Interspecies avian brain chimeras reveal that large brain size differences are influenced by cell-interdependent processes.

Like humans, birds that exhibit vocal learning have relatively delayed telencephalon maturation, resulting in a disproportionately smaller brain prenatally but enlarged telencephalon in adulthood relative to vocal non-learning birds. To determine if this size difference results from evolutionary changes in cell-autonomous or cell-interdependent developmental processes, we transplanted telencephala from zebra finch donors (a vocal-learning species) into Japanese quail hosts (a vocal non-learning species) during the early neural tube stage (day 2 of incubation), and harvested the chimeras at later embryonic stages (between 9-12 days of incubation). The donor and host tissues fused well with each other, with known major fiber pathways connecting the zebra finch and quail parts of the brain. However, the overall sizes of chimeric finch telencephala were larger than non-transplanted finch telencephala at the same developmental stages, even though the proportional sizes of telencephalic subregions and fiber tracts were similar to normal finches. There were no significant changes in the size of chimeric quail host midbrains, even though they were innervated by the physically smaller zebra finch brain, including the smaller retinae of the finch eyes. Chimeric zebra finch telencephala had a decreased cell density relative to normal finches. However, cell nucleus size differences between each species were maintained as in normal birds. These results suggest that telencephalic size development is partially cell-interdependent, and that the mechanisms controlling the size of different brain regions may be functionally independent.

An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

The genomic analysis of erythrocyte microRNA expression in sickle cell diseases.

BACKGROUND: Since mature erythrocytes are terminally differentiated cells without nuclei and organelles, it is commonly thought that they do not contain nucleic acids. In this study, we have re-examined this issue by analyzing the transcriptome of a purified population of human mature erythrocytes from individuals with normal hemoglobin (HbAA) and homozygous sickle cell disease (HbSS). METHODS AND FINDINGS: Using a combination of microarray analysis, real-time RT-PCR and Northern blots, we found that mature erythrocytes, while lacking ribosomal and large-sized RNAs, contain abundant and diverse microRNAs. MicroRNA expression of erythrocytes was different from that of reticulocytes and leukocytes, and contributed the majority of the microRNA expression in whole blood. When we used microRNA microarrays to analyze erythrocytes from HbAA and HbSS individuals, we noted a dramatic difference in their microRNA expression pattern. We found that miR-320 played an important role for the down-regulation of its target gene, CD71 during reticulocyte terminal differentiation. Further investigation revealed that poor expression of miR-320 in HbSS cells was associated with their defective downregulation CD71 during terminal differentiation. CONCLUSIONS: In summary, we have discovered significant microRNA expression in human mature erythrocytes, which is dramatically altered in HbSS erythrocytes and their defect in terminal differentiation. Thus, the global analysis of microRNA expression in circulating erythrocytes can provide mechanistic insights into the disease phenotypes of erythrocyte diseases.

Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.

Anatomical identification of extracellularly recorded cells in large-scale multielectrode recordings.

This study combines for the first time two major approaches to understanding the function and structure of neural circuits: large-scale multielectrode recordings, and confocal imaging of labeled neurons. To achieve this end, we develop a novel approach to the central problem of anatomically identifying recorded cells, based on the electrical image: the spatiotemporal pattern of voltage deflections induced by spikes on a large-scale, high-density multielectrode array. Recordings were performed from identified ganglion cell types in the macaque retina. Anatomical images of cells in the same preparation were obtained using virally transfected fluorescent labeling or by immunolabeling after fixation. The electrical image was then used to locate recorded cell somas, axon initial segments, and axon trajectories, and these signatures were used to identify recorded cells. Comparison of anatomical and physiological measurements permitted visualization and physiological characterization of numerically dominant ganglion cell types with high efficiency in a single preparation.

Efficacy of Pharmacokinetics-Directed Busulfan, Cyclophosphamide, and Etoposide Conditioning and Autologous Stem Cell Transplantation for Lymphoma: Comparison of a Multicenter Phase II Study and CIBMTR Outcomes.

Busulfan, cyclophosphamide, and etoposide (BuCyE) is a commonly used conditioning regimen for autologous stem cell transplantation (ASCT). This multicenter, phase II study examined the safety and efficacy of BuCyE with individually adjusted busulfan based on preconditioning pharmacokinetics. The study initially enrolled Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients ages 18 to 80 years but was amended due to high early treatment-related mortality (TRM) in patients > 65 years. BuCyE outcomes were compared with contemporaneous recipients of carmustine, etoposide, cytarabine, and melphalan (BEAM) from the Center for International Blood and Marrow Transplant Research. Two hundred seven subjects with HL (n = 66) or NHL (n = 141) were enrolled from 32 centers in North America, and 203 underwent ASCT. Day 100 TRM for all subjects (n = 203), patients > 65 years (n = 17), and patients ≤ 65 years (n = 186) were 4.5%, 23.5%, and 2.7%, respectively. The estimated rates of 2-year progression-free survival (PFS) were 33% for HL and 58%, 77%, and 43% for diffuse large B cell lymphoma (DLBCL; n = 63), mantle cell lymphoma (MCL; n = 29), and follicular lymphoma (FL; n = 23), respectively. The estimated rates of 2-year overall survival (OS) were 76% for HL and 65%, 89%, and 89% for DLBCL, MCL, and FL, respectively. In the matched analysis rates of 2-year TRM were 3.3% for BuCyE and 3.9% for BEAM, and there were no differences in outcomes for NHL. Patients with HL had lower rates of 2-year PFS with BuCyE, 33% (95% CI, 21% to 46%), than with BEAM, 59% (95% CI, 52% to 66%), with no differences in TRM or OS. BuCyE provided adequate disease control and safety in B cell NHL patients ≤ 65 years but produced worse PFS in HL patients when compared with BEAM.

Polyploidy and Mitotic Cell Death are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells

Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.

Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.

Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Non-Pharmacological Approaches for Pain Management in Sickle Cell Disease: Development of a Mindfulness-Based Intervention

Background: Sickle Cell Disease (SCD) is a genetic hematological disorder that affects more than 7 million people globally (NHLBI, 2009). It is estimated that 50% of adults with SCD experience pain on most days, with 1/3 experiencing chronic pain daily (Smith et al., 2008). Persons with SCD also experience higher levels of pain catastrophizing (feelings of helplessness, pain rumination and magnification) than other chronic pain conditions, which is associated with increases in pain intensity, pain behavior, analgesic consumption, frequency and duration of hospital visits, and with reduced daily activities (Sullivan, Bishop, & Pivik, 1995; Keefe et al., 2000; Gil et al., 1992 & 1993). Therefore effective interventions are needed that can successfully be used manage pain and pain-related outcomes (e.g., pain catastrophizing) in persons with SCD. A review of the literature demonstrated limited information regarding the feasibility and efficacy of non-pharmacological approaches for pain in persons with SCD, finding an average effect size of .33 on pain reduction across measurable non-pharmacological studies. Second, a prospective study on persons with SCD that received care for a vaso-occlusive crisis (VOC; N = 95) found: (1) high levels of patient reported depression (29%) and anxiety (34%), and (2) that unemployment was significantly associated with increased frequency of acute care encounters and hospital admissions per person. Research suggests that one promising category of non-pharmacological interventions for managing both physical and affective components of pain are Mindfulness-based Interventions (MBIs; Thompson et al., 2010; Cox et al., 2013). The primary goal of this dissertation was thus to develop and test the feasibility, acceptability, and efficacy of a telephonic MBI for pain catastrophizing in persons with SCD and chronic pain.

Methods: First, a telephonic MBI was developed through an informal process that involved iterative feedback from patients, clinical experts in SCD and pain management, social workers, psychologists, and mindfulness clinicians. Through this process, relevant topics and skills were selected to adapt in each MBI session. Second, a pilot randomized controlled trial was conducted to test the feasibility, acceptability, and efficacy of the telephonic MBI for pain catastrophizing in persons with SCD and chronic pain. Acceptability and feasibility were determined by assessment of recruitment, attrition, dropout, and refusal rates (including refusal reasons), along with semi-structured interviews with nine randomly selected patients at the end of study. Participants completed assessments at baseline, Week 1, 3, and 6 to assess efficacy of the intervention on decreasing pain catastrophizing and other pain-related outcomes.

Results: A telephonic MBI is feasible and acceptable for persons with SCD and chronic pain. Seventy-eight patients with SCD and chronic pain were approached, and 76% (N = 60) were enrolled and randomized. The MBI attendance rate, approximately 57% of participants completing at least four mindfulness sessions, was deemed acceptable, and participants that received the telephonic MBI described it as acceptable, easy to access, and consume in post-intervention interviews. The amount of missing data was undesirable (MBI condition, 40%; control condition, 25%), but fell within the range of expected missing outcome data for a RCT with multiple follow-up assessments. Efficacy of the MBI on pain catastrophizing could not be determined due to small sample size and degree of missing data, but trajectory analyses conducted for the MBI condition only trended in the right direction and pain catastrophizing approached statistically significance.

Conclusion: Overall results showed that at telephonic group-based MBI is acceptable and feasible for persons with SCD and chronic pain. Though the study was not able to determine treatment efficacy nor powered to detect a statistically significant difference between conditions, participants (1) described the intervention as acceptable, and (2) the observed effect sizes for the MBI condition demonstrated large effects of the MBI on pain catastrophizing, mental health, and physical health. Replication of this MBI study with a larger sample size, active control group, and additional assessments at the end of each week (e.g., Week 1 through Week 6) is needed to determine treatment efficacy. Many lessons were learned that will guide the development of future studies including which MBI strategies were most helpful, methods to encourage continued participation, and how to improve data capture.

IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.