Mechanisms of CD8+ T Cell Mediated Virus Inhibition in HIV-1 Virus Controllers

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

A miR-34a-Numb Feedforward Loop Triggered by Inflammation Regulates Asymmetric Stem Cell Division in Intestine and Colon Cancer.

Emerging evidence suggests that microRNAs can initiate asymmetric division, but whether microRNA and protein cell fate determinants coordinate with each other remains unclear. Here, we show that miR-34a directly suppresses Numb in early-stage colon cancer stem cells (CCSCs), forming an incoherent feedforward loop (IFFL) targeting Notch to separate stem and non-stem cell fates robustly. Perturbation of the IFFL leads to a new intermediate cell population with plastic and ambiguous identity. Lgr5+ mouse intestinal/colon stem cells (ISCs) predominantly undergo symmetric division but turn on asymmetric division to curb the number of ISCs when proinflammatory response causes excessive proliferation. Deletion of miR-34a inhibits asymmetric division and exacerbates Lgr5+ ISC proliferation under such stress. Collectively, our data indicate that microRNA and protein cell fate determinants coordinate to enhance robustness of cell fate decision, and they provide a safeguard mechanism against stem cell proliferation induced by inflammation or oncogenic mutation.

Antigen Drives Regulatory B10 Cell Development and Function

B cells mediate immune responses via the secretion of antibody and interactions with other immune cell populations through antigen presentation, costimulation, and cytokine secretion. Although B cells are primarily believed to promote immune responses using the mechanisms described above, some unique regulatory B cell populations that negatively influence inflammation have also been described. Among these is a rare interleukin (IL)-10-producing B lymphocyte subset termed “B10 cells.” B cell-derived IL-10 can inhibit various arms of the immune system, including polarization of Th1/Th2 cell subsets, antigen presentation and cytokine production by monocytes and macrophages, and activation of regulatory T cells. Further studies in numerous autoimmune and inflammatory models of disease have confirmed the ability of B10 cells to negatively regulate inflammation in an IL-10-dependent manner. Although IL-10 is indispensable to the effector functions of B10 cells, how this specialized B cell population is selected in vivo to produce IL-10 is unknown. Some studies have demonstrated a link between B cell receptor (BCR)-derived signals and the acquisition of IL-10 competence. Additionally, whether antigen-BCR interactions are required for B cell IL-10 production during homeostasis as well as active immune responses is a matter of debate. Therefore, the goal of this thesis is to determine the importance of antigen-driven signals during B10 cell development in vivo and during B10 cell-mediated immunosuppression.

Chapter 3 of the dissertation explored the BCR repertoire of spleen and peritoneal cavity B10 cells using single-cell sequencing to lay the foundation for studies to understand the full range of antigens that may be involved in B10 cell selection. In both the spleen and peritoneal cavity B10 cells studied, BCR gene utilization was diverse, and the expressed BCR transcripts were largely unmutated. Thus, B10 cells are likely capable of responding to a wide range of foreign and self-antigens in vivo.

Studies in Chapter 4 determined the predominant antigens that drive B cell IL-10 secretion during homeostasis. A novel in vitro B cell expansion system was used to isolate B cells actively expressing IL-10 in vivo and probe the reactivities of their secreted monoclonal antibodies. B10 cells were found to produce polyreactive antibodies that bound multiple self-antigens. Therefore, in the absence of overarching active immune responses, B cell IL-10 is secreted following interactions with self-antigens.

Chapter 5 of this dissertation investigated whether foreign antigens are capable of driving B10 cell expansion and effector activity during an active immune response. In a model of contact-induced hypersensitivity, in vitro B cell expansion was again used to isolate antigen-specific B10 clones, which were required for optimal immunosuppression.

The studies described in this dissertation shed light on the relative contributions of BCR-derived signals during B10 cell development and effector function. Furthermore, these investigations demonstrate that B10 cells respond to both foreign and self-antigens, which has important implications for the potential manipulation of B10 cells for human therapy. Therefore, B10 cells represent a polyreactive B cell population that provides antigen-specific regulation of immune responses via the production of IL-10.

Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate.

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCgamma and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.

The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.

Differentiation of mouse induced pluripotent stem cells (iPSCs) into nucleus pulposus-like cells in vitro.

A large percentage of the population may be expected to experience painful symptoms or disability associated with intervertebral disc (IVD) degeneration - a condition characterized by diminished integrity of tissue components. Great interest exists in the use of autologous or allogeneic cells delivered to the degenerated IVD to promote matrix regeneration. Induced pluripotent stem cells (iPSCs), derived from a patient's own somatic cells, have demonstrated their capacity to differentiate into various cell types although their potential to differentiate into an IVD cell has not yet been demonstrated. The overall objective of this study was to assess the possibility of generating iPSC-derived nucleus pulposus (NP) cells in a mouse model, a cell population that is entirely derived from notochord. This study employed magnetic activated cell sorting (MACS) to isolate a CD24(+) iPSC subpopulation. Notochordal cell-related gene expression was analyzed in this CD24(+) cell fraction via real time RT-PCR. CD24(+) iPSCs were then cultured in a laminin-rich culture system for up to 28 days, and the mouse NP phenotype was assessed by immunostaining. This study also focused on producing a more conducive environment for NP differentiation of mouse iPSCs with addition of low oxygen tension and notochordal cell conditioned medium (NCCM) to the culture platform. iPSCs were evaluated for an ability to adopt an NP-like phenotype through a combination of immunostaining and biochemical assays. Results demonstrated that a CD24(+) fraction of mouse iPSCs could be retrieved and differentiated into a population that could synthesize matrix components similar to that in native NP. Likewise, the addition of a hypoxic environment and NCCM induced a similar phenotypic result. In conclusion, this study suggests that mouse iPSCs have the potential to differentiate into NP-like cells and suggests the possibility that they may be used as a novel cell source for cellular therapy in the IVD.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

On the Origin of Natural Antibody

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

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.

Cryptococcal cell morphology affects host cell interactions and pathogenicity.

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

Impact of simian immunodeficiency virus infection on chimpanzee population dynamics.

Like human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus of chimpanzees (SIVcpz) can cause CD4+ T cell loss and premature death. Here, we used molecular surveillance tools and mathematical modeling to estimate the impact of SIVcpz infection on chimpanzee population dynamics. Habituated (Mitumba and Kasekela) and non-habituated (Kalande) chimpanzees were studied in Gombe National Park, Tanzania. Ape population sizes were determined from demographic records (Mitumba and Kasekela) or individual sightings and genotyping (Kalande), while SIVcpz prevalence rates were monitored using non-invasive methods. Between 2002-2009, the Mitumba and Kasekela communities experienced mean annual growth rates of 1.9% and 2.4%, respectively, while Kalande chimpanzees suffered a significant decline, with a mean growth rate of -6.5% to -7.4%, depending on population estimates. A rapid decline in Kalande was first noted in the 1990s and originally attributed to poaching and reduced food sources. However, between 2002-2009, we found a mean SIVcpz prevalence in Kalande of 46.1%, which was almost four times higher than the prevalence in Mitumba (12.7%) and Kasekela (12.1%). To explore whether SIVcpz contributed to the Kalande decline, we used empirically determined SIVcpz transmission probabilities as well as chimpanzee mortality, mating and migration data to model the effect of viral pathogenicity on chimpanzee population growth. Deterministic calculations indicated that a prevalence of greater than 3.4% would result in negative growth and eventual population extinction, even using conservative mortality estimates. However, stochastic models revealed that in representative populations, SIVcpz, and not its host species, frequently went extinct. High SIVcpz transmission probability and excess mortality reduced population persistence, while intercommunity migration often rescued infected communities, even when immigrating females had a chance of being SIVcpz infected. Together, these results suggest that the decline of the Kalande community was caused, at least in part, by high levels of SIVcpz infection. However, population extinction is not an inevitable consequence of SIVcpz infection, but depends on additional variables, such as migration, that promote survival. These findings are consistent with the uneven distribution of SIVcpz throughout central Africa and explain how chimpanzees in Gombe and elsewhere can be at equipoise with this pathogen.

Quality of Life and Neurocognitive Functioning in Children with Sickle Cell Disease: Investigating the Feasibility of a Computerized Cognitive Training Program

Children with sickle cell disease (SCD) have a high risk of neurocognitive impairment. No known research, however, has examined the impact of neurocognitive functioning on quality of life in this pediatric population. In addition, limited research has examined neurocognitive interventions for these children. In light of these gaps, two studies were undertaken to (a) examine the relationship between cognitive functioning and quality of life in a sample of children with SCD and (b) investigate the feasibility and preliminary efficacy of a computerized working memory training program in this population. Forty-five youth (ages 8-16) with SCD and a caregiver were recruited for the first study. Participants completed measures of cognitive ability, quality of life, and psychosocial functioning. Results indicated that cognitive ability significantly predicted child- and parent-reported quality of life among youth with SCD. In turn, a randomized-controlled trial of a computerized working memory program was undertaken. Eighteen youth with SCD and a caregiver enrolled in this study, and were randomized to a waitlist control or the working memory training condition. Data pertaining to cognitive functioning, psychosocial functioning, and disease characteristics were obtained from participants. The results of this study indicated a high degree of acceptance for this intervention but poor feasibility in practice. Factors related to feasibility were identified. Implications and future directions are discussed.

Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex.

The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.