In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Endogenous Retroviruses and the Immune System

Initial sequencing of the human and mouse genomes revealed that substantial fractions were composed of retroelements (REs) and endogenous retroviruses (ERVs), the latter being relics of ancestral retroviral infection. Further study revealed ERVs constitute up to 10% of many mammalian genomes. Despite this abundance, comparatively little is known about their interactions, beneficial or detrimental, with the host. This thesis details two distinct sets of interactions with the immune system. Firstly, the presentation of ERV-derived peptides to developing lymphocytes was shown to exert a control on the immune response to infection with Friend Virus (FV). A self peptide encoded by an ERV negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and resulted in an impaired immune response. However, CD4+ T cell-mediated antiviral activity was fully preserved and repertoire analysis revealed a deletional bias according to peptide affinity, resulting in an effective enrichment of high-affinity CD4+ T cells. Thus, ERVs exerted a significant influence on the immune response, a mechanism that may partially contribute to the heterogeneity seen in human immune responses to retroviral infections. Secondly, a requirement for specific antibodies was shown in the control of ERVs. In a range of mice displaying distinct deficiencies in antibody production, products from the intestinal microbiota potentially induce ERV expression. Subsequent recombinational correction of a defective murine leukaemia virus (MLV) results in the emergence of infectious virus. In the long term, this leads to retrovirus-induced lymphomas and morbidity. ERVs, therefore, provide a potential link between the intestinal microbiota and a range of pathologies, including cancer. Finally, a new computational tool, REquest, was developed for use in the above studies. REquest allows the mining of retroelement (RE) and ERV expression data from the majority of commercially available human and murine microarray platforms and allows rapid hypothesis testing with publicly available data

Analysis of MHC-I homologues in molluscum contagiosum and human cytomegalovirus

Molluscum contagiosum virus (MOCV) and human cytomegalovirus (HCMV) each encode two MHC-I homologues: mc080 and mc033 in MOCV, UL18 and UL142 in HCMV. Codon optimised versions of the mc080 and mc033 genes were cloned in to a second-generation lentivirus system, vaccinia virus and a replicationdeficient adenovirus (Ad) vector. MOCV mc033 and mc080 were expressed from the Ad vector as endoglycosidase H (EndoH) sensitive glycoproteins with apparent molecular weights of 64kDa and 44kDa respectively, both localised predominantly to the ER. MC033 had no effect on either MHC-I or HLAE expression, nor did it impact NK cells or T cell function. MC080 downregulated cell surface expression of classical HLA-I and HLA-E in a TAP independent, although not MICA/B. This downregulation of HLA-1 correlated with protection against CD8+ T cell activation, thus MC080 is here identified as a novel viral T cell evasion function. MC080 was capable of supressing or activating NK cell, depending on context and consistent with MC080’s control of HLA-I and HLA-E. UL142 expression was enhanced by optimising codon usage and through provision of an alternative leader sequence. Using an Ad vector, UL142 was expressed as a heavily glycosylated 105kDa protein that localised to the ER, cell surface and was released into the supernatant in an EndoH-resistant form. When over-expressed from the Ad vector UL142 suppressed full length MICA, but not in the context of a productive HCMV infection where that function was performed by the US12 gene family. Preliminary allogenic functional NK cell assays showed UL142 to suppress NK cell function in one of four donors. However, cell-associated and secreted UL142 both activated NK cell degranulation when the assay was moved to an autologous setting. The results suggest that UL142 can modulate NK cell function independently of MICA

Investigations of tetraspanin functions using large extracellular loops

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.This thesis describes our characterization of a specific tetraspanin domain: the large extracellular loop (LEL). Tetraspanins are involved in cellular migration, adhesion, and metastasis, sperm-egg fusion, and viral infectivity. The large extracellular loop domain is the major extracellular domain of tetraspanins and the binding of a monoclonal antibody against the tetraspanin CD9 serves to inhibit fertilization, consistent with the CD9-null mouse model. The first area of focus in this thesis is the characterization of the murine CD9-LEL domain. We present a methodology to express and purify the mCD9-LEL to homogeneity. Biophysical characterization of the mCD9-LEL protein reveals that it is an autonomously folding, [alpha]-helical dimer. Mutagenesis over much of the mCD9-LEL protein reveals that it is composed of two subdomains: a dimerization subdomain and a variable subdomain proposed to mediate heterotypic interactions. These results suggest both a means for exploring endogenous tetraspanins functions and a mechanism by which tetraspanins may oligomerize. Surprisingly, we were not able to detect oligomerization of the intact CD9 molecule, in discordance with our biophysical data on the mCD9-LEL.(cont.) In the latter part of this thesis, we expand our methodology to purify and characterize three different tetraspanins-LELs, the hCD9-LEL, the hCD63-LEL, and the hCD8 1-LEL. These tetraspanins-LELs all exhibit similar characteristics to the mCD9-LEL, consistent with a published crystal structure of the hCD81-LEL. Lastly, we demonstrate the ability of our tetraspanin-LEL proteins to bind integrins, to inhibit sperm-egg fusion, and to inhibit hepatitis C viral infectivity. Taken as a whole, these studies present novel, biophysically validated tetraspanins-LELs that lend insight into endogenous tetraspanins functions.by Christopher C. Liu.Ph.D

The role of T cell specific factors and RNA Polymerase II pausing in HIV-1 replication in CD4+ T cells

In order to eradicate HIV-1 infection the virus needs to be specifically eliminated from latently infected memory CD4+ T cells. There does not seem to be a single mechanism that promotes HIV-1 latency. RNA Polymerase II (RNAP II) pausing, chromatin structure, tissue specific transcriptional repressors and transcriptional interference have been implicated in regulating HIV-1 transcription. The transcription factor B Lymphocyte-Induced Maturation Protein 1 (Blimp-1) is expressed in B and T cells and upregulated in patients chronically infected with HIV-1. I hypothesized that Blimp-1 is a T cell intrinsic factor that binds to HIV-1 LTR, inhibits HIV-1 transcription and contributes to HIV-1 latency. Blimp-1 is expressed in primary peripheral blood CD4+ T cells and is further induced by T cell activation. Importantly, Blimp-1 is highly expressed in memory CD4+ T cells compared to naïve CD4+ T cells. Ectopic expression of Blimp-1 in CD4+ T cells represses HIV-1 transcription, whereas decreasing Blimp-1 in memory CD4+ populations activates HIV-1 transcription. Reduction of Blimp-1 in infected primary T cells increases RNAP II processivity and histone H3 acetylation. Blimp-1 binds downstream of the HIV-1 5'-LTR to the interferon-stimulated response element (ISRE) in resting primary CD4+ T cells and strongly represses Tat-dependent HIV-1 transcription. Upon T cell activation, Blimp-1 is released from the HIV-1 ISRE and this correlates with significant increase in HIV-1 transcription. These results demonstrate that Blimp-1 acts to limit HIV-1 transcription in memory CD4+ T cells and promotes the establishment and maintenance of latency. I also examined whether neighboring host promoters could impact HIV-1 transcription. Using a set of inducible cell lines I observed that neighboring promoters have minimal impact on HIV-1 transcription and that enabling release of paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus. The implications of my results in the different mechanisms regulating HIV-1 latency are discussed

Factors affecting replication and cross-species transmission of feline immunodeficiency virus

In order to successfully invade a new species, lentiviruses must overcome restriction factors, dominant blocks to replication, and be able to make use of available host factors such as entry receptors for replication. Human immunodeficiency virus (HIV-1) infection is blocked at a post-entry stage by rhesus macaque TRIM5α, an effect that is enhanced by host factor cyclophilin A (CypA), but largely evades restriction by the human TRIM5α variant. HIV-1 replication is also inhibited by simian APOBEC3G but is able to evade restriction by human APOBEC3G by inducing its degradation through expression of accessory protein Vif. Viral entry and tissue tropism are determined by an interaction of the viral Env glycoprotein with a cell surface receptor and a seven transmembrane domain co-receptor. The feline immunodeficiency virus (FIV) infects diverse felid species including the African lion, where infection has likely been endemic since at least the late Pleistocene, and the domestic cat, a more recent host. For domestic cat strains of FIV (FIV-Fca), entry is mediated by host proteins CD134 and CXCR4, but the identity of receptors in non-domestic strains of FIV is unknown. This thesis demonstrates that two strains of FIV isolated from lions (FIV-Ple subtypes B and E) differ in their receptor tropism and that subtype E shares entry receptors with FIV-Fca. The findings suggest that alternative receptor usage is a strategy employed by FIV in this species and has implications for the disputed pathology and tissue tropism of infection in African lions. Next, we tested the hypothesis that species which have harboured lentiviral infection for a long time are better able to prevent viral replication than recent hosts. Whilst we found that TRIM5α is non-functional in all felid species tested, evidence of potent APOBEC3 activity was found and, in lion cells, potently restricts production of infectious FIV. Moreover, lion primary T-cells prevent replication of diverse FIV strains and restrict primate lentiviruses at post-entry stages, suggesting that co-evolution with lentiviruses has driven the selection of broad-ranging restriction factors. Structural and biophysical analyses suggest that whilst FIV’s interaction with CypA appears to be conserved in affinity with HIV-1, the interaction does not appear to be crucial for replication and, in the absence of restriction by TRIM5α, the role of the capsid-CypA interaction is discussed. Overall the study explains the permissivity of domestic cat cells to retroviral infection and identifies FIV infection of lions as an example of host adaptation driven by current lentiviral infection

Sequence analysis of the merozoite surface protein 7 (PvMSP-7) multigene family: vaccine candidates for Plasmodium vivax

P. vivax is found predominantly in Asia and the emergence of resistance to antimalarial drug and insecticides are major challenges to control of vivax malaria. Control is further complicated by the dormant liver stage of P. vivax, which produces an asymptomatic parasite reservoir. Malaria vaccine development is recognised as the most efficient control intervention globally. Currently, malaria vaccine development is concentrated in P. falciparum and RTS,S has been the most promising vaccine. This has encouraged similar initiatives to develop a P. vivax vaccine. The subject of this thesis is the Plasmodium vivax merozoite surface protein 7 (PvMSP-7), which forms a multi-protein complex with other merozoite surface proteins and plays a principal role in erythrocyte invasion. Studies in P. falciparum have shown that targeting MSP-7 can impair erythrocyte invasion and regulate disease severity. For this reason, PvMSP-7 is a plausible vaccine candidate. However, several questions remain to be addressed before a vaccine can be developed; (i) the antigenic variation and expression pattern of the PvMSP-7 multigene family, (ii) which PvMSP-7 paralog is the most promising target, and (iii) which domain of the protein is most antigenically relevant. The main aim of this thesis is to characterise the structural and expression variation in PvMSP-7 paralogs in the Thai clinical setting, to pinpoint the optimal PvMSP-7 antigen for malaria vaccine development. The conclusion of the thesis is that, of the 13 PvMSP-7 paralogs, PvMSP-7A is the most promising malaria vaccine candidate, being least polymorphic across parasite populations, expressed throughout the bloodstream infection cycle, and containing the greatest number of immunogenic B-cell epitopes. These immunogenic epitopes may confer a two-fold advantage in eliciting immunity and by impairing host cell invasion. This thesis provides a basis for the development of PvMSP-7A as an experimental vaccine, leading to the sustainable prevention of vivax malaria across the world

Going viral : an integrated view on virological data analysis from basic research to clinical applications

Viruses are of considerable interest for several fields of life science research. The genomic richness of these entities, their environmen- tal abundance, as well as their high adaptability and, potentially, pathogenicity make treatment of viral diseases challenging. This thesis proposes three novel contributions to antiviral research that each concern analysis procedures of high-throughput experimen- tal genomics data. First, a sensitive approach for detecting viral genomes and transcripts in sequencing data of human cancers is presented that improves upon prior approaches by allowing de- tection of viral nucleotide sequences that consist of human-viral homologs or are diverged from known reference sequences. Sec- ond, a computational method for inferring physical protein contacts from experimental protein complex purification assays is put for- ward that allows statistically meaningful integration of multiple data sets and is able to infer protein contacts of transiently binding protein classes such as kinases and molecular chaperones. Third, an investigation of minute changes in viral genomic populations upon treatment of patients with the mutagen ribavirin is presented that first characterizes the mutagenic effect of this drug on the hepatitis C virus based on deep sequencing data.Viren sind von beträchtlichem Interesse für die biowissenschaftliche Forschung. Der genetische Reichtum, die hohe Vielfalt, wie auch die Anpassungsfähigkeit und mögliche Pathogenität dieser Organismen erschwert die Behandlung von viralen Erkrankungen. Diese Promotionsschrift enthält drei neuartige Beiträge zur antiviralen Forschung welche die Analyse von experimentellen Hochdurchsatzdaten der Genomik betreffen: erstens, ein sensitiver Ansatz zur Entdeckung viraler Genome und Transkripte in Sequenzdaten humaner Karzinome, der die Identifikation von viralen Nukleotidsequenzen ermöglicht, die von Referenzgenomen ab- weichen oder homolog zu humanen Faktoren sind. Zweitens, eine computergestützte Methode um physische Proteinkontakte von experimentellen Proteinkomplex-Purifikationsdaten abzuleiten welche die statistische Integration von mehreren Datensätzen erlaubt um insbesondere Proteinkontakte von flüchtig interagierenden Proteinklassen wie etwa Kinasen und Chaperonen aus den Daten ableiten zu können. Drittens, eine Untersuchung von kleinsten Änderungen viraler Genompopulationen während der Behandlung von Patienten mit dem Mutagen ribavirin die zum ersten Mal die mutagene Wirkung dieses Medikaments auf das Hepatitis C Virus mittels Tiefensequenzdaten nachweist

Isolation and characterization of membrane vesicles secreted by human renal cells

Most cells release membrane vesicles for various purposes including, but not limited to, intercellular communication and disposal of membrane and soluble proteins. These vesicles are secreted into urine coming from the cells lining the urinary tract and bladder epithelium. These vesicles are a promising source of biomarkers for various cardiovascular and renal diseases. This thesis pursues twofold objectives, one being the development and improvement of an isolation method for urinary membrane vesicles and the second being proteomic characterization of the content of these vesicles. These objectives are important to realise the clinical potential of these vesicles. An alternative method for removal of contaminant high-abundant proteins was developed which preserves the activity of vesicular proteins. Moreover, lipid-affinity and lectin-affinity-based novel methods to enrich membrane vesicles from minimally processed urine were evaluated and developed. More than 600 proteins were identified in urinary membrane vesicles using shotgun proteomic analysis. Post-translational modification (PTM) proteomics was carried out to identify the PTM status of vesicular proteins. Many different PTMs like glycosylation, ubiquitination and palmitoylation were assessed. Surface glycan profiles of these vesicles were elucidated using fluorophore-linked lectin assay (FLLA) employing 18 different lectins. Lectin blotting, lectin-affinity chromatography using multiple lectins and hydrazide chemistry based enrichment of glycoproteins were carried out. As a result, 108 glycoproteins were identified. Immuno-affinity chromatography was used to enrich and identify ubiquitin-conjugated proteins present in urinary membrane vesicles. A number of potential palmitoylated proteins were identified as well. Computational prediction and validation methods were applied to these protein lists. In conclusion, novel methods to isolate urinary membrane vesicles were developed. In addition, a thorough proteomic characterisation of contents of urinary membrane vesicles was achieved. This work will serve as platform for further characterization of urinary membrane vesicles