IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

Background: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC). Methodology/Principal Findings: Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1β and IL-6 ("conv. mix"). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC. Conclusion: Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy. © 2013 Schilling et al

Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG

Background: This randomized phase III trial was designed to demonstrate the superiority of autologous peptide-loaded dendritic cell (DC) vaccination over standard dacarbazine (DTIC) chemotherapy in stage IV melanoma patients. Patients and methods: DTIC 850 mg/m2 intravenously was applied in 4-week intervals. DC vaccines loaded with MHC class I and II-restricted peptides were applied subcutaneously at 2-week intervals for the first five vaccinations and every 4 weeks thereafter. The primary study end point was objective response (OR); secondary end points were toxicity, overall (OS) and progression-free survival (PFS). Results: At the time of the first interim analysis 55 patients had been enrolled into the DTIC and 53 into the DC-arm (ITT). OR was low (DTIC: 5.5%, DC: 3.8%), but not significantly different in the two arms. The Data Safety & Monitoring Board recommended closure of the study. Unscheduled subset analyses revealed that patients with normal serum LDH and/or stage M1a/b survived longer in both arms than those with elevated serum LDH and/or stage M1c. Only in the DC-arm did those patients with (i) an initial unimpaired general health status (Karnofsky = 100) or (ii) an HLA-A2+/HLA-B44− haplotype survive significantly longer than patients with a Karnofsky index <100 (P = 0.007 versus P = 0.057 in the DTIC-arm) or other HLA haplotypes (P = 0.04 versus P = 0.57 in DTIC-treated patients). Conclusions: DC vaccination could not be demonstrated to be more effective than DTIC chemotherapy in stage IV melanoma patients. The observed association of overall performance status and HLA haplotype with overall survival for patients treated by DC vaccination should be tested in future trials employing DC vaccine

Direct Type I IFN but Not MDA5/TLR3 Activation of Dendritic Cells Is Required for Maturation and Metabolic Shift to Glycolysis after Poly IC Stimulation

We thank the Rockefeller University Center for Clinical and Translational Science (UL1 TR000043 NCATS, NIH) for technical support

Boosting regulatory T cell function by CD4 stimulation enters the clinic

Understanding tolerance mechanisms at the cellular and molecular level holds the promise to establish novel immune intervention therapies in patients with allergy or autoimmunity and to prevent transplant rejection. Administration of mAb against the CD4 molecule has been found to be exceptionally well suited for intentional tolerance induction in rodent and non-human primate models as well as in humanized mouse models. Recent evidence demonstrated that regulatory T cells (Treg) are directly activated by non-depleting CD4 ligands and suggests Treg activation as a central mechanism in anti-CD4-mediated tolerance induction. This review summarizes the current knowledge on the role of Treg in peripheral tolerance, addresses the putative mechanisms of Treg-mediated suppression and discusses the clinical potential of harnessing Treg suppressive activity through CD4 stimulation

Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy

© 2015. Oncotarget. Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teffcells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery

Alloantigen-specific T-cell hyporesponsiveness induced by dnIKK2 gene-transfected recipient immature dendritic cells

AbstractImmature dendritic cells (iDCs) have been shown to be able to induce peripheral T-cell tolerance through distinct pathways. Here, we investigated the tolerogenic property of recipient iDCs whose maturation was arrested by a dominant negative mutant of inhibitor of nuclear factor kappa-B kinase 2 (dnIKK2) gene. We found that dnIKK2-iDCs presented a typical semi-mature morphology and expressed lower levels of CD80 and CD86, slightly higher MHC-II than untransfected iDCs. The expression of these molecules had no significant change even dnIKK2-iDCs were pulsed by donor antigen. In primary mixed leukocyte reaction (MLR), dnIKK2-iDCs exhibited impaired ability to stimulate allogeneic T-cells, but induced CD4+CD25− T-cell formation. In co-culture MLR, these CD4+CD25− T-cells suppressed T-cell alloreaction in an antigen-specific manner. Besides, CD4+CD25− T-cells inhibited IL-2 and IFN-γ release, whereas promoted IL-10 and TGF-β secretion. These data suggested recipient dnIKK2-iDCs could maintain peripheral tolerance through down-regulating costimulatory molecule expressions and inducing CD4+CD25− T-cell formation

The unfinished legacy of liver transplantation: Emphasis on immunology

Liver transplantation radically changed the philosophy of hepatology practice, enriched multiple areas of basic science, and had pervasive ripple effects in law, public policy, ethics, and theology. Why organ engraftment was feasible remained enigmatic, however, until the discovery in 1992 of donor leukocyte microchimerism in long-surviving liver, and other kinds of organ recipients. Following this discovery, the leukocyte chimerism-associated mechanisms were elucidated that directly linked organ and bone marrow transplantation and eventually clarified the relationship of transplantation immunology to the immunology of infections, neoplasms, and autoimmune disorders. We describe here how the initially controversial paradigm shift mandated revisions of cherished dogmas. With the fresh insight, the reasons for numerous inexplicable phenomena of transplantation either became obvious or have become susceptible to discriminate experimental testing. The therapeutic implications of the "new immunology" in hepatology and in other medical disciplines, have only begun to be explored. Apart from immunology, physiologic investigations of liver transplantation have resulted in the discovery of growth factors (beginning with insulin) that are involved in the regulation of liver size, ultrastructure, function, and the capacity for regeneration. Such studies have partially explained functional and hormonal relationships of different abdominal organs, and ultimately they led to the cure or palliation by liver transplantation of more than 2 dozen hepatic-based inborn errors of metabolism. Liver transplantation should not be viewed as a purely technologic achievement, but rather as a searchlight whose beams have penetrated the murky mist of the past, and continue to potentially illuminate the future. Copyright © 2006 by the American Association for the Study of Liver Diseases

Infectious Tolerance: Human CD25+ Regulatory T Cells Convey Suppressor Activity to Conventional CD4+ T Helper Cells

Regulatory CD4+CD25+ T cells (Treg) are mandatory for maintaining immunologic self-tolerance. We demonstrate that the cell-cell contact–mediated suppression of conventional CD4+ T cells by human CD25+ Treg cells is fixation resistant, independent from membrane-bound TGF-β but requires activation and protein synthesis of CD25+ Treg cells. Coactivation of CD25+ Treg cells with Treg cell–depleted CD4+ T cells results in anergized CD4+ T cells that in turn inhibit the activation of conventional, freshly isolated CD4+ T helper (Th) cells. This infectious suppressive activity, transferred from CD25+ Treg cells via cell contact, is cell contact–independent and partially mediated by soluble transforming growth factor (TGF)-β. The induction of suppressive properties in conventional CD4+ Th cells represents a mechanism underlying the phenomenon of infectious tolerance. This explains previously published conflicting data on the role of TGF-β in CD25+ Treg cell–induced immunosuppression

In-depth immune-oncology studies of the tumor microenvironment in a humanized melanoma mouse model

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHCP using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model. Keywords: Chipcytometry multiplex immunohistochemistry melanoma humanized mice tumor microenvironment flow cytometr