Дисертації з теми "CD30L T cell"

ZTLs vermitteln die Eliminierung von infizierten und entarteten Zellen durch Apoptose. Neuste Erkenntnisse unserer Gruppe haben gezeigt, dass eine Subpopulation der CD8+ T-Zellen, anstelle der zytotoxischen Marker das Oberflächenmolekül CD40L exprimiert. Die Expression von CD40L ist bislang als Schlüsselmolekül für die CD4+ T-Zell vermittelte Hilfe bekannt, welche durch Bindung an den CD40 Rezeptor auf anderen Immunzellen induziert wird. Das von den CD4+ T–Zellen ausgehende CD40L Signal ist besonders für die T-Zell abhängige B-Zell Aktivierung und die Bildung von Keimzentren essentiell, in denen B-Zellen heranreifen und hochaffine Antikörper produzieren um den Organismus vor eindringenden Erregern zu schützen. Aufgrund der CD40L-assoziierten Helferfunktion sollte in dieser Arbeit untersucht werden, welche Auswirkungen die Interaktion von CD8+CD40L+ T-Zellen mit B Zellen hat. In in vitro Studien konnte gezeigt werden, dass 50% der antigen-spezifischen CD8+ T-Zellen nach Aktivierung durch B-Zellen CD40L hochregulieren. Sowohl auf RNA- als auch auf Proteinebene induzierten CD8+CD40L+ T-Zellen einen B-Zell Phänotyp, der stark dem von CD4+ T-Zellen stimulierten B-Zellen ähnelte. In Infektionsversuchen mit dem B-Zell-trophen Virus MHV-68 konnte gezeigt werden, dass transgene Mäuse mit CD40L defizienten CD8+ T-Zellen im Vergleich zu Kontrolltieren eine signifikante Reduktion der Keimzentrums-B-Zellen in den Lymphknoten der oberen Halsregion aufweisen. Eine genauere Betrachtung des B-Zell Repertoires von IgG Gedächtniszellen ergab jedoch, dass die Sequenzen der IGHJ3 Genfamilie bevorzugt für die Modifikation der CDR3 Region in Mäusen mit CD40L defizienten CD8+ T-Zellen verwendet wird, die eine entscheidende Rolle bei der Antigenerkennung spielt. Zusammengefasst kann mit dieser Arbeit zum ersten Mal gezeigt werden, dass CD8+CD40L+ T-Zellen Helferfunktionen durch Unterstützung der B-Zell Aktivierung und Bildung von Keimzentren übernehmen können.
CTLs are important for the elimination of infected and degenerated cells by inducing apoptosis of the target cells. Recently our group identified a sub-population of CD8+ T cells expressing CD40L instead of common CTL markers. To that date, transient CD40L expression on T cells has been only described as a function of activated CD4+ T cells, which displays this key molecule for CD4+ T cell mediated help by binding to the CD40 receptor on other immune cells. Particularly, CD40L signaling provided by CD4+ T cells is indispensable for T cell dependent B cell activation and GC responses, which generate B cells secreting high affinity antibodies that protect the host from invading pathogens. Due to its associated helper functions, this thesis aimed to dissect whether CD40L positive CD8+ T cells are restricted to cytotoxic killing or if this sub-population possesses similar properties as CD4+ T cells when interacting with B cells. In vitro co-culture experiments showed that 50% of murine antigen specific CD8+ T cells up-regulated CD40L upon activation by antigen presenting B cells. When compared to CD40L deficient CD8+ T cells, the interaction of CD8+ CD40L+ T cells induced remarkable changes in B cells on the RNA and protein level and triggered a B cell phenotype resembling that of B cells primed by CD4+ T cells. By the infection of mice with the B cell trophic virus MHV-68, it was found that E8IcrexCD40Lflox transgenic mice lacking CD40L only on matured CD8+ T cells, exhibited a significant decrease of GC B cells in superficial cervical lymph nodes at the acute state of infection compared to WT mice. A closer look into the memory B cell repertoire revealed a preferred usage of the murine IGHJ3 gene family that modifies the CDR3 and thus the recognition groove of the B cell antibody in E8IcrexCD40Lflox mice. In summary, this work provides sufficient evidence that CD8+ CD40L+ T cells adopt helper-like functions by supporting B cell activation and subsequent GC formation.

Les lymphocytes T CD8+ spécifiques de l'antigène sont impliqués dans la réponse immunitaire adaptative et jouent un rôle essentiel dans la protection de l'hôte contre l'infection par des pathogènes intracellulaires. Cette protection de longue durée dépend de la génération de réponses lymphocytaires T CD8+ mémoires, hautement fonctionnelles en termes de fréquence et de fonctionnalité, après réinfection.Après présentation de l'antigène, une cellule T CD8 naïve subit une forte expansion clonale, générant une population hétérogène de cellules activées qui est dominée, au sommet de l'expansion, par des effecteurs CD8 de courte durée (SLEC). Cette expansion est suivie d'une phase de contraction massive par apoptose. Quelques cellules survivent à cette phase de contraction et finissent par se différencier en cellules mémoire hautement compétentes. Les processus par lesquels et le moment où se différencient les précurseurs de mémoire (MPECs) restent largement inconnus, tout comme les étapes ultérieures de leur maturation en cellules mémoire pleinement fonctionnelles. Les signaux d'aide provenant des cellules T CD4+ sont clairement requis tout au long du processus de maturation des MPEC.Notre équipe a montré que les lymphocytes T CD4+ régulateurs FoxP3+ (Tregs) favorisent la maturation des MPEC en limitant l'exposition à l'IL-2 et en fournissant des signaux inhibiteurs, mais ce n'est probablement qu'une facette de l'aide complexe et multiforme apportée par les cellules T CD4+ au MPEC. Les Tregs agissent sur des MPEC préexistants. Les réponses mémoire B et CD8+ partagent des caractéristiques communes, telles que l'expression du facteur de transcription Bcl-6. Les lymphocytes T CD4+ folliculaires (Tfh) sont les principaux producteurs de la cytokine IL-21. Bien que les mécanismes par lesquels les Tfh induisent l’expression de Bcl-6 dans les cellules B doivent être clarifiés, ils pourraient inclure l’IL-21 et l’interaction CD40-CD40L.Dans ce projet de thèse, nous avons étudié le rôle potentiel des Tfh dans l'initiation de la différenciation mémoire T CD8+, dans des modèles de souris transgéniques permettant une déplétion transitoire et sélective des Tfh, infectées par la bactérie recombinante Listeria monocytogenes-OVA.Nous avons montré que dès 2 jours après l'infection, les MPECs très précoces peuvent être identifiés par l’expression du récepteur de chimiokine CXCR5. Ces précurseurs précoces, qui ont un phénotype effecteur, se développent et migrent temporairement à la jonction des zones T et B, où ils interagissent avec les Tfh puis perdent leur expression CXCR5.Cette interaction avec les Tfh, considérés jusqu'à présent comme des auxiliaires exclusifs des cellules B, est nécessaire pour que les MPECs CD8+ deviennent des cellules mémoire compétentes sensibles à l'IL-21, capables de générer des réponses effectrices secondaires efficaces.Cette étude dévoile les premières étapes cruciales dans la génération de la mémoire CD8+, identifie CXCR5 comme le premier marqueur connu des MPECs CD8+, révèle l’implication fondamentale des Tfh dans le CD4 help et indique une coordination possible, via les Tfh, entre les voies de différentiation mémoire CD8+ et B. Ces résultats peuvent avoir des implications pour la conception du vaccin et de l'immunothérapie
Antigen-specific CD8 T cells are involved in the adaptive immune response and play a critical role in protecting the host from infection by intracellular pathogens. This long-lasting protection depends on the generation of memory CD8+ T cell responses, which are highly functional in terms of frequency and functionality, after secondary infection.Following antigen activation, a naive CD8 T cell undergoes strong clonal expansion, generating a heterogeneous population of activated cells that is dominated, at the peak of expansion, by short-lived CD8 effectors (SLECs). This expansion is followed by a phase of drastic contraction through massive apoptosis. A few cells survive this contraction phase and eventually become highly competent memory cells. Precisely when and how these memory precursors (MPECs) are generated is largely unknown, and so are the subsequent steps of their maturation into fully functional memory cells. Help signals from CD4+ T cells are clearly required throughout the MPEC maturation process.Our team has previously shown that FoxP3+ regulatory CD4+ T cells (Tregs) favor MPECs maturation by limiting exposure to IL-2 and by providing inhibitory signals, but this is probably only one facet of the complex and multifaceted help provided by CD4+ T cells to MPEC, and Tregs act on pre-existing MPECs.B-cell memory and CD8+ T cell memory share some common features, such as the expression of the transcription factor Bcl-6. Tfh are major producers of the cytokine IL-21. The mechanisms by which Tfh induces Bcl-6 in B-cells need to be clarified, they might include IL-21 and CD40-CD40L.In this PhD project, we have investigated the potential role of Tfh on the initiation of CD8 memory differentiation, in transgenic mice models, allowing transient and selective depletion of Tfh cells, infected by recombinant Listeria monocytogenes-OVA.We have shown that as early as 2 days after infection, very early memory precursors can be identified by their expression of the chemokine receptor CXCR5. These early precursors, which have an effector phenotype, expand and temporarily migrate to the junction of T-cell and B-cell zones, where they interact with follicular CD4 T cells (Tfh) then lose their CXCR5 expression.Remarkably, this interaction with Tfh, hitherto considered as exclusive B-cell helpers, is required for memory precursors to become competent memory cells responsive to IL-21 and capable of mounting efficient cytotoxic secondary effector responses.This study thus unveils critical early steps in the generation of CD8 memory, identifies CXCR5 as the earliest known marker of CD8 memory precursors, reveals a major helper role for Tfh, and points to possible coordination, through Tfh, between the pathways of CD8 and B-cell memory generation. These findings may have implications for vaccine and immunotherapy design

T Zellen können in zwei Hauptpopulationen mit unterschiedlichen Aufgaben unterschieden werden. CD4+ T Zellen exprimieren im Zuge ihrer Aktivierung CD40L, welches ein zentraler kostimulatorischer Rezeptor zur Induktion von B-Zell basierter humoraler Immunität, APC Aktivierung und einer effizienten Effektor CD8+ T Zell Entwicklung ist („Helfer-Funktion“). Im Gegensatz dazu sind die zytotoxischen CD8+ T Zellen dazu vorbestimmt, infizierte oder maligne Zellen direkt abzutöten. Jedoch wurde eine Fraktion von CD8+ T Zellen identifiziert, die nach Aktivierung CD40L exprimiert. Bisher ist nicht verstanden, wie in solchen CD8+ T Zellen a) die CD40L Expression reguliert ist, b) wann und wie die Fähigkeit CD40L zu exprimieren implementiert wird und c) was die Folgen für das Immunsystem sind. In dieser Arbeit konnten wir zeigen, dass sowohl in CD4+ als auch in CD8+ T Zellen die CD40L Expression durch DNA-Methylierung regulatorischer Regionen des CD40LG Lokus reguliert wird. Die Demethylierung zentraler Elemente wird im Thymus implementiert, manifestiert sich mit der T-Zell Reifung und geht mit einer zunehmenden Stabilität der CD40L Expression einher. Erhöhte Expression von CD5 und NUR77 in CD40L+ CD8+ SP Thymozyten weisen auf eine positive Selektion mit hoher Affinität gegen Selbst-peptide während der Reifung im Thymus hin, welche das weitere Schicksal der CD40L exprimierenden CD8+ T Zellen beeinflusst. Naive CD40L+ CD8+ T Zellen besitzen ein anderes TCR Repertoire als CD40L- CD8+ T Zellen und reifen im Zuge ihrer Aktivierung bevorzugt zu Gedächtniszellen mit Zytokin- und Chemokinrezeptorprofilen von Tc2, Tc17 und Tc22 Zellen heran. Mit ihrem nicht-zytotoxischen Phänotyp und ihrer Genexpressionsignatur ähneln diese Zellen stark Helfer-CD4+ T Zellen und können von den klassisch zytotoxischen Tc1 und Tc17+1 Zellen durch ihre IL-6R und fehlende SLAMF7 Expression sowie der Expression von Markern die auf eine Fähigkeit in die Haut zu wandern schließen lassen, unterschieden werden. Zusammenfassend zeigen wir hier, dass naive CD8+ T Zellen von den frühsten Entwicklungsstadien im Thymus an nicht homogen sind und die Fähigkeiten über CD40L Expression eine Helferfunktion auszuüben beziehungsweise über die Sekretion zytolytischer Moleküle Zielzellen abzutöten unabhängig vom CD4+ or CD8+ T-Zell Status sind. Zellen mit Zytokin- und Genexpressionsignaturen, die mit denen der CD8+ Helfer-T Zellen übereinstimmen, wurden von uns und anderen in Geweben (Haut, Lunge) identifiziert und tragen zu den verschiedensten autoinflammatorischen Erkrankungen bei. Diese Arbeit insinuiert daher die Notwendigkeit einer grundlegenen Neubewertung der CD8+ T Zell Fähigkeiten und Funktionen in Immunantworten.
The T cell compartment consists of two major subsets with diverse assignments. CD4+ T cells express CD40L upon activation, a central co-stimulatory receptor to induce B cell mediated humoral immunity, activate APCs and prime efficient effector CD8+ T cell development (“helper function”). In contrast, cytotoxic CD8+ T cells are predetermined to kill infected or malignant cells directly. However, a fraction of CD8+ T cells expressing CD40L upon activation was identified. So far, it is not understood in CD8+ T cells a) how CD40L expression is regulated, b) when and how the ability of CD40L expression is implemented and c) what are the implications for the immune system. In this thesis, we found that CD40L expression is regulated by DNA-methylation of regulatory regions of the CD40LG locus in CD4+ as well as CD8+ T cells. The de-methylation of central elements is implemented in the thymus and increases with T cell maturation reflected by enhanced stability of CD40L expression. Elevated CD5 and NUR77 expression of CD40L+ CD8+ SP thymocytes suggests that high affine detection of self-peptides during positive selection in the thymus implements CD40L expression ability and predetermines the fate of the CD40L imprinted CD8+ T cells. CD40L+ naïve CD8+ T cells differ in their TCR repertoire from their CD40L- counterparts and preferentially mature into memory cell subsets with cytokine and chemokine receptor profiles of Tc2, Tc17 and Tc22 cells. With their non-cytotoxic phenotype and gene expression signatures, the CD40L+ memory CD8+ T cell subsets Tc2, Tc17 and Tc22 widely resemble helper CD4+ T cells and can be distinguished from classical cytotoxic Tc1 and Tc17+1 cells by their IL-6R and absent SLAMF7 expression and their skin migratory phenotype. Altogether, we demonstrate that from the earliest developmental stages in thymus onwards naive CD8+ T cells are not homogenous and the abilites to provide “CD40L based help” or “cytotoxicity mediated killing” are independent of the CD4+ or CD8+ T cell status. Cells with helper-type CD8+ T cell cytokine and gene-expression signatures were found at barrier sites (skin, lung) by us and others where they contribute to multiple autoinflammatory diseases. Therefore, this work insinuates the need to revisite CD8+ T cell capablities and function in immune responses.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
91
英文摘要 Hodgkin''s disease is a type of malignant lymphoma, characterized by the presence of abnormal cells, named Reed-Sternberg cells, in patient’s lymph nodes. CD30 was originally described as a marker of Hodgkin/Reed-Sternberg cells. CD30 and its cognate ligand, CD153, belong to members of the TNFR and TNF superfamilies, respectively. CD30 is expressed on the surface of Hodgkin/Reed-Sternberg (H-RS) cell lines (KM-H2), while expression of CD153 can be induced on the surface of peripheral blood T cells by anti-CD3 or PHA activation. In this study, we addressed the effect of CD30 reverse signaling on T cells. By co-cultures of KM-H2 and PBMC activated by anti-CD3 or PHA, we observed T cell proliferation was inhibited. The inhibition was not dependent on cytokines or substances released from KM-H2, because KM-H2 cells were fixed with paraformaldehyde before the co-culture. We further study the effect of CD30 on T cell proliferation with CD30-expressing Chinese Hamster Ovary (CHO) cells to. In the presence of CD30-positive CHO cells, PHA-treated PBMC failed to achieve significant proliferation. Similar effects were observed if PHA-treated PBMC were cultured in the medium containing chimeric CD30-Fc fusion proteins. Taken together, we discover the inhibitory effect of CD30 reverse signaling on CD153-positive T cells. Furthermore, in order to characterize the protein expression profile in response to CD30 reverse signaling, proteomic technology was used. Several candidate spots were found to be likely regulated by CD30 engagement. One of these spots was identified as Mn-SOD by the use of MALDI-TOF MS. We conclude that H-RS cells are able to inhibit the proliferation of activated T cells through the CD30-CD153 interaction, which may lead to a microenvironment in favor of the growth and survival of the tumor cells.

CD30 is a member of the TNFR superfamily that was initially identified on Reed-Sternberg cells of Hodgkin's disease and is widely expressed in other lymphomas as well as in a number of autoimmune diseases. On normal cells, CD30 is expressed primarily on activated CD8⁺ T cells and is induced by two distinct pathways, an IL-4 dependent pathway and an IL-4-independent pathway via CD28. The precise role of CD30 has been controversial, but it has been implicated in a number of T cell functions, including costimulation, cytokine production, cell survival and cytotoxicity, although much of the published work to date has been carried out in cell lines. In an attempt to elucidate the role of CD30 in normal T cells, the function of primary lymphocytes derived from CD30-deficient mice was studied. In the absence of CD30, proliferation and activation were normal, with CD30[sup -/-] cells exhibiting levels of proliferation and expression of activation markers comparable to that of wild type cells. As well, among those cytokines examined, production by activated CD30-deficient cells was normal, although production of IL-4 was reduced compared to wild type. 2C-transgenic CD30-deficient cells were unable to kill specific target cells to the same extent as wild type effectors, although expression of effector molecules including perforin, granzyme B and FasL was normal, as was killing of targets when the requirement for TCR recognition was bypassed. Finally, although the reduction of effector function suggested that memory development may also be effected, 2C/CD30[sup -/-] effectors were able to develop into memory-like cells to the same extent as wild type cells. Although the deletion of CD30 has little effect on a number of T cell functions, particularly activation and proliferation, it appears that CD30 does play a role in the regulation of later events such as cytotoxic effector function and the maintenance of IL-4 production.

GITR and CD30 are T cell costimulatory members of the TNFR superfamily known to regulate T cell responses. Elucidating the mechanisms whereby these receptors modulate T cell responses is crucial for maximizing their potential for immunotherapy. In this thesis, I examine the role of GITR and CD30 on CD8 T cell responses to influenza virus. I show that CD8 T cell intrinsic GITR is required for both maximal primary and secondary CD8 T cell expansion to influenza, while in contrast, CD30 is dispensable for anti-influenza CD8 T cell responses. GITR does not impact on CD8 T cell proliferation or homing, however, it mediates CD8 T cell survival signaling. GITR induces TRAF2/TRAF5 dependent, but TRAF1 independent, NF-κB activation, resulting in the upregulation of the pro-survival molecule Bcl-xL. Furthermore, I show that GITR on CD8 T cells can augment viral clearance and confer protection from death upon severe influenza infection of mice. Similarly, CD30 also elicits protection from death upon severe influenza infection, although the cells responsible for this effect remain to be elucidated. In this thesis, I also show that in unimmunized mice GITR expression is upregulated to higher than basal levels on a population of CD8 memory phenotype cells in the bone marrow. In contrast, CD8 memory phenotype T cells in the spleen and LN have GITR levels similar to that on naïve T cells. The upregulation of GITR in the bone marrow is IL-15 dependent and therefore, GITR serves as a marker for cells that have recently received an IL-15 signal. Furthermore, GITR is required for the persistence, but not for the homeostatic proliferation of CD8 memory phenotype T cells in the bone marrow. Therefore, GITR plays a key role for CD8 T cell intrinsic responses to influenza, as well as for the persistence of CD8 memory phenotype T cells.

碩士
國立臺灣大學
免疫學研究所
106
Regulatory T cells are first discovered to regulate immune response through suppression to other immune cells. Upon facing environmental stimuli, it has been suggested that Treg cells show adaptive properties, even reported to provide a helper function in a mouse model. However, little is unknown about the helper activity of human Treg cells. The aims of this study are to identify human CD40L-expressing Treg cells along with their phenotypic and functional characteristics. In this study, we isolated human Treg cells from healthy donors and identified CD40L-expressing Treg cells after strong CD3/TCR stimulation. We found Treg cells from tumor-infiltrating lymphocytes (TILs) of oral cancer showed stronger tendency to express CD40L under restimulation. Through DNA methylation analyses, both CD40L+ and CD40L- Treg cells were hypomethylated at FOXP3 promoter and Treg-specific demethylated region (TSDR), suggesting that both subsets are stable Treg cells. According to the expression patterns of Treg-associated markers, CD40L+ Treg cells exhibited similar phenotype to CD40L- counterpart. As for the suppression function, CD40L+ Treg cells showed superior suppressive activity on proliferation and cytokine production of Tconv cells. Nonetheless, the suppressive activities of both Treg subsets were equivalent in suppression assay with monocytes. CD40L+ Treg cells displayed consistently higher cytokine productivity than CD40L- Treg cells in both suppression systems. Furthermore, CD40L+ Treg cells promoted CD83 expression on dendritic cells, a marker for dendritic cell maturation. Consistent with the phenotype, CD40L+ Treg-treated dendritic cells possessed better capacity to promote allogeneic CD8+ T cell activation and proliferation. In summary, these results indicated that human CD40L-expressing Treg cells might have positive effects on immune response apart from their suppressive function.

Le CD40 ligand (CD40L) est un régulateur important de la réponse immunitaire et un contributeur clé dans les maladies auto-immunes. Nous avons rapporté précédemment que le CD40L se liait à l’intégrine α5β1, toutefois, les conséquences fonctionnelles de cette interaction demeurent inconnues. Les lymphocytes T sont au centre de la pathogénèse des maladies auto-immunes. Ils expriment, lors de celles-ci, des quantités aberrantes d’intégrines β1 faisant en sorte que la liaison CD40L/α5β1 pourrait être d’une haute importance dans les réponses inflammatoires. Dans cette étude, nous avons démontré que la forme soluble du CD40L (sCD40L) se liait aux lymphocytes T primaires ainsi qu’aux cellules Jurkat E6.1 et ce, dépendamment de l’intégrine α5β1. L’interaction du CD40L avec l’α5β1 lymphocytaire a induit l’activation des voies anti-apoptotiques dont les MAPKs (les protéines kinases mitogène activée) et les PI3 kinases (PI3K). La liaison du sCD40L à l’α5β1 n’a pas induit son changement structural ni son adhésion à la FN (fibronectine). Ceci pourrait avoir des conséquences directes sur la survie des cellules T lors de la progression des maladies inflammatoires. Ces résultats soulignent l’impact de l’interaction CD40L/α5β1 sur la fonction biologique des lymphocytes T et ils pourraient expliquer leur survie et leur persistance au niveau des sites d’inflammations durant les maladies auto- immunes.
CD40 ligand (CD40L) is a critical regulator of the immune response and a key contributor to autoimmune diseases. We have previously reported that CD40L binds to α5β1, albeit the functional consequences of this interaction remain elusive. T cells are central to the pathogenesis of autoimmune diseases and express high levels of β1 integrins in disease conditions, making the CD40L/α5β1 interaction a potential axis of significant importance in inflammatory complications. Here, we show that soluble CD40L (sCD40L) binds to freshly isolated primary T cells and to Jurkat E6.1 T cells in a α5β1-dependant manner. This leads to the activation of key survival proteins, including the mitogen-activated protein kinases (MAPKs) and the Akt PI3 kinase (PI3K). Binding of sCD40L to α5β1 does not induce its conformational change nor allow adherence to fibronectin. These results highlight the impact of the CD40L/α5β1 interaction in T-cell function and may explain the link between sCD40L and T-cell survival and persistence in inflammatory diseases.

Indiana University-Purdue University Indianapolis (IUPUI)
The immune system protects the body from foreign organisms. T cells and B cells are integral components of the ability of the immune system to generate focused immune responses. The development of specialized subsets of T helper cells is governed by transcription factors. Previous work demonstrated a requirement for the transcription factor PU.1 in the development of IL-9-secreting Th9 cells. Work in this dissertation demonstrates that the Th9 subset is not stable in vitro, and that PU.1 expression decreases during long-term culture. To examine a role for PU.1 in Th9-independent immunity we examined a model of multiple sclerosis termed experimental autoimmune encephalomyelitis (EAE). Mice that lack PU.1 expression in T cells (Sfpi1lck-/- mice) demonstrated more severe disease with attenuated recovery compared to control mice, and this was accompanied by an increase of T cells in the central nervous system. We also observed that following multiple routes of immunization Sfpi1lck-/- mice had increased numbers of T follicular helper (Tfh) cells and increased germinal center responses. This correlated with increased expression of the cytokine IL-21 and the surface protein CD40L in T cells that lacked PU.1 expression and resulted in increased numbers of germinal center B cells and antigen-specific antibody titers compared to control mice. The increased germinal center B cells and antibody titers were attenuated with blocking CD40L antibody but not with neutralizing IL-21 antibody. These results suggest that PU.1 limits the expression of CD40L on Tfh cells to regulate the humoral immune response. Together, the data in this dissertation demonstrate Th9-independent functions of PU.1. Moreover, this work shows that transcription factors promoting the development of one subset of T helper cells can simultaneously have negative effects on distinct T cell lineages.

The goal of this body of research was to elucidate the mechanism by which CD4+ T cells provide help for CD8+ cytotoxic T lymphocyte (CTL) responses in different immunization types. The establishment of diseases, such as chronic infections and cancers, is attributed to severe loss of or dysfunctions of CD4+ T cells. Even in acute infections, CD4+ T cell deficiency leads to poor memory responses. While the role of CD4+ T cells is being increasingly appreciated in these diseases, the timing and nature of CD4+ T help and associated molecular mechanisms are not completely understood. Growing evidence suggests that, depending on the type of infections or immunizations, the requirements of CD4+ T cells can vary for optimal CD8+ CTL responses. In order to understand the modulatory effects of CD4+ T cells for optimal CD8+ CTL responses, two distinct immunization types were chosen. These include: 1) non-inflammatory dendritic cell (DC) immunization, which fails to provide inflammatory/danger signals; and 2) inflammatory adenovirus (AdV) immunization, which provides profound inflammatory/danger signals. This allowed us to study CD4+ T cell’s participation under different inflammatory conditions. The studies described in Chapters 2 and 3 of this thesis were performed to further understand the concept of how CD4+ T cells mediate optimal CD8+ CTL responses. This has been called the “new dynamic model of CD4+ T helper – antigen (Ag)-presenting cells (Th-APCs),” proposed in 2005 by our laboratory. The study described in Chapter 2 shows that Th-APCs participate not only in augmenting CTL-mediated immune responses, perhaps during early phase, but also in regulating cellular immunity, perhaps during a later phase. Through enhanced IL-2, CD80 and CD40L singnaling, and weaker peptideMHC I (pMHC) signaling, Th-APCs stimulated naïve CD8+ T cells to differentiate into effector CTLs, capable of developing into, central memory CTLs. Th-APC-stimulated CD4+ T cells behaved like Th cells in function, augmenting the overall magnitude of CTL responses. In contrast, Th-APCs were able to kill DCs and other Th-APCs, predominantly through perforin-mediated pathway. The experiments described in Chapter 3 revealed a novel co-operative role of cognate Th-CTL interactions, contrary to previously known immune-regulatory mechanisms among Th-Th or CTL-CTL interactions. In our experiments, Th cells, via CD40L, IL-2, and acquired pMHC-I signaling, enhanced CTL survival and transition into functional memory CTLs. Moreover, RT-PCR, flow cytometry and western blot analysis demonstrate that increased survival of Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of FasL and TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (NFATc1, Bcl-10, Casp-3, Casp-4, Casp-7) genes/ molecules. Finally, helped CTLs were also able to induce protection against highly metastasizing tumor challenge, explaining why memory CTLs generated under cognate Th1’s help show survival and recall advantages. The studies in Chapter 4 showed how the precursor frequency (PF) of CD8+ T cells impacts CD4+ T helper requirements for functional CTL responses. At endogenous PF, CD4+ T helper signals were necessary for both primary and memory CTL responses. At increased PF, CD4+ T help, and its CD40L but not IL-2 signal became dispensable for primary CTL responses. In contrast, memory CTL responses required CD4+ T cell signals, largely in the form of IL-2 and CD40L. Thus, these results could impact the development of novel immunotherapy against cancers, since their efficacy would be determined in part by CD4+ T help and CD8+ T cell PF. Finally, the study showed the importance of CD4+ T cells for multiple phases of AdV transgene product-specific CTL responses. These include: a) cognate CD4+ T cells enhanced CTL responses via IL-2 and CD40L signaling during primary, maintenance and memory phases; b) polyclonal CD4+ T environment enhanced the survival of AdV-specific CTL survival, partially explaining protracted CTL contraction phase; and c) during the recall phase, the CD4+ T environment, particularly memory CD4+ T cells, considerably enhanced not only helped, but also unhelped, memory CTL expansion. Thus, these results suggest the participation of both cognate and polyclonal CD4+ T cells for multiple phases of AdV-specific CTLs. Taken together, the current work delineated the critical roles of CD4+ T cells in different stages of CTL responses and in the development of anti-tumor immunity. The results presented here will significantly advance our current understanding of immunity to cancers, autoimmunity and chronic infections, since pathogenesis of these diseases is largely determined by CD4+ T helper functions. As most immunization procedures use the principle that is based on functions of memory cells, the knowledge gained from this work will also have a major impact on designing vaccines against intractable diseases, including cancers and chronic infections. Moreover, in advanced tumors, vaccines developed using this knowledge may act synergistically with other cancer treatments such as irradiation, chemotherapy and microsurgery, minimizing their side effects and prolonging the lives of patients.