It is well recognized that regulatory T cells (Tregs) are immunosuppressive, by which they prevent systemic autoimmunity throughout life. Beyond this stereotypical function, however, a growing body of evidence demonstrates that Tregs in distinct tissues, including the visceral adipose tissue, dystrophic muscle, the flu-infected lung, and wounded skin can acquire unique functions directed by their local environment. Tregs in these tissues can employ a wide variety of mechanisms to accumulate and acquire tissue-specific function, including conversion from conventional T cells, canonical T cell receptor (TCR)-dependent expansion and non-canonical, TCR-independent, cytokine-dependent expansion. Intriguingly, the niche-specific function of tissue Tregs can be independent of, and mutually exclusive of, their immunosuppressive capacity. Together, this recent literature reveals that Tregs can accumulate in discrete tissue sites through non-canonical mechanisms, and in response to niche-specific cues can acquire distinct functions, which distinguish them from their peripheral, lymphoid Treg counterparts. Other tissue Treg populations remain to be identified and characterized. Moreover, it is unknown whether other tissue Tregs rely on non-canonical mechanisms of accumulation, and exhibit functions distinct from the typical Treg immunosuppressive role. Tregs are known to accumulate in the CNS during infection, injury and inflammation. The CNS is an organ with distinctive architecture that maintains a regulated interaction with the peripheral immune system due to its critical function and poor regenerative capacity. While it is known that Tregs broadly protect against excessive tissue pathology in the diseased CNS, the origin, localization, function, mechanism of accumulation, and gene signature of CNS-infiltrating Tregs have not been studied, likely due to the challenge of isolating these rare cells and distinguishing them from circulating cells left over after perfusion. Here, we establish a safe model of CNS infection using encephalomyocarditis virus and employ a series of methods to locate, monitor and isolate CNS-infiltrating Tregs free from contamination from the circulation. We show that a distinct population of thymus-derived Tregs accumulates within the cerebrospinal fluid (CSF) of the EMCV-infected CNS, independently of lymph node priming. Tregs function in this unique niche to limit excessive tissue pathology. While CNS Tregs maintain expression of core Treg signature genes, including FoxP3, their global transcriptome is more similar to that of conventional T cells (Tcons) harvested from the infected CNS than to that of peripheral Tregs. Bioinformatics analysis reveals that genes shared by CNS Tcons and CNS Tregs are also shared by Tregs and Tcons from injured muscle and from the visceral adipose tissue of aged mice, indicating that tissue inflammation and injury, rather than viral infection per se, contribute to CNS Treg accumulation, function and phenotype. Additionally, we observe that CNS Treg accumulation during infection is associated with a simultaneous increase in meningeal/choroid plexus dendritic cells (m/chDCs), which are professional antigen presenting cells that localize to the gates of the CNS. Splenic cDC and peripheral lymphoid Treg homeostasis are linked, and both populations can be artificially increased by treatment with the DC-poietin and adjuvant, Ftlt3L. Therefore, we hypothesized that CNS Tregs and m/chDCs may also be linked and could also be manipulated by Flt3L treatment. Indeed, treatment with Flt3L in conjunction with EMCV infection results in enhanced CNS Treg and m/chDC accumulation, independent of Flt3 receptor expression on Tregs. In an effort to determine if dendritic cells mediate CNS Treg increase during infection, we turned to a DC-ablative mouse model in which all CD11c-expressing cells express the catalytic subunit of diphtheria toxin and are depleted. Surprisingly, while splenic cDCs are completely abrogated in these mice, a portion of m/chDCs persists, unaffected. Moreover, CNS Tregs accumulate normally in these mice during infection. This data suggests an unappreciated heterogeneity in m/chDCs, and indicates that those that remain unaffected in these mice may mediate CNS Treg accumulation during infection. While characterizing m/chDC heterogeneity, we found that m/chDCs comprise three distinct subsets with unknown potential. Whereas m/chDCs were previously considered to be a homogeneous, CD45hiB220-CD11c+MHCII+ population, we have found them to contain three subsets, distinguishable by IRF8 and FcR-γ expression. This finding paves the way for further study of the origin, localization, and division of labor between these three m/chDC subsets. In summary, our studies clarify the distinct compartmentalization, lymph node-independent accumulation, and inflammation-associated gene signature of CNS Tregs. Most importantly, these findings have implications for neuro-immune cross-talk, particularly at the interface of the CSF and brain parenchyma. That is, neural progenitors extend their apical domains into the CSF of the ventricles, and therefore may be subject to regulation by CSF-borne Tregs. Further, while many studies have focused on the differences between tissue Treg subsets, we find a core set of genes expressed by CNS Tregs, injured muscle Tregs and VAT Tregs. This data suggests that common mechanisms may be used for therapeutic manipulation of these cells.

Despite the actions of central tolerance during thymic selection, it is clear that the peripheral T cell repertoire contains significant numbers of self-reactive T cells. The immune system needs to curtail the risk of autoimmune disease by controlling the activity of these self-reactive T cells. Various mechanisms are in place to achieve this control (peripheral tolerance). Activation of CD4+ T cells requires two signals; engagement of the T cell receptor (TCR) with an appropriate peptide:MHC complex (signal 1), and the aggregate effect of multiple signals generated following ligation of costimulatory and coinhibitory molecules (signal 2). Both signals are required for the generation of a productive T cell response and both are provided by the professional antigen presenting cell, the dendritic cell (DC). T cells are fully activated upon receiving both signal 1 and 2, but are rendered tolerant when they receive only signal 1. This can be exploited therapeutically through the administration of peptides to induce tolerance in peptidereactive T cells. Administration of peptide with an adjuvant provides both signal 1 and 2, and leads to a sustained T cell response against the administered peptide (immunity). However, if the same peptide is administered in soluble form, only signal 1 is provided, leading to the establishment of T cell tolerance. The studies in this thesis explore the role of both signal 1 and signal 2 in peptide-induced T cell tolerance. Previous data from our laboratory have highlighted PD-1 and RANKL as costimulatory molecules which could play a role in peptide-induced T cell tolerance. Here we show that PD-1, an important coinhibitory molecule, plays a vital role in restraining peripheral T cell expansion under conditions leading to T cell immunity. However, in contrast to data from other studies, we demonstrate that PD-1 plays no role in the induction, establishment or maintenance of peptide-induced T cell tolerance. We show that the costimulatory receptor ligand pair RANK:RANKL plays a role in the balance between T cell tolerance and immunity; as administration of anti-RANKL was seen to potentiate both tolerance and immunity. We also explored the effect of altering the affinity of a peptide for MHC on the induction of peptide tolerance. We demonstrate that use of a peptide with a high-affinity for MHC induces tolerance via a novel, non-deletional mechanism of peptide-tolerance induction. Importantly, we show that the high-affinity peptide can form peptide- MHC complexes which persist in a biologically relevant form for fourteen days following peptide administration. We suggest that this leads to chronic stimulation of peptide-reactive T cells which promotes acquisition of a novel tolerant phenotype. Collectively the work described in this thesis demonstrates the important roles both signal 1 and 2 play in therapeutic-tolerance induction and how the qualitative and quantitative alteration of these signals can alter T cell fate and/or responsiveness.

A balanced immune response is required to mediate clearance of a virus infection without immune-mediated disease. CD4 and CD8 T cells are capable of both exerting antiviral effector functions and regulating the immune response. The regulatory T cell (Treg) subset of CD4 T cells helps to modulate immune activation and inflammation. During respiratory syncytial virus (RSV) infection in mice, conventional CD4 T-cell-mediated cytokine production has been shown to contribute to immune-mediated pathology. I demonstrate that Tregs are critical to control immunopathology during RSV infection. This was demonstrated through diphtheria toxin (DT)-mediated Treg elimination in a mouse strain expressing the DT receptor (DTR) under the control of the Foxp3 promoter. However, these mice were unable to maintain extended Treg depletion limiting the effectiveness of this model. In addition, DT-treated wild-type (WT) mice were found to be a necessary control for adverse DT-induced disease. In humans, I have shown that activated Tregs are reduced in the peripheral blood of RSV-infected infants compared to controls. RSV-infected infants also exhibited an increased proinflammatory cytokine response in nasal aspirates. However, the alarmin cytokine IL-33, which has been shown to mediate Treg homeostasis, was the only cytokine that exhibited reduced protein levels in RSV-infected infants compared to controls. Thus, severe RSV infection in infants may be due to lack of proper Treg-mediated immune regulation. Similar to RSV, regulation of the T cell response during chronic viral infection with lymphocytic choriomeningitis virus (LCMV) is vital to prevent immune-mediated pathology. During LCMV and human chronic viral infections, CD4 and CD8 T cells exhibit T cell exhaustion where they lose the ability to exert effector functions. However, a functional CD4 and CD8 T cell response is required for viral clearance. During human chronic viral infection, an association between increased CD4 and CD8 T cell function and enhanced viral control has been identified that can be influenced by genetic factors. I aimed to identify the contribution of the host genetic factors that contribute to enhanced CD8 T cell function and viral control using the LCMV model. I found that increasing the major histocompatibility complex (MHC) diversity resulted in enhanced viral control in both a C57BL and BALB genetic background. Thus, induction of a broader T cell response was associated with enhanced viral control. However, mice expressing a heterozygous MHC on the C57BL background also exhibited mortality following chronic viral infection. Both CD4 and CD8 T cells were shown to contribute to this mortality and exhibited reduced T cell exhaustion during LCMV infection in these mice. Heterozygous MHC expression on the C57BL mouse background was also associated with an increased T helper (Th)-1 skewed CD4 T cell response compared to mice on the BALB background. Furthermore, CD4 T-cell-mediated IFN-γ production contributed to both CD8 T cell effector activity and mortality during chronic LCMV infection. Thus, both T cell epitope diversity and host genetics contribute to LCMV-induced mortality. Collectively, my data highlight both the need for effective immune-meditated viral control and regulation of T-cell-mediated pathology during both acute and chronic viral infections.

Les lymphocytes T régulateurs Foxp3+ jouent un rôle crucial dans l'établissement de la tolérance au soi, le contrôle des réponses inflammatoires et le maintien de l'homéostasie du système immunitaire. La compréhension des mécanismes moléculaires impliqués dans la fonction de ces cellules représente un défi important. Chez le rat, la déficience en Themis1, une nouvelle molécule de la signalisation du TCR, associée à un locus de 117kb d'origine BN induit un défaut fonctionnel des Tregs et le développement spontané d'une maladie inflammatoire des intestins. Au sein de ce locus, le rat BN présente deux polymorphismes non-synonymes, un au niveau du gène C3 et un au niveau du gène Vav1 (R63W). Ce dernier est un candidat potentiel du fait du rôle joué par Vav1 dans l'activation des lymphocytes T et de sa régulation par Themis1. Dans ce travail de thèse, j'ai étudié l'effet de la déficience en Themis1 associé au polymorphisme R63W de Vav1 chez la souris de fond génétique C57BL/6 sur les fonctions des lymphocytes T régulateurs. J'ai montré que la déficience en Themis1 associé au polymorphisme R63W induit un défaut fonctionnel des Tregs in vitro et in vivo dans un modèle de colite. Ce défaut est associé à une production accrue de cytokines pro-inflammatoires par ces Tregs. J'ai également mis en évidence que l'association de ces deux mutations induit une sensibilité accrue à la colite induite par le DSS. Au niveau moléculaire, j'ai mis en evidence que ce défaut fonctionnel est associé à une réduction de la signalisation du TCR impliquant les vois Erk et NF-ĸB. De plus, j'ai montré que l'inhibition d'une phosphatase de la signalisation du TCR, SHP-1, permet de restaurer les fonctions suppressives des Tregs Vav1R63W-Themis1-/-. Cette étude souligne l'importance de l'intégrité du hub de la signalisation impliquant Vav1, Themis1 et SHP-1 dans la plasticité des lymphocytes T régulateurs et dans le maintien de leur fonction suppressive. Ainsi ce hub de signalisation représente une cible thérapeutique pour augmenter les fonctions des Tregs dans le cadre des maladies inflammatoires ou réduire leurs fonctions suppressives pour favoriser les réponses immunes anti-tumorales dans le cadre du cancer. / Regulatory T cells (Treg) are of paramount importance for restraining excessive immune responses and their manipulation holds enormous therapeutic potential. Our recent results using a congenic rat model suggested that the integrity of Vav1/Themis1 T-cell receptor signaling hub plays a crucial role in Treg suppressive function. Indeed, Themis1 deficiency in BN, but not in LEW rats, led to the development of inflammatory bowel disease (IBD), linked to a defect in Treg suppressive function. Genetic studies revealed that this phenotype depended on a 117 Kb genomic locus, containing the R63W polymorphism on Vav1 that impacted its expression and functions. To test the importance of the Vav1/Themis1 TCR signaling hub in Treg function, we generated Themis1-T-/- mice expressing conditionally Themis1 in thymocytes, but not in peripheral T cells. In contrast to regular germline Themis1 knockout mice, these mice were not lymphopenic and exhibited normal proportions of CD4+ T cells in the thymus and in peripheral lymphoid organs. Next, Themis1-T-/- mice were crossed with Vav1R63W mice to assess the impact of these combined mutations on Treg suppressive functions. Using in vitro approaches, together with in vivo analyses of IBD, we showed that suppressive activity of Treg was impaired in Themis1-deficient mice harboring the mutated Vav1; this defect is linked to higher production of IL-17 and IFNg. Functional studies showed that Themis1-deficient associated with the mutated Vav1 induced a defect in Erk and P65 phosphorylation after TCR engagement. Interestingly, the inhibition of the SHP-1 phosphatase restore the functional defect of Tregs. Together, these data showed that Themis1, Vav1 and SHP-1 cooperate in the signaling hub to regulate the suppressive function of regulatory T cells. Thus, this signaling hub represents a therapeutic target to enhance the suppressive functions of Tregs in the context of autoimmune and inflammatory diseases or to decrease their functions to favor anti-tumoral immune responses.

Typ 1 NKT Zellen oder iNKT Zellen (invariante Natürliche Killer T Zellen) stellen eine Subpopulation der abT Zellen dar, die sich durch mehrere charakteristische Eigenschaften aus- zeichnet. Ihr Hauptmerkmal ist die Expression eines semi-invarianten T Zellrezeptors (TCR), der die Bindung von CD1d:Glycolipid Komplexen ermöglicht, wohingegen ‚klassische‘ T Zellen an Komplexe aus MHC (Haupthistokompatibilitätskomplex) Molekülen und Peptiden binden. Die während der Reifung im Thymus durch Transkriptionsfaktoren festgelegte Voraktivierung der iNKT Zellen ermöglicht das unmittelbare Freisetzen von Cytokinen bei Antigenkontakt, wodurch iNKT Zellen die adaptive Immunantwort stark beeinflussen können: Sie tragen sowohl zur Regulation von Autoimmunerkrankungen als auch der Bekämpfung von Krebs und Infektionen bei. Der iNKT TCR setzt sich aus einer invarianten a-Kette (AV14/AJ18 in der Maus bzw. AV24/AJ18 im Menschen) und einer charakteristischen Auswahl an b-Ketten (vorwiegend BV8S2, BV7 und BV2 in der Maus und BV11 im Menschen) zusammen. Das Cerebrosid a-Galactosylceramid (aGC, KRN7000) stellt eines der potentesten Antigene für iNKT Zellen dar. Die Präsentation dieser Antigenklasse erfolgt durch CD1d Moleküle, die, abgesehen von tiefen hydrophoben Bindungstaschen, strukturell MHC I Molekülen ähneln, jedoch nicht polymorph sind und außerhalb des MHC Locus codiert sind. Die, zwischen Maus und Mensch hochkon- servierte, Interaktion von iNKT TCR und CD1d:aGC Komplex zeichnet sich bei potenten Antigenen durch die eingeschränkte Nutzung der Antigenspezifität bestimmenden Regionen aus: CDR1a, CDR3a und CDR2b. Die den CDR3b definierende V-D-J Umlagerung der b-Kette stellt im iNKT TCR den Bereich der höchsten Variabilität dar, beeinflusst jedoch nur die Bindung schwächerer Antigene. Natürlich auftretende Variabilität innerhalb der a-Kette kann durch Abweichungen von der kanonischen V-J Umlagerung am Beginn des CDR3a entstehen und beeinflusst ebenfalls die Bindung des iNKT TCR. Die iNKT Zellpopulation in F344 Ratten ähnelt in Frequenz und Korezepotorexpression derjenigen des Menschen. Ratten besitzen ein CD1D Gen, welches hoch homolog zu denen der Maus ist und zwei dem BV8S2 Gensegment der Maus homologe BV Segmente (BV8S2 und BV8S4), die in F344 Ratten beide funktionell sind. Eine Besonderheit der Ratte ist jedoch das Auftreten einer AV14 Multigenfamilie von bis zu zehn Gensegmenten. Diese unterscheiden sich neben dem HV4 vor allem in ihren CDR2 Sequenzen und werden anhand dieser Unterschiede in zwei Gruppen (Typ 1 und 2) eingeteilt. Zusätzlich wurde in der iNKT Zellpopulation eine hohe Frequenz an natürlich auftretenden A93G Substitutionen in der TCR↵ Kette beschrieben und es wurde gezeigt, dass, im Gegensatz zur Kreuzreaktivität zwischen iNKT TCR und CD1d von Maus und Mensch, iNKT Zellen der Ratte nicht an Maus CD1d binden. Die Besonderheiten des Ratten iNKT TCR und deren Auswirkungen auf die TCR Expression und Ligandenbindung der Ratten iNKT Zellpopulation wurden in der vorliegenden Arbeit untersucht. Durch in dieser Arbeit durchgeführte in vitro Mutagenesestudien konnten Position 68 in der vierten Hypervariablen Schleife (HV4↵) und Position 93 zu Be- ginn des CDR3↵ als entscheidende Modulatoren der CD1d Bindung im iNKT TCR von Ratte und Maus identifiziert werden, wobei auch speziesspezifische Unterschiede aufgedeckt werden konnten. Die Spezieskreuzreaktivität des Ratten iNKT TCR selbst hing stark von einer A93G Substitution im TCRa ab. Bei Untersuchungen der b-Kette zeigte sich, dass sowohl BV Segmente als auch CDR3b Region die Ligandenbindung in differenziellem Zusammenspiel beeinflussen, was bei Paarung mit unterschiedlichen AV14 Segmenten verschieden ausgeprägt sein konnte. Weiterhin wurden humane CD1d Dimere generiert und zum ersten Mal die Bindung von Ratten CD1d an humane iNKT TCR gezeigt. Weiterhin wurde in dieser Arbeit das TCR Repertoire von iNKT Zellen der F344 Ratte und deren CD1d Bindungseigenschaften charakterisiert. Hierzu wurde die bereits etablierte Methode der in vitro Expansion von iNKT Zellen aus der Rattenmilz weiterentwickelt, was die Langzeitkultur und -expansion der sortierten iNKT Zellpopulation ermöglichte. Bei Untersuchung der TCR Expression konnte gezeigt werden, dass die Auswahl der im Ratten iNKT TCR genutzten BV Gensegmente ähnlich limitiert ist wie in der Maus. Neben der dominanten Nutzung der BV8S4 und BV8S2 Gensegmente wurden hauptsächlich BV8S1, BV14 und BV7 gefunden. Bei Untersuchungen der CD1d Dimerbindung der iNKT Zellpopulation konnte der Einfluss der na- türlich auftretenden A93G Substitution in der iNKT TCRa Kette bestätigt werden. Außerdem zeigte sich hier ebenfalls der Einfluss des BV Gensegments auf die Ligandenbindung, wobei BV8S4 negative Zellen im Vergleich zu BV8S4 positiven Zellen eine stärkere Ratten CD1d Dimerbindung zeigten. / Type 1 NKT cells, also called iNKT cells (invariant Natural Killer T cells), are a subpopulation of abT cells with characteristic features. Their hallmark is the expression of a semi-invariant T cell receptor (TCR) which binds CD1d:glycolipid complexes. This is in contrast to ‚conventional‘ T cells which bind complexes of MHC (major histocompatibility complex) molecules and peptides. iNKT cells show a transcription factor dependend, preactivated phenotype which is obtained during their development in the thymus. This allows for immediate cytokine release after antigen encounter and enables iNKT cells to strongly influence the adaptive immune re- sponse: They are not only able to help regulating autoimmune diseases, but also contribute to the clearance of cancer and infections. The iNKT TCR is composed of an invariant a-chain (AV14/AJ18 in mice, AV24/AJ18 in humans) and a limited number of � chains (mostly BV8S2, BV7 and BV2 in mice and BV11 in humans). a-galactosyceramide (aGC, KRN7000), a cerebroside, is one of the most potent iNKT cell antigens known so far. iNKT cell antigens are presented by CD1d, a non polymorphic molecule which is encoded outside the MHC locus. CD1d molecules resemble MHC I structurally but possess deep hydrophobic pockets as antigen binding grooves. The iNKT TCR interaction with CD1d:aGC complexes is highly conserved between mice and humans. Its binding mode is characteristic for the restricted CDR (complementarity determining region) usage and relies mostly on CDR1a, CDR3a and CDR2b. The only highly variable region within the iNKT TCR, the CDR3b defining diverse V-D-J rearrangement of the TCRb chain, has been shown to only influence binding to less potent antigens. Natural variability within the TCRa chain is usually only possible due to non canonical AV14/AJ18 rearrangements at the beginning of CDR3a and has been shown to indirectly influence ligand binding. The iNKT cells in F344 rats resemble human iNKT cells in terms of frequency and coreceptor expression profile. Rats have one CD1D gene, highly homologous to those found in mice, and two BV8S2 homologs (BV8S2 and BV8S4 ), which are both functional in F344 rats. Peculiar for the rat is the presence of an AV14 gene family with up to ten members, which have been grouped by their CDR2↵ sequences into Type 1 and 2, and a high frequency of A93G substitutions within the iNKT TCRa chains. Additionally, in contrast to the species cross-reactive interaction of iNKT TCR and CD1d from mice and humans, a lack of mouse CD1d binding by the rat iNKT TCR has been demonstrated. Those characteristic features of the rat iNKT TCR and its influence on TCR expression and ligand binding of rat iNKT cells were investigated in this study. Due to in vitro mutagenesis studies conducted in this thesis, position 68 within the fourth hypervariable loop of the TCRa chain (HV4a) as well as position 93 at the start of CDR3a could be identified as strong modulators of CD1d binding within the iNKT TCR of rat and mouse with specific features for both species. Furthermore, species cross-reactivity of rat iNKT TCR was found to be strongly enhanced due to the naturally occuring A93G substitution within the rat TCRa chain. The analysis of the rat iNKT TCRb chain showed that BV segments as well as the highly diverse CDR3b region influence CD1d binding in a complex interplay, which also differs depending on the paired a-chain. Furthermore, human CD1d dimers were generated and for the first time binding of rat CD1d to human iNKT TCRs could be shown. Additionally, the TCR repertoire of F344 rat iNKT cells and its ligand binding has been characterized in this study. For this purpose, the already established in vitro expansion of rat iNKT cell from splenocytes was further developed, allowing long term culture and expansion of purified iNKT cell populations. Analysis of the TCR expression did reveal a restricted BV segment usage, similar to mouse iNKT cells. Besides the predominant usage of BV8S2 and BV8S4 gene sements, mainly BV8S1 BV14 and BV7 were found. CD1d dimer binding studies confirmed the influence of the A93G substitution on ligand binding properties of the rat iNKT cell population and the influence of different BV segments on CD1d binding could also be seen by comparison of BV8S4 positive and negative iNKT cell subpopulations, where BV8S4 usage led to a lower rat CD1d binding profile.

In Pseudomonas aeruginosa, which causes severe secondary infections in immunocompromised patients, virulence factor expression is regulated by quorum sensing signal molecules known as acyl homoserine lactones (AHLs). One of the major AHLs produced by P. aeruginosa, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), has also been shown to alter the function of a range of mammalian cells. The goals of experiments reported in this thesis were to use murine models to investigate the effects of in vivo exposure to OdDHL on TH responses, define the direct effects of OdDHL on TH cells and to explore the mechanism by which OdDHL alters the function of TH cells. It was found that in vivo exposure to OdDHL led to changes in cytokine and antibody subclass production indicative of a shift towards the underlying TH bias of the mouse strain studied. Such shifts may play a role in infections with P. aeruginosa, as strong TH1 or TH2 responses have been associated with worsening prognosis for the host, while more balanced responses have been associated with decreases in both infection and pathology. These results suggest that treatments targeting the immunomodulatory activities of OdDHL may be of benefit in the clinical setting in the future. Direct analysis of TH cells in defined in vitro systems revealed that exposure to OdDHL led to uniform decreases in cytokine production and proliferation. These decreases in cytokine production were found to be the result of OdDHL acting on both TH cells and the antigen presenting cells (APCs) that activate them, and only occurred when cells were exposed to OdDHL within 4 hours of stimulation. These findings suggest that OdDHL is acting on a molecular target common to several cells types, and that in TH cells and APCs, this target is involved in the early stages of TH cell activation. Experiments in which T cells were activated with mitogens that bypass the cell membrane revealed that OdDHL is not acting on the cell membrane or membrane-associated activation factors, suggesting that OdDHL is instead inhibiting TH cell function through interactions with one or more intracellular signalling molecules.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are well-recognized for their immunosuppressive activity, which is mediated through an intracellular receptor and transcription factor, aryl hydrocarbon receptor (AhR). Laboratory animals exposed to TCDD are less resistant to infection and have severely impaired humoral and cell-mediated immune responses. This dissertation addressed the hypothesis that exposure to TCDD disrupts early events during the activation of CD4⁺ T cells, leading to their premature loss from the spleen. Initially, ovalbumin (OVA)-specific CD4⁺ T cells from transgenic DO11.10 mice were used to monitor the effects of TCDD on activated antigen-specific T cells. A graft-versus-host (GVH) model, in which T cells from C57B1/6 (B6) mice are injected into C57B1/6 x DBA/2 Fl (Fl) mice, was used to study the role of AhR specifically in the T cells in response to TCDD. B6 donor T cells (from AhR[superscript +/+] or AhR[superscript -/-] mice) respond to DBA/2 antigens in Fl mice and a CD4-dependent CTL response is generated. In both models, exposure to TCDD significantly decreased the number of responding CD4⁺ T cells in the spleen beginning on day 4 after initiation of the response. Exposure to TCDD altered the phenotype of OVA-specific CD4⁺ T cells beginning on day 2 after immunization with OVA. These studies also suggested that apoptosis was not the primary mechanism responsible for the loss of CD4⁺ T cells from the spleen in TCDD-treated mice. Exposure to TCDD induced AhR-dependent changes in the phenotype of B6 donor CD4⁺ T cells such that a subpopulation of CD25⁺ cells was increased in TCDD-treated Fl mice, and these cells had in vitro functional characteristics consistent with regulatory T (Treg) cells. Exposure to TCDD increased the frequency of donor CD4⁺ T cells producing interleukin (IL)-2. In addition, increased expression of CD25 in TCDD-treated mice was correlated with increased signaling through the IL-2 receptor. However, IL-2 alone was not sufficient to mimic the potent immunosuppressive effects of TCDD. These results suggest that TCDD suppresses T cell immunity in part by inducing and/or expanding a subpopulation of Treg cells by a mechanism that may involve IL-2. / Graduation date: 2006

Binding of the antigen receptor on T cells initiates a rapid series of signaling events leading to an immune response. To fully understand T cell mediated immunity, underlying regulatory properties of the receptor network must be understood. Monitoring dynamic protein signaling events allows for network analysis. Unfortunately, dynamic data acquisition is often extremely time-consuming and expensive with conventional methods; the number of proteins monitored at the same time on the same sample is limited. Furthermore, with conventional, multi-well plate assays it is difficult to achieve adequate resolution at sub-minute timescales. Microfluidics is a capable alternative, providing uniformity in sample handling to reduce error between experiments and precision in timing, an important factor in monitoring phosphorylation events that occur within minutes of stimulation. We used a two-module microfluidic platform for simultaneous multi-time point stimulation and lysis of T cells to investigate early signaling events with a resolution down to 20 seconds using only small amounts of cells and reagents. The device did not elicit adverse cellular stress in Jurkat cells. The activation of 6 important proteins in the signaling cascade upon stimulation with a soluble form of α-CD3 in the device was quantified and compared under a variety of conditions. First, in comparison to manual pipetting, the microdevice exhibits significantly less error between experiments. Secondly, a comparison between Jurkat cells and primary T cells shows similar dynamic trends across the 6 proteins. Finally, we have used the device to compare properties of long-term vs, short-term cultured primary T cells. As expected, older cells present a much weakened response to antigenic cues, as measured with TCR response markers. This modular microdevice provides a flexible format for investigating cell signaling properties through the use of soluble cue stimuli.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 79-94).

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.