The rate that HIV-infected individuals progress to AIDS and death varies greatly. Viral load taken during the asymptomatic phase of the disease is one of the best-known predictors of HIV progression rate and transmission risk, and is known to be in uenced by both host and environmental factors. However, the role that the virus itself plays in determining the viral load is less clear. Previous studies have attempted to quantify the amount the viral genome in uences viral load, or the heritability of viral load, using transmission pairs and phylogenetic signal in small sample sizes, but have produced highly disparate estimates. E cient and accurate methods to estimate heritability have been utilised by quantitative geneticists for years, but are rarely applied to non-pedigree data. Here, I present a novel application of a population-scale method based in quantitative genetics to estimate the heritability of viral load in HIV using a viral phylogeny. This new phylogenetic method allows the inclusion of more samples than ever previously used, and avoids confounding e ects associated with transmission pair studies. This new method was applied to the two largest HIV subtypes found in the UK, subtypes B and C, using sequences and clinical data from UK-wide HIV databases. For subtype B (n=8,483) and C (n=1,821), I estimated that 5.7% (CI 2.8{8.6%) and 29.7% (CI 14.8{44.7%) of the variance in viral load is determined by the viral genome, respectively. These estimates suggest that viral in uence on viral load varies greatly between subtypes, with subtype C having much larger viral control over viral load than subtype B. I expanded the phylogenetic method to test whether the component of the viral load determined by the virus has changed over time. In subtype B, I foundevidence of a small but signi cant decrease in the viral component of viral load of -0.05 log10 copies/mL/yr. I built a stochastic, individual-based model capable of simulating a realistic HIV epidemic, with heritable viral loads that in uence transmission and disease progression, capable of generating data sets to assess the accuracy of phylogenetic methods. This was successfully used to generate epidemics approximating those in a small African village and a Western `men who have sex with men' community under a variety of conditions. To test the accuracy of the new phylogenetic heritability estimation method, simulated datasets were generated with the heritability of viral load set at values of 30%, 50%, 70%, and 90%. Unfortunately, complications in the heritability equation used prevented full assessment of the new phylogenetic method on the simulated data. Future development of the model will enable simulation of realistic viral loads under varying heritability values, enabling simulation of data sets that can be used to test this and other heritability estimation methods. This new phylogenetic method allows accurate estimation of heritability in large datasets, and has provided valuable insight into the viral in uence on viral load in HIV.

A common characteristic of all chemical allergens, both respiratory and skin allergens, is the ability to form stable associations with proteins; the resulting hapten-protein complex being sufficient to provoke an immune response. There is evidence to suggest that selective binding of chemicals with proteins or peptides may impact on the quality of immune response that will develop. In the investigations described here, we have taken a reductionist approach to protein reactivity and evaluated the binding characteristics of 20 of the most commonly reported chemical respiratory allergens towards defined peptides with a single reactive amino acid of interest. The hypothesis is that it is possible to identify and characterize different forms of chemical allergens as a function of preferential peptide binding.Utilizing the standardized reaction conditions of a direct peptide reactivity assay (DPRA), the reactivity of respiratory allergens for cysteine and lysine peptides was evaluated. Activity in the DPRA is reported as the percent depletion of peptide following 24 h incubation. An important and intriguing observation was that, when compared with skin sensitizers, chemical respiratory allergens exhibited a preferential reactivity for lysine. This preference was characterized quantitatively as a ratio of the mean depletion of lysine compared with cysteine (Lys:Cys ratio). The Lys:Cys ratio was observed to be robust and reproducible over time.A limitation of many in chemico methods for hazard identification is the lack of a metabolic component that allows for the identification of pro-haptens. In order to address this limitation, reported here is the use of the peroxidase peptide reactivity assay (PPRA), which utilizes a horseradish peroxidase/hydrogen peroxide (HRP/P) enzymatic system as a proxy for oxidative metabolism. Additionally, reactivity in the PPRA is characterized after a 24 h reaction time utilizing a concentration-response model (thus, permitting consideration of dose-response relationships defined as an EC15 value). Unexpectedly, the preferences for lysine observed with chemical respiratory allergens in the DPRA were lost or blunted in PPRA. The EC15 values demonstrated that relative reactivity between chemical respiratory allergens varied by up to 4 orders of magnitude. The identification of quantitative differences in reactivity could prove useful as a guide to evaluate potency in the future, should reliable metrics become available.To characterize the selectivity of binding by chemical respiratory allergens, the DPRA was modified to allow for the evaluation of reactivity to histidine, tyrosine and arginine. Confirming our previous observations, each of the respiratory sensitizers was observed to react to both lysine and cysteine, with in most instances, a preference for the former. Reactive promiscuity was a function of the other peptides with histidine being the most reactive followed by arginine and tyrosine. To model more complex reactive conditions, a novel modification was made to the DPRA to allow competition for lysine and cysteine to be assessed in a single reaction mixture. The results of these competitive reactivity experiments identified a range of binding patterns to lysine and cysteine that in some cases resulted in different binding being expressed.At present, there are no methods available to reliably identify potential chemical respiratory allergens. The work presented here has demonstrated that respiratory allergens can be identified as potential sensitizers based on their ability to react with lysine and cysteine. More importantly, the balance of reactivity to these two peptides can provide a means of discriminating between respiratory and skin sensitizers.

Within the paradigm of HIV-1 infection, macrophages play a crucial role as long-lived viral reservoirs. However, cell-free virus infection is inefficient and is unlikely to explain the levels of infection observed in vivo. To investigate the hypothesis that macrophages might be infected via direct contact with HIV-1-infected T cells, macrophage and HIV-1-infected T cell cocultures were imaged in real time. I observed that macrophages preferentially phagocytosed HIV-1-infected T cells and, using long-term culture assays, I established that following coculture the macrophage became productively infected. Phagocytosis of HIV-1-infected cells occurred independently of viral tropism; however, productive infection following T cell phagocytosis was restricted by viral tropism. Imaging flow cytometry showed that macrophages primarily phagocytose dying HIV-1-infected T cells. However, a significant population of HIV-1-infected 'healthy' cells were also taken up. Furthermore, ICAM-1 was identified as mediating the uptake of HIV-1-infected T cells. These results indicate that apoptosis plays a significant, but not sufficient, role in the mechanism for recognition and uptake of HIV-1-infected T cells. The response of macrophages to HIV-1 infection remains controversial. Using both primary macrophages and a monocyte/macrophage NFκB reporter line assay, I demonstrated that macrophages are activated in response to HIV-1-infected T cells. In addition, during coculture with HIV-1-infected T cells, macrophages upregulated secretion of Th1 cytokines, with associated dysregulation of regulatory cytokines. Finally, data presented suggest that polarisation of macrophages towards M1 and M2 phenotypes alters the susceptibility to HIV-1 infection in the cell-to-cell route.

Food allergies are an increasing health problem throughout the developed world. Among these, peanut allergy is particularly significant, due to its exceptional severity and frequent lifelong duration. Much of its aetiology remains unclear. In particular, it remains unknown why, unlike other food allergies, peanut allergy incidence correlates poorly with average dietary peanut consumption. A popular explanation for this discrepancy is that peanut allergy is more common in regions where predominantly dry-roasted (DR) peanuts are consumed, leading to speculation that DR-induced chemical modifications may contribute to pathological T_h2 responses in humans. Yet to date, no research group has demonstrated an enhanced immunogenicity of DR peanuts relative to raw in a murine model of sensitisation. This thesis begins with the hypothesis that dry-roasting does indeed alter the chemical composition of peanut proteins in such a way as to increase immunogenicity and allergenicity. To test this hypothesis robustly, I have first addressed flaws in previous studies by developing a methodology to thoroughly characterise samples of raw and DR peanut protein, as well as purifying samples of individual peanut allergens. Using these samples, I have demonstrated an enhanced immunogenicity of DR peanut protein relative to raw, in intragastric, subcutaneous and epicutaneous models of mouse sensitisation, and furthermore, that such enhanced responses feature a pronounced T_h2 bias and functional IgE production. I will present evidence that this difference is not caused by either protein aggregation or the presence of other non-protein substances, but is due to an intrinsic property of the DR peanut proteins. I will go on to clarify candidate molecular mechanisms of this effect, examining several putative receptors and probing the effects of roasting on dendritic cell binding and interactions of peanut proteins. I conclude in light of these investigations that the dry-roasting hypothesis remains the most plausible explanation for the epidemiological distribution of peanut allergy, although many additional questions remain regarding the nature of the chemical modifications produced by roasting and the molecular basis of their recognition by the immune system.

In recent years the glycan shield of the HIV-1 envelope spike (Env) has emerged as a potential target for microbicide and vaccine design. The densely packed glycans on its surface include an intrinsic population of under-processed oligomannose structures, and a number of lectins and broadly neutralising antibodies (bnAbs) have been isolated which are reactive to these ‘non-self’ glycan structures. The potential value of these agents in therapeutic or vaccine contexts depends upon the prevalence of their glycan targets in nature and their resilience to sequence mutation. Here the prevalence of oligomannose-type glycans on recombinant gp120 was demonstrated across a panel of isolates, revealing subtle cross clade differences. Alanine scanning of all potential N-glycosylation sites (PNGSs) within a model gp120 demonstrated the overall stability of the oligomannose population, but highlighted regions of glycan clusters where individual glycans act to limit the processing of their neighbours. This was formally demonstrated for the N332 ‘site of vulnerability’, where deletion of nearby glycosylation sites led to altered glycan processing at the N332 site. A panel of N332-dependent bnAbs was screened for their ability to tolerate such changes in glycan processing, with differing results. While some displayed promiscuous binding, others were more sensitive to glycan microheterogeneity. Site-specific glycosylation analysis of the PGT135 epitope revealed that an intolerance of certain glycoforms may explain its limited breadth. While a greater understanding of Env glycan microheterogeneity and bnAb promiscuity is required, these findings reveal insights into the architecture of the HIV-1 glycan shield that suggest it is a conserved and robust target for drug and vaccine design.

Long-term non-progressors (LTPNs) in HIV infection target the structural protein Gag more frequently than individuals who progress to disease. However, the targeting of Gag per se does not always distinguish these two groups. Various factors have been put forth as likely explanations for this discrepancy including differences in the breadth and magnitude of observed responses, the HLA type of the host, the nature of the individual epitopes targeted and the ability of the virus to mutate these antigenic regions. The purpose of this thesis was to examine, using PBMCs isolated from HIV-2 infected LTNPs and CTL clones established in vitro, the clonotypic architecture and quality of an immunodominant HIV-2 Gag-specific response directed towards the HLA-B*3501-restricted epitope NPVPVGNIY (NY9: Gag245-253). The data presented in this thesis show that in spite of the expression of multiple inhibitory receptors on the surface of NY9-specific CD8+ T-cells, the NY9-response, which is a clonotypically 'private' response, bears a signature characterised by an increased cytotoxic sensitivity and the production of an array of cytokines, most notably IFN-γ and MIP-1β. Moreover, the results of this thesis indicate that the NY9-specific CD8+ T-cells are able to cross-recognise and lyse target B-cells pulsed with the corresponding HIV epitope PY9 and its variants at functional avidities (EC50) that are close to those exhibited by PY9-specific T-cells. However, not all mobilised TCR clonotypes are equally sensitive or equally cross-reactive. When individual CTL clones were studied it emerged that dominant clonotypes within the NY9-specific CD8+ T-cell memory pool possessed a higher avidity for tetramer and sensitivity for antigen than subdominant ones and demonstrated a better cross-reactive potential towards variants of the HIV-2 epitope. Hence, future HIV vaccine strategies may benefit from the inclusion of epitopes like NY9, the presentation of which appears to mobilise CD8+ T-cells with superior functional profiles.

Introduction. HIV-associated neurocognitive disorders appear to remain common despite access to antiretroviral therapy (ART). Penetration of ART into the central nervous system (CNS) is highly variable between drugs and between individuals. Cerebrospinal fluid (CSF) concentrations of many antiretroviral medications fall below the minimum inhibitory concentration for wild type virus. HIV-1 RNA can be detected in the CSF at greater levels than in plasma (CSF/plasma discordance), however the clinical significance of this is unclear, and the degree of difference considered pathological varies. Whether the CNS can act as a sanctuary site leading to persistent HIV detection in plasma is not known. Methods. The PARTITION study recruited HIV positive adults from 13 UK clinical sites. Paired CSF and plasma was collected from patients undergoing LP for clinical indication (group A) and subjects with unexplained viraemia despite ART (group B). The study aimed to determine a) the prevalence of CSF/plasma discordance and factors associated with this occurrence, and b) the prevalence of HIV-1 RNA detection in CSF in those with HIV-1 RNA persistence in plasma. A sensitive assay detected HIV-1 RNA below 50 copies/ml in a subgroup and a cytometric bead array determined CSF biomarkers. A matrix of clinical features and CSF/plasma biomarkers was related to cognitive decline in subjects from the CHARTER study. Drug concentrations in CSF and plasma were measured by mass spectrometry assays and related to host genetic factors in subjects in the PARTITION study and a Vietnamese cohort with tuberculous meningitis. Results. CSF/plasma HIV discordance occurred in 13% of this cohort and was associated with nadir, but not current, CD4 cell count. CSF/plasma discordance occurred in 7 of 40 (18%) of subjects with ongoing viral detection in plasma vs. 0 of 39 of those without. Residual HIV-1 RNA detection below 50 copies/ml was also associated with CSF HIV-1 RNA detection. Resistance associated HIV mutations were detected in CSF of subjects with CSF/plasma discordance. CSF/plasma discordance above 0.5log10 was associated with raised profiles of inflammatory CSF proteomic biomarkers compared to those without discordance. In the CHARTER cohort, cognitive decline over 18 months was associated with lower concentrations of CSF TNFa and plasma IGF1/2. CSF concentrations of efavirenz were associated with the CYP2B6 c.516G>T single nucleotide polymorphism. Efavirenz metabolites were mainly glucuronidated in CSF, were present at neurotoxic levels, and were related to degree of blood brain barrier permeability. Host genetic factors were did not relate to CSF DRV concentrations. Conclusions. CSF/plasma discordance is a frequent occurrence, likely related to processes established during advanced immunosuppression not fully reversed by ART. It is associated with CSF HIV resistance and raised CSF biomarkers, even at levels 0.5-1log10 which have been considered non-significant in some studies. Potentially neurotoxic CSF concentrations of efavirenz relate to host genetic factors.

Le syndrome de Sézary (SS) est un variant leucémique et érythrodermique de lymphomes T cutanés épidermotropes. Son diagnostic repose à la fois sur des critères cliniques, la présence de lymphocytes T à noyau atypique cérébriforme sur un frottis sanguin et la mise en évidence dans la peau, les ganglions et le sang d’un clone lymphocytaire T CD4+. Notre laboratoire a identifié KIR3DL2 comme premier marqueur membranaire spécifique des cellules tumorales de Sézary. KIR3DL2 peut ainsi être utilisé pour le diagnostic et le suivi des patients atteints du SS. Toutefois, aucune étude n’a démontré de lien entre sa structure de récepteur inhibiteur et sa fonction dans les lymphocytes tumoraux de Sézary, et plus particulièrement son implication possible dans les mécanismes régulant la prolifération et/ou la résistance à l’apoptose des cellules tumorales.Au cours de ce travail deux axes ont été développés :- Un premier axe visant à mieux comprendre la fonction de KIR3DL2 et les mécanismes de signalisation intracellulaire initiés lors de son engagement par l’anticorps AZ158 dans les lymphocytes T tumoraux de Sézary. Nos résultats mettent en évidence un rôle de corécepteur inhibiteur pour KIR3DL2 dans les cellules tumorales de Sézary. En effet, l’engagement de KIR3DL2 inhibe la prolifération et l’AICD induites par la stimulation CD3, cette inhibition étant corrélée à une modulation négative des signaux médiés par le TCR. Ainsi, KIR3DL2 ne se comporte pas comme une unité de signalisation indépendante dans les cellules tumorales de Sézary, contrairement à ce qui est observé dans les cellules NK.- Un second axe portant sur l’évaluation d’une nouvelle fonction de KIR3DL2 comme récepteur pour les ODN CpG. Ainsi, nous rapportons pour la première fois un effet direct de l’ODN CpG sur les cellules tumorales T CD4+ de Sézary. En effet, nous avons observé un effet apoptotique de l’ODN CpG-C caspases-dépendant sur les lignées et les cellules tumorales circulantes. De plus, le traitement des cellules tumorales de patients Sézary avec l’ODN CpG-C conduit à une inhibition de l’activation constitutive du facteur de transcription STAT3.La réalisation de cette étude a permis de mieux comprendre la fonction et les mécanismes initiés à partir de KIR3DL2 dans les cellules tumorales T CD4+ de Sézary. De plus, ce travail ouvre de nouvelles perspectives thérapeutiques basées sur le ciblage direct et spécifique des cellules tumorales de Sézary pouvant être associé à une stimulation des acteurs immuns grâce à l’action des ODN CpG. / Sézary syndrome (SS) is an aggressive leukemic and erythrodermic variant of cutaneous T-cell lymphoma. It is characterized by the presence of a clonal CD4+ T lymphocyte population in the skin, lymph nodes and peripheral blood. Our laboratory has previously identified the NK cell receptor KIR3DL2 as a valuable diagnostic and prognostic marker for the detection of the tumoral T cell burden of Sézary syndrome patients. However, the function of this receptor on the malignant T lymphocyte population remained unexplored. The specific expression of KIR3DL2 by SS patients malignant cells prompted us to investigate its possible influence on mechanisms regulating the tumoral cells outgrowth and apoptosis process.To this aim, two axes were developed. The first axis aimed to highlight the function of KIR3DL2 on the malignant T lymphocyte population and to elucidate the intracellular signaling mechanisms initiated by engagement of the receptor with the monoclonal antibody AZ158. Our results show that KIR3DL2 can exert an inhibitory co-receptor function in malignant Sézary cells. Indeed, triggering of KIR3DL2 inhibits the CD3-mediated proliferation and cell death of the CD4+ KIR3DL2+ cells, this inhibition being correlated to a down-modulation of the TCR-mediated signals. Thus, KIR3DL2 does not behave as an independent signaling unit in Sézary cells, unlike NK cells.The second axis aimed to evaluate a new function of KIR3DL2 as CpG ODN receptor. We show for the first time a direct effect of CpG ODN on tumoral CD4+ T Sézary cells. Thus, we observed a caspase-dependent apoptotic effect of CpG ODN-C on Sézary cell lines and circulating malignant T cells. This process of cellular death is correlated to a dephosphorylation of the transcription factor STAT3, which is found constitutively phosphorylated and activated in Sézary cells.This study has provided new insights into the function and the intracellular signaling pathways initiated by KIR3DL2 in malignant Sézary T cells. Furthermore, this work opens new therapeutic perspectives based on the direct and specific targeting of tumor cells that could be associated to immune cell stimulation through the use of ODN CpG.

Self-reactive CD4+ helper T cells (Th) are key causal agents in the pathogenesis of many autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a CD4+T cell model of the demyelinating autoimmune disease multiple sclerosis (MS). It has been shown that EAE is caused by CD4+ T-cells that produce pro-inflammatory cytokines IFN-γ (Th1) and IL-17 (Th17). As such, understanding how these Th cells are generated and controlled is essential. There is debate as to whether Th1 and Th17 cells act independently in EAE or if there is plasticity between these two subtypes, and whether the capacity to switch from Th1 to Th17 confers pathogenic capacity. T-bet was first described as the master transcription factor for Th1 cells, and is thought to have a critical role in EAE even though IFN-γ, the Th1 archetypal cytokine, has been shown to be redundant. More recent work has shown that T-bet is expressed in multiple immune cell types, and it remains unclear in what cells the expression of T-bet is required for EAE. Considerable efforts have been put into understanding the role of T-bet in EAE pathogenesis, with a view to modulate cells expressing T-bet for therapy. The hypothesis of this work was that T-bet has multifaceted roles in EAE, in initiating and directing an immune response in innate antigen presenting cells such as dendritic cells (DC) as well as programming pathogenic effector CD4+ T cell (Teff) response to antigen. T-bet-/- mice were studied using different models of EAE to dissect the role of T-bet in disease pathogenesis. Active immunisation of C57BL/6 mice with the immunodominant peptide from myelin oligodendrocyte glycoprotein (MOG35-55) showed that T-bet-/- mice developed EAE with an IL-17 dominated profile and critically, T-bet-/- mice were able to produce GM-CSF which has recently been described as a key cytokine for EAE. T-bet-/- cells were not able to transfer EAE in a model of passive transfer EAE, where CD4+ T cells were polarised towards a Th1 profile in vitro. Illustrating that T-bet is required in CD4+ T cells for Th1 mediated EAE. DC driven EAE showed that T-bet-/- DC were able to activate CD4+ T cells in vitro and cause EAE upon co-transfer into host mice with transgenic CD4+ T cells. Thus, it has been shown that T-bet is not required in EAE. This work represents a step further towards understanding the disease mechanisms involved in EAE and suggests T-bet is not an appropriate therapeutic target for the treatment of MS.

L'inflammation allergique de la peau est un état dans lequel l'hôte réagit de manière excessive à des allergènes en induisant une inflammation de type Th2. Un certain nombre d’acteurs cellulaires et moléculaires ont été impliqués dans cette pathologie, mais la façon dont ils agissent dans le réseau inflammatoire demeure largement inconnue. Les basophiles ont été reconnus pour leurs fonctions effectrices en allergie, cependant, comment ils sont recrutés et activés, ainsi que comment ils interagissent avec d'autres cellules dans l’inflammation allergique cutanée demeurent mal caractérisés. L'objectif de ce travail de thèse est d'étudier le recrutement, l'activation et la fonction des basophiles dans le réseau inflammatoire de la peau allergique. En employant un modèle expérimental murin de dermatite de contact allergique, combiné à des outils génétique de souris, d’immunologie et d’approches biologiques cellulaires/moléculaires, l’étude de ma thèse a démontré que l’IL-3 joue un rôle crucial dans l’extravasation des basophiles dans la peau allergique des souris (Partie I), et que l’invalidation des basophiles chez les souris Mcpt8DTR entraîne une réduction systémique des éosinophiles et des neutrophiles (Partie II). Ces études fournissent ainsi de nouvelles connaissances sur le recrutement, l'activation et la fonction des basophiles dans l'inflammation allergique de la peau. / Allergic skin inflammation is a state in which the host overreacts to otherwise innocuous allergens by inducing T helper type 2 inflammation. A number of cellular and molecular players have been implicated in the generation of allergic skin inflammation, but how they act and crosstalk in the inflammatory network remains largely unknown. Basophils have been recognized for their effector functions in allergy, however, how they are recruited to the inflamed tissue, get activated and crosstalk with other cells in inflammatory skin remain still incompletely understood. The objective of this PhD study is to investigate the recruitment, activation and function of basophils in the inflammatory network of allergic skin. Using experimental mouse model for allergic contact dermatitis, combined with mouse genetic tools, immunology and cellular/molecular biology approaches, the study of my thesis discovered that IL-3 plays a crucial role in basophil extravasation to mouse allergic skin (Part I), and that the depletion of basophils in Mcpt8DTR mice leads to a systemic reduction of eosinophils and neutrophils (Part II). These studies provide novel insights into the recruitment, activation and function of basophils in allergic skin inflammation.