Dissertations / Theses on the topic 'Human lymphocyte'

Extracellular adenosine 5′-triphosphate (ATP) is an agonist for the P2Z receptor of human leukaemic lymphocytes and opens a Ca ²⁺-selective ion channel, which also conducts Ba²⁺, Sr²⁺ and the small fluorescent dye, ethidium⁺. A wide range of receptor agonists, many of which raise cytosolic [Ca²⁺] activate phospholipase D (PLD). In the present study, it was shown that both ATP and 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) stimulated PLD activity in a concentration-dependent manner, and the inhibitory effects of suramin, oxidised ATP, extracellular Na⁺ and Mg²⁺ suggested that the effect of these agonists is mediated by P2Z receptors. The role of divalent cations in ATP-stimulated PLD activity was investigated. Several agonists (eg ATP, thapsigargin, ionomycin) stimulated a rise in cytosolic [Ca²⁺] in human lymphocytes, but only ATP and ionomycin stimulated PLD activity. When Ca²⁺ influx was prevented by EGTA, the majority of ATP-stimulated and all of ionomycin-stimulated PLD activity was inhibited. Preloading cells with the Ca²⁺ chelator, BAPTA, reduced cytosolic [Ca²⁺] and, paradoxically, ATP-stimulated PLD activity was potentiated. ATP-stimulated PLD activity was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Furthermore, both ATP-stimulated PLD activity and ATP-stimulated ¹³³Ba²⁺ influx showed a linear dependence on extracellular [Ba²⁺]. Thus it was concluded that ATP stimulated PLD activity in direct proportion to the influx of divalent cations through the P2Z ion channel and this PLD activity was insensitive to changes in bulk cytosolic [Ca²⁺]. The calmodulin (Ca²⁺/CaM) inhibitor, trifluoperazine (TFP) inhibited ionomycin- and ATP-stimulated PLD activity and ATP-stimulated apoptosis, but had no effect on PLD activity already activated by ATP. However, TFP inhibited ATP-stimulated Ca²⁺, Ba²⁺ and ethidium⁺ fluxes, at concentrations below those which inhibit Ca²⁺/CaM, suggesting that TFP inhibits the P2Z receptor. Similarly, the isoquinolinesulphonamide, KN-62, a selective inhibitor of Ca²⁺/CaM-dependent protein kinase II (CaMKII), also prevented ATP-stimulated apoptosis, but had no effect on pre-activated PLD. In addition, KN-62, and an analogue, KN-04, which has no effect on CaMKII, potently inhibited ATP-stimulated Ba²⁺ influx (IC₅₀ 12.7 ± 1.5 and 17.3 ± 2.7 nM, respectively), ATP-stimulated ethidium⁺ uptake (IC₅₀ 13.1 ± 2.6 and 37.2 ± 8.9 nM, respectively), ATP-stimulated phospholipase D activity (50% inhibition 5.9 ± 1.2 and 9.7 ± 2.8 nM, respectively) and ATP-induced shedding of the surface adhesion molecule, L-selectin (IC₅₀ 31.5 ± 4.5 and 78.7 ± 10.8 nM, respectively). They did not inhibit phorbol ester- or ionomycin-stimulated PLD activity or phorbol ester-induced L-selectin shedding. Neither KN-62 nor KN-04 (both 500 nM) have any effect on UTP-stimulated Ca²⁺ transients in fura-2-loaded human neutrophils, a response which is mediated by the P2Y₂ receptor, neither did they inhibit ATP-stimulated contractile responses mediated by the P2X₁ receptor of guinea pig urinary bladder. Thus, KN-62 and KN-04 are almost equipotent as P2Z inhibitors with IC₅₀s in the nanomolar, indicating that their actions cannot be due to CaMKII inhibition, but rather that they are potent and direct inhibitors of the P2Z receptor. Extracellular ATP-induced shedding of L-selectin from lymphocytes into the medium is a Ca²⁺-independent response. L-selectin is either cleaved by a metalloproteinase or a PLD with specificity for glycosylphosphatidylinositol (GPI). The novel hydroxamic acid-based zinc chelator, Ro-31-9790 blocks ATP-induced L-selectin shedding, but was without effect on ATP-induced Ba²⁺ influx or ATP-stimulated PLD activity. Furthermore, another zinc chelator, 1,10-phenanthroline, an inhibitor of a GPI-PLD, potentiated rather than inhibited ATP-stimulated PLD activity, suggesting that ATP-induced L-selectin shedding and ATP-stimulated PLD activity are independent of each other. Although extracellular ATP is the natural ligand for the lymphocyte P2Z receptor, it is less potent than BzATP in stimulating Ba²⁺ influx. Concentration-response curves for BzATP- and ATP-stimulated ethidium⁺ influx gave EC₅₀s 15.4 ± 1.4 µM and 85.6 ± 8.8 µM, respectively. The maximal response to ATP was only 69.8 ± 1.9% of that for BzATP. Hill coefficients were 3.17 ± 0.24 and 2.09 ± 0.45 for BzATP and ATP respectively, suggesting greater positive cooperativity for BzATP than for ATP in opening the P2Z-operated ion channel. A rank order of agonist potency of BzATP > ATP = 2MeSATP > ATPγS was observed for agonist-stimulated ethidium⁺ influx, while maximal influxes followed a rank order of BzATP > ATP > 2MeSATP > ATPγS. When ATP (300 -1000 µM) was added simultaneously with 30 µM BzATP (EC₉₀), it reduced both ethidium⁺ and Ba²⁺ fluxes by 30 - 40% relative to values observed with BzATP alone. KN-62, previously shown to be a specific inhibitor of the lymphocyte P2Z receptor, was a less potent antagonist of BzATP-induced fluxes than ATP, when maximal concentrations of both agonists (50 and 500 µM respectively) were used. However, when BzATP (18 µM) was used at a concentration equiactive with a maximally effective ATP concentration, KN-62 showed the same inhibitory potency for both agonists. The ecto-ATPase antagonist, ARL-67156, inhibited both ATP- and BzATP-stimulated Ba²⁺ influx, suggesting that the lower efficacy of ATP compared with BzATP was not due to preferential hydrolysis of ATP. Thus, the natural ligand, ATP, is a partial agonist for the P2Z receptor while BzATP is a full agonist. Moreover the competitive studies show that only a single class of P2-receptor (P2Z class) is expressed on human leukaemic lymphocytes. Both ATP- and BzATP-stimulated PLD activity were significantly inhibited (P < 0.05) when cells were suspended in iso-osmotic choline Cl medium. Choline⁺ was found to be a permeant for the P2Z ion channel, since ATP induced a large uptake of [¹⁴C]choline⁺ (60 to 150 µmol/ml intracellular water) during a 5 min incubation, which remained in the cells for several hours, and ATP was used to load cells with these levels of choline⁺. Intracellular choline⁺ inhibited ATP-, BzATP-, PMA- and ionomycin-stimulated PLD activity. Brief exposure of lymphocytes to ATP increased the subsequent basal rate of ethidium⁺ uptake, and this was prevented by intracellular choline⁺. It is proposed that P2Z-mediated Ca²⁺ influx in lymphocytes activates PLD leading to significantly changes of the phospholipid composition of the plasma membrane, which subsequently produces a permeability lesion, which in turn contributes to cell death.

Summary of the work

Abdominal aortic aneurysm (AAA) is a chronic degenerative disease that usually affects men over 65 years with an estimated prevalence of 5%. Aneurysm rupture represents a catastrophic event which carries a mortality rate of almost 90%. Current therapeutic options for AAAs measuring 5.5 cm in diameter or larger are based on prophylactic surgery, including conventional open reconstruction and endovascular stent-graft insertion. For patients with small asymptomatic AAAs (4.0 up to 5.5 cm in diameter), evidence from two recent large randomized controlled trials indicates no long-term survival benefit from immediate elective surgical repair as compared to imaging surveillance until aneurysm expands to 5.5 cm. This highlights the need for development of novel medical management strategies, including selective pharmacologic approaches, directed at preventing aneurysm expansion. In this regard, it is expected that a detailed knowledge of the pathobiology of human AAA lesion and a better understanding of pathophysiological mechanisms underlying initiation and progression of aneurysmal degeneration, particularly the specific involvement of T lymphocytes, will have special relevance to this challenging issue.

Inflammatory and helper T-cell responses in abdominal aortic aneurysm :controversial issues

Innate and inflammatory responses to endovascular versus open AAA repair. The occurrence of early acute systemic inflammatory responses after conventional open AAA repair is widely recognized and is thought to lead to the development of organ dysfunction and multiple organ failure, responsible for a large proportion of morbidity and mortality associated with aortic surgery. New therapeutic strategies designed to avoid ischemia-reperfusion injury related to aortic cross-clamping and to minimize the degree of tissue damage have thus been developed recently. Specifically, the advent of endovascular techniques has radically extended management options for patients with AAA. Although the method is believed to offer a clear short-term benefit over open repair, notably as regards restricted perioperative haemodynamic parameter fluctuations, reduced blood loss, briefer duration of surgery, shorter hospital stay, and lower 30-day mortality and complication rates, conflicting data are available regarding the exact nature and extent of the inflammatory events arising after such endoluminal procedures ;while several authors have indeed reported that endovascular AAA repair can determine a less intense and extensive inflammatory response, others have unexpectedly observed that the method may elicit a strong inflammatory response, the so-called « postimplantation syndrome ».

Adaptive cellular immune responses in human aneurysmal aortic lesion.

The inflammatory nature of AAA disease has long been suggested by the presence of a great number of CD4+ T lymphocytes in the outer media and adventitia of human AAA lesion. Interestingly, such infiltrating T-cell populations may have significant implications in the process of aneurysm dilation, since cytokines produced by T cells, notably IFN-gamma, have previously been shown to modulate production of matrix-degrading enzymes by resident macrophages and to induce apoptosis of medial SMCs. Through these key pathological mechanisms, T cells could potentially contribute to orchestrate aortic wall connective tissue disordered remodeling and degradation, and promote extensive disruption of elastic media, ultimately leading to aneurysmal degeneration. Nevertheless, despite their relative abundance in human AAA wall tissues, there is limited and controversial information as regards the functional profile of lesional lymphocytes, the exact nature of aortic wall adaptive cellular responses, and the etiologic role of T cells and their cytokines in initiation and progression of the aneurysmal process. Indeed, both Th1-type and Th2-type responses have been identified in human studies and experimental animal models of AAA.

Aims of the work

The main objectives of our work were to explore the innate and adaptive cellular immune responses in human AAA. In the first part of our work, we aimed to examine prospectively innate and inflammatory responses arising in a non-randomised cohort of patients undergoing endovascular versus open AAA repair. In the second part of our work, we focused our efforts on characterizing the nature of adaptive cellular immune responses and the phenotypic and functional repertoire of T cells in human AAA wall tissues obtained from a consecutive series of patients undergoing open AAA repair. Specifically, we sought to determine whether type 1 or type 2 responses occur predominantly in advanced AAA lesion.

Main experimental findings

Limited inflammatory response after endovascular AAA repair. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or insertion of endograft, and after 1, 3, 6, 12, 24, 48, and 72 hours. We first examined the acute phase reaction and liberation of complement cascade products using turbidimetric method and nephelometry. We found that endovascular repair produced lower postoperative CRP, leucocytosis, neutrophilia, and C3d/C3 ratio as compared to open surgery. We next analyzed surface expression of activation markers on peripheral CD3+ T cells using flow cytometry. We observed a strong upregulation of CD38 after open but not endovascular repair. Analysis of CD69 and CD25 molecules revealed no perioperative fluctuations in any group. We then investigated release of various circulating soluble cell adhesion molecules, proinflammatory cytokines, and chemokines using enzyme-linked immunosorbent assays. We demonstrated that both procedures are characterized by similar increases in ICAM-1 and IL-6 levels. Finally, tendency towards high levels of TNF-alpha and IL-8 was detected in endovascular repair, but data failed to reach statistical significance.

Predominance of type 1 CD4+ T cells in human aneurysmal aortic lesion. We have developed a tissue enzymatic digestion and cell extraction procedure to isolate intact mononuclear cells from aortic wall segments. This original cell isolation protocol enabled us to examine ex vivo the presence, phenotype, and cytokine secretion profile of infiltrating T lymphocytes freshly isolated from human AAA tissues for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+ T cells display a unique activated memory phenotype, as assessed by an increased expression of CD69 and HLA-DR activation antigens, downregulation of CD62L molecule, and predominant expression of the CD45RO isoform characteristics of memory cells. In addition, we identified the presence in human aneurysmal aortic wall lesion of CD4+ T cells producing high levels of IFN-gamma but not IL-4, reflecting their type 1 nature. In an additional series of experiments, cytokine gene expression was determined in whole aneurysmal and non-diseased aortic samples using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. The molecular basis of type 1 or type 2 dominant responses was further specified by analyzing mRNA levels of transcription factors specifically involved in Th1 or Th2 differentiation such as T-bet and GATA-3. We demonstrated that aneurysmal aortic specimens exhibit high transcript levels of IFN-gamma but not IL-4, and consistently overexpressed the IFN-g-promoting cytokine IL-12 and the type 1-restricted transcription factor T-bet, further establishing the prominent type 1 nature of aortic wall responses. Moreover, such selective tissue expression of IL-12 and T-bet in the vessel microenvironment points to a potential role for these signals in directing aortic wall responses towards a type 1 phenotype.

Conclusions

Our findings indicate that endovascular AAA repair is associated with a lesser degree of acute phase reaction, peripheral T-cell activation, and release of complement proteins as compared to conventional open surgery, suggesting that the innate and inflammatory responses to AAA repair are significantly attenuated by the endovascular approach as compared to the traditional open reconstruction. These results support the view that the endoluminal procedure represents an attractive alternative to open surgery for the treatment of large aneurysms. On the other hand, we have demonstrated that Th1 cell infiltrates predominate in human end-stage AAA lesion. These observations are relevant for helping clarify the pathobiology of human AAA tissues and defining prospects for the prevention of aneurysm expansion. Indeed, identification of such infiltrating populations of IFN-gamma-producing CD4+ T cells not only provide new insights into the pathogenesis of the disorder, but could also serve as a basis for the development of novel medical management strategies directed at preventing aneurysm formation and progression, including therapeutic approaches based on the modulation of aortic wall responses and designed to selectively target T-cell activation and cytokine production. In this respect, the present work provides experimental evidence in support of the emerging concept that, although multifactorial, aneurysm disease may be regarded as a Th1-driven immunopathological condition, and suggests that strategies targeting IFN-gamma could be a particularly exciting and fruitful avenue for further investigation. Ongoing clinical and basic research in these areas can be expected to yield design of promising pharmacologic approaches to control AAA expansion. From a clinical perspective, such efforts have the potential to dramatically influence both the outcome and management of this common and life threatening condition.

Doctorat en sciences médicales
info:eu-repo/semantics/nonPublished

Bien que la description qualitative et phénoménologique de la migration cellulaire soient décrites de façon minutieuse dans son approche holistique, des mécanismes de base et connus depuis longtemps n’ont pas encore été explorés quantitativement avec une approche "bottom-up". Un de ces exemples est le comportement migratoire en deux dimensions des lym- phocytes T effecteurs humains. Alors que leur comportement in vivo est déjà connu depuis longtemps et décrit qualitativement, une approche quantitative in vitro offre de nombreuses perspectives. Les interactions des intégrines LFA-1 et VLA-4 avec leurs ligands respectifs ICAM-1 et VCAM-1 ont déjà été étudiées et sont les principales molécules impliquées dans la migration des lymphocytes T effecteurs. Du fait de leur importance dans l’organisation de la mobilité, ces deux protéines sont les principaux objets d’étude de cette thèse. La majorité des travaux précédents ont été réalisés en observant les lymphocytes T dans des tissus vi- vants dont la composition et la densité des molécules d’adhésion ne peuvent ni être déterminées ni contrôlées de façon précise. Nous avons developpés des substrats artifi- ciels permettant d’imiter et de contrôler ces caractéristiques adhésives afin d’examiner et de relier les propriétés physiques des cellules tels que la vitesse ou l’index de mi- gration orientée, avec une réponse cellulaire donnée ainsi que d’associer un type de mouvement avec des interactions intégrines-ligands spécifiques. Pour aller plus loin, nous avons à nouveau analysé la conformation des intégrines puis l’avons modulé pour altérer leur affinité et changer les propriétés précédemment décrites
The ability of T-lymphocytes to migrate to sites of inflammation towards all different types of tissues based on the interplay between biochemical and mechanical signaling is unique among human cells and underlines the importance of their complex motility apparatus relying on multiple stimuli. A crucial part within the leukocyte adhesion cascade is the firm attachment of the immune cell to the inner wall of the blood vessel and the subsequent migration along its surface until crossing the endothelial cell barrier. These migrational steps are guided not only by the shear stress to which the cell is exposed to by the flow of blood, but also by expression of adhesion molecules, the most important among them are ICAM-1 and VCAM-1 and their integrin counterparts LFA-1 and VLA-4, expressed by the immune cell. These proteins are crucial not only in a mechanically anchoring sense, but they also play a part in an intracellular signaling process leading to a change in migrational direction, overall cell affinity and phenotype. Few is known about how all components shape the movement behaviour on a quantitative level, raising questions about hierarchy, affinity and density of the involved proteins. Besides enhancing the general knowledge of the mechanisms of T-cell migration, the role of ICAM-1 and VCAM-1 in various diseases makes this study a promising endeavour. The approach taken in this thesis is to dissect and recompose the important adhesion molecules on a laminal flow chamber to link the cell’s response to them to specific movement properties and answer the questions addressed above

CD23 is a multi-functional protein which exists in membrane-bound and soluble forms. Its functions include acting as the low affinity receptor for IgE and generating pro-inflammatory cytokine release in monocytes. CD23 has been found to interact with αvβ5 and this interaction greatly enhances growth of the B cell precursor cell line SMS-SB. This interaction may have a role in the development of normal human B cells and in cancer as the integrin is expressed on both precursor and ALL cells but not on normal mature B cells. One of the aims of this investigation was to expand on the finding that CD23 peptides containing an RKC motif had the same positive growth effect on SMS-SB cells as CD23. Other B cell lines – representative of both precursor and mature stages – were studied to ascertain whether this proliferative effect was dependent upon cell differentiation stage and/or presence of the αvβ5 integrin. It was found that peptides containing the basic RKC motif were mitogenic only for precursor B cells which were expressing αvβ5. Details of these peptides and their varying effects on the different cell lines are in Chapter 4. Stimulation of SMS-SB cells, presumably via the αvβ5, results in signalling through PI3K and subsequent phosphorylation of Akt. The growth of SMS-SB cells observed following stimulation with peptides containing the RKC motif was abrogated by the PI3K inhibitor LY294002 and western blotting revealed that phosphorylation of Akt was enhanced by stimulation with RKS containing peptides. Among CD23’s receptors is the integrin αvβ3. This integrin can form a signalling complex with CD47. Ligation of CD47 by anti-CD47 antibodies induces apoptosis in some cell lines. To determine whether a pattern exists between response to this stimulation and expression of αvβ3 integrin, cell lines with and without the integrin were tested. It was found that the myeloma cell lines KMS11 and H929 were responsive to this stimulus. Since these cell lines differ in their expression of αvβ3 (H929 cells express αvβ3 whereas KMS11 do not) it does not appear that any connection between the presence of the integrin and response via CD47 exists and therefore this signalling mechanism would appear to occur independently of the complex formed by CD47 and αvβ3.

Lymphocyte infiltration of the liver drives the progression of all inflammatory liver diseases. Lymphocyte homing to the liver occurs within the hepatic sinusoids. The hepatic sinusoidal endothelial cells (HSEC) that line these channels have a unique phenotype and lack conventional adhesion molecules, making these cells a potential organ specific target for therapy. I carried out a detailed analysis of the expression of CLEVER-1 in the human liver and show that it is expressed on sinusoidal endothelium in situ and in vitro and at other sites of lymphocyte recruitment in the inflamed liver including neovessels, portal associated lymphoid tissue and vessels supplying hepatocellular carcinomas. The expression of CLEVER-1 on HSEC in vitro was upregulated by hepatocyte growth factor. Flow based adhesion assays with HSEC demonstrated that CLEVER-1 mediated lymphocyte transmigration and that this activity was subset specific with preferential activity for B cells and T regulatory (Treg) cells. Using confocal imaging I was able to show that CLEVER-1 mediates Treg migration via the transcellular route through HSEC. This is the first report of transcellular migration in the liver. This study identifies CLEVER-1 as a novel lymphocyte recruitment signal to the liver and a potential target for therapy in chronic liver diseases.

The immune system is responsible for defending a host animal from a wide variety of threats. Manipulation of the immune system can result in beneficial outcomes such as immunity to pathogens, or deleterious outcomes such as autoimmunity. Advances in our understanding of how the immune system develops and functions have benefited greatly from studies in animals, particularly in mice where the genetics are well known and a multitude of reagents are readily available for experimental use. Although much has been learned from animal experimentation, it must be cautioned that animals are not humans. Unforeseen outcomes and complications often arise when translating research obtained in animal models to treatment of human patients. A small animal model in which the human immune system can be established and manipulated experimentally in vivo would be valuable for the study of human immune responses in infectious diseases, transplantation, and autoimmunity, and ultimately for translation of these findings to the human patient. Contribution to this model is the overall goal of this thesis. The severe combined immunodeficiency (Prkdcscid, termed scid) mutation was discovered in 1983 in the C.B-17 strain of mice. Scid mice lack functional T and B lymphocytes and are unable to mount immune responses or reject allogeneic or xenogeneIc grafts. However, C.B-17-scid mice do develop normal or even elevated innate immune function. Based on the ability of scid mice to accept xenografts, human peripheral blood mononuclear cells have been injected to generate "Hu-PBL-scid mice." These Hu-PBL-scid mice have been proposed as an in vivo model of the human immune system. Although this model was described over 12 years ago, there are a number of obstacles that impede its ability to recapitulate human immunity. The Hu-PBL-scid mouse model established using C.B-17-scid mice as recipients is hindered by 1) low levels of human leukocyte engraftment, 2) engraftment of predominately memory/activated human T cells with specificity directed against host murine MHC antigens, 3) rapid transition of engrafted human lymphocytes to a functionally anergic state, and 4) a paucity of knowledge providing an understanding of the mechanisms that underlie engraftment and function of the human lymphocytes in the immunodeficient hosts. This thesis was initiated at the time the NOD-scid mouse became available for establishment of Hu-PBL-scid mice, and this model has subsequently been termed "Hu-PBL-NOD-scid." The NOD-scid mouse was designed as an improved recipient for human PBL based on its innate immune characteristics. Defects in innate immunity in wild-type NOD/Lt mice include reduced NK cell activity, defects in macrophage development and function, and a lack of hemolytic complement. NOD-scid mice retained these characteristics, and engraft human cells at higher levels compared to C.B-17-scid mice. However, the ratio of CD4+ to CD8+ T cells in Hu-PBL-NOD-scid mice remained skewed towards the CD8+ population, similar to that of the Hu-PBL-C.B-17-scid mouse. My thesis was based on the overall hypothesis that additional genetic manipulation of the recipient strain would enhance further the engraftment and function of human cells. Based on the engraftment results obtained in NOD-scid mice, we performed these genetic manipulations using the NOD-scid strain as the reference. We first hypothesized that removal of MHC expression would lower human anti-mouse xenoreactivity and enhance engraftment of naïve T cells, a cell population not readily detectable in Hu-PBL-C.B-17-scid or Hu-PBL-NOD-scid mice. Towards this goal, the NOD-scid mouse expressing a targeted mutation for beta-2 microglobulin (B2mnull) mouse was created by Dr. Leonard Shultz at the Jackson Laboratory. Because B2m is required for expression of MHC class I and for development of functional NK cells, we predicted that NOD-scid-B2mnull mice would first exhibit a normalized CD4:CD8 T cell ratio resulting from reduced CD8 engraftment due to decreased human anti-mouse MHC class I reactivity. Since MHC class I molecule expression is required for the development of NK cells, we further predicted that there would be a reduction of NK cell activity, permitting enhanced engraftment. Data presented in this thesis demonstrates that human PBL engraft in NOD-scid-B2mnull mice at levels higher than NOD-scid mice, and with an increased CD4:CD8 T cell ratio. The mechanism(s) responsible for the increased engraftment of human cells in these mice became a major focus of this thesis. This thesis is composed of three Specific Aims. Specific Aim 1 was to determine the mechanism(s) underlying human cell engraftment and function in Hu-PBL-scid mice. These data are contained in Part 1 of the Results. Specific Aim 2 was to elucidate the costimulation interactions between human T cells and murine host APCs that control the level of engraftment and activation state of the human lymphocytes. These data are contained in Part 2 of the Results. Specific Aim 3 was to utilize these fundamental observations to initiate studies into the induction of primary human immune responses in Hu-PBL-scid mice. Although the goal of these latter studies was not attained, the data from the experiments performed is provided in Part 3 of the Results, and is discussed relative to future directions for this arm of the project. In Part 1 of the Results, we utilized in vitro and in vivo techniques to study the underlying mechanisms regulating human cell engraftment and function in scid mice. We observed that the absence of mouse MHC class I antigens in NOD-scid-B2mnull mice does not reduce the stimulatory capacity of APCs toward human T cells in vitro. We further demonstrated that naïve human T cells persist for at least 2 weeks in the peritoneal cavity of Hu-PBL-NOD-scid and Hu-PBL-NOD-scid-B2mnull mice. However, in both strains of recipients, the human cells progress to an anergic phenotype as documented by cell surface molecule expression and by functional activity. We further documented that the increased engraftment of human CD4+ T cells observed in NOD-scid-B2mnull mice is due predominantly to the ablation of host NK cell activity. Increased engraftment of human CD4+ cells in NOD-scid-B2mnull mice can be recapitulated in NOD-scid mice by antibody-mediated depletion of residual host NK cells. Finally, we demonstrated that expression of MHC class II molecules by recipient mice facilitated stimulation and engraftment of human cells. However, mouse MHC class II expression is not required for human cell engraftment into scid mice. In Part 2 of the Results, we addressed the cellular and costimulatory interactions of engrafted human cells in scid mice. Costimulatory interactions between T cells and APC are now known to be critical for the proper activation and functioning of cells in the immune system. Understanding the role of costimulatory interactions between human T cells, human APCs, and mouse APCs became a major focus of this thesis. We demonstrated that human CD4+ T cells are required for the engraftment of human CD8+ T cells. The mechanism by which human CD4+ cells mediate this "helper" activity requires expression of CD154. Antibody-mediated blockade of CD154 in vivo abrogates human cell engraftment in scid mice. The role of host APCs in the engraftment of human lymphocytes was demonstrated by blocking host CD40 with antibody. Preventing human T cell CD154 interaction with host CD40 on murine APCs blocked human cell engraftment in scid mice, demonstrating the importance of "trans-costimulation" in human T cell activation. This "trans-costimulation" appeared to be mediated by B7 expression on mouse APCs. We further demonstrated that in vivo depletion of human CD8+ T cells in Hu-PBL-NOD-scid mice leads to increased levels of human CD4+ T cells, elevated human immunoglobulin in the serum, and increased incidence of EBV-related lymphoproliferative disorders. These observations suggested that human CD8+ T cells are able to regulate human CD4+ T cell help and provide "surveillance" activity for EBV-related lymphoproliferative disorders. In Part 3 of the Results, we used the Hu-PBL-scid mouse to initiate experiments designed to generate primary human immune responses in vivo. These experiments were based on our observation that few if any human APCs survive in scid mice, and on reports that dendritic cells (DC) are required for activation of naïve T cells and initiation of a primary immune response. We used recombinant viruses expressing HIV-1 proteins that are being developed as potential vaccines for HIV as our immunizing reagent. For APCs, we used DCs from NOD-scid mice expressing human MHC class II molecules as the source of APCs presenting antigen to the engrafted human T cells in the scid mice. Our attempts to induce a primary immune response using DC from human MHC-transgenic NOD-scid mice were unsuccessful, as were direct immunization protocols. The results section, however, does highlight deficiencies that could be approached experimentally in future studies. In summary, the results presented in this thesis advance our understanding of the fundamental mechanisms controlling human cell engraftment in scid mice. This information supports the long-term goal of establishing a functional human immune system in a small animal model. We have identified many of the cell interactions and factors that regulate human cell engraftment and function in scid mice, and we have provided insights into host characteristics that will provide optimized engraftment of naïve human T cells. The studies led to the novel observations of the regulation of human CD4+ T cells by human CD8+ T cells, B cell activation, and progression of latent EBV infection to lymphoproliferative disorders in vivo. These studies further provide new information regarding "trans-costimulation", a previously unrecognized mechanism of T cell activation. These results provide data on the fundamental mechanisms that underlie obstacles to the goal of achieving engraftment of a functional human immune system in scid mice.

Memory T cell recruitment to the non-lymphoid tissue of the intestine (the gut lamina propria) requires the interactions between integrin a4B7 and its ligand Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1), which is exclusively expressed in the gut microvasculature. T cell homing to small intestine also requires the expression of chemokine receptor CCR9 on T cells, whose ligand - the gut chemokine CCL25 - is abundantly expressed in the small intestine. The a4B7 integrin and chemokine receptor CCR9 are known as the "homing" molecules whose expression is "imprinted" on T cells by intestinal dendritic cells during antigen presentation. In the first part of this thesis, data from in vitro studies showed that other than retinoic acid, soluble tissue-derived factors such as the gut chemokine CCL25 and T cell growth factor IL-2 also contributed to the induction of gut-homing receptor acquisition. Moreover, the expression of a4B7 integrin and CCR9 appeared to be modulated differently. The present study suggests that under the influence of tissue-derived factors, T cell acquisition of a4B7 integrin is the "default" response at steady state, and that only fully activated T cells are able to express high levels of CCR9. In the second part of this thesis, T cell gut homing was investigated in the context of disease, i.e. inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). IBD is characterized by excessive lymphocyte infiltration to the intestine. By quantitatively analyzing peripheral blood and intestinal lymphocyte subsets, proportional changes in the circulating gut-homing T cells (a4B7+) was found to be associated with CD. In addition, my data suggest that downregulation of T cell receptor zeta chain (TcRC) correlates to disease severity in both CD and UC. Measuring TcRC expression may thus provide an additional tool for the monitoring and management of IBD.

La différenciation terminale des lymphocytes B qui se déroule dans les centres germinatifs des organes lymphoïdes secondaires est l’étape ultime de la réponse T dépendante et aboutit à la production de plasmocytes (PC) à longue durée de vie qui sécrètent des anticorps hautement affins spécifiques de l’antigène et caractéristiques de la réponse immune adaptative. La transition d’une cellule B naïve vers un PC est gouvernée par un réseau de régulation génique bien décrit et est largement influencée par l’intégration de stimuli externes qui contrôlent le devenir des cellules B tels que l’interaction BCR-antigène et les cytokines produites par les cellules T. La stimulation précoce des lymphocytes B humains activés par IL-2, induit la différenciation en PC via une signalisation ERK prolongée entraînant la baisse d’expression de BACH2, un facteur de transcription clef des cellules B. La répression transitoire de BACH2 est suffisante pour déclencher la différenciation en plasmablastes en l’absence d’IL-2, suggérant ainsi qu’il joue un rôle de « verrou moléculaire » de la différenciation en PC. Il est à noter que cette répression forcée de BACH2 aboutit à la production de plasmablastes caractérisés par un phénotype lymphoplasmocytaire. Ce travail de recherche s’est focalisé sur la caractérisation des mécanismes moléculaires régulant l’expression de BACH2 via la voie de signalisation ERK induite par IL-2. Nous avons identifié ELK-1 comme un médiateur de la répression de BACH2 par la voie IL-2/ERK, comme l’atteste sa capacité à se lier avec un élément de régulation d’un enhancer localisé dans l’intron 1 de BACH2, induisant ainsi la répression de l’enhancer et déverrouillant la différenciation en PC. La caractérisation de cet enhancer de BACH2 a confirmé qu’il est régulé de manière dynamique au cours de la différenciation terminale B et qu’il est localisé dans une région sujette aux mutations suggérant qu’il pourrait être impliqué dans la lymphomagenèse
The terminal differentiation of B cells, which takes places within germinal centres of secondary lymphoid organs, is the ultimate step of a T cell dependent response and results in the generation of long-lived plasma cells (PCs) that secrete protective, antigen-specific, high-affinity antibodies as part of adaptive immunity. The transition of a naive B cell into a PC is governed by a well-characterised gene regulatory network and is heavily influenced by the integration of externally received signals, including BCR-antigen binding and T cell help, such as cytokines which guide B cell fate. The early IL-2 priming of human primary activated B cells triggers PC differentiation through sustained ERK signalling resulting in the down regulation of B cell transcription factor BACH2. Transient BACH2 repression is sufficient to trigger plasmablast differentiation in the absence of IL-2 suggesting that it acts as a key lock of PC differentiation. Importantly, this enforced BACH2 repression results in the generation of plasmablasts with a lymphoplasmacytic phenotype. The focus of this thesis was to characterise the molecular mechanisms regulating BACH2 expression via the IL-2 ERK transduction pathway. We identify ELK-1 as the mediator of IL-2 ERK induced BACH2 downregulation as it binds to a regulatory enhancer element located within intron 1 of BACH2 instigating its repression and unlocking the PC programme triggering differentiation. The characterisation of this BACH2 enhancer confirms that it is dynamically regulated during PC differentiation and is located within a region targeted for mutation suggesting that it may have a potential role in lymphomagenesis

MALA-2 (Murine Lymphocyte Activated Antigen-2) is a murine cell surface antigen that is detected at high concentration on activated, proliferating lymphocytes, but only weakly on resting lymphocytes. It is thought to play an important role in lymphocyte activation since the rat monoclonal antibody YN1/1.7.4 which recognizes MALA-2 is capable of inhibiting the mixed lymphocyte reaction. Considering the central role of lymphocyte activation to the generation and maintenance of the immune response, I undertook the purification and biochemical characterization of MALA-2. In these studies, MALA-2 was isolated and purified to homogeneity using immobilized YN1/1.7.4 monoclonal antibody and sodium docecylsulphate-polyacrylamide gel electrophoresis. Biochemical characterization studies revealed that MALA-2 is a Mr 95-100 kD glycoprotein containing a protein backbone of approximately 66 kD, and N-linked carbohydrate chains amounting to a Mr of approximately 35 kD. Two dimensional gel electrophoresis suggested that MALA-2 has an isoelectric point of 4.9. Although it was previously suspected that MALA-2 might be associated with the transferrin receptor on the cell surface, this was shown not to be the case on NS-1 cells. Additionally, ³²P-orthophosphate labelling of MALA-2 on NS-1 or MBL-2 cells could not be detected. Finally, the partial amino acid sequence of MALA-2 was determined by sequencing trypsin-generated peptides from purified MALA-2. Computer-assisted homology comparisons of the MALA-2 partial amino acid sequences with other known sequences showed that MALA-2 shared its most consistent homology with a class of proteins known as the immunoglobulin superfamily. Subsequent to this study, the partial amino acid sequences obtained within this study were used to construct oligonucleotide probes. These probes were used for the screening of cDNA libraries, facilitating the successful cloning of the MALA-2 gene. This, in turn, resulted in the identification of MALA-2 as the murine counterpart of the human ICAM-1 molecule, a protein known to play a significant role in intercellular adhesion and lymphocyte activation within the immune system. Significantly, results obtained from the biochemical characterization of MALA-2 carried out in this thesis have been confirmed by the subsequent nucleotide sequence data from the cloning of MALA-2.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Cytotoxicity is the capacity for immune cells to kill infected or malignant cells in order to eliminate pathogens and tumours through different mechanisms including the exocytosis of perforin-containing cytosolic granules. This crucial property is usually restricted to specialized innate and adaptive lymphocytes such as natural killer (NK) cells and CD8 T cells. T lymphocytes differentiate in the thymus and are delivered to the peripheral blood as naive T cells committed to either the CD8 or the CD4 lineage. CD8 T cells are programmed to acquire cytotoxic effector functions under the control of the transcription factor (TF) Runx3. The fate of CD4 T cells is to acquire multiple helper functions through the action of the TF ThPOK that promotes CD4 helper functions and restricts the CD8 cytotoxic program. However, this restriction is not absolute as cytotoxic CD4 (CD4CTX) T cells differentiate in vivo, indicating that the multipotency of human naive CD4 T cells includes the ability to acquire perforin expression and potent cytotoxicity in vitro and ex vivo. This cytotoxic potential correlates with outcome in human pathology and mediates protection against viral challenge and tumour eradication in murine models. CD4CTX T cells are terminally differentiated effector memory T cells that accumulate during cytomegalovirus chronic infection and ageing. They are phenotypically and functionally related to T helper type 1 (Th1)-effector memory cells. However, whether they belong to the Th1 pathway or constitute a separate specialized helper T cell subset is unknown. In this work, we show that CD4CTX T cell differentiation is an integral part of the Th1 pathway. Indeed, CD4 T cells acquire cytotoxic potential early in the memory differentiation process as central memory Th1 but not Th2 and Th17 cells are epigenetically primed to develop a cytotoxic program. The expression of perforin and other cytotoxic genes present a stepwise increase profile that is specific of the Th1 differentiation pathway. This profile has been recapitulated in an in vitro model of effector CD4 T cell differentiation in which naive CD4 T cells acquire cytotoxicity one to two weeks after polyclonal stimulation when cultured in presence of Th1 cytokines. The molecular regulation of CD4CTX T cells is poorly understood and most available data have been generated in mice. These data include the observation of intraepithelial CD4CTX T cells in the mouse gut after loss of ThPOK expression and subsequent up-regulation of a Runx3-dependent cytotoxic program. Other candidate regulators of CD4 T cell cytotoxic function include the TF regulating Th1 and CD8CTX T cells differentiation such as Runx3, T-bet and Eomesodermin (Eomes). We show that the transcriptional program of human CD4CTX T cells is enriched in CD8-lineage genes. However, by contrast to CD4CTX T cells from the mouse intestine, human circulating CD4CTX T cells maintain the expression of ThPOK and even up-regulate this TF upon differentiation from naive CD4 T cells. Surprisingly, this sustained expression of ThPOK was compatible with the establishment of a T-bet- and Runx3-dependent cytotoxic transcriptional program. The specific knockdown of T-bet or Runx3 but not Eomes resulted in impaired cytotoxic differentiation whereas ThPOK knockdown enhanced perforin expression and cytotoxicity. We propose that CD4CTX T cells constitute the terminal stage of Th1 memory differentiation and that ThPOK, Runx3 and T-bet co-regulate this process by instructing a cytotoxic transcriptional network largely shared with CD8CTX T cells. The modulation of this network is a potential target for novel immunotherapeutic strategies in viral infections and cancer.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

AIMS: The aim of the current project was to determine if lycopene could modulate T-cell function and activity. METHODS: 1) Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults and incubated with lycopene-enriched liposomes (0.0-1.2 μg/ml) then activated with or without the mitogens Con A, anti-CD3 and LPS and cultured for various lengths of time before measuring CD3, CD4, CD8, CD69, CD11a and CD25 expression.  the production of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, TGF-β and TNF-α were also measured along with the percentage of FoxP3 and IL-10 positive T-regulatory cells.  2) Ex-vivo expression of CD11a, CD49d, CD54, CD3 and CD69, as well as Con A stimulated proliferation were measured in subjects before and after a 12 week tomato-food and lycopene intervention study. RESULTS: Lycopene reduced PBMC proliferation and CD69 expression irrespective of the type of T-cell considered.  Lycopene reduced IL-2, IL-10, IL-17 and IFN-γ but increased IL-1β and TNF-α production.  lycopene also reduced the number of CD4⁺/CD25⁺ cells present as well as those positive for IL-10. Other parameters were not affected. CONCLUSION: Lycopene reduces T-cell activity by mechanisms dependent on early cell activation but this modulation is not specific to T-cell types.  the data support a potential beneficial effect of lycopene in the reduction of atherosclerosis through the modulation of T-cell function.

Endogenous posterior uveitis (EPU) is currently treated with immunosuppressive drugs, the uses of which are limited by systemic side effects and the development of refractoriness towards the treatment. EPU is thought to be the result of an autoimmune response towards sequestered antigens in the retina. In animal models disease can be prevented by inducing tolerance to an initiating antigen. Knowing the role each antigen plays in human disease will be pivotal in developing a strategic approach to tolerance therapy in humans. There is an urgent need for the production of human sequence autoantigens to further our understanding of the diseases and develop novel therapies. In this project we produced the putative candidate autoantigens S-antigen and recoverin and used these to investigate the cellular and humoral immune responses in EPU. Recoverin was expressed in E. coli BL21 (DE3) using the expression vector pET14b. The cDNA was obtained from RT-PCR of healthy lymphocytes. The recombinant recoverin was purified with nickel chelation chromatography then reverse HPLC. The purified recoverin was used to develop an ELISA. S-antigen was expressed to CHO cells transfected with the expression vector pcDNA3.1zeo into which human S-antigen cDNA had been cloned. The S-antigen was purified, but not to the degree that it could be used for human cellular assays. Therefore, cellular assays investigating proliferation, and IL-10 and TGF-b secretion were carried out using an S-antigen synthetic peptide panel. We demonstrated that certain peptides could stimulate PBMC to produce IL-10 without proliferating, raising the possibility that certain epitopes may induce regulatory rather than pathogenic responses.

Differentiation within the lymphoid system produces effector cells which are involved in a variety of immune functions. For T cells these include the provision of help, suppression, cytolytic activity and the regulation of cooperative cellular interactions. The primary function of B lineage cells is the production of specific antibody. Understanding the regulation of normal lymphocyte proliferation and differentiation may lead to a better appreciation of those factors which result in the development of malignancy. The non-Hodgkin's lymphomas are neoplasms of the immune system, the majority of which are B cell in origin. Despite advances in immunology and molecular biology, little is known about the mechanisms involved in B cell activation, proliferation and differentiation or about those events leading to their malignant transformation. The advent of monoclonal antibody technology a decade ago has revolutionized our ability to identify and characterize cell surface antigens. Because the activation and control of proliferation of B cells was already known to involve structures at the cell surface, it was logical to utilize monoclonal antibodies to identify additional cell surface molecules that might be important in the function of normal B lymphocytes and that might allow normal and various types of neoplastic B cells to be distinguished. To achieve this goal, we developed monoclonal antibodies that showed differential reactivity between large actively dividing lymphoma cells and small inactive (quiescent) lymphocytes. These were tested for their ability to inhibit various T and B lymphocyte functions (i.e. responses to anti-µ, lipopolysaccharide, phytohemagglutinin and the mixed lymphocyte response) as well as for their reactivity with cell suspensions from a variety of malignant and nonmalignant hematopoietic tissues. From these studies emerged the following: 1) Cell surface molecules other than Immunoglobulin are involved in regulating the activation of normal B cells. This was shown by the discovery that monoclonal antibodies to both lymphocyte function associated antigen (LFA-1) and certain HLA class II determinants were able to inhibit the activation of peripheral blood mononuclear cells by the B cell mitogens anti-p and LPS. This inhibition was shown to be mediated via effects of these antibodies on T cells and/or monocytes. 2) B lymphoma cells appear to express unique cell surface antigens (defined by monoclonal antibodies LM-26 and LM-155) not detectable on cells of other lineages, and absent from normal resting or activated B lymphocytes. Future investigations will attempt to define the mechanisms by which the indirect involvement of LFA-1 and HLA class II molecules in B cell activation in vitro suggests new regulatory interactions not previously identified. Further studies will be required to define the mechanisms underlying these interactions and their significance in vivo. Similarly, the structure and function of the antigens detected by LM-26 and LM-155 remains to be determined. Nevertheless, the expression of apparently unique molecules on B lymphoma cells holds new promise for the diagnosis, classification and treatment of this group of diseases.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus which causes acute infectious mononucleosis and is etiologically associated with malignant lymphoproliferative disorders including Burkitt's lymphoma, nasopharyngeal carcinoma, B-cell lymphomas in immunocompromised hosts, Hodgkin's disease, T cell lymphomas, and smooth muscle tumors in allograft recipients. The medical significance of EBV is underscored by its potent growth transforming effects on human B-lymphocytes in-vitro and the potentially oncogenic consequences of infection in-vivo. The majority of EBV-associated malignancies occur in the setting of chronic infection and strong virus-specific humoral immunity, suggesting that cellular immunity is primarily responsible for preventing the outgrowth of EBV-transformed B cells in-vivo. Similarly, primary EBV infection in adolescents and adults stimulates an intense cytotoxic-T-lymphocyte (CTL) response which coincides with a marked reduction in the number of infected B cells in the peripheral blood. Evidence of previous EBV infection can be confirmed by the presence of EBV-specific, HLA-restricted memory T cells in the peripheral blood which inhibit the outgrowth of newly EBV-transformed B cells and efficiently lyse established autologous B-lymphoblastoid cell lines. Worldwide, EBV is responsible for substantial morbidity, comparable to measles, mumps and hepatitis virus, for which vaccines exists. Accordingly, the potential public health impact of an EBV vaccine has reinforced our efforts to identify the immunodominant virus-encoded T-cell epitopes which stimulate naive CTL effectors during acute infection and maintain memory CTL surveillance during convalescence. The EBV-encoded antigens against which the memory CTL response is directed have been partially defined, and include most of the EBV latent proteins (EBNA-2, 3a, 3b, 3c, LP, and LMP-l, 2a, 2b) consistently expressed by in-vitro EBV-transformed B lymphocytes (type-III latency). Importantly, all EBV-associated malignancies express EBNA-1, and as yet no EBNA-1-specific memory CTL have been convincingly demonstrated. Additionally, many EBV-specific CTL lines and clones have been described which do not recognize any of the known latent proteins or other EBV protein antigens tested thus far. Thus while much is known about CTL-mediated immunity against EBV, our knowledge of EBV-derived CTL epitopes remains incomplete. In contrast to the EBV-specific memory CTL response, very little is known about the source of viral epitopes recognized during the primary CTL response to EBV. In this regard, acute infectious mononucleosis represents an ideal model system to study virus-specific, cell-mediated immunity. Acute IM is a self-limited illness characterized by the appearance of "atypical" lymphocytes (CD3+/CD8+/HLADR+), including both virus-specific and alloreactive CTL, which undoubtedly contribute to virus elimination and provide CTL precursors for life-long immunity to EBV. Like other herpesvirus, EBV can undergo either lytic or latent cycle replication. During primary EBV infection many lytic cycle genes are expressed which are likely responsible for stimulating the intense cellular immune response associated with acute infectious mononucleosis. During convalescence a minor population of circulating B cells remain latently infected, harbor multiple EBV episomes, and express only EBNA-1 and possibly LMP-2a (type-I latency). Thus, latency type-I infected B cells in-vivo express a much more restricted spectrum of latent proteins and are therefore not subject to elimination by the same virus-specific CTL as are type-III EBV latently infected cells. Accordingly, many mechanisms have been proposed to explain EBV persistence including; restricted expression of EBV latent genes, reduced levels of cellular adhesion molecules, downregulation of MHC class-I molecules, absence of EBNA-1 T-cell-epitopes, and most recently, EBNA-1-mediated inhibition of antigen processing. While these mechanisms may contribute to ineffective T cell surveillance against latency type-I EBV- infected cells, B cells expressing the full spectrum of latent proteins (type-III) also exist transiently in vivoand maintain detectable humoral and CTL responses to most latent proteins. Our first goal was to identify the virus-encoded immunodominant antigens recognized by in-vivoactivated MHC class-I restricted CTL isolated from college students experiencing primary EBV infection, manifested as acute IM. Following a prodromal period of several weeks, newly EBV infected patients present with signs and symptoms of acute IM, including elevated numbers of activated CD8+ T cells in their peripheral blood, many of which, like memory CTL, are EBV-specific and HLA-restricted. In order to address the issue of EBV persistence and the immune control of EBV-induced lymphoproliferation, we also studied the long-term EBV-specific memory CTL response in these same individuals. Blood from acute IM patients and healthy EBV seropositive donors served as a source of peripheral blood lymphocytes to generate bulk CTL cultures and autologous target cells. The infecting strain of EBV was determined for each patient by DNA-PCR amplification of virus from saliva. Lymphocytes were isolated from whole blood by Ficoll-Paque density centrifugation and T- and B-cell enriched populations were obtained by AET-sheep red cell rosette selection. Autologous B cell blasts served as a source of target cells and recombinant vaccinia virus constructs were used to introduce individual EBV latent genes into target cells. Expression of individual EBV genes in target cells was confirmed by both western blot and immunofluorescence. Primary CTL responses to EBV were evaluated in standard 5lCr release assays using freshly isolated, T-cell enriched PBL from acute IM patients as effector cells. EBV-specific memory CTL responses were evaluated with bulk CTL culture generated by in-vitro restimulation with autologous B-LCLs. FACS analyses were routinely performed on bulk cultures of effector CTL populations in order to more clearly characterize their phenotype. Lastly, monoclonal antibody blocking studies and cold target competition assays were performed in order to accurately identify the viral antigen and MHC components responsible for target cell recognition. Our results based upon evaluation of 35 acute IM patients and 32 convalescent patients demonstrate that the virus-specific primary CTL response is broadly directed against the full spectrum of latent proteins, including EBNA1 and the viral coat glycoprotein gp350, while the memoryCTL response, which essentially lacks EBNA1 reactivity, is directed primarily against the EBNA 3 family of proteins (3A, 3B, 3C). Importantly, the immunodominant response by both primary and memory CTL was directed against the EBNA3 proteins. CTL from 7 of the 35 acute IM patients evaluated recognized EBNA1 expressing targets, and in 4 of these 7 patients, EBNA1 was an immunodominant antigen. Similarly, CTL from 7 of 35 acute IM patients recognized gp350 transfected targets, while no gp350-specific memory CTL responses were observed. While the phenotype of in-vivo primed CTL effectors were CD8+/HLA-DR+/CD11b+, the major subpopulation of memory CTL were CD8+/HLA-DR+/CD11b-. The CD11b "memory marker" reached peaked levels on the first sample day for all patients and gradually declined to baseline levels over a period of several months. In contrast, the CD11b marker was quickly shed from in vitropropogated CTL, over a period of 5-10 days. Target cell lysis by in-vivoactivated CTL was almost completely blocked by antibody directed againt [against] class-I molecules (BBM.1), whereas the effect of blocking target cell lysis by anti-CD8 mAb varied between 40-75%. These findings are consistent with an absolute need for class-I restricted antigen presentation, and imply that CD8 was variably required, likely for the lower affinity TCR/ Ag combinations. Cell lysis mediated by in-vitro-restimulated memory CTL was also largely inhibited by anti-class-I mAb, while anti-CD8 mAb was only mild/moderately effective in blocking target cell lysis, in keeping with the concept that memory CTL bear higher avidity TCR which can recognize antigen independent of CD8. Our detection of only one EBNA1-specific memory CTL response among the 32 patients tested supports the theory that latently infected B cells in-vivo, expressing only EBNA1, escape CTL recogition and thus might serve as a reservoir for viral persistence and/or reactivation. The rare ability to detect an EBNA1-specific memory CTL responses remains a relatively unexplained phenomenon and may involve a number of tolerizing mechanisms including the induction of anergy by presentation of EBNA-1 in the absence of costimulation, clonal deletion of low affinity T cells, the absence of dominant T cell epitopes within EBNA1 or a result of the recently described inhibiting properties of EBNA-1 on antigen processing and presentation. Alternatively, the absence of detectable EBNA1-specific memory CTL may be the result of insufficient or inappropriate restimulation of memory CTL in vitro. We addressed this possibility by attempting to selectively restimulate and expand EBNA1-specific CTL from acute IM patients by using EBNA1 expressing B cells blasts as a stimulus. Effector cells generated in this manner killed target cells in an MHC class-I restricted manner but were specific for an unspecified vaccinia antigen. Interestingly, the phenotype of the effector cells was predominantly CD3+/CD4-/CD8-/γδ T cells. In summary, our findings suggest that a multitude of previously unrecognized, EBV-specific CTL are present in the peripheral blood during acute IM, and include EBNA-1-specific CTL. The importance of accurately defining the in-vivo immune response to EBV is underscored by the ever-growing list of EBV associated malignancies. In addition to providing insights into the oncogenesis and potential treatment of NPC, a newly described link between precursor lesions and EBV infection raises the possibility that heightened immunity to EBV or EBV-infected cells may prevent the development of NPC. An obvious expectation would include extension of such knowledge to other EBV associated malignancies such as B and T cell lymphomas, Hodgkin's lymphomas, and smooth muscle tumors. First however, existing gaps in knowledge regarding the immune response to EBV and EBV-associated malignancies must be closed. Details about the viral gene products which are involved in stimulating a broadly protective, virus-specific immune response in a large number of individuals is fundamental to the design of an effective EBV vaccine. Since the presence of activated CD8+ T cells correlates with the rapid decline of EBV infected B cells in the peripheral blood, a concise description of the EBV-specific CTL response in the setting of acute infection will be necessary for the rational design of an effective acute IM vaccine. Increased understanding of viral escape mechanisms is also likely to contribute to therapeutic modalities to treat autoimmune disorders.

Le système immunitaire humain est majoritairement commandé par les cellules dendritiques et les lymphocytes T auxiliaires. Lorsque les cellules dendritiques détectent un pathogène, elles vont instruire les lymphocytes T auxiliaires afin qu’ils adoptent le phénotype approprié à la menace rencontrée. Les lymphocytes T auxiliaires peuvent être divisés en plusieurs sous-populations, caractérisées par la production de cytokines spécifiques. Chaque sous-population de lymphocyte T auxiliaire possède des fonctions propres et est impliquée dans l’élimination de pathogènes distincts. Si les réponses des lymphocytes T auxiliaires ne sont pas finement régulées, ils peuvent devenir pathogéniques, et dans ce cas, considérés comme cibles potentielles pour des thérapies. Dans ce contexte, j’ai concentré mon travail de doctorat sur l’étude de la diversité des sous- populations de lymphocytes T auxiliaires et de leur régulation. Premièrement, j’ai démontré que les cellules dendritiques activées par la TSLP sont capables d’induire la polarisation de lymphocytes T folliculaires. Ensuite, j’ai participé à la construction d’un modèle mathématique capable de prédire la réponse lymphocytaire T auxiliaire en fonction de signaux dérivés des cellules dendritiques. Ce modèle nous a permis d’identifier un rôle spécifique pour l’IL-12p70, dépendant du contexte IL-1, dans l’induction d’IL-17F sans IL-17A. Enfin, j’ai monitoré huit populations de lymphocytes T auxiliaires et folliculaires dans le sang périphérique de patients atteints de dermatite atopique traités par Dupilumab, une immunothérapie ciblant la sous-unité alpha du récepteur de l’IL-4 et j’ai pu montré que la diminution du pourcentage de lymphocytes Th17 correlait avec l’amélioration du score clinique EASI. Globalement, mon travail sur la diversité de phénotypes Th apporte une ressource mécanistique importante, avec une potentielle application en immunothérapie
Human immunity is essentially driven by dendritic cells and T helper cells. When dendritic cells detect a pathogen, they will instruct T helper cells to adopt the adapted phenotype for the specific threat encountered. T helper cells are subdivided in multiple subsets, characterized by particular sets of cytokines. Each T helper subset has specific functions and is involved in the clearance of distinct pathogens. If T helper responses are not precisely regulated, they can become pathogenic, in this case T helper pathways can be considered as potential targets for therapy. In this context, I focused my PhD work on studying T helper cell subset diversity and regulation. First, I demonstrated the ability of TSLP-activated dendritic cell to induce T follicular helper cell polarization. Then I participated in building a mathematical model capable of predicting T helper cell response to dendritic-cell derived signals. This model allowed us to identify the specific role of IL-12p70, in an IL-1 context, to induce IL-17F without IL-17A. Finally, I monitered eight T helper and T follicular helper cell populations in peripheral blood from atopic dermatitis patients treated with Dupilumab, an immunotherapy targeting the IL-4 receptor alpha subunit, and was able to show a correlation between decrease of Th17 cell percentage and improvement of EASI clinical score. Overall, my work on Th phenotype diversity provides key mechanistic insight with potential application in immunotherapy

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Bachelors
Burnett School of Biomedical Sciences
Molecular Biology & Microbiology