Abstract not available

T helper (Th) cell activation and development is one of the most critical events in regulating the adaptive immune response. Understanding its regulation could be of great therapeutical value as many severe diseases are associated with failure in controlling T cell activation and development. However, the regulation of T cell activation appears to be one of the most complex set of cellular and molecular interactions known in the immune system. There is therefore an urgent need for tools to unravel this complexity, and to make use of the quantitative experimental data. To address this issue, mathematical and computational models, based on rigorous biophysical and kinetic data, were developed to study the specific role of some of the major costimulatory molecules involved in Th cell activation, and others developed to investigate proposed theories about mechanisms involved in Th cell differentiation. The simulations of costimulation reveal new implications for the function of the costimulatory molecules CD28 and CTLA-4, and their ligands B7-1 and B7-2, and show how binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects, all profoundly influence complex formation at the immunological synapse. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be, as has been previously proposed, due to the different binding properties of B7-1 and B7-2. Simulations of Th cell development reveal that both instructive and selective processes are likely to be involved in Th cell differentiation. In addition, further simulations indicate that Th2 cells are more likely to become dominant by inhibiting Th1 cells (negative selection), rather than selecting their own growth (positive selection). This thesis also includes an experimental work in which the immunomodulatory role of the bacterial signalling molecule N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) was analysed. This study strongly suggests that OdDHL suppresses Th cell activation and development, and that it is likely targeting the intracellular signalling events involved in the early stages of Th cell activation.

[Truncated abstract] HIV infection compromises the ability of the host to mount effective immune responses. In untreated HIV disease, immune activation drives high rates of cell turnover and apoptosis, ultimately leading to abnormal and dysregulated cellular function. Immune activation may also induce the expansion of CD4+ regulatory T (Treg) cell populations capable of suppressing anti-HIV responses. Treatment with antiretroviral therapy (ART) allows the recovery of CD4+ T cell numbers in most patients. Persistent deficiencies in the number and function of CD4+ T cells seen in a proportion of individuals may reflect elevated numbers of Treg cells or an imbalanced regulatory-to-effector cytokine milieu. Furthermore, some patients develop paradoxical illnesses associated with the recovery of cellular function, known as immune restoration disease (IRD). The first part of this thesis addresses the role of CD4+ Treg cells in untreated and treated HIV disease. The second part addresses the phenotype of immune cells that express IL-10 and its receptor in untreated and treated patients, and the role of IL-10 in mycobacterial IRD. Firstly, several cell surface markers were evaluated to find a flow cytometry assay that could be used routinely to identify CD4+ Treg cells in HIV-infected patients. I tested CD25, GITR, CTLA-4, NRP-1 and LAG-3, but their expression did not mirror the expression of FoxP3, an intracellular transcription factor specific to CD4+ Treg cells (Chapter 2). Two published studies then described the use of CD127 to identify CD4+FoxP3+ Treg cells in humans. Using CD127, I determined the proportions and numbers of CD4+ Treg cells in untreated HIV-infected patients and in patients in their first year of ART. Proportions of CD4+ Treg cells correlated with the proportions of activated (HLA-DRHI) CD4+ T cells and with plasma HIV RNA levels in untreated patients, but showed an inverse correlation with CD4+ T cell count. In both untreated and treated patients, the proportions and numbers of FoxP3+ cells that expressed CD8 were significantly higher than in uninfected donors. This was clearest in patients with CD4+ T cell counts below 300/'L (Chapter 3). This body of work suggests that the frequencies of CD4+ Treg cells are directly related to the level of HIV-associated immune activation. Phenotyping of FoxP3+CD4+ Treg cells in untreated and treated patients and in uninfected donors revealed that co-expression of CD45RO, CD28, CTLA-4 and markers of activation were similar in all HIV-infected patients and controls. ii FoxP3+CD8+ T cells exhibit lower levels of CD45RO, CD28 and CTLA-4, but higher expression of PD-1 and CD57 (Chapter 4). This suggests that FoxP3+CD8+ T cells may have a reduced functional capacity. It is unclear whether they have regulatory activity by virtue of FoxP3 expression. ... Both patients produced higher levels of IFN? compared with IL-10 in response to mycobacterial antigens. In contrast, patients who experienced uneventful immune reconstitution produced higher levels of IL-10 (Chapter 6). Part 1 of this thesis highlights the importance of using specific cellular markers to identify CD4+ Treg cells, and confirms CD127 as a valuable marker for routine monitoring of blood Treg cells. Part 2 of this thesis demonstrates the important regulatory role of IL-10 in patients receiving ART.

Multiple checkpoints control the survival and activation of auto-reactive B cells. The discovery of the TNF family cytokine BAFF has been crucial to understanding peripheral B cell tolerance mechanisms. Homeostatic levels of BAFF are tightly regulated to maintain tolerance in the periphery. Chronically increased levels of BAFF lead to the survival of autoreactive B cells. Autoimmune patients display elevated serum BAFF levels. BAFF Tg mice model this situation with systemically high levels of BAFF and the subsequent development of two separate but related autoimmune syndromes; systemic lupus erythematosus (SLE) and Sj??gren???s syndrome (SS). The work conducted in this thesis further investigates the defects in tolerance down-stream of self-reactive B cell survival, which may contribute to autoimmune disease development in BAFF Tg mice. Expansion of the Marginal zone (MZ) B cell population correlates with the pathogenesis of several models of autoimmune disease. BAFF Tg mice are unique in that they not only display an increased splenic MZ B cell population, but also MZ B cells are found in the salivary glands of mice developing SS. The examination of genes differentially regulated between MZ and Follicular (Fo) B cells led to the investigation of sphingosine-1-phosphate receptor biology. The expression of S1P receptors was shown to be required for the positioning of MZ B cells in the spleen. Chronic BAFF stimulation alters the retention of MZ B cells through the alteration of S1P receptors and decreased integrin activation. The alteration of S1P receptors and increased ligand sensitivity leads to the accumulation of MZ B cells in the inflamed salivary glands of BAFF Tg mice. This works provides a potential mechanism for the tissue specificity seen in systemic autoimmune disease. The provision of T cell help to auto-reactive B cells is thought to underlie the development of SLE. BAFF Tg mice deficient in T cells surprisingly developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did however require signals through the toll-like receptor (TLR)-associated signalling adaptor, MyD88, which controlled the production of pathogenic autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, which requires TLR signalling and is independent of T cell help. It is likely that autoimmune patients with elevated levels of BAFF show a similar basis for disease.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 283-321).

<p>T lymphocytes are primarily involved in adaptive, cell-mediated, immune reactions. In this thesis T cells were studied regarding central and peripheral differentiation and homeostatic mechanisms for maintanance of the immune repertoire.</p><p>The influence by mature T cells on thymic development was studied in C.B-17 <i>scid/scid</i> (SCID) mice, devoid of mature T and B cells, and whose thymocyte development is arrested at the early pro-T cell stage. When mature syngeneic T cells were injected the developmental block was overcome and there was an accumulation of CD4⁺CD8⁺ thymocytes. This event was accompanied by the maturation of medullary epithelial cells in thymus which seemed to be driven by CD8⁺ T cells. In the periphery there was initially a spontaneous T-cell proliferation and later, the majority of the donor T lymphocytes showed a memory phenotype with high expression of CD44 and with an early onset of proliferation and cytokine production upon stimulation. This stable pool of memory type of cells sustained for more than a year following treatment.</p><p>Treating SCID mice with allogeneic T cells results in graft-versus-host disease (GVHD). Severe GVHD was dependent on the MHC-haplotype of the donor cells and was accompanied by profound alterations of the TCR-Vβ repertoire and with high production of IFN-γ.</p><p>Kappa light chain (κ)-deficient mice have only half the number of B cells as their normal counterparts but normal levels of immunoglobulins. When T cells from κ-deficient mice were stimulated <i>in vitro</i> there was a bias towards production of B-cell stimulatory type 2 cytokines. This is proposed as a mechanism for the homeostatic control of serum immunoglobulin levels in κ-deficient mice.</p>

<p>Antibodies have the ability to influence their own production in a process called antibody feedback regulation. Depending on the type of antigen and the subclass of the antibody, the outcome of feedback regulation can be complete suppression or several hundred-fold enhancement of the antibody response.</p><p>IgE and IgG3 enhance responses to soluble protein antigens. Previous results suggest that IgG3-mediated enhancement of antibody responses is dependent on complement and not Fc receptors for IgG. However, the Fc receptor-deficient animals used did not completely lack the IgG3-binding FcγRI. We re-examined the role of this receptor in a new mouse strain completely lacking FcγRI and found that IgG3-mediated enhancement was unperturbed, thus confirming a role for complement. </p><p>To investigate the early responses resulting in IgE-mediated enhancement of antibody responses we used biotinylated antigen and found that mature follicular B cells and to a lesser extent transitional type 2 B cells capture IgE/antigen complexes. Adoptive transfer of CD4+ T cells expressing a transgenic TCR specific for ovalbumin demonstrated that these T cells localize near the B-cell follicle after 6-12 hours and that IgE, in contrast to IgG3, significantly increased specific T cell proliferation. After 3 days the T cells had gone through several rounds of divisions and showed an activated phenotype. Additional cell transfer studies identified CD23+ B cells as the responsible effector cells. These results indicate that the mechanism underlying IgE-mediated enhancement is rapid transport of IgE/antigen complexes by follicular B cells into B-cell follicles, followed by antigen presentation by CD23+ B cells to naïve CD4+ T cells. IgG3, inducing poor T cell responses, is more likely to depend on lowering the threshold for B-cell activation by co-ligating the B-cell receptor with the complement receptor 2/CD19 complex on the surface of the B cell.</p>

MicroRNAs (miRNAs), an abundant class of ~22 nucleotide non-coding RNAs, are thought to play an important regulatory role in animal and plant development at the posttranscriptional level. Many miRNAs cloned from mouse bone marrow cells are differentially regulated in various hematopoietic lineages, suggesting that they might influence hematopoietic lineage differentiation. Some human miRNAs are linked to leukemias: the miR-15a/miR-16 locus is frequently deleted or down-regulated in patients with B-cell chronic lymphocytic leukemia and miR-142 is at a translocation site found in a case of aggressive B-cell leukemia. miR-181, a miRNA upregulated only in the B cell lineage of mouse bone marrow cells, promotes B cell differentiation and inhibits production of CD8⁺ T cells when expressed in hematopoietic stem/progenitor cells. In contrast miR-142s inhibits production of both CD4⁺ and CD8⁺ T cells and does not affect B cells. Collectively, these results indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. / Singapore-MIT Alliance (SMA)

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 106-113).

Les principaux échecs des greffes de cellules souches allogéniques (HSCT) sont la rechute et les infections, accompagnées ou non de GvHD (Graft versus Host Disease, maladie du greffon contre lhôte), manifestations qui pourraient être partiellement attribuées à un déficit immunitaire (rechute, infections) ou à une réaction immunitaire exacerbée envers le receveur (GvHD) (Baron, Storer et al. 2006). Létude de la reconstitution du système immunitaire, et particulièrement, lymphocytaire, savère dès lors capitale dans le développement des HSCT. Nous avons investigué la reconstitution immune de 50 patients traités par HSCT nonmyéloablative classique vs HSCT nonmyéloablative déplétée en lymphocytes T CD8+. 50 patients ont été randomisés : greffon déplété en CD8 (n=22) vs non manipulé (n=28). Lâge médian était de 57 ans au moment de la greffe (range 36-69). Le régime de conditionnement consistait en une irradiation corporelle totale de 2 Gy avec ou sans ajout de Fludarabine. 20 patients ont reçu une greffe de donneur familial, 14 de donneurs non familiaux HLA identiques, et 16 de donneurs non familiaux présentant une disparité HLA. La reconstitution immunitaire la première année après HCT a été monitorée par cytométrie en flux, analyse de la diversité du répertoire du TCR (spectratyping), quantification de sjTREC (signal joint T cell receptor excision circle, marqueur de la thymopoïèse). La déplétion des CD8 a réduit la reconstitution des taux de CD8 durant les 6 premiers mois postgreffe (P<0.0001) mais na pas présenté dimpact significatif sur la récupération des autres populations cellulaires. Les concentrations de sjTREC et des taux de CD3 ont augmenté parallèlement entre le jour 100 et le jour 365 après greffe (P=0.006 et P=0.022, respectivement), suggérant ainsi la néoproduction de lymphocytes T par le thymus, même chez ces patients âgés. Les facteurs associés à une concentration conséquente de TREC un an après greffe incluent 1° le choix dun donneur non familial HLA-matched (P=0.029), 2° de hautes concentrations de lymphocytes T dans le greffon (P=0.002), et 3° labsence de GVHD chronique (P<0.0001). Nos données suggèrent un modèle biphasique de reconstitution du pool lymphocytaire T: 1) une expansion des T matures du greffon en périphérie durant les 3 premiers mois ;2) une néoproduction active par voie intrathymique assurant la reconstitution du système immunitaire à plus long terme. Combien de temps cette néosynthèse intrathymique perdure-t-elle ? Quels facteurs laffectent ? Est-elle associée à une diversité accrue du répertoire lymphocytaire ? Ces questions nous ont amenés à étudier la reconstitution immunitaire à long terme (entre 1 et 6.5 ans) de 73 patients après minigreffe (211 points au total). Nous avons observé un maintien de la thymopoïèse réenclenchée au cours de la première année postgreffe chez les patients âgés de moins de 50 ans et de 50-60 ans. Cette reprise de la thymopoïèse na pas été mise en évidence au sein du groupe des plus de 60 ans. Ainsi, une application clinique concrète à cette observation pourrait être ladministration de greffons particulièrement riches en lymphocytes T à ce type de patients, puisquils seront plus susceptibles de développer une lymphopénie persistante postgreffe par absence de réenclenchement de la voie thymodépendante. Les facteurs associés à une reconstitution thymodépendante à long terme après minigreffe étaient : 1°labsence de cGvHD (P<0.0001); 2°lâge du receveur (P<0.0001); 3° la concentration en lymphocytes T dans le greffon (P=0. 0.0038) ; 4°laugmentation de la diversité HLA (P=0.0001). Enfin, une tendance non significative à une augmentation parallèle de la diversité du répertoire TCR et des concentrations en sjTREC a été mise en évidence. Cette analyse doit être confirmée par létude dun plus grand nombre de sujets. Afin déliminer au maximum les influences extrathymiques sur les taux de TREC périphériques, nous comptons mesurer pour chaque patient des TREC précoces (BTREC) et un TREC tardif (sjTREC), et calculer un ratio reflétant exactement le nombre de divisions intrathymiques. Cette méthode a été validée dans notre centre au cours dune expansion de lymphocytes T in vitro au moyen de billes anti-CD3 anti-CD28, expansion durant laquelle nous avons pu observer une diminution des sjTREC et des BTREC, mais pas du ratio sjTREC/BTREC. Lanalyse des ratios de TREC des patients greffés est actuellement à létude. Une troisième étude a été également menée afin déclaircir le lien entre GvHD, thymopoïèse et présence de lymphocytes T régulateurs (TRegs) chez 64 patients après HCT. Lémergence dun nouveau marqueur spécifique des TRegs, le CD127, a permis pour la première fois une isolation sans équivoque des TRegs (Liu, Putnam et al. 2006). Nous navons pas pu mettre en évidence de différence significative entre lapparition dune cGvHD chez les patients présentant après greffe des taux de TRegs supérieurs ou inférieurs à la médiane (P=0.13). Inversement, loccurrence de cGvHD na pas paru significativement affecter les concentrations en TRegs après greffe (P=0.1). Nous avons également mis en évidence une corrélation positive significative entre le taux de sjTREC/ml et le taux de TReg/ul au J100 (R=0.46, P=0.007) et à 1 an R=0.47, P=0.001). Afin de déterminer lorigine précise de ces TReg après greffe (thymus du donneur?), nous réalisons actuellement des mesures de sjTREC et de chimérisme sur les populations cellulaires triées au moyen du triple marquage CD4+CD25+CD127- (cellules T classiques vs régulatrices).