected toxicity profile. This case series, taken together with emerging case reports of efficacy provide 'proof of concept' for the utility of PI in antibody mediated autoimmune disease. A prospective clinical trial protocol is in development. Disclosures Off Label Use: The drug to be discussed is bortezomib and its use in managing refractory autoimmune haematological disorders (off label use). Catalano:Celgene, Roche, Gilead: Honoraria. Opat:Janssen Pharmaceuticals: Other: Provision of subsidized medications only. Shortt:Janssen Pharmaceuticals: Other: Provision of subsidized medications only.
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Sun, Li, Naqvi, Naqvi, Chu, Xu, Song, Li, and Yan. "Succinate Coenzyme A Ligase Beta-Like Protein from Trichinella spiralis Suppresses the Immune Functions of Rat PBMCs in Vitro and Inhibits the Secretions of Interleukin-17 in Vivo." Vaccines 7, no. 4 (November 2, 2019): 167. http://dx.doi.org/10.3390/vaccines7040167.
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: Succinate Coenzyme A ligase beta-like protein (SUCLA-β) is a subunit of Succinyl-coenzyme A synthetase, which is involved in substrate synergism, unusual kinetic reaction in which the presence of SUCLA-β for one partial reaction stimulates another partial reaction. Trichinella spiralis is a parasitic nematode, which may hinder the development of autoimmune diseases. Immunomodulatory effects of SUCLA-β from Trichinella spiralis in the parasite-host interaction are unidentified. In this study the gene encoding T. spiralis SUCLA-β was cloned and expressed. Binding activities of recombinant T. spiralis SUCLA-β (rTs-SUCLA-β) to rat peripheral blood mononuclear cells (PBMCs) were checked by immunofluorescence assay (IFA) and the immuno-regulatory effects of rTs-SUCLA-β on cell migration, cell proliferation, nitric oxide (NO) production and apoptosis were observed by co-incubation of rTs-SUCLA-β with rat PBMCs in vitro, while cytokine secretions in rTs-SUCLA-β treated rats were evaluated in vivo. Furthermore, phagocytosis of monocytes was detected by flow cytometry and effects of rTs-SUCLA-β-induced protective immunity on T. spiralis adult worms and muscle larva were evaluated in rats. The IFA results revealed that rTs-SUCLA-β could bind to rat PBMCs. Treatment of PBMCs with rTs-SUCLA-β significantly decreased the monocyte phagocytosis, cell migration and cell proliferation, while NO production and apoptosis of PBMCs were unaffected. Results of the in vivo study showed that the IL-17 secretion decreased significantly after rTs-SUCLA-β administration in rats, while no significant effects were observed on the secretions of IFN-γ, IL-9, TGF-β and IL-4. Moreover, significant reduction of T. spiralis muscle larvae burden and significant increase in anti-rTs-SUCLA-β immunoglobulin level of IgG, IgG1 and IgG2a was observed in rTs-SUCLA-β-administered rats. The results indicated that rTs-SUCLA-β may be a potential target for controlling T. spiralis infection by suppressing the immune functions of the rat PBMCs and by reducing the parasite burden. Additionally it may also contribute to the treatment of autoimmune diseases and graft rejection by suppressing IL-17 immune response in the host.
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Bar, Haim, and Seojin Bang. "A mixture model to detect edges in sparse co-expression graphs with an application for comparing breast cancer subtypes." PLOS ONE 16, no. 2 (February 11, 2021): e0246945. http://dx.doi.org/10.1371/journal.pone.0246945.
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We develop a method to recover a gene network’s structure from co-expression data, measured in terms of normalized Pearson’s correlation coefficients between gene pairs. We treat these co-expression measurements as weights in the complete graph in which nodes correspond to genes. To decide which edges exist in the gene network, we fit a three-component mixture model such that the observed weights of ‘null edges’ follow a normal distribution with mean 0, and the non-null edges follow a mixture of two lognormal distributions, one for positively- and one for negatively-correlated pairs. We show that this so-called L2 N mixture model outperforms other methods in terms of power to detect edges, and it allows to control the false discovery rate. Importantly, our method makes no assumptions about the true network structure. We demonstrate our method, which is implemented in an R package called edgefinder, using a large dataset consisting of expression values of 12,750 genes obtained from 1,616 women. We infer the gene network structure by cancer subtype, and find insightful subtype characteristics. For example, we find thirteen pathways which are enriched in each of the cancer groups but not in the Normal group, with two of the pathways associated with autoimmune diseases and two other with graft rejection. We also find specific characteristics of different breast cancer subtypes. For example, the Luminal A network includes a single, highly connected cluster of genes, which is enriched in the human diseases category, and in the Her2 subtype network we find a distinct, and highly interconnected cluster which is uniquely enriched in drug metabolism pathways.
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Massa, Margherita, Stefania Croce, Rita Campanelli, Carlotta Abbà, Elisa Lenta, Chiara Valsecchi, and Maria Antonietta Avanzini. "Clinical Applications of Mesenchymal Stem/Stromal Cell Derived Extracellular Vesicles: Therapeutic Potential of an Acellular Product." Diagnostics 10, no. 12 (November 24, 2020): 999. http://dx.doi.org/10.3390/diagnostics10120999.
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In the last decade, the secreting activity of mesenchymal stem/stromal cells (MSCs) has been widely investigated, due to its possible therapeutic role. In fact, MSCs release extracellular vesicles (EVs) containing relevant biomolecules such as mRNAs, microRNAs, bioactive lipids, and signaling receptors, able to restore physiological conditions where regenerative or anti-inflammatory actions are needed. An actual advantage would come from the therapeutic use of EVs with respect to MSCs, avoiding the possible immune rejection, the lung entrapment, improving the safety, and allowing the crossing of biological barriers. A number of concerns still have to be solved regarding the mechanisms determining the beneficial effect of MSC-EVs, the possible alteration of their properties as a consequence of the isolation/purification methods, and/or the best approach for a large-scale production for clinical use. Most of the preclinical studies have been successful, reporting for MSC-EVs a protecting role in acute kidney injury following ischemia reperfusion, a potent anti-inflammatory and anti-fibrotic effects by reducing disease associated inflammation and fibrosis in lung and liver, and the modulation of both innate and adaptive immune responses in graft versus host disease (GVHD) as well as autoimmune diseases. However, the translation of MSC-EVs to the clinical stage is still at the initial phase. Herein, we discuss the therapeutic potential of an acellular product such as MSC derived EVs (MSC-EVs) in acute and chronic pathologies.
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Palomar, Amaya Pérez del, Alberto Montolío, José Cegoñino, Sandeep Kumar Dhanda, Chit Tong Lio, and Tanima Bose. "The Innate Immune Cell Profile of the Cornea Predicts the Onset of Ocular Surface Inflammatory Disorders." Journal of Clinical Medicine 8, no. 12 (December 2, 2019): 2110. http://dx.doi.org/10.3390/jcm8122110.
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Ocular surface inflammatory disorder (OSID) is a spectrum of disorders that have features of several etiologies whilst displaying similar phenotypic signs of ocular inflammation. They are complicated disorders with underlying mechanisms related to several autoimmune disorders, such as rheumatoid arthritis (RA), Sjögren’s syndrome, and systemic lupus erythematosus (SLE). Current literature shows the involvement of both innate and adaptive arms of the immune system in ocular surface inflammation. The ocular surface contains distinct components of the immune system in the conjunctiva and the cornea. The normal conjunctiva epithelium and sub-epithelial stroma contains resident immune cells, such as T cells, B cells (adaptive), dendritic cells, and macrophages (innate). The relative sterile environment of the cornea is achieved by the tolerogenic properties of dendritic cells in the conjunctiva, the presence of regulatory lymphocytes, and the existence of soluble immunosuppressive factors, such as the transforming growth factor (TGF)-β and macrophage migration inhibitory factors. With the presence of both innate and adaptive immune system components, it is intriguing to investigate the most important leukocyte population in the ocular surface, which is involved in immune surveillance. Our meta-analysis investigates into this with a focus on both infectious (contact lens wear, corneal graft rejection, Cytomegalovirus, keratitis, scleritis, ocular surgery) and non-infectious (dry eye disease, glaucoma, graft-vs-host disease, Sjögren’s syndrome) situations. We have found the predominance of dendritic cells in ocular surface diseases, along with the Th-related cytokines. Our goal is to improve the knowledge of immune cells in OSID and to open new dimensions in the field. The purpose of this study is not to limit ourselves in the ocular system, but to investigate the importance of dendritic cells in the disorders of other mucosal organs (e.g., lungs, gut, uterus). Holistically, we want to investigate if this is a common trend in the initiation of any disease related to the mucosal organs and find a unified therapeutic approach. In addition, we want to show the power of computational approaches to foster a collaboration between computational and biological science.
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Peritt, David, Kim Campbell, Amy Krutsick, Janine Huber, Ulrich Thienel, Agatha Schwarz, Akira Maeda, and Thomas Schwarz. "Generation of Regulatory T Cells through Intravenous Delivery of Autologous Apoptotic Cells." Blood 104, no. 11 (November 16, 2004): 2390. http://dx.doi.org/10.1182/blood.v104.11.2390.2390.
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Abstract Extracorporeal photopheresis (ECP) is approved for the palliative treatment of skin manifestations associated with cutaneous T cell lymphoma. As reported in the literature, ECP has shown promise as a treatment for such immune-mediated inflammatory disorders as graft versus host disease, transplantation rejection, and autoimmune diseases. ECP involves the reinfusion of autologous, apoptotic peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen (8-MOP) and UVA light. The biological mechanism of action of ECP, however, remains unresolved. We have evidence to suggest that delivery of ECP-treated apoptotic cells modulates immune responses, possibly through generation of regulatory T cells. When co-incubated with ECP-treated cells, activated dendritic cells produce reduced levels of proinflammatory cytokines, such as IL-12, while TGFβ levels were modestly increased. Activation of CD4+ T cells in the presence of allogeneic dendritic cells and ECP-treated cells promotes generation of a population of T cells that can suppress proliferation of, and IFNγ production by, naïve syngeneic T cells. To confirm these findings in vivo, we employed a murine contact hypersensitivity model. ECP-treated or control spleen and lymph node cells from mice sensitized with the hapten dinitrofluorobenzene (DNFB) were injected intravenously into naïve recipients. Compared to controls, mice that received ECP-treated cells demonstrated significantly less ear swelling following sensitization and challenge with DNFB. Suppression of ear swelling was specific for DNFB and cell-mediated, as demonstrated by the ability to transfer DNFB tolerance to naïve mice, which could appropriately respond to the unrelated hapten oxazalone. Transfer of this tolerance was abrogated by depletion of either CD4+ or CD25+ T cell populations. Collectively, these results suggest that delivery of ECP-treated cells promotes the generation of regulatory T cells that are capable of modulating immune responses. Therakos sponsored Phase II trials for the prevention and treatment of GvHD are concluding and an international blinded pivotal phase III study is planned for 2005.
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Knobler, Robert M., Ulrike Just, Gabriele Klosner, Florian Klinglmueller, Martin Bilban, Zoya Kuzmina, Hildegard T. Greinix, and Franz Trautinger. "Analysis of the Effect On the Expression of Global Gene Expression Profiles in Lymphocyte Subpopulations Treated by Extracorporeal Photopheresis." Blood 120, no. 21 (November 16, 2012): 4841. http://dx.doi.org/10.1182/blood.v120.21.4841.4841.
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Abstract Abstract 4841 Introduction After the initial introduction of extracorporeal photopheresis (ECP) for the therapy of Sezary syndrome (CTCL) it has been found to have significant clinical effect on other T-cell mediated diseases including graft-versus-host disease (GvHD), organ transplant rejection, systemic sclerosis and other autoimmune disorders. To obtain information about modifications in gene expression patterns before and after ECP and to define gene sets with important changes in expression we analysed gene expression profiles in major lymphocyte subsets of patients before and after treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) irradiation. Patients, Materials, and Methods For this initial study 6 female patients suffering from chronic GvHD under treatment with ECP were included. Affymetrix® Human Genome U133 Plus 2.0 Arrays were used to compare global gene expression profiles in CD4+ and CD8+ lymphocytes before and after ECP. Lymphocyte subsets were isolated before and immediately after exposure to 8-MOP/UVA during a standard ECP treatment cycle. Total RNA was isolated from each cell sample and processed and analyzed according to standard procedures. Results Preliminary data suggest a significant effect in CD4+ and CD8+ lymphocytes in patients with chronic GvHD on gene transcription after 8-MOP/UVA exposure. In CD4+ cells 20 times more gene transcription can be detected when compared to CD8+ cells. These findings underline the possible key role of CD4+ cells in the mechanisms of action of ECP. Recent research in animal models found evidence for a role of CD4+ regulatory T cells in the immunomodulatory activity of ECP. Conclusion The scientific approach of this study has the advantage of exploring global gene regulatory effects of ECP without any experimental bias based on earlier evidence or on a mechanistic hypothesis. Its results offer the advantage of finding new and so far unexplored effects of ECP on the treated cells; information which should be able to generate significant data to help define key targets for subsequent research into the mechanism of action of ECP. Disclosures: No relevant conflicts of interest to declare.
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Djordjevic, Vidosava, Tatjana Cvetkovic, Ivana Stojanovic, Vladan Cosic, Slavica Kundalic, Lilika Zvezdanovic, and Tatjana Jevtovic-Stoimenov. "Inter-dependence between cytokines and NO/NOS system in resting and activated endothelial cells." Jugoslovenska medicinska biohemija 23, no. 3 (2004): 241–47. http://dx.doi.org/10.2298/jmh0403241d.
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Cytokines are a heterogeneous and multifunctional group of molecules synthetized in various human cells. Structurally they are peptides (often glycosylated) used by cells for intercellular communication and control the inner environment of the cells in which they operate. Cytokines are produced by the cells involved in the immune response, inflammation, hemopoiesis, healing and systemic response to injury. Immunity, inflammatory reactions and haemostasis involve close interactions between immunocompetent cells and vascular endothelium. Vascular cells are both a target for cytokines and their source. The spectrum of endothelial cell responses challenged by cytokines is wide and varied, with different cytokines activating distinct, but overlapping, sets of functions. Under normal resting conditions endothelial cells constitutively express certain protective genes with the purpose to maintain the endothelial cells in their quiescent phenotype by inhibiting NF-kB activation and exerting antiapoptotic functions. In this status endothelial cells can exhibit their barrier and anticoagulant functions even in the presence of low levels of stimulants. When the endothelial cells are exposed to numerous stimuli such as TNF, IL-1, endotoxin or xenoreactive antibodies and complement, which are usually associated with infections, graft rejection or autoimmune diseases such as, vasculitis, NF-kB induces the expression of adhesion molecules such as E-selectin, chemokines such as IL-8 and procoagulant molecules such as TF. Besides the induction of expression of a functional programme related to thrombosis and inflammation, IL-1 and TNF also induce production of autocoids including nitric oxide (NO). Both the inducible form of NO synthase (iNOS) type II and the constitutive (type III) isoform of NOS are present in endothelial cells catalyzing the conversion of arginine into citruline and NO. The formation of NO is an ubiquitous biochemical pathway involved in the regulation of neurotransmission, vasodilatation, immunity and cytotoxicity. During inflammatory reaction NO produced by endothelial cells exerts its autocrine function through the inhibition of cytokine-induced expression of adhesion molecules and cytokine production by endothelial cells. Also, it has a protective role in inflammation through the inactivation of NADPH oxidase and the consequent impairment of superoxide production for cell mediated injury. On the other hand, there is considerable evidence that NO contributes to tissue destruction in inflammatory and immune diseases being a key component of the cytostatic/cytotoxic function of the immune system. The damage to target cells by NO released from activated macrophages or endothelial cells may involve both necrotic and apoptotic pathways of cell death.
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Fan, Huahua, Xiaona Huo, Juan Sun, Yiming Yang, and Xiao Li. "Efficient Induction and Expansion Of CD8+CD28+Foxp3+ Regulatory T Cells By TGF-beta1 and Rapamycin." Blood 122, no. 21 (November 15, 2013): 190. http://dx.doi.org/10.1182/blood.v122.21.190.190.
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Abstract Background Although extensive studies have focused on the CD4+CD25+Foxp3+Tregs in recent years, CD8+Tregs have also been reported to play important roles in maintenance of immune tolerance. Adoptive transfer of CD8+Tregs in rodents can prevent or treat autoimmune diseases, allograft rejection or graft-versus-host disease. Objective Several approaches for induction of Ag-specific CD8+Tregs have been reported, but there is currently no reliable protocol for the ex vivo induction and large-scale expansion of human polyclonal CD8+CD28+Foxp3+Tregs. Our research was designed to investigate an effective method to induce and expand polyclonal CD8+CD28+Foxp3+Tregs in vitro on a large scale to meet the clinical demand. Methods CD8+ T lymphocytes were isolated from adult peripheral blood with immunomagnetic beads and cultured for a week using anti-CD3/CD28 antibody-coated beads and IL-2 in combination with TGF-beta1 and rapamycin. Expanded CD8+Tregs were re-stimulated with fresh anti-CD3/CD28 antibody-coated beads and cytokines for other cycles. The phenotype of CD8+Tregs was analyzed by flow cytometry. In the suppression assay, CFSE progressive dilution was used as readout of responder cells proliferation. For in vivo experiments, the mouse models of collagen-induced arthritis (CIA) were established. 2x10E6 ex vivo induced and expanded human CD8+CD28+Foxp3+Tregs were transferred into CIA recipient mouse with the onset of RA. Clinical and histopathologic scores, cytokine and the secretion of anti-type-two collagen antidody in serum were analyzed. Results A large number of CD8+CD28+Foxp3+Tregs could be efficiently induced and expanded from CD8+ T lymphocytes in polyclonal stimulation culture system added TGF-beta1 and rapamycin. By repeated stimulation of CD8+ T cells for 4 weeks, we could generate larger than 1x10E10 CD8+CD28+Foxp3+Tregs without loss of their suppressive function from every 1x10E6 CD8+ T cells, which can typically be isolated from 5-10ml of peripheral blood. The expanded CD8+Tregs expressed high level of Foxp3, CD28, CD25, PD-1, CD103, CD62L, CCR7, CTLA4, CD39 and CD73, secreted small amount of IL-2, IFN-gamma, IL-10 and TGF-beta, did not secret IL-17A, displayed a partially anergic phenotype, and could inhibit the proliferation of autologous or allogeneic CD4 effective T lymphocytes activated by anti-CD3/CD28 antibody. We found that the suppression of CD8+CD28+Foxp3+Tregs in proliferation of effective T lymphocytes was mainly dependent on cell contact but not dependent on their cytotoxicity. CD8+CD28+Foxp3+Tregs did not secret IL17A or secret small amount of IFN-gamma in the presence of inflammatory cytokines such as IL-1beta and IL-6 or IL-21 and IL-23. Furthermore, CD8+CD28+Foxp3+Tregs are a kind of induced regulatory T cells, because their Treg-cell-specific demethylated region (TSDR) has a methylation status. CD8+CD28+Foxp3+Tregs treatment could significantly alleviate the severity of arthritis, and reduce the cartilage destruction of the joints of CIA mice and the level of total anti-type-two collagen IgG antibody in serum as well. Conclusion we have developed a simple method using TGF-beta1 and rapamycin to induce and expand highly efficient human polyclonal CD8+CD28+Foxp3+Tregs with suppressive capacities from CD8+ T cells on a large scale. This procedure of CD8+Tregs expansion we used fits with the currently available clinical grade isolation tools with the objective of facilitating easy translation into clinical practice. This approach may facilitate the clinical application of CD8+Treg-based immunotherapy in transplantation and autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.
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Sun, Zhengda, Haval Shirwan, Narendra Singh, Nadir Askenasy, and Esma S. Yolcu. "A Novel Approach to Prevent GvHD: Donor Cells Engineered to Display on Their Surface a Recombinant Form of FasL Protein Effectively Prevent Lethal GvHD in a Mouse Model." Blood 112, no. 11 (November 16, 2008): 3519. http://dx.doi.org/10.1182/blood.v112.11.3519.3519.
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Abstract Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders and malignancies. BMT can also be used as a cell-based immunomodulatory approach to induce tolerance to foreign and auto-antigens for the prevention and/or treatment of foreign graft rejection and autoimmune disorders. The routine application of allogeneic BMT as a therapeutic intervention in the clinic, however, is complicated by graft-versus-host (GVH) reaction, which is the major cause of graft-versus-host disease (GVHD) with potential life-threatening complications. T cells specific for alloantigens are the primary culprit of GVHD. Although elimination of T cells from the donor bone marrow inoculum can curtail GVH reaction, it results in compromised engraftment. Thus, strategies targeting specific and effective elimination of only the pathogenic T cells may have important implications for routine application of BMT to the clinic for the treatment of a variety of diseases. The main objective of this study was to use a novel and practical approach, designated as ProtEx™, to engineer bone marrow cells to display on their surface a modified form of FasL protein with potent apoptotic activity as an immunomodulatory agent and test the capacity of the engineered cells to engraft in allogeneic recipients without complications of GVHD. ProtEx™ technology involves generation of chimeric molecules with a core streptavidin (SA), modification of cell membrane with biotin, and the display of chimeric molecules on the cell surface taking advantage of strong noncovalent interaction (10−15 M) between biotin and SA. This technology allows for rapid (~ 2 hr) and efficient (100% of the targeted cells) display of exogenous proteins of interest on any cell without compromising the function of the cell or the proteins. In this study, ProtEx™ was used to display SA-FasL protein on C57BL/6 T cells and BMCs and these cells were tested for elimination of alloreactive T cells as well as prevention of acute GVHD following transplantation into lethally (1000 cGy) irradiated F1 (C57BL/6xBALB/c) mice. We hypothesized that mature T cells in the BM inoculum displaying FasL will respond to the host alloantigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. In support of this hypothesis, we demonstrated specific elimination of C57BL/6 T cells engineered to display SA-FasL on their surface in response to BALB/c antigen presenting cells in mixed lymphocyte cultures. Transplantation of unmodified 10×106 BMCs comixed with 20×106 splenocytes of C57BL/6 mice into lethally irradiated F1 recipients (n=12) resulted in lethal GVHD in all recipients within 34 days. In marked contrast, transplantation of cells engineered to display SA-FasL on splenocytes only (n=9) or splenocytes and BMCs (n=9) effectively prevented acute GVHD in all recipients that survived over 80 days of an observation period. The importance of FasL-mediated apoptosis in immune homeostasis and tolerance combined with our ability to use a practical approach, ProtEx™, to display SA-FasL with potent apoptotic activity on the surface of BMC or mature T cells at the protein level to prevent acute GVHD is significant and may have immediate clinical application.
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Hoebe, Kasper, Edith Janssen, and Bruce Beutler. "Identification of a Novel Toll-Like Receptor-Independent Immunoadjuvant Pathway That Depends upon Programmed Cell Death." Blood 104, no. 11 (November 16, 2004): 775. http://dx.doi.org/10.1182/blood.v104.11.775.775.
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Abstract Molecules of microbial origin, and synthetic derivatives of these molecules, have long been used for their immuno-adjuvant effect, and as the key sensors of microbial infection, Toll-like receptors (TLRs) are thought to be essential for adjuvanticity. To the contrary, we now demonstrate the existence of a robust, TLR-independent pathway for adjuvant effect: one that is actually far stronger than the TLR-dependent pathway. Activation of Toll-like receptors (TLRs) and the subsequent production of cytokines such as type I interferon leads to the maturation of dendritic cells (DCs) with upregulation of MHC molecules and costimulatory molecules such as CD40, CD80 and CD86, allowing for optimal interaction between DCs and T-cells. We have determined that TLR signal transduction is minimally dependent upon two adapter proteins, MyD88 and TRIF. In compound homozygous mutant (DKO) mice that lack functional MyD88 and TRIF, there is complete abrogation of all TLR signaling. Such animals therefore comprise a unique model with which to study TLR-independent immune responses. We have now used DKO mice to determine whether an adaptive immune response can be obtained in the absence of TLR signaling. As expected, adjuvanticity obtained via “classical” microbial adjuvants such as complete Freund’s adjuvant or LPS was completely absent in DKO mice. However, subcutaneous administration of syngeneic murine cells expressing ovalbumin and rendered apoptotic by exposure to ultraviolet light resulted in a strong T-cell response in vivo, with impressive production of interferon-g by CD8+ cells and efficient killing of EL-4 cells that expressed CD8-specific OVA peptides, both in wildtype and DKO mice. Adjuvanticity was observed only in the context of apoptosis, in that living cells, not exposed to ultraviolet light before injection, induced little or no response. Moreover, the mixture of the protein antigen with apoptotic cells was insufficient to induce an adaptive immune response; rather, only cells that expressed the protein prior to induction of apoptosis were stimulatory. These results indicate the existence of a specific, cell death-dependent mechanism for adjuvanticity that is TLR-independent and induced by endogenous molecules. We propose that this new adjuvant pathway is of fundamental importance to immune responses at large. We believe that it is required for initiation of the adaptive immune response witnessed in the context of allograft rejection, graft-versus-host disease, and autoimmune diseases as well.
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Awada, Hassan, Reda Z. Mahfouz, Jibran Durrani, Ashwin Kishtagari, Deepa Jagadeesh, Alan Lichtin, MD, Brian T. Hill, et al. "Long-Term Experience with Large Granular Lymphocytic Leukemia Evolving after Solid Organ and Hematopoietic Stem Cell Transplantation." Blood 134, Supplement_1 (November 13, 2019): 1226. http://dx.doi.org/10.1182/blood-2019-130167.
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T-cell large granular lymphocyte leukemia (T-LGLL) is a clonal proliferation of cytotoxic T lymphocytes (CTL). T-LGLL mainly manifest in elderly and is associated with autoimmune diseases including rheumatoid arthritis (RA), B cell dyscrasias, non-hematologic cancers and immunodeficiency (e.g., hypogammaglobulinemia). LGL manifestations often resemble reactive immune processes leading to the dilemmas that LGLs act like CTL expansion during viral infections (for example EBV associated infectious mononucleosis). While studying a cohort of 246 adult patients with T-LGLL seen at Cleveland Clinic over the past 10 years, we encountered 15 cases of overt T-LGLL following transplantation of solid organs (SOT; n=8) and hematopoietic stem cell transplantation (HSCT; n=7). Although early studies reported on the occurrence of LGL post-transplant, these studies focused on the analysis of oligoclonality skewed reactive CTL responses rather than frank T-LGLL. We aimed to characterize post-transplantation T-LGLL in SOT and HSCT simultaneously and compare them to a control group of 231 de novo T-LGLL (cases with no history of SOT or HSCT). To characterize an unambiguous "WHO-defined T-LGLL" we applied stringent and uniform criteria. All cases were diagnosed if 3 out of 4 criteria were fulfilled, including: 1) LGL count >500/µL in blood for more than 6 months; 2) abnormal CTLs expressing CD3, CD8 and CD57 by flow cytometry; 3) preferential usage of a TCR Vβ family by flow cytometry; 4) TCR gene rearrangement by PCR. In addition, targeted deep sequencing for STAT3 mutations was performed and charts of bone marrow biopsies were reviewed to exclude other possible conditions. Diagnosis was made 0.2-27 yrs post-transplantation (median: 4 yrs). At the time of T-LGLL diagnosis, relative lymphocytosis (15-91%), T lymphocytosis (49-99%) and elevated absolute LGL counts (>500 /µL; 93%) were also seen. Post-transplantation T-LGLL were significantly younger than de novo T-LGLL, (median age: 48 vs. 61 yr; P<.0001). Sixty% of post-transplantation T-LGLL patients were males. Fifteen% of patients had more cytogenetic abnormalities compared to de novo T-LGLL, had a lower absolute LGL count (median: 4.5 vs. 8.5 k/µL) and had less frequent neutropenia, thrombocytopenia and anemia (27 vs. 43%, 33 vs. 35% and 20% vs. 55%; P=.01). TCR Vb analysis identified clonal expansion of ≥1 of the Vb proteins in 60% (n=9) of the patients; the remaining 40% (n=6) of the cases had either a clonal process involving a Vb protein not tested in the panel (20%; n=3) or no clear expansion (20%; n=3). Signs of rejection were observed in 20% (n=3/15) and GvHD in 13% (n=2/15) of the patients. Post-transplantation, 27% of cases presented with neutropenia (absolute neutrophil count <1.5 x109/L; n=4), 33% with thrombocytopenia (platelet count <150 x109/L; n=5) and 25% with anemia (hemoglobin <10 g/dL; n=3). T-LGLL evolved in 10 patients (67%; 10/15) despite IST including cyclosporine (n=5), tacrolimus (n=4), mycophenolate mofetil (n=5), cyclophosphamide (n=1), anti-thymocyte globulin (n=1), and corticosteroids (n=6). Lymphadenopathy and splenomegaly were seen in 13% (n=2) and 33% (n=5) of the patients. Other conditions observed were MGUS (20%; n=3) and RA (7%; n=1). Conventional cytogenetic showed normal karyotype in 89% (n=11, tested individuals 13/15). Somatic STAT3 mutations were identified in 2 patients. Sixty% of cases (n=9) were seropositive for EBV when tested at different time points after transplant. Similarly, 53% (n=8) were seropositive for CMV, of which, 5 were positive post-transplantation and 3 pre-/post-transplantation. The complexity of T-LGLL expansion post-transplantation might be due to several mechanisms including active viral infections, latent oncogenic viral reactivation and graft allo-antigenic stimulation. However, in our cohort graft rejection or GvHD was encountered in a few patients (2 allo-HSCT recipients). Autoimmune conditions were present in 50% of SOT recipients (n=4/ 8, including RA, ulcerative colitis, systemic lupus erythematosus). Some of our patients also had low immunoglobulin levels. Overt EBV (post-transplant lymphoproliferative disorder) and CMV reactivation was diagnosed in only 27% (4/15) of the patients. In sum we report the long term follow up of a cohort of T-LGLL and emphasize the expansion of T-LGLL post-transplant highlighting the difficulty in assigning one unique origin of LGLL. Disclosures Hill: Genentech: Consultancy, Research Funding; Takeda: Research Funding; Celegene: Consultancy, Honoraria, Research Funding; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Consultancy, Honoraria; Amgen: Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TG therapeutics: Research Funding; AstraZeneca: Consultancy, Honoraria. Majhail:Atara Bio: Consultancy; Mallinckrodt: Honoraria; Nkarta: Consultancy; Anthem, Inc.: Consultancy; Incyte: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
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Afzal, Amber, Maryna Tarbunova, George Despotis, and Brenda J. Grossman. "Comparison of Outcomes between the Cellex and Uvar-Xts Closed-System Extracorporeal Photopheresis (ECP) Devices When Used for Graft-Versus-Host Disease; A Single Center Experience." Blood 132, Supplement 1 (November 29, 2018): 4686. http://dx.doi.org/10.1182/blood-2018-99-115122.
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Abstract BACKGROUND: Graft versus host disease (GVHD) is a significant contributor to non-relapse mortality after allogeneic stem cell transplant (SCT). Steroids are first line therapy and extracorporeal photopheresis (ECP) is used as second line therapy for steroid refractory or intolerant patients. ECP has immunomodulatory effects rather than immunosuppressive effect which decreases the risk of infection, and may decreases the risk of disease relapse. There are two ECP instruments approved in the US for the treatment of Cutaneous T Cell Lymphoma (CTLC), however both have been used "off-label" to prevent or treat solid organ transplant rejection, acute and chronic GVHD, and other autoimmune diseases. Until recently the UVAR-XTS instrument has been the most commonly used closed system in the US until the CELLEX was approved in 2009. The CELLEX and the UVAR-XTS procedures are similar in that buffy coat is collected, exposed to methoxsalen (UVADEX®) ex vivo and then UVA light before returning the treated cells back to the patient in a closed looped system. The instruments differ in the collection of the buffy coat; collection is continuous by the CELLEX, and it is intermittent by UVAR-XTS. In addition CELLEX has been shown to be more efficient in collecting mononuclear cells when compared to the UVAR -XTS. Little clinical data is available comparing the clinical efficacy of the two instruments in the setting of GVHD. We designed a single institution retrospective study to compare the efficacy and safety of the two instruments. The primary outcomes analyzed were >/=50% reduction of steroid dose between the initial and final dose during the study period, and the number and type of adverse events that occurred during the ECP procedures. METHODS: Study Population: We reviewed charts of allogeneic stem cell transplant patients who received ECP for the treatment of acute or chronic GVHD between 1/2009 and 12/2015. The patients were divided into two groups based on the type of ECP instrument that was used. One group was exclusively treated with UVAR-XTS while the second group with CELLEX. Patients who received treatments with both instruments were excluded. The frequency of steroid dose reduction by >/=50%, and toxicity was compared between the two instruments while adjusting for age, gender, GVHD severity, number of organs involved by GVHD, type of allogeneic stem cell transplant, conditioning regimen, number of other immunosuppressants, type of anticoagulants (ACDA vs heparin), type of access line, and baseline blood counts. Statistical Analysis: The baseline characteristics of the patients in the two groups were compared using Chi square, Fischer's exact test for categorical variables and one-way analysis of variance (ANOVA) for continuous variables. Chi square analysis was used to determine the difference in frequency of >/=50% steroid dose reduction and adverse events between the two groups. Logistic multivariate regression was used to evaluate the potential interactions of all significant covariates. A p value of less than 0.05 was considered to be statistically significant. Statistical analyses were performed using STATA14 software (StataCorp, College Station, TX). RESULTS: We identified 242 allogeneic SCT recipients who received ECP for acute or chronic GVHD in the study period, 146 of whom met the selection criteria. 69 patients had all procedure performed with UVAR-XTS and 77 patients had all procedures performed with CELLEX. There was no significant difference in age, gender, percent of acute GVHD, anticoagulant used, type of transplant, number of organs involved, number of immunosuppressants, vascular access and baseline blood counts between the two treatment cohorts as shown in table 1. Although year of transplant and conditioning regimen were different between the two cohorts, neither of these covariates influenced the impact of instrument on the primary outcome. In multivariate analysis, the patients who underwent ECP with CELLEX were 3 times more likely to have >/=50% steroid dose reduction (p =0.01). The total number of adverse events was similar between the instruments (p = 0.73). (Table 2) CONCLUSION: More than twice as many patients with GVHD treated with the CELLEX had a steroid dose reduction by >/=50% reflecting clinical improvement when compared to the patients treated with the UVAR-XTS. Similar safety profile was observed between the two instruments based on a similar number of adverse events. Disclosures No relevant conflicts of interest to declare.
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Logan, Aaron C., Agnieszka Czechowicz, Benjamin V. Kelley, Theingi M. Thway, Ivan Magana, Mark R. Krampf, Jessica Poyser, et al. "Anti-CD117 (c-Kit) Monoclonal Antibodies Deplete Human Hematopoietic Stem Cells and Facilitate Their Replacement in Humanized NOD/SCID/IL2Rγ−/− Mice: A Non-Toxic Conditioning Regimen for Allotransplantation." Blood 120, no. 21 (November 16, 2012): 4099. http://dx.doi.org/10.1182/blood.v120.21.4099.4099.
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Abstract Abstract 4099 Engraftment of allogeneic hematopoietic stem cells (HSC) requires conditioning to overcome immunologic and anatomic barriers preventing access to the marrow niche. Most patients who undergo allogeneic hematopoietic cell transplantation (allo-HCT) are prepared with cytotoxic chemotherapy and/or radiation to eliminate these barriers, and to facilitate eradication of malignant cells, if present. Many non-malignant conditions, such as primary immunodeficiencies, hemoglobinopathies, and autoimmune diseases may be successfully treated by transplantation of allogeneic HSC, but the toxicity of conventional conditioning regimens is, in many cases, prohibitive. Targeted elimination of barriers to the HSC niche would be a preferable approach. Signaling via the c-Kit receptor (CD117) is critical for the maintenance of pluripotent HSC. Anti-CD117 monoclonal antibodies (mAbs) deplete HSC and facilitate engraftment of donor HSC in a mouse model of severe combined immunodeficiency (SCID) (Czechowicz et al., Science, 2007). Patients with SCID are highly susceptible to infections, but also have limited immunologic barriers to alloengraftment, making this patient population ideal for studying targeted stem cell depletion to facilitate allo-HSC engraftment. We identified a clinical grade humanized anti-human CD117 mAb (anti-hCD117) as a potential candidate for this purpose. Anti-hCD117 significantly inhibited mitosis in human cord blood and bone marrow derived HSC (Lin−CD34+CD38−CD90+CD45RA−) in liquid and methylcellulose culture containing Flt3 ligand, stem cell factor (SCF), thrombopoietin (TPO), IL-3, and IL-6. To assess in vivo activity of anti-hCD117, we employed it alone, or in combination with alemtuzumab (anti-CD52), to deplete human stem and differentiated cells from hematopoietically humanized NOD/scid/IL2Rg−/− (HuNSG) mice. Pups were conditioned with 100cGy and then humanized by injection of 2000–4000 human HSC into the facial vein on day p2 or intrahepatically on day p4–5. After permitting hematopoietic stabilization for 4–6 months, we confirmed multi-lineage xenochimerism in the peripheral blood (PB) and bone marrow (BM) prior to mAb treatment. After a single treatment with anti-hCD117, mice were depleted of total human leukocytes a median 60% (35–100%; n=11) in the PB and 100% (84–100%; n=10) in the BM at 6 weeks after treatment, with >80% depletion of human myeloid cells in both compartments. Partial recovery of human chimerism was observed at 16 weeks, consistent with recovery of some LT-HSC after anti-hCD117 therapy. The addition of anti-CD52 facilitated clearance of human lymphoid cells not eradicated by anti-hCD117. Human HSC and progenitor cells (Lin−CD34+CD117+; HS/PC) in the bone marrow decreased from 0.4% (0–1.7%) to 0% (0–0.1%; n=10) 6 weeks after treatment with anti-hCD117. We then modeled a human transplant by treating HuNSG mice with anti-hCD117, anti-CD52, or both, to deplete their primary human graft. After monitoring mAb catabolism by ELISA, mice received a second (non-HLA matched) human CD34+ HS/PC graft modified to express the green fluorescent protein using a lentivector. After overnight prestimulation in XVIVO-15 supplemented with SCF, Flt3 ligand, TPO, and IL-3, human CD34+ HS/PC were exposed for 18 hours to lentivector at 1×108 TU/mL. Cells were washed and 80,000 transduced CD34+ HS/PC were injected IV into untreated and mAb-conditioned HuNSG mice. After 6 weeks, PB was evaluated and demonstrated GFP+hCD45+cells in 3/5 (60%) mice treated with anti-hCD117 + anti-CD52, 0/5 mice treated with either anti-hCD117 or anti-CD52 alone, and 1/5 untreated mice. Anti-hCD117 is a promising reagent for depletion of human HSC and facilitation of allo-HSC engraftment. Although anti-hCD117 alone capably depletes human CD34+CD117+ HS/PC and myeloid chimerism in HuNSG mice, the addition of anti-CD52 facilitates engraftment, possibly by reducing alloreactive rejection by T cells from the primary graft. Additional HuNSG mice are receiving second human transplants following mAb conditioning to further explore the utility of combining anti-hCD117 and anti-CD52 for this purpose. These studies will lead the way to minimally toxic allogeneic HSC transplant regimen, and in a broader view, to the application of targeted biological therapies that deplete endogenous stem cells and facilitate their replacement with allogeneic or gene-corrected stem cells. Disclosures: Thway: Amgen, Inc.: Employment. Magana:Amgen, Inc.: Employment. Weissman:Amgen, Inc.: Equity Ownership.
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Marinelli Busilacchi, Elena, Andrea Costantini, Nadia Viola, Benedetta Costantini, Antonella Poloni, Jacopo Olivieri, Francesco Saraceni, Giorgia Mancini, and Attilio Olivieri. "In Vitro Comparison of Different TKI Activity in T-Cell Populations: Selective Sparing of Treg By Nilotinib." Blood 128, no. 22 (December 2, 2016): 5774. http://dx.doi.org/10.1182/blood.v128.22.5774.5774.
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Abstract Introduction Chronic Graft Versus Host Disease (cGVHD) is a major complication of allogeneic stem-cell transplantation and is characterized by frequent multi-organ involvement that resembles the autoimmune diseases. Donor-derived CD4+ and CD8+ T lymphocytes have classically been considered to be the main effector cells mediating GVHD pathogenesis. Indeed, removal of T cells from transplant inocula almost completely prevents GVHD developing, at the price of increased incidences of graft rejection and disease recurrence. However recent studies suggest that B cells might also play an important role in the biology of cGVHD. The role of Treg lymphocytes in the pathogenesis of cGVHD is still controversial and the tyrosine kinase inhibitor′s (TKI) role in the modulation of this pathway is not yet fully characterized. In vitro data confirm that TKIs regulates both innate and adaptive immune response by interacting with many cell population such as T-cells, B-cells, dendritic cells, mast cells and macrophages. According to these observations, we investigated the TKI′s immunomodulatory effects (Nilotinib, Dasatinib, Imatinib, Ponatinib) on lymphocyte populations. Materials and Methods Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Biocoll. Cells were cultured in RPMI 1640 at a concentration 1x106 cell/well. Nilotinib, Imatinib, Dasatinib and Ponatinib were added to cell cultures at serial concentration (Imatinib:1μM,10μM,50μM; Nilotinib:0.5μM,2μM,10μM; Dasatinib:50nM,100nM,200nM; Ponatinib:1nM,10nM,50nM,100nM) on the first day. Six-color flow cytometry analysis (Facs Canto II) was performed on the cells harvested after 96 h cultures using conjugated antibodies (CD3,CD4,CD16,CD56,CD3,CD25,CD19,CD45RA,FoxP3,CD127,7-Aminoactinomycin-D), for cell cycle analysis cells were stained with propidium iodide. For cytokine analysis, supernatants were collected and analyzed for cytokines according to the instruction of Bio-Plex Pro Human Cytokine 17-plex Assay with Bio-Plex (Bio-Rad). Results A significant decrease of cytotoxic T cells viability was observed when cells were cultured in presence of Imatinib (50μM,p<0.01), Ponatinib (10nM,p<0.05) and Dasatinib (100nM,p<0.01). On the contrary, exposure to Nilotinib didn′t induce cell death. Increasing concentrations of all the tested TKI significantly inhibited T cell proliferation in a dose-dependent manner; the effect become statistically significant starting from Imatinib (1μM,p<0.05), Dasatinib (50nM,p<0.01), Ponatinib (50nM,p<0.01) and Nilotinib (0.5μM,p<0.01). Exposure to Imatinib, Dasatinib and Ponatinib induced a statistically significant decrease (p<0.01) of Treg cells proportion, even at the lowest drug concentration in culture; Nilotinib induced Treg decrease only at concentrations exceeding 2μM (p<0.01), higher than those usually achieved in clinical practice. A significant increase of naive Treg apoptosis was observed after exposure to Dasatinib (50nmM,p<0.01), Ponatinib (50nM,p<0.01) and Imatinib (50μM,p<0.01); exposure to Nilotinib has no effect on this population. Both Nilotinib and Dasatinib induced a profound inhibition of pro-inflammatory cytokine production (in particular TNFα, IFNγ, IL13 and IL17) when added to the cell cultures (p<0.05); slower decrease in supernatant cytokine concentration was observed in presence of either Imatinib (50μM,p<0.05) and Ponatinib (50nM,p<0.05). Increasing concentrations of all TKIs except Nilotinib induced a significant decline of NK cells (p<0.01) and B cell (p<0.01). Conclusion The present study focuses the peculiar Nilotinib activity on lymphocyte′s regulation: this TKI, at therapeutic concentrations in vitro, interact with innate and adaptive immune response show anti-inflammatory properties. Unlike other TKIs, Nilotinib determine inflammatory cytokines reduction, preserving T cell population and Treg. These data support the potential use of Nilotinib in cGVHD Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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He, Shan, Fang Xie, Qing Tong, Kazuhiro Mochizuki, Yongnian Liu, Philip E. Lapinski, Shin Mineishi, et al. "Histone Methyltransferase Ezh2 Controls T-Cell Immunity by Regulating Bioenergetic Metabolism." Blood 120, no. 21 (November 16, 2012): 953. http://dx.doi.org/10.1182/blood.v120.21.953.953.
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Abstract Abstract 953 Adoptive T cell therapy has the potential to enhance antitumor immunity and improve vaccine efficacy, of which a key challenge is to generate sufficient numbers of T cells that can persist in vivo after transfer. Cellular metabolism plays important roles in regulating T cell proliferation and survival. T cells responding to antigen activation dramatically upregulate both glycolysis and oxidative phosphorylation (OXPHOS), leading to increased production of adenosine triphosphate (ATP) and metabolic intermediates that are required for cell growth and proliferation. Without sufficient support for their demands, activated T cells may be deleted or become quiescent. Thus, better understanding of the mechanism that regulates cellular metabolism in T cell response will lead to new strategies to improve the efficacy of adoptive T cell therapy. Here we explore the functional impact of an epigenetic pathway in cellular metabolism in antigen-driven T cells and tumor immunity. Using genetic approaches and experimental mouse models, we demonstrate that Ezh2, which is a histone methyltransferase that represses the transcription of cohorts of developmental regulators, promotes the survival and expansion of antigen-driven T cells through regulating bioenergetic metabolism. Conditional deletion of Ezh2 caused selective apoptosis in T cells upon activation with alloantigens in vivo and in vitro or with T cell receptor (TCR)-ligation in vitro. Ezh2 deficiency resulted in markedly increased expression of proapoptotic gene Bim, but had no significant impact on the expression of other Bcl-2 family members (e.g., anti-apoptotic genes Bcl-2 and Bcl-xL,). Genetic inactivation of Bim only slightly improved the survival of alloantigen-activated Ezh2-deficient T cells, suggesting that Ezh2 may control T-cell immunity largely through a Bim-independent mechanism. This differs from our recent observations showing that Bim is required for increased apoptosis in activated T cells treated with a pharmacologic inhibitor of Ezh2 and histone methylation 3-Deazaneplanocin A (Blood, 2012). Our prior studies and others suggest that impaired cellular metabolism may lead to increased apoptosis of antigen-activated T cells. We observed that upon TCR-ligation Ezh2 null T cells were incapable to upregulate OXPHOS as compared to wild-type (WT) T cells, which was accompanied with reduced ATP levels and increased reactive oxygen species (ROS). Neutralization of ROS by N-acetylcysteine significantly improved the survival of TCR-activated Ezh2 null T cells. Interestingly, overexpression of WT Ezh2 in TCR-activated Ezh2 null T cells, but not enzymatically inactive H689A Ezh2 mutant or nuclear localization-inactive Ezh2 mutant, restored the ability of Ezh2-deficient T cells to upregulate OXPHOS, reduced ROS levels, and rescued their survival capability in vitro. These results suggest that Ezh2 is important for regulating bioenergetic metabolism in activated T cells. Furthermore, the nuclear but not cytoplasmic Ezh2 is required to regulate bioenergetic metabolism in activated T cells during clonal expansion phase, although Ezh2 in the cell cytoplasm could be involved in regulating actin polymerization. In mouse models of graft-versus-host disease (GVHD) and leukemia, transfer of donor T cells lacking Ezh2 failed to mediate GVHD and anti-leukemia activity in mice receiving allogeneic bone marrow transplantation. In addition, Ezh2 deficiency also ablated the ability of adoptively transferred antigen-specific CD8 T cells to control tumor growth in mice with established melanoma. Importantly, the absence of Ezh2 did not impair the development of effector T cells producing IFN-γ, granzyme B, Fas ligand and Trail, ruling out the possibility that impaired T-cell immunity of Ezh2 null T cells results from defective effector differentiation. Our findings identify the critical role of Ezh2 in regulating bioenergetic metabolism in antigen-driven T cells, therefore for the first time linking the epigenetic pathway to cellular metabolism in T cell response. Thus, Ezh2 and its-regulated bioenergetic metabolism may represent novel targets to improve the efficacy of adoptive T-cell immunotherapy. Modulation of Ezh2 and its activity may have broad implications in the treatment of many other inflammatory disorders, such as graft rejection after organ transplantation, GVHD and autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.
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Reich-Zeliger, Shlomit, Esther Bachar-Lustig, and Yair Reisner. "Effective Deletion of Anti-Donor Host Memory Effector Cells by Anti-3rd Party Veto CTLs: Implications to Tolerance Induction in Presensitized Bone Marrow Recipients." Blood 104, no. 11 (November 16, 2004): 44. http://dx.doi.org/10.1182/blood.v104.11.44.44.
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Abstract Recently we demonstrated that veto CTLs enhance engraftment of mismatched T cell depleted BM in recipient mice following reduced intensity conditioning. This desirable tolerance induction can be further enhanced by combining veto CTLs with CD4+CD25+ cells and Rapamycin. While these results are encouraging, they were largely based on models in which the resistant effector T cells mediating the allorejection are naive CTLp. However, considering that many patients undergoing BMT are presensitized by transfusions of different blood products, memory T cells could play an important role in graft rejection and, therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in BMT. Clearly, memory T cells respond under less stringent conditions to foreign antigens, compared to their naïve counterparts. In particular, they are programmed to be activated promptly, with a reduced requirement for costimulatory signals and therefore they might be more resistant to veto cells. To address this question we used the 2C mouse model, the CD8 T cells of which express a transgenic TCR against H-2d. The CD8 T cells bearing the TCR transgene can be followed by FACS using staining with a clontypic antibody (1B2) against the transgene. In this model, addition of veto CTLs was shown to inhibit expansion of CD8+1B2+ effector cells by induction of apoptosis which can be monitored by annexin V staining. Thus, in a total of 10 experiments the addition of 5% veto cells to 3 day MLR culture of naive 2C effector cells in the presence of H-2d stimulator cells, led to 76%±9% inhibition of expansion. In order to compare the sensitivity of memory cells in the same model, memory cells were established by immunizing 2C transgenic mice with 1x106 irradiated splenocytes from Balb/c donors (H-2d origin). Six weeks later, splenocytes were harvested and after Ficoll separation were shown to be enriched with memory CD8 T cells(CD44+high CD45Rb+ CD62L+, average in 16 different experiments was 73%±11). Upon addition of 5% veto cells to MLR culture of memory 2C spleen cells in the presence of stimulator cells, 78%±7% inhibition of 2C expansion was found. This veto activity was associated with increased apoptosis of allospecific memory CD8 T cells. Thus, in the absence of veto cells the CD8+1B2+ memory cells exhibited a low level of Annexin V (6%±3%) while in the presence of 5% veto cells, a high level of Annexin V (25%±9%) was detected. The deletion of the 2C memory effectors, as previously shown for naive 2C cells, is largely dependent on the presence of Fas-FasL interaction, as indicated by using memory cells from 2C- lpr mice that lack Fas receptor on the cell surface. Upon addition of veto cells to MLR culture with 2C memory spleen cells from lpr mice, only a minor reduction of expansion (5.5%±6% in the presence of 10% veto CTLs) was detected. In conclusion, these results suggest that veto cells can delete memory effector cells as efficiently as exhibited on naive effector cells and by a similar Fas-FasL dependent mechanism. This finding might have significant implications not only for BMT, but also for the treatment of autoimmune diseases in which memory T cells play a major role.
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Garcia-Bernal, David, Miguel Blanquer, Jose Antonio del Rio, Enrique Correal, Javier Lopez, Maria Juliana Majado, and Jose M. Moraleda. "In Vitro Study of New Photochemotherapeutic Compounds for Extracorporeal Photopheresis." Blood 120, no. 21 (November 16, 2012): 2144. http://dx.doi.org/10.1182/blood.v120.21.2144.2144.
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Abstract Abstract 2144 Introduction: Extracorporeal photopheresis (ECP) is a cell-based immunomodulatory therapy involving the separation of autologous mononuclear cell fraction followed by ex-vivo administration of 8-methoxypsoralen (8-MOP) and UVA irradiation before reinfusion. ECP is efficient for the treatment of cutaneous T-cell lymphomas, multiple skin disorders, autoimmune diseases, solid organ transplant rejection and graft versus host disease (GVDH). During UVA irradiation phase, 8-MOP binds covalently to leukocytes' DNA leading to cell cycle arrest and apoptosis. These pre-apoptotic leukocytes are reintroduced into the peripheral circulation where they are phagocytosed by immature dendritic cells, which play a crucial role in the induction of peripheral tolerance through the induction of T regulatory cells subsets, production of anti-inflamatory cytokines such as IL-10 and TGF-b and the deletion of effector antigen-specific CD4+ and CD8+ T cells into lymph nodes. Our aim in the present work was to compare the therapeutic effectiveness of 8-MOP with other four new compounds (BB01 to BB04). Methods: Mononuclear cells (MNC) were isolated by ficoll density gradient, incubated with increasing concentrations of 8-MOP, BB01, BB02, BB03 and BB04 and irradiated with UVA light (2 J/cm2). MNC apoptosis percentage was measured by flow cytometry (Annexin-V and 7-AAD staining) after 48h of culture at 37°C. Mixed lymphocyte cultures (MLC) with either myeloid immature (iDC) or mature dendritic cells (mDC) and UVA irradiated MNC treated with the different compounds were performed. After 48h of MLC CCL21-stimulated migration of iDC and mDC was studied. In other assays, after 6 days of MLC the proliferation of treated MNC (BrdU incorporation) and the production of pro-inflammatory and anti-inflammatory cytokines were analyzed. Results: After incubation of MNC with the different compounds + UVA there was a significative increase of apoptosis percentage using BB02 (from 50 ng/ml, p<0.001) and BB01 (from 200 ng/ml, p<0.001) over that achieved using 8-MOP, while BB03 and BB04 induced significatively less apoptosis. Furthermore, in comparation with 8-MOP, there was a significant upregulation of the CCL21-promoted migration of iDC (p<0.05) and lower migration of mDC (p<0.001) respectively, when these DC were co-cultured with MNC treated with BB02 + UVA. On the other hand, MNC proliferation achieved after MLC with allogeneic iDC or mDC was partially inhibited after treatment with all tested compounds + UVA. These inhibition was higher using BB02 and BB01, although the difference was no statistically significant when compared to 8-MOP. In addition, in MLC using treated MNC and stimulator iDC there was a reduction in the release of pro-inflammatory cytokines (IFN-g, IL-6 and IL-17A) and an increase of anti-inflammatory cytokines such as IL-10 and TGF-b that was higher using BB02 + UVA. Conclusions: Compounds BB01 and specially BB02 were more efficient than 8-MOP in the induction of apoptosis and the upregulation of chemokine-promoted migration of iDC. They were also better in the inhibition of the proliferation of MNC stimulated by iDC and mDC, and in the induction of anti-inflammatory cytokine production. This higher in vitro activity might be relevant to increase the therapeutic potential of ECP. Acknowledgments: Work financed by the Spanish Ministry of Science and Innovation (Ref: BFU2010–19599). Spanish Net of Cell Therapy (TerCel) Carlos III Institute. Disclosures: No relevant conflicts of interest to declare.
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Meng, Lijun, Zhenjiang Bai, Shan He, Kazuhiro Mochizuki, Janaki Purushe, Hongxing Sun, Jian Wang, et al. "The Notch Ligand DLL4 Derived from Human Dendritic Cells Is Critical for Promoting T Helper (Th)1 and Th17 Cell Differentiation." Blood 126, no. 23 (December 3, 2015): 3431. http://dx.doi.org/10.1182/blood.v126.23.3431.3431.
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Abstract Dendritic cells (DCs) are important for primary T cell responses, and cytokines produced by DCs are thought to be essential for promoting T helper (Th)1 and Th17 differentiation. However, DCs can drive effector differentiation independent of cytokines. In mouse models of graft-versus-host-disease (GVHD), which is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT), we found that DC-derived Notch ligand Dll4 was important for CD4+ Th1 and Th17 cell differentiation. Blocking Dll4 led to decreased production of IFN-g and IL-17 in mice receiving allo-HSCT and inhibition of GVHD. However, the human counterparts of murine DLL4+ DCs and their function in alloreactive T cells have never been investigated. We report here the identification of human DLL4+ DCs and the critical role of DLL4 in DC-regulation of Th1 and Th17 cell differentiation. Flow cytometric analysis revealed that CD1c+ DCs and plasmacytoid DCs (pDCs) from the peripheral blood (PB) of healthy donors (n=18) did not express DLL4. However, 24 hours after stimulation with Toll-like receptor (TLR) agonists, PB DCs from healthy donors produced high levels of DLL4 on their surface. Pam3 (TLR1/2 stimulus), Poly I:C (TLR3 stimulus), LPS (TLR4 stimulus) and R848 (TLR7/8 stimulus) induced high levels of DLL4 expression on the surface of 50% to 80% of CD1c+ DCs. CpG oligodeoxynucleotides (TLR9 agonists) did not increase DLL4 in CD1c+ DCs, likely due to their lacking of TLR9. pDCs increased DLL4 expression when activated by R848 (16.0% ± 2.7%) and to a less extent by CpG oligodeoxynucleotides (8.6% ± 0.8%). Thus, activation of TLR signaling induces high levels of DLL4 in CD1c+ DCs and pDCs, with R848 being the most potent stimulus. Functional analysis using mixed lymphocyte reaction revealed that R848-activated CD1c+ DCs and pDCs induced greater proliferation of allogeneic CD4+ T cells and production of more IFN-g- and IL-17-producing effector cells compared to unstimulated CD1c+ DCs and pDCs. Blocking DLL4 using a neutralizing antibody decreased Notch signaling in T cells stimulated with activated DCs and led to production of 2- and 3-fold less Th1 cells and Th17 cells compared to IgG control, suggesting the importance of DLL4 in DC-regulation of effector differentiation. Molecular mechanism investigation revealed that SATAT3 and NFkB were crucial for inducing DLL4 in human DCs. Inhibiting STAT3 alone using its specific inhibitor S31-201 dramatically decreased DLL4 expression in activated PB DCs. Promoter reporter assays showed that STAT3 was required for activating DLL4 transcription. Inhibiting NFkB using its inhibitor PDTC also decreased the expression of DLL4 on the surface of R848-stimulated PB DCs. However, DCs derived from monocytes induced by GM-CSF and IL-4, which had activation of NFkB but did not express active STAT3 following stimulation by R848 + LPS, were DLL4 negative despite their upregulation of costimulatory molecules (e.g., CD40, CD80, CD83, and CD86). Thus, activation of STAT3 is critical for inducing DLL4 in human DCs, whereas active NFkB is important but not sufficient for inducing DLL4 in PB DCs. Finally, given the importance of alloreactive Th1 and Th17 cells in mediating GVHD in human allogeneic HSCT recipients, we further obtained PB from patients (n=7) undergoing allo-HSCT between 21 and 39 days after transplantation when these patients were fully engrafted and no longer pancytopenic. As compared to healthy donors, HSCT recipients had an averaged 12-fold higher frequency of DLL4+ CD1c+ DCs. These results indicate that upregulation of DLL4 on the surface of DCs is associated with alloreactive inflammatory conditions in HSCT patients. In summary, our findings show that DLL4 surface expression on human DCs is critical for the priming of human Th1 and Th17 responses and may have significant implication in better understanding of T cell-mediated inflammatory conditions such as chronic infection, autoimmune diseases, tumor rejection and GVHD after allo-HSCT. Disclosures No relevant conflicts of interest to declare.
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Zaimoku, Yoshitaka, Bhavisha A. Patel, Sachiko Kajigaya, Xingmin Feng, Lemlem Alemu, Diego Quinones Raffo, Emma M. Groarke, and Neal S. Young. "Deficit of Circulating CD19+CD24hiCD38hi Regulatory B Cells in Severe Aplastic Anemia." Blood 134, Supplement_1 (November 13, 2019): 1219. http://dx.doi.org/10.1182/blood-2019-127053.
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Background: Immune aplastic anemia (AA) is caused by cytotoxic T cells (CTLs) that destroy hematopoietic stem and progenitor cells. Regulatory T cells (Tregs) are reduced in AA and increase in response to immunosuppressive therapy (IST; Solomou E et al, Blood 2007). Recent studies suggested an immune regulatory role of regulatory B cells (Bregs). Human CD19+CD24hiCD38hi Bregs suppress Th1 response of CD4+ T cells as well as IFN-γ production by CD8+ CTLs (Mauri C, Menon M, J Clin Invest 2017). The quantity and/or function of Bregs are impaired in autoimmune diseases, malignancies, chronic graft-versus-host disease, and during rejection of transplanted organs. Methods: We investigated B cell phenotypes including CD24hiCD38hi Bregs in previously untreated severe AA (SAA) and very severe AA (VSAA) patients, and healthy individuals aged 18 years and older, and tested their correlation with severity and response to IST. Absolute numbers of lymphocyte subsets, including CD19+ B cells, CD8+ T cells, CD4+ T cells, and NK cell (TBNK), were quantified in fresh blood. Percentages of B cell subsets among total CD19+ B cells, including CD24hiCD38hi Bregs, CD24loCD38lo mature naïve B cells, CD24hiCD38lo memory B cells and CD24loCD38hi plasma cells/plasmablasts, were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs). Blood samples were obtained from patients close to time of diagnosis and before institution of definitive therapy. All patients were treated with horse anti-thymocyte globulin, cyclosporine, and eltrombopag between 2012 and 2018 at the Hematology Branch, NHLBI (clinicaltrials.gov NCT01623167). Results: TBNK analysis revealed no significant difference in total B cell counts in 104 AA patients compared to 40 healthy individuals (median, 137/μl [IQR, 73-212] vs 163/μl [106-242], P=.11); NK cells were significantly decreased in patients with AA, as previously reported (Gascon P et al, Blood 1986). Total B cell count did not correlate with severity of AA (P=.89) nor with overall response at six months (P=.93). CD8+ T cells and NK cells were lower in VSAA patients compared to SAA patients. None of the TBNK subsets was predictive of overall response in six months after IST. When we assessed the phenotype of B cells among 60 AA patients whose cryopreserved PBMCs were available, CD24hiCD38hi Bregs were markedly decreased as compared to 29 healthy individuals (0.31% [0.14-0.85%] vs 1.9% [1.3-3.6%], P=3×10-7; Figure, Table), while there was no significant difference in other B cell phenotypes. Among these 60 patients, the percentage of CD24hiCD38hi Bregs was especially decreased in VSAA patients compared to SAA (0.18% [0.11-0.34%] vs 0.50% [0.17-1.4%], P=.017). In contrast, CD24loCD38lo mature naïve B cells were higher in VSAA than in SAA (69% [58-86%] vs 60% [42-70%], P=.024). CD24hiCD38hi Breg frequency was positively associated with neutrophil and reticulocyte counts (correlation coefficients [r], 0.34 and 0.26, respectively), while the frequency of CD24loCD38lo mature naïve B cells was negatively correlated (r, -0.34 and -0.40). CD24loCD38lo mature naïve B cells before IST were significantly lower in 47 patients who achieved overall responses at six months compared to 13 non-responders (64% [42-71%), vs 73% [58-88%], P=.014), but CD24hiCD38hi Breg frequency was not correlated with IST responses. At six months after IST, CD24hiCD38hi Bregs in AA patients had recovered to levels present in healthy individuals (2.3% [0.98-4.8%]), in both 34 responders and five non-responders; non-responders showed non-significant increased CD24loCD38lo mature naïve B cells at six months (P=.068). Discussion: A deficit of circulating CD24hiCD38hi Bregs in immune AA with recovery after IST, as occurs with Tregs, suggests Bregs may contribute to the immune pathophysiology in AA. We unexpectedly observed a higher percentage of CD24loCD38lo mature naïve B cells to be associated with more severe disease and a lower probability of responses to IST. B cell phenotype analysis may be beneficial for monitoring of AA and predicting outcomes of therapy. Disclosures No relevant conflicts of interest to declare.
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Hu, Yongxian, Yanjun Gu, Lixia Sheng, Kangni Wu, Jimin Shi, Yamin Tan, Xiaohong Yu, and He Huang. "Decitabine Facilitates the Generation of Functional Regulatory γδT Cells Via Foxp3 Gene Demethylation and NF-κB up-Regulation." Blood 120, no. 21 (November 16, 2012): 3281. http://dx.doi.org/10.1182/blood.v120.21.3281.3281.
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Abstract Abstract 3281 Regulatory γδT cells (γδTregs) are capable of suppressing T-cell activation and proliferation (Rita et al Journal of immunology 2009). Our previous studies demonstrated the immunosuppressive features of γδTregs and further supported the use of γδTregs as a promising therapeutic strategy for the adoptive immunotherapy of allograft rejection, post-transplantation graft-versus-host disease (GVHD) and autoimmune diseases (Hu et al ASH abstract 2011). To this end, large-scale, efficient expansion of γδTregs represents a fundamentally important prerequisite for their clinical application. Here we explored the efficacy of decitabine (a DNA hypomethylating agent) for the generation of functional γδTregs and its underlying mechanisms. Human peripheral blood mononuclear cells (PBMCs) were cultured with interleukin (IL)-2, IL-15, TGF-β1 and zoledronic acid (ZOL). Decitabine was added to aliquots of PBMCs. Frequency and absolute number of γδTregs were investigated by flow cytometry (FACS) and trypan blue live-cell counts after cultivation. Our results showed a higher frequency (61.9±10.3% versus 38.4±7.8%, p<0.01) and absolute number of γδTregs [(3.1±0.1)×106 versus (5.9±0.2)×106, p<0.01] were seen in cells treated with decitabine plus ZOL/IL-2/IL-15/TGF-β1 (referred to as decitabine-induced γδTregs below) than in cell without decitabine induction (referred to as common γδTregs) as shown in Fig. 1. These data demonstrated that decitabine and ZOL/cytokines synergized to induce stable γδTregs via improved Foxp3 expression. Next we analyzed the underlying mechanisms of decitabine on γδTreg induction. Genomic DNA was isolated from γδTregs sorted by FACS. The Foxp3 gene methyaltion status of upstream enhancer CpG islands from −5995 to −5778 and from −5031 to −4792, proximal promoter CpG island from -139 to -15 and non-coding DNA sequence 3 (CNS3) CpG island from 3748 to 3971 was analyzed by bisulfite sequencing. We found decitabine-induced γδTregs displayed efficient CpG demethylation of the upstream enhancer from −5031 to −4792, and of CNS3 from 3748 to 3971, but no differences were detected in the other CpG islands investigated in contrast to common γδTregs (Fig. 2A). We subsequently investigated if DNA demethylation of CpG islands could influence upstream enhancer or CNS3 activity to promote Foxp3 transcription. The upstream enhancer from −5031 to −4792 and CNS3 from 3748 to 3971 were methylated using SssI (CpG) methylase, and methylated or unmethylated sequences were inserted into demethylated pFoxP3Pro, respectively. Vectors were added to γδT cells and electroporated using the T-023 program of the Nucleofector. Dual fluorescence-based transient reporter assays showed an obvious reduction in luciferase activity after methylation of both CpG islands, but not with the demethylated CpG islands (Fig. 2B), indicating that demethylation of the upstream enhancer and CNS3 can promote Foxp3 expression via increased enhancer activity. Transcription factors (TF) play important roles in gene expression. To test whether decitabine enhanced specific TFs expression to influence γδTreg induction, we performed TF CHIP assays in FACS-sorted common γδTregs versus decitabine-induced γδTregs. The expression levels of 245 TFs were evaluated and the result showed that NF-κb expression in decitabine-induced γδTregs was up-regulated 50 times in contrast to that in common γδTregs. NF-κb up-regulation was further comfirmed by western blot analysis from 6 different healthy volunteers. Finally, We evaluated the immunosuppressive function of the enriched γδTregs on Carboxyfluorescein Diacetate (CFSE)-labeled hPBMC proliferation and activation stimulated with anti-CD3/anti-CD28 monoclonal antibodies for 5 days in vitro. As shown in Fig. 3, both common γδTregs and decitabine-induced γδTregs exerted dose-dependent suppression of proliferation, but decitabine-induced γδTregs had a greater suppressive effect than common γδTregs. In conclusion, these data demonstrate for the first time that decitabine facilitates the generation of functional γδTregs via foxp3 gene demethylation and NF-κb up-regulation. Futhermore, our results lay the foundation for large-scale availability of induced γδTregs for potential clinical applications, and reveal novel targets for regulating Foxp3 expression in γδT cells. Disclosures: No relevant conflicts of interest to declare.
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Miller, Weston, Caleb E. Wheeler, Angela Panoskaltsis-Mortari, Allan D. Kirk, Christian P. Larsen, Bruce R. Blazar, and Leslie S. Kean. "Prevention of Acute GvHD During MHC Haploidentical BMT: Evaluating the Efficacy of T-Cell Costimulation Blockade Using a Novel Rhesus Macaque Transplant Model." Blood 114, no. 22 (November 20, 2009): 2455. http://dx.doi.org/10.1182/blood.v114.22.2455.2455.
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Abstract Abstract 2455 Poster Board II-432 Introduction: While hematopoietic stem cell transplantation (HSCT) offers a cure for many hematologic diseases, it remains plagued by often fatal graft-versus-host disease (GvHD). Despite the inadequacy of current GvHD prevention strategies, especially for MHC-mismatched HSCT, the pace of the clinical introduction of novel therapeutics has been slow, likely due to the lack of a suitable translational model to rigorously test the immunologic and clinical impact of novel biologic therapies. Among the most promising of these therapies include those that block T cell costimulation blockade. While they have been used for both autoimmune disease and to prevent rejection of solid organ transplants, costimulation blockade reagents have not yet been evaluated for efficacy in preventing clinical GvHD. Here we describe a novel primate model of MHC-mismatched GvHD, that has allowed us, for the first time, to evaluate the mechanisms controlling GvHD in a primate translational system, and to evaluate the efficacy of costimulation blockade for the prevention of primate GvHD, even across haplo-MHC barriers. Methods: Using DNA microsatellite-based pedigree analysis and MHC haplotype determination, we have developed the first MHC-defined Rhesus macaque HSCT system. MHC haplo-identical transplant pairs were chosen, and recipients prepared for transplant with TBI (8 Gy, as a single dose, with lung shielding to 6 Gy). Animals were either treated with no immunosuppression post-transplant (controls) or with a costimulation blockade-based regimen which included CD28/B7 blockade with abatacept (20mg/kg every 7 days), CD40/CD154 blockade with the 3A8 anti-CD40 monoclonal antibody (maintenance dosing at 5mg/kg twice weekly) as well as sirolimus to maintain serum trough levels between 5-10 ng/mL. Either leukopheresis-derived peripheral blood stem cells or bone marrow was used for transplant (average total nucleated cell dose = 9.3 +/-2.7×108/kg; average CD3+ cell dose = 1.1 +/- 0.88 ×108/kg) Donor engraftment was measured by microsatellite analysis, and GvHD was graded clinically using standard scales. The immune phenotype after transplant was determined by multicolor cell- and serum-based flow cytometric analyses. Results: Seven haploidentical transplants have been completed. Three controls received no immunosuppression. These animals demonstrated rapid and complete donor engraftment, with donor T cell activation and proliferation occurring within one week of transplant, coincident with the onset of severe clinical GvHD, which predominantly targeted the GI tract. Flow cytometric analysis showed loss of CD127 expression on both CD4+ and CD8+ T cells, consistent with their rapid clonal expansion and differentiation. Multiplexed luminex cytokine analysis demonstrated high-level secretion of the inflammatory cytokines IFNγ, and IL18, as well as the counter-regulatory cytokine IL-1RA. Importantly, no rise in TNF, IL-1b, nor IL17 was measured despite severe GvHD. In contrast, four treated animals received a haplo-identical BMT in the setting of abatacept/anti-CD40 and sirolimus for GvHD prophylaxis. All of these recipients demonstrated rapid donor engraftment, but, unlike the controls, they were protected against clinical GvHD—they displayed neither the skin rash nor the profuse diarrhea noted in the control animals. Flow cytometric analysis demonstrated maintenance of CD127 expression on both CD4+ and CD8+ T cells. Furthermore, luminex analysis revealed that expression of IFNγ, IL18 and IL-1RA were all normal in the setting of GvHD prophylaxis with costimulation blockade and sirolimus. Conclusions: We have established a robust model of haplo-identical HSCT and GvHD using an MHC-defined Rhesus macaque colony. This model has allowed us to begin to determine the mechanisms underlying GvHD during primate haplo-identical BMT and to assess the efficacy of novel regimens to prevent this disease. We find that unprotected primate GvHD is characterized by rapid T cell proliferation, with concomitant loss of expression of CD127 on both CD4+ and CD8+ T cells. In addition, it is associated with a cytokine storm, including high level secretion of IFNγ, IL18 and IL-1RA into the serum. Finally, we find that CD28/CD40-directed costimulation blockade in combination with sirolimus can effectively inhibit both the clinical and cellular hallmarks of GvHD during haplo-identical BMT, and thus may deserve close clinical scrutiny as a possible prophylaxis strategy during these high risk transplants. Disclosures: No relevant conflicts of interest to declare.
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Matos, Tiago R., Hongye Liu, Masahiro Hirakawa, Ana Cristina Alho, and Jerome Ritz. "Maturation and Phenotypic Diversity of Human CD4+ Regulatory T Cells in Umbilical Cord Blood and Peripheral Blood from Healthy Donors." Blood 126, no. 23 (December 3, 2015): 2357. http://dx.doi.org/10.1182/blood.v126.23.2357.2357.
Full textAbstract:
Abstract Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) are required for maintenance of immune tolerance and immune homeostasis. Quantitative or functional Treg deficiency has been correlated with autoimmune disease, allergy, allograft rejection and graft versus host disease. Conversely, increased Treg can suppress tumor immunity resulting in tumor progression. Treg express a large number of cellular markers that reflect their level of maturation, functionality, activation and migratory capacity. Nevertheless, it has not previously been possible to integrate the expression of these various markers and correlate their expression with human Treg differentiation in vivo. Methods: We used single cell mass cytometry (CyTOF) to simultaneously study 36 phenotypic and functional markers of human Treg in samples obtained from umbilical cord blood (CB) (n=4) and healthy adult donors (n=10). Wanderlust trajectory detection algorithm was used to analyze temporal positioning of Treg across development. To quantify Treg heterogeneity we used ACCENSE; an analysis tool that combines a nonlinear dimensionality reduction algorithm (t-SNE) with spectral clustering algorithm and automated cell classification into subpopulations. Results: Using Wanderlust to characterize Treg maturation, the majority of CB Treg were naive (CD45RA+) and CB memory Treg (CD45RA-) were poorly differentiated with minimal expression of activation (HLA-DR) and pro-apoptotic (CD95) markers (Figure 1A). Adult Treg contained few naive cells and mature Treg effector cells expressed high levels of activation and pro-apoptotic markers. (Figure 1B). Using Wanderlust together with spearman correlation, 5 discrete stages of Treg maturation were identified; 1) recent thymic emigrants (RTE), 2) naive, 3) effector, 4) activated and 5) terminal effector. RTE Treg defined by expression of CD45RA, CD31 and CCR7, also expressed markers of proliferation (KI67) and functionality (Tbet, PDL-1, Helios) at low levels but lacked functional CTLA4 and TIM-3. Naive Treg lacked expression of CD31 but expressed other markers characteristic of RTE. Effector Treg expressed increased levels of CD95, CTLA-4, CCR7, GITR, Helios and FoxP3 but lacked HLA-DR. Activated effector Treg expressed the highest levels of FoxP3, Helios and Ki67, along with functional markers (CD28, CXCR3, ICOS, GITR, CD39, CTLA-4, TIM-3) and homing molecules (vascular endothelial CCR5, gut addressin ACT-1, skin addressins CCR4, CLA). Activated Treg express the highest levels and diversity of functional markers along with the ability to migrate to different tissues. Lastly, terminal effector Treg express a more restricted set of functional and homing markers (CD28, CTLA-4, ICOS and CCR5) with less diversity. Markers of exhaustion (PD-1 and TIM-3) are also expressed by effector, activated and terminal effector Treg. Pro-apoptotic markers (CD95high BCL2low) are primarily expressed by activated and terminal effector Treg. Using ACCENSE to evaluate Treg diversity allowed further identification of discrete Treg sub-populations within each maturation state. RTE and naive Treg appear very homogeneous and appear as a single cluster in both CB and adults. In contrast, effector, activated and terminal effector Treg are more heterogeneous and are visualized as 9 distinct clusters (Figure 1C, D). This clustering reflects distinct subsets of memory Treg that co-express various combinations of functional markers in our panel. All 9 Treg effector populations are present in CB, but at much lower levels. Treg effector cell diversity is maintained but does not increase as Treg mature and expand in adults and RTE/naive Treg become less prevalent. Conclusion: Our study is the first to quantify human Treg heterogeneity based on expression of a large set of activation, proliferation, tissue homing and functional markers in conjunction with stages of Treg maturation and differentiation. These studies define 5 stages of Treg maturation and 10 clusters of Treg diversity based on differential expression of phenotypic and functional markers. Similar approaches can be applied to describe maturation and diversity of other cell populations. Further application of this CyTOF panel can be used to study Treg maturation and diversity after hematopoietic stem cell transplantation and in immune and inflammatory diseases, to identify specific defects that may contribute to immune dysfunction. Disclosures No relevant conflicts of interest to declare.
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Sarkar, Debalina, Gongxian Liao, Cox Terhorst, and Roland W. Herzog. "Synergistic Effect of Flt3L and Rapamycin On Immune Tolerance Induction Via Plasmacytoid Dendritic Cells and Treg." Blood 120, no. 21 (November 16, 2012): 2209. http://dx.doi.org/10.1182/blood.v120.21.2209.2209.
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Abstract Abstract 2209 In vivo induction and expansion of Treg is a powerful tool to limit unwanted immune responses and promote tolerance. For example, we have been successful inducing tolerance to factors VIII and FIX in hemophilic mice when the coagulation factor antigen was administered with the mTOR inhibitor rapamycin (J Thromb Haemost 7:1523 and 9:1524, Front Microbiol 2:244). Rapamycin, a macrocyclic triene antibiotic, is an immunosuppressant used to avoid transplant rejection. It suppresses the mTOR1 (and upon prolonged exposure also mTOR2) signaling pathway. Importantly, while mTOR blockage results in deletion of Teff, Treg can be induced and expanded because they are able to utilize alternative (STAT) signaling pathways. Others have shown that existing Treg can be expanded in vivo upon administration of Fms-like tyrosine kinase ligand-3 (Flt3L), a cytokine that drives generation of dendritic cells (DC) from hematopoietic progenitor cells and DC proliferation. This link between DC homeostasis and Treg is evident from the low Treg numbers found in Flt3L-deficient mice and from prevention of graft vs host disease upon treatment with Flt3L. This raises the question of whether a combined approach of rapamycin administration and Flt3L-induced DC generation would result in an optimal immune tolerance protocol. Interestingly, it has been reported that rapamycin blocks Flt3L-induced differentiation of progenitor cells into DC, indicating that Flt3L signaling in DC occurs through the mTOR pathway. However, we find in mice transgenic for an ovalbumin-specific CD4+ T cell receptor (but deficient in recombinase activating gene, rag-2) that ova peptide antigen administration results in substantially enhanced deletion of Teff and in induction of CD4+CD25+FoxP3+CD62L+GITR+ Treg when combined with these two drugs. This was accomplished by repeated administration (twice per week) of a cocktail of the 3 components. Antigen plus either drug causes Teff deletion, while rapamycin is required for Treg induction (which is further enhanced by Flt3L). Antigen, rapamycin, and Flt3L all impact changes in the numbers and frequencies of DC subsets in the spleen during the regimen. The combination all 3 components most potently directs a substantial (3–5 fold, P<0.001) increase in CD11cloPDCA+ plasmacytoid DC numbers (but not of conventional CD11chiPDCA− DCs). While pDCs are known to provide innate anti-viral responses, they also play an important role in immune tolerance. Consequently, when pDC were partially depleted with anti-PDCA, Treg induction was significantly impaired. Furthermore, the protocol caused an increase in the frequency of Indoleamine-pyrrole 2,3-dioxygenase (IDO)-expressing pDCs (which is known to activate resting Treg for suppressor activity). Finally, FLt3L-induced expansion of Treg (but not of DCs) is less effective in GITR-L −/− mice. Combined, these data demonstrate that i) Flt3L and rapamycin can be used synergistically for induction of T cell tolerance, ii) pDCs can be expanded within a rapamycin regimen, iii) and Fl3tL-induced pDC expansion facilitates Treg induction, which is partially dependent on GITR-L (a co-stimulatory molecule primarily expressed by pDCs that promotes cross talk to Treg by engagement of the GITR receptor). In order to establish relevance of this protocol for treatment of disease, we intravenously injected a F.VIII protein/rapamycin/Flt3L cocktail into hemophilia A mice (C57BL6/129 F8e16 −/−) twice per week for 1 month. Subsequently, mice received 1 month of factor replacement therapy (1 IU human FVIII, IV, once per week). Control mice without prior immune modulatory regiment or that received non-specific immune suppression (rapamycin and Flt3L only) formed high-titer inhibitors against FVIII (70–80 BU), which was significantly suppressed to ∼10 BU (P<0.001, n=5 per group). Importantly, inhibitor titers were only mildly reduced (to ∼40 BU) when Flt3L was omitted from the tolerogenic cocktail, thereby confirming the synergistic effect of flt3L and rapamycin in tolerance induction. This approach combines expansion of regulatory antigen presenting and T cells and should be of broad relevance for cell and organ transplantation as well as for treatment of inherited protein deficiencies and of autoimmune diseases. Disclosures: No relevant conflicts of interest to declare.