Toll-Like Receptor Stimulation by MicroRNAs in Acute Graft-vs.-Host Disease

Acute graft-vs.-host disease (aGVHD) is a frequent complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), accounting for substantial morbidity and mortality associated with this treatment modality. The pathogenesis of aGVHD involves a complex cascade of humoral and cellular interactions in which donor T cells target HLA mismatched host tissues, causing tissue injury through secretion of pro-inflammatory cytokines and induction of direct cytotoxicity. Toll-like receptors (TLRs) are key components of the innate immune system that recognize endogenous danger-associated molecular patterns (DAMPs) and exogenous pathogen-associated molecular patterns (PAMPs). Patients receiving conditioning chemotherapy and/or whole-body irradiation prior to all-HSCT are prone to gastrointestinal damage and translocation of microbiota across compromised intestinal epithelium, resulting in release of PAMPs and DAMPs. These “danger signals” play critical roles in disease pathogenesis by both initiating and propagating aGVHD through dendritic cell maturation and alloreactive T cell responses. There are 10–15 TLRs identified in mammalian species, a subset of which recognize single-stranded RNA (ssRNA) and serve as a key component of viral immunity. Recently, ssRNAs other than those of viral origin have been investigated as potential ligands of TLRs. MicroRNAs (miRs) are short (19–24 nt) non-coding RNAs that play critical roles in a variety of diseases. While traditionally miRs post-translationally modulate gene expression, non-canonical functions such as regulating TLR stimulation by acting as TLR ligands have been described. Here, we review the role of TLRs in aGVHD pathogenesis, the function of miRs in TLR stimulation, and the recent literature describing miRs as TLR ligands in aGVHD

Discovery and functional implications of a miR-29b-1/miR-29a cluster polymorphism in acute myeloid leukemia

We previously reported that microRNA (miR)-29b is down-regulated and has a tumor suppressor role in acute myeloid leukemia (AML). However, little is known about the mechanisms responsible for miR-29b expression downregulation in AML. In this work we screened for mutations that could affect miR-29b expression. Using Sanger sequencing, we identified a germline thymidine (T) base deletion within the miR-29b-1/miR-29a cluster precursor in 16% of AML patients. Remarkably we found a significant enrichment for the presence of the miR-29 polymorphism in core binding factor (CBF) newly diagnosed AML patients (n = 61/303; 20%) with respect to age, sex and race matched controls (n = 43/402:11%, P < 0.01). Mechanistically, this polymorphism affects the expression ratio of mature miR-29b and miR-29a by dampening the processing of miR-29a. RNA immunoprecipitation assays showed reduced DROSHA binding capacity to the polymorphism with respect to the controls. Finally, we showed that this polymorphism negatively impacts the ability of miR-29b-1/miR-29a cluster to target MCL-1 and CDK6, both known miR-29 targets

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD

Characterization of Natural Autoantibodies: A case for Functional Significance

Natural autoantibodies are defined as self-reactive antibodies present in healthy individuals in the absence of purposeful immunization or any known antigenic stimulation. While most NAAbs are of the IgM isotype, T cell dependent isotypes such as IgG and IgA NAAbs have been reported. Natural antibodies are conserved through evolution; they have been detected in sharks and humans, the phylogenetic limits of vertebrates capable of producing antibodies. The phylogenetic conservation of NAAbs suggests that they play an important, but overlooked, role in the maintenance of physiological homeostasis of the immune system.My project involved the characterization of two NAAbs. The first part of my dissertation elucidates the production and functional characterization of an anti-T Cell Receptor NAAb, OR3. OR3 is an IgM heterohybridoma made from the peripheral blood B cells of a patient with Rheumatoid Arthritis. The results show that OR3 specifically binds to the Complementarity Determining Region 1 segment of TCRV b8 regions, is specific for a subset of T cells and blocks the antigen activation of these T cells. Importantly, OR3 does not induce apoptosis or necrosis of T cells. My results support our hypothesis that anti-TCR NAAbs are immunomodulatory and indicate that autoantibodies present in humans may have significant functional activity in the regulation of T cell responses.The second part of the dissertation describes the phenomenon of NAAbs to human delta opioid receptor. I affinity purified IgG antibodies to human DOR from intravenous immunoglobulin, a therapeutic blood product that contains purified IgG isolated from the plasma of thousands of healthy donors. The anti-DOR NAAbs bind to DOR protein, and initiate an activating signaling cascade, in a manner similar to a receptor specific agonist. Interestingly, there is a significant difference in the agonistic activity of the autoantibodies compared to the synthetic DOR agonist DPDPE. Unlike DPDPE, these autoantibodies display significant immunomodulatory activity as evinced by changes in CCR5 and CXCR4 chemokine receptor expression. The presence of functional autoantibodies in IVIG shows that they are a part of the normal healthy immune repertoire in humans. This work also presents data supporting the interconnecting network between the neuroendocrine and immune systems

Targeting Wnt signaling in acute myeloid leukemia stem cells

Not available

Serum MicroRNA-155 in Acute Graft-Versus-Host-Disease (aGVHD)

Allogeneic hematopoietic stem cell transplant (alloHSCT) is a curative treatment for many hematologic malignancies. Unfortunately, about 30-50% of all recipients undergoing alloHSCT develop acute graft-versus-host-disease (aGVHD), which is associated with high morbidity and mortality [1,2]. Treatment of aGVHD involves the use of immune suppressive drugs such as high dose of steroids that leads to further immunosuppression and risk for opportunistic infections. Often patients are refractory to steroids therapy making the prognosis dismal. Thus, it is critical to identify robust biomarkers to detect aGVHD before onset of clinical symptoms so that therapeutic strategies can be implemented that may result in better treatment responses and less toxicity.&nbsp