Tim-1 (T cell immunoglobulin mucin domain 1) is a transmembrane protein expressed by many cell types, including activated T cells and B cells. Antibodies to Tim-1 have been shown to decrease severity of airway hyperreactivity and Th2 cytokine production in mice. Current literature suggests Tim-1 functions as a co-stimulatory molecule. We hypothesize that Tim-1 signals in lymphocytes, and that Tim-1 signaling modulates allergic airway disease. Chapter one provides a brief overview of current literature exploring identification of the Tim family of receptors, genetic associations between TIM-1 polymorphisms and human diseases, Tim-1 expression, Tim-1 ligands, studies of antibodies to Tim-1 in various mouse models of human disease, and signaling events downstream of Tim-1 engagement. Chapter two provides detailed experimental methodology. Chapter three details the characterization of Tim-1 deficient mice. Tim-1 deficient mice do not exhibit defects in lymphocyte or myeloid cell development, as determined by numbers of cells present in bone marrow, thymus, spleen, and lymph nodes. C57BL/6 Tim-1 deficient female mice appear to develop an increased number of lymph node cells and also develop anti-double stranded DNA antibodies. Chapter four explores the impact of Tim-1 deficiency in a murine allergic airway disease model, which demonstrated that Tim-1 deficient mice developed increased lung inflammation and increased antigen-specific Th2 cytokine production that was evident in mice backcrossed to both BALB/c and C57BL/6 backgrounds. These phenotypes were not evident using purified naïve CD4+ T cells polarized in vitro. As Tim-1 expression is not restricted to CD4+ T cells, adoptive transfer experiments were performed to determine whether the phenotype observed was due to the deficiency of Tim-1 on CD4+ T cells, non-CD4+ T cells, or Tim-1 deficiency on both CD4+ T cells and non-CD4+ T cells. Chapter five explores the impact of Tim-1 deficiency in a chronic Leishmania major intradermal infection model. Tim-1 deficient mice crossed to both BALB/c and C57BL/6 backrounds demonstrated similar parasite burden over the course of time, but in vitro restimulation of lymph node cells revealed a striking increase in cytokine production that extended to Th1, Th2, and Th17 lineages. Tim-1 signaling in murine B cell lines is explored in Chapter six. A Tim-1 monoclonal antibody conjugated to beads induces phosphorylation of Tim-1 and recruitment of the Src family kinase Fyn. This phosphorylation of Tim-1 is reduced in Fyn-deficient B cell lines. Chapter seven discusses the significance of these findings, relates current literature to these results, and provides some avenues for further exploration of Tim-1 function and signaling.

In recent years, there has been a worldwide increase in obesity, which parallels a rise in pathologies, including type 2 diabetes, collectively termed the metabolic syndrome. Chronic, low-grade inflammation has been implicated as a major link between these diseases. Recent work showed the presence of a unique subset of CD4+Foxp3+ regulatory T cells residing in visceral adipose tissue (VAT Treg) with PPAR-g being the key transcription factor responsible for their phenotype and function in controlling adipose tissue inflammation and, thereby, insulin sensitivity. VAT Tregs inversely correlated with insulin resistance. In contrast, there was a dramatic age-associated increase in frequency of VAT Tregs in lean animals, correlating with continued insulin sensitivity, despite significant increases in body and adipose tissue weights. This increase in Treg frequencies was not observed in other lymphoid and non-lymphoid tissues, including the subcutaneous fat depot. We characterized this unique age-associated increase in VAT Tregs through the use of adoptive transfer models, in vivo labeling and tracking systems, parabiosis, and analysis of the T cell receptor (TCR) repertoire used by VAT Tregs. Our findings indicate that the progressive increase in VAT Tregs is not due to conversion of conventional CD4+ T cells nor to substantial infiltration of Tregs from the circulation and secondary lymphoid organs. However, by analyzing the TCR repertoire on a single-cell level we uncovered a striking oligo-clonal expansion of VAT Tregs, suggesting their accumulation results from in situ proliferation. We further showed that this accumulation is dependent on major histocompatibility complex (MHC) class II, but not on CD1d. Finally, we showed that IL-33 was able to induce proliferation of VAT Tregs. In parallel, we extended our analysis of TCR repertoire to the Treg population residing in skeletal muscle. In acute and chronic models of muscle injury, muscle-resident Tregs underwent a substantial clonal expansion, with a particular clone being detected in multiple individuals. Taken together these studies highlight the importance of proliferation as a mechanism of Treg accumulation in tissues in response to acute and chronic inflammation.

As infecções humanas com a filaria Mansonella ozzardi ocorrem em focos situados em regiões tropicais e subtropicais da América Central e do Sul e frequentemente coexistem com outras doenças endêmicas tropicais. Na Amazônia brasileira, as infecções são geralmente assintomáticas e a maior parte delas, consequentemente, deixam de ser diagnosticada. As filarioses crônicas, geralmente não tratadas, podem criar um ambiente imunorregulador, caracterizado pela expansão de linfócitos T produtores de IL-10, que mediam a supressão de respostas proliferativas de células T frente a antígenos específicos bem como a antígenos não-relacionados. Neste trabalho, utilizamos marcadores de ativação celular (CD69 e HLA-DR) e de atividade reguladora (CD39, CTLA-4, OX40, GITR, LAG3, PD-1, LAP-TGF-β e TNFRII) para caracterizar populações de células mononucleares de sangue periférico (PBMCs) em indivíduos infectados por M. ozzardi bem como em controles saudáveis de uma área endêmica deste parasito na Amazônia Brasileira. A análise de PBMCs, por citometria de fluxo multiparamétrica de 49 pacientes infectados por M. ozzardi, mostrou que pacientes e controles apresentam proporções similares de Treg clássicas circulantes, no entanto, indivíduos infectados apresentam um aumento da proporção de células CD4+ e células T reguladoras (Tregs) que expressam a molécula CD39. Células Treg CD39+ parecem definir uma população distinta entre as Treg, pois ao compararmos os marcadores de regulação e ativação entre Tregs CD39+ e CD39- encontramos proporções aumentas destes marcadores nas Treg CD39+. O bloqueio dessa molécula em condições de reestimulo celular aumenta a produção de citocinas inflamatórias e diminui a produção de IL-10 confirmando seu papel regulador. / Human infections with the filarial parasite Mansonella ozzardi are common in areas of tropical and subtropical Central and South America and often coexist with other endemic tropical diseases, such as malaria. In the Amazonian Basin of Brazil, infections are typically asymptomatic; most of them will remain undiagnosed. These chronic, untreated filarial infections are potentially associated with a regulatory immune environment, dominated by IL-10-producing T-cells, which mediate the suppression of T-cell proliferation in response to filarial and non-related antigens. Here, we used markers of cell activation (CD69 and HLA-DR) and regulatory activity (CD39, CTLA-4, OX40, GITR, and TNFRII) to characterize peripheral-blood mononuclear cell (PBMCs) subpopulations in individuals infected with |M. ozzardi and in healthy controls living in an area of M. ozzardi endemicity in the Brazilian Amazon. Multiparameter flow cytometry analysis of PBMCs from 49 malaria patients showed that patients and controls have similar proportions of classic circulating Tregs, however, the proportion of CD4 + cells and Tregs expressing the CD39 (an ectonucleotidase that regulates the balance of immune responses through Phosphohydrolysis of ATP, an inflammatory molecule in adenosine, an anti-inflammatory molecule), is increased in infected patients. CD39+Treg cells seem to define a distinct population among Tregs, compare activation and regulatory markers between CD39+ and CD39- Tregs - we found increased proportions of these markers in the CD39+ Tregs. Blocking this molecule under cellular restimulation conditions increases production of inflammatory cytokines and decreases IL-10 production, improving its regulatory role.

No description available.

Many events associated with Human Immunodeficiency Virus (HIV) infection/replication occur in and around the germinal centers (GCs) of secondary lymphoid tissues where follicular dendritic cells (FDCs) reside, suggesting that this microenvironment may contribute unique signaling that is important to viral progression. My research focused on characterizing signaling, both positive and negative, contributed by FDCs that affects HIV infection and replication. Specifically, I determined if FDC signals could induce the expression of latent HIV in T cells and if so, to characterize the signaling pathways involved. Moreover, I also examined the ability of FDCs to produce inhibitory signals that might block active virus expression. I approached these problems using FDCs from tonsils and coculturing these with primary CD4+ T cells or latently-infected Jurket cells with a GFP reporter. Results indicated that FDCs dramatically augmented HIV production of these cells. FDC signaling was costimulatory in nature and was mediated by soluble TNFα. However, when ex vivo latently infected T cells were treated with PMA/ionomycin or IL2/IL7, little virus expression was observed until FDCs were added, which greatly increased virus production. The transcription factor NFAT is important for the reactivation of latent HIV. Inhibition studies as well as ELISA suggested that JAK/STAT signaling pathway was involved in virus reactivation. Because FDCs produce prostaglandins (PGs) E2 and I2, I determined the effect of PGE2 and PGI2 analogs on HIV infected T cells. Results indicated that both the PGE2 and PGI2 analogs inhibited proliferation and activation-induced cell death of HIV infected T cells in a dose- and time-dependent manner. Additionally, it was shown that indomethacin and CAY10404, cyclooxygenase and cyclooxygenase-2 inhibitors, partially restored HIV production in the presence of FDCs, suggesting that FDC-produced PGE2 and PGI2 may inhibit virus replication. Thus, FDCs produce PGs that can block virus gene expression in T cells, which may be ideal for viral persistence. Therefore, FDC signaling appears to both promote and inhibit HIV production. A better understanding of FDC signaling and regulation in GCs may suggest new treatment strategies that would be beneficial to infected subjects.

No description available.

No description available.

CD4 T cell homeostasis and competency are critical for the effectiveness of antiviral immunity. However, CD4 T cells derived from people living with HIV (PLWH) and individuals with chronic HCV infection often exhibit an inflammaging phenotype, evidenced by persistent inflammation, immune activation, exhaustion, senescence, and cellular apoptosis. Despite intensive investigations, the molecular mechanisms underlying CD4 T cell dysfunction in antiretroviral therapy (ART)-controlled PLWH and HCV-infected patients remain poorly understood. By investigating the roles of non-coding (nc)RNA transcripts in regulating the functions of CD4 T cells derived from PLWH and HCV-infected patients, we demonstrated that long non-coding (lnc)RNA - growth arrest-specific transcript 5 (GAS5) - is downregulated and plays a crucial role in regulating CD4 T cell functions through and beyond the microRNA (miR)-21-mediated signaling network. Our data suggest that disrupting the GAS5-miR21 axis may restore CD4 T cell homeostasis and competency during latent HIV infection and prevent premature CD4 T cell aging or immune senescence. Moreover, our results also showed that TRF2, a component of the shelterin complex maintaining the integrity of telomeres, is post-transcriptionally inhibited, which is one of the major forces driving cellular dysregulation in CD4 T cells from PLWH and HCV patients. Importantly, our study identified miR-23a as the key regulator of TRF2 translational expression by targeting its 3’UTR in CD4 T cells and that targeting miR-23a may restore the TRF2 protein level, and thereby reconstitute CD4 T cell homeostasis and competency to rescue CD4 T cells from premature aging and immunosenescence during latent HIV infection. The findings from these studies improved our understanding and knowledge of how ncRNA-mediated networks regulate the functions of CD4 T cells during chronic viral (HIV and HCV) infections. Understanding such mechanisms is important for developing therapeutic approaches to reverse the inflammaging phenotype observed in CD4 T cells from ART-controlled PLWH and chronically HCV-infected patients to improve their immunological functions and quality of life.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: Reactivation of human cytomegalovirus (HCMV) infection in individuals infected with human immunodeficiency virus (HIV) may lead to life-threatening end-organ diseases (EOD). The EOD becomes clinically apparent when a critical number of cells in the affected organs become damaged as a consequence of HCMV-infection. Treatment of the HCMV-associated disease at this point may not be effective. Therefore, early detection of HCMV reactivation may be useful to guide pre-emptive therapy. Aim: The aim of this study was to determine whether there is a point at which the HCMV-specific cellular immune response breaks down, as determined by the interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay, and HCMV reactivation occurs in HIV-positive, antiretroviral therapy (ART)-naïve individuals in a South African setting. This was done in relation to the CD4+ T cell count and the HCMV viral load as determined by real-time polymerase chain reaction (qPCR). Materials and methods: Fifty-two (52) HIV-infected, ART-naïve subjects were recruited from primary healthcare centres that they attended for the management of their HIV infection. Heparinised blood samples were collected to quantify the HCMV-specific cellular immune response using the IFN-γ-ELISPOT assay and to determine the HCMV IgG serostatus. Ethylenediaminetetraacetic acid (EDTA) blood samples were collected for the determination of the CD4+ T cell counts and the HCMV viral loads. Results: All 52 subjects recruited were confirmed to be HIV-HCMV co-infected based on their HCMV IgG serostatus. The results of 34 subjects with completed data sets were analysed. The CD4+ T cell counts of these subjects ranged from 10 to 784 cells/μl. Twenty-two (22) (65%) subjects had positive HCMV IFN-γ-ELISPOT results with 94% having no detectable HCMV viral loads. All subjects (28) with a CD4+ T cell count above 100 cells/μl had undetectable HCMV viral loads. Two of the six subjects with CD4+ T cell counts <100 cells/μl had detectable HCMV viral loads. There was no statistically significant association between the CD4+ T cell counts and the HCMV IFN-γ-ELISPOT results. Conclusion: No specific point could be determined when there is loss of integrity of the HCMV-specific cellular immune response in HIV-positive individuals. Low CD4+ T cell counts did not correlate with HCMV IFN-γ-ELISPOT results suggesting that the HCMV-specific cellular immunity did not necessarily break down at low CD4+ T cell counts. Nevertheless, a CD4+ T cell count above 100 cells/μl appeared to be protective against viraemia as determined by the HCMV viral load qPCR. The IFN-γ-ELISPOT assay was employed as a tool to determine the integrity of the HCMV-specific cellular immune response in HIV-positive individuals. However, the IFN-γ-ELISPOT assay should be used in conjunction with the CD4+ T cell count and the HCMV viral load qPCR to determine when there is loss of integrity of the HCMV-specific cellular immune response and HCMV reactivation occurs. This may assist clinicians in their choice of management and appropriate pre-emptive treatment in the HIV-HCMV co-infected individual at a risk for HCMV reactivation. / AFRIKAANSE OPSOMMING: Inleiding: Heraktivering van menslike sitomegaalvirus (MSMV) in menslike immuniteitsgebreksvirus (MIV)-MSMV ko-geïnfekteerde individue kan lei tot dodelike end-orgaan siektes (EOS). Die EOS word klinies duidelik wanneer 'n kritieke aantal selle in die organe beskadig raak as gevolg van die MSMV-infeksie. Behandeling van die MSMV-verwante siekte op hierdie punt mag moontlik nie meer effektief wees nie. Daarom kan die vroeë opsporing van MSMV heraktivering nuttig wees in die gebruik van voorkomende terapie. Doel: Die doel van hierdie studie is om die punt te bepaal wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word met behulp van die interferon gamma (IFN-γ) ensiem-gekoppelde immunospot (ELISPOT) toets en MSMV heraktivering voorkom in MIV-positiewe, antiretrovirale terapie (ART)-naïewe individue in' n Suid-Afrikaanse instelling. Dit word gedoen in verhouding met die CD4+ T sel telling en die MSMV virale lading. Materiale en metodes: Twee-en-vyftig (52) MIV-geïnfekteerde, ART-naïewe pasiënte is vanaf primêre gesondheidsentrums, wat hul bywoon vir die behandeling van hul MIV infeksie, genader. Gehepariniseerde bloedmonsters is gebruik om die MSMV-spesifieke sellulêre immuun reaksie met behulp van die IFN-γ-ELISPOT toets en die MSMV IgG serostatus te bepaal. Etileendiamientetra-asynsuur (EDTA) bloed monsters is versamel vir die bepaling van hul CD4+ T sel telling en hul MSMV virale lading met behulp van die ―real-time‖ polimerase kettingreaksie (qPKR) waardes. Resultate: Al 52 pasiënte is bevestigde MIV-MSMV ko-infeksies, gebasseer op hul serologiese status. Die resultate van 34 pasiënte met voltooide data is ontleed. Die CD4+ T sel tellings van hierdie pasiënte het gewissel 10-784 selle/μl. Twee-en-twintig (22) (65%) pasiënte het positiewe MSMV IFN-γ-ELISPOT resultate met 94% wat ‗n negatiewe qPKR resultate. Alle pasiënte (28) met 'n CD4+ T-seltelling bo 100 selle/μl het' n negatiewe qPKR resultate. Twee van die ses pasiënte met 'n CD4+ T-seltelling <100 selle/μl het waarneembare MSMV virale ladings oor die qPKR. Daar was geen statisties beduidende assosiasie tussen die CD4+ T sel tellings en die MSMV IFN-γ-ELISPOT resultate nie. Gevolgtrekking: Geen spesifieke punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word kon in MIV-positiewe individue bepaal word nie. Lae CD4+ T sel tellings het nie ooreengestem met die MSMV IFN-γ-ELISPOT resultate nie en dui daarop dat die MSMV-spesifieke sellulêre immuniteit nie noodwendig afgebreek word teen 'n lae CD4+ T sel tellings nie. Tog blyk 'n CD4+ T-seltelling bo 100 selle/μl om beskerming teen viremie te bied. Die meriete van die gebruik van die IFN-γ-ELISPOT toets die integriteit van die MSMV-spesifieke sellulêre immuun response in MIV-positiewe individue te bepaal, is waargeneem in die opgehoopte data. Tog kan die gebruik van die IFN-γ-ELISPOT toets in samewerking met die CD4+ T sel telling en die MSMV virale lading meer voordelig in die bepaling van 'n punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afbreek en herstel plaasvind. Dit kan help om klinici in hul keuse van bestuur en gepaste voorkomende behandeling in die MIV-MSMV mede-geïnfekteerde individu op 'n risiko vir herstel.

HIV remains an incurable viral infection and a significant global health concern. Despite the advent of antiretroviral therapy, there are 36.9 million recorded cases of HIV worldwide, with an additional 1.8 million new infections recorded in 2017 alone. An HIV cure is therefore one of several priorities within the field, and will require HIV “reservoir” cells—comprised of latently-HIV infected CD4+ T cells and productively-infected, tissue resident macrophages—to be selectively killed in vivo. HIV reservoir cells are rarely found within the peripheral circulation, residing instead within inaccessible tissue sanctuaries. Consequently, their characterization has been limited to in vitro laboratory models. To complicate matters further, a definitive cellular surface marker of HIV infected cells has yet to be identified. Impairment of the type I interferon (IFN1) response has been observed during HIV infection, however, making it a unique intracellular maker of HIV-infected cells. The recent development of oncolytic viruses (OV) designed to selectively kill IFN-defective cancer cells also suggests that these IFN1 defects possess therapeutic value. It was therefore hypothesized that the impairment of the IFN1 response in HIV-infected CD4+ cells and macrophages could serve as a target for oncolytic virus-mediated killing. The induction of several antiviral IFN-stimulated proteins, including PKR and ISG15, was inhibited in HIV-infected monocyte-derived macrophages (MDM) following stimulation with IFNα or a synthetic RNA. Consequently, HIV-infected MDM were more susceptible to infection and killing by the oncolytic Maraba virus, MG1. Importantly, MG1-mediated killing required the presence of replication-competent OV, and could not be potentiated by UV-inactivated MG1 or supernatants from MG1-infected cells. The ability of MG1 to target the HIV reservoir was further confirmed using alveolar macrophages collected from the lungs of cART-suppressed individuals living with HIV. These findings indicate that IFN1 defects are a feature of HIV infected cells, which can be exploited for selective killing by OV. This project is therefore unique in that it demonstrates that HIV reservoir cells can be eradicated in a targeted manner by exploiting an intracellular marker of HIV infection. As MG1-based cancer therapies are currently being explored in Phase I/II clinical trials, there is potential for this approach to be adapted for use within the HIV cure field.