Der Einfluss von Chemotherapeutika mit Hypoxie-induzierbarer Faktor (HIF)-modulierendem Potential sowie eines Verlustes des HIF-Regulators von Hippel-Lindau (VHL) auf den Phänotyp und die Funktion dendritischer Zellen

Der Transkriptionsfaktor Hypoxie-induzierbarer Faktor (HIF) gibt dem Organismus die Möglichkeit, sich auf zellulärer Ebene an unterschiedliche Sauerstoffverhältnisse anzupassen. Vor allem Tumorzellen weisen aufgrund ihres ungeregelten Wachstums und der daraus resultierenden unzureichenden Durchblutung (hypoxisches Milieu) eine erhöhte HIF-Expression auf. Die erhöhte HIF-Expression stellt somit ein interessantes Ziel in der Tumortherapie dar. Dendritische Zellen (DCs) besitzen eine bedeutende Rolle in der Generierung und Modulierung von Antitumor-Immunantworten. Aus diesem Grund ist es überaus wichtig zu wissen, welche Effekte Antitumor-Agenzien, im Besonderen HIF-Inhibitoren, auf DCs und somit auch auf die Generierung von adäquaten Immunantworten besitzen.rnIm ersten Teil dieser Arbeit wurde aus diesem Grund der Einfluss der Antitumor-Agenzien Geldanamycin (GA) und Topotecan (TPT) auf den Phänotyp und die Funktion von DCs untersucht. Hierfür wurden Monozyten aus humanen, mononukleären, peripheren Blutzellen isoliert und unter DC-differenzierenden Konditionen kultiviert. Diese immaturen monozytenabgeleiteten DCs (Mo-DCs) wurden mithilfe eines Reifungscocktails ausgereift. Die Applikation der Antitumor-Agenzien erfolgte während der Differenzierungs- bzw. Ausreifungsphase. Abhängig vom Reifungsgrad der Mo-DCs konnte ein differentieller Einfluss von GA bzw. TPT auf die DC-Aktivierung beobachtet werden. Eine Behandlung von unstimulierten Mo-DCs mit GA resultierte in einer partiellen DC-Aktivierung basierend auf einem noch unbekannten Mechanismus. Ebenso führte eine Behandlung von unstimulierten Mo-DCs mit TPT zu einer funktionellen Aktivierung der DCs, die mit einer vermehrten AKT-Expression korrelierte. Die jeweilige Koapplikation der Antitumor-Agenzien mit dem DC-Reifungscocktail führte zu einer reduzierten DC-Aktivierung, die sich in einer verminderten NF-κB-Aktivierung, einer verringerten Oberflächenexpression der getesteten kostimulatorischen Moleküle, einer verringerten Migrationsfähigkeit und einem reduzierten Zellstimulierungspotential widerspiegelte.rnDie autosomal dominant vererbte Tumorerkrankung von Hippel-Lindau (VHL) wird häufig durch genetische Mutationen des als HIF-Negativregulator fungierenden VHL-Gens hervorgerufen. Patienten, die an dem VHL-Syndrom erkrankt sind, weisen oft benigne oder maligne Tumore und Zysten in den verschiedensten Organsystemen auf. Wie schon zuvor erwähnt, besitzen DCs eine essentielle Rolle in der Initiierung und Aufrechterhaltung von Antitumor-Immunantworten. Deshalb wurde im zweiten Abschnitt der vorliegenden Arbeit untersucht, inwieweit ein partieller Verlust von VHL Auswirkungen auf die Ausprägung desrnPhänotyps und der Funktion von DCs hat. Mittels Cre/lox-Technologie wurden transgene Mäuse mit einem heterozygoten Verlust von Exon 1 bzw. Exon 2 des VHL-Gens generiert. Aus diesen Mäusen wurden Knochenmarkszellen isoliert und unter DC-differenzierenden Konditionen kultiviert. Die immaturen knochenmarkabgeleiteten DCs (BM-DCs) wurden mit LPS ausgereift. Weder der heterozygote Verlust von Exon 1 noch von Exon 2 des VHL-Gens bewirkte eine Veränderung der Oberflächenmarkerexpression, der in vitro-Migrations- undrnEndozytosekapazität, sowie der allogenen T-Zellstimulierungskapazität. Allerdings zeigten Mäuse mit einem partiellen Verlust von Exon 2 im Vergleich zu Kontrollmäusen nach Immunisierung und Provokation mit dem Modellallergen OVA eine verminderte Atemwegshyperreaktion, die möglicherweise auf die beobachtete Abnahme der Migrationsfähigkeit in vivo und die verminderte OVA-spezifische T-Zellstimulierungskapazität der DCs zurückzuführen ist.

Induktion tolerogener dendritischer Zellen zur Suppression von experimenteller autoimmuner Enzephalomyelitis und Kontaktallergie

Dendritische Zellen (DC) spielen als professionelle antigenpräsentierende Zellen (APC) eine zentrale Rolle in der Aktivierung und Regulierung antigenspezifischer Immunantworten. Aus diesem Grund wird der therapeutische Einsatz von DC zur Behandlung von Autoimmunerkrankungen und Allergien sowie zur Tumorbekämpfung erforscht. Im ersten Teil der vorliegenden Arbeit untersuchten wir das Potenzial einer biolistischen DNA-Vakzinierung zur Induktion tolerogener DC in vivo. Im Tiermodell der Myelin-Oligodendrozyten-Glykoprotein Peptid 35-55 (MOGp35-55) induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) sollte mittels präventiver biolistischer Kovakzinierung von Plasmid-DNA kodierend für MOG und die immunregulatorischen Zytokine TGFβ oder IL-10 eine protektive Immunität induziert werden. Die MOG-Expression stand dabei entweder unter der Kontrolle des ubiquitär aktiven CMV-Promotors oder des murinen Fascin-Promotors, um eine ektopische MOG-Expression spezifisch in dermalen DC und Langerhanszellen zu erreichen. Dass MOGp35-55-präsentierende DC nach biolistischer DNA-Vakzinierung von der Haut in die drainierenden Lymphknoten migrieren und dort T-Zellen aktivieren, konnte im Vorfeld anhand einer substanziellen Proliferation von MOGp35-55-reaktiven 2D2 T-Zellen nachgewiesen werden. Im präventiven Ansatz der MOGp35-55-induzierten EAE zeigten Mäuse, die mit MOG-kodierenden Plasmiden biolistisch transfiziert wurden, eine leicht reduzierte EAE-Symptomatik. Die Kotransfektion von MOG und TGFβ führte zu einer Verstärkung der EAE-Suppression – unabhängig davon, ob die MOG-Expression unter der Kontrolle des CMV- oder des Fascin-Promotors stand. Interessanterweise resultierte die Koapplikation von MOG- und IL-10-kodierender Plasmid-DNA nur bei DC-fokussierter MOG-Expression zu reduzierter EAE-Symptomatik. Für biolistische DNA-Vakzinierungen stellt somit der Fascin-Promotor eine potente Alternative zu viralen Promotoren dar. Entsprechend der milderen EAE-Symptome beobachteten wir bei behandelten EAE-Mäusen einen geringeren Grad an Demyelinisierung sowie eine reduzierte Infiltration des ZNS mit IFNγ-produzierenden CD4+ Th1- und IL-17-produzierenden CD4+ Th17-Zellen. Desweiteren zeigten Milzzellen ex vivo nach MOGp35-55-Restimulation eine inhibierte Proliferation und eine signifikant reduzierte IFNγ- und IL-17-Zytokinproduktion. Überraschenderweise ging die antigenspezifische Immunsuppression nicht mit der Expansion von Foxp3+ regulatorischen T-Zellen einher. Da die Milzen aber erhöhte Mengen an CD8+IFNγ+ T-Zellen aufweisen, könnte ein zytotoxisch-suppressiver Mechanismus für die Inhibition der Th1- und Th17-Immunantwort verantwortlich sein. Nachfolgende Untersuchungen sind notwendig, um die induzierten immunologischen Mechansimen mittels biolistischer DNA-Vakzinierung aufzuklären. Der zweite Teil der Arbeit befasst sich mit der Generierung von tolerogenen DC in vitro. Dafür wurden murine Knochenmarkszellen unter DC-differenzierenden Bedingungen in Gegenwart des synthetischen Glucocorticoids Dexamethason (DEX) kultiviert. Die DEX-Zugabe führte zur Differenzierung von APC mit geringer CD11c-Expression. DEX-APC waren in vitro weitestgehend gegen LPS stimulierungsresistent und zeigten eine reduzierte Expression von MHC-II und den kostimulatorischen Molekülen CD80, CD86 und CD40. Ihrem tolerogenen Phänotyp entsprechend besaßen DEX-APC ein geringeres syngenes T-Zellstimulierungspotenzial als unbehandelte BM-DC. Anhand der erhöhten Oberflächenexpression von CD11b, GR1 und F4/80 besteht eine phänotypische Ähnlichkeit zu myeloiden Suppressorzellen. Die Fähigkeit von DEX-APC in vivo antigenspezifische Toleranz zu induzieren, wurde durch einen therapeutischen Ansatz im murinen Krankheitsmodell der Kontaktallergie überprüft. Die therapeutische Applikation von DEX-APC führte hierbei im Vergleich zur Applikation von PBS oder unbehandelten BM-DC zu einer signifikant reduzierten Ohrschwellungsreaktion. Zusammenfassend demonstrieren die Ergebnisse dieser Arbeit, dass potente tolerogene DC sowohl in vivo als auch in vitro induziert werden können. Dass diese Zellpopulation effektiv antigenspezifische Immunreaktionen supprimieren kann, macht sie zu einem vielversprechenden Werkzeug in der Behandlung von Autoimmunerkrankungen und Allergien.rn

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients

FOXP3-expressing naturally occurring CD4(+)CD25(high) T regulatory cells (Treg) are relevant in the control of autoimmunity, and a defect in this cell population has been observed in several human autoimmune diseases. We hypothesized that altered functions of peripheral Treg cells might play a role in the immunopathogenesis of myasthenia gravis, a T cell-dependent autoimmune disease characterized by the presence of pathogenic autoantibodies specific for the nicotinic acetylcholine receptor. We report in this study a significant decrease in the in vitro suppressive function of peripheral Treg cells isolated from myasthenia patients in comparison to those from healthy donors. Interestingly, Treg cells from prednisolone-treated myasthenia gravis patients showed an improved suppressive function compared with untreated patients, suggesting that prednisolone may play a role in the control of the peripheral regulatory network. Indeed, prednisolone treatment prevents LPS-induced maturation of monocyte-derived dendritic cells by hampering the up-regulation of costimulatory molecules and by limiting secretion of IL-12 and IL-23, and enhancing IL-10. In addition, CD4(+) T cells cultured in the presence of such tolerogenic dendritic cells are hyporesponsive and can suppress autologous CD4(+) T cell proliferation. The results shown in this study indicate that prednisolone treatment promotes an environment that favors immune regulation rather than inflammation.

Impact of the CTLA-4/CD28 axis on the processes of joint inflammation in rheumatoid arthritis

OBJECTIVE: The importance of the costimulatory molecules CD28 and CTLA-4 in the pathologic mechanism of rheumatoid arthritis (RA) has been demonstrated by genetic associations and the successful clinical application of CTLA-4Ig for the treatment of RA. This study was undertaken to investigate the role of the CTLA-4/CD28 axis in the local application of CTLA-4Ig in the synovial fluid (SF) of RA patients. METHODS: Quantitative polymerase chain reaction was used to analyze the expression of proinflammatory and antiinflammatory cytokines in ex vivo fluorescence-activated cell sorted CTLA-4+ and CTLA-4- T helper cells from the peripheral blood and SF of RA patients. T helper cells were also analyzed for cytokine expression in vitro after the blockade of CTLA-4 by anti-CTLA-4 Fab fragments or of B7 (CD80/CD86) molecules by CTLA-4Ig. RESULTS: CTLA-4+ T helper cells were unambiguously present in the SF of all RA patients examined, and they expressed increased amounts of interferon-γ (IFNγ), interleukin-17 (IL-17), and IL-10 as compared to CTLA-4- T helper cells. The selective blockade of CTLA-4 in T helper cells from the SF in vitro led to increased levels of IFNγ, IL-2, and IL-17. The concomitant blockade of CD28 and CTLA-4 in T helper cells from RA SF by CTLA-4Ig in vitro resulted in reduced levels of the proinflammatory cytokines IFNγ and IL-2 and increased levels of the antiinflammatory cytokines IL-10 and transforming growth factor β. CONCLUSION: Our ex vivo and in vitro results demonstrate that the CTLA-4/CD28 axis constitutes a drug target for not only the systemic, but potentially also the local, application of the costimulation blocking agent CTLA-4Ig for the treatment of RA.

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

A novel regulatory macrophage induced by a helminth molecule instructs IL-10 in CD4+ T cells and protects against mucosal inflammation

Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4(+) T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4(+) T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation.

Studies on gene expression within germinal centers

During T cell dependent immune responses, the acquisition of B cell memory from naïve cells takes place within a highly specialized microenvironment: The germinal centers (GC) of the secondary lymphoid organs. The GC reaction is a tightly regulated process in which the balance between survival and death is mediated by signals transduced through ligation of critical costimulatory molecules such as CD40 and CD154. While most cognate receptor-ligand interactions occur between T-cells and antigen (Ag)-presenting cells (APC) such as B-cells, evidence of homotypic B cell interactions has emerged. Despite the progress in our understanding of the reaction, several questions remain: (1) What determines the concomitant expression of CD40 and its ligand CD154 by GC B-cells? (2) Which molecules are responsible for inducing GC-B cell survival? and (3) how can cognate T-cell help be recruited into the organized structure of GCs? ^ Because the expression of costimulatory and survival molecules is predominant at distinct Ag-dependent maturation stages, we hypothesized the existence of stage specific gene expression responsible for the regulation of the GC reaction. Our studies reveal several novel genes whose expression may be critical for the GC reaction. The discovery of AKNA reveals the mechanism behind homotypic B cell CD40 and CD40 ligand interactions, which can explain the costimulatory signaling to GC B cells in the absence of T cells. Additionally, the identification of the pro-survival molecule PRELI may provide a novel mechanism for the survival of Ag-selected B cells. We propose that PRELI and its phylogenic homologues represent a novel family of proteins responsible for the protection of cells against caspase-independent apoptosis. Furthermore, we show that GC B cells actively participate in the recruitment of T cells through the secretion of CC and CxC chemokines, thus supporting their mutual involvement in cognate interactions. ^

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Apoptosis resistance of nonobese diabetic peripheral lymphocytes linked to the Idd5 diabetes susceptibility region

Defects in lymphocyte apoptosis may lead to autoimmune disorders and contribute to the pathogenesis of type 1 diabetes. Lymphocytes of nonobese diabetic (NOD) mice, an animal model of autoimmune diabetes, have been found resistant to various apoptosis signals, including the alkylating drug cyclophosphamide. Using an F2 intercross between the apoptosis-resistant NOD mouse and the apoptosis-susceptible C57BL/6 mouse, we define a major locus controlling the apoptosis-resistance phenotype and demonstrate its linkage (logarithm of odds score = 3.9) to a group of medial markers on chromosome 1. The newly defined gene cannot be dissociated from Ctla4 and Cd28 and in fact marks a 20-centimorgan region encompassing Idd5, a previously postulated diabetes susceptibility locus. Interestingly, we find that the CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and the CD28 costimulatory molecules are defectively expressed in NOD mice, suggesting that one or both of these molecules may be involved in the control of apoptosis resistance and, in turn, in diabetes susceptibility.

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

Fully competent dendritic cells as inducers of T cell anergy in autoimmunity

Mature immunologically competent dendritic cells are the most efficient antigen-presenting cells that powerfully activate T cells and initiate and sustain immune responses. Indeed, dendritic cells are able to efficiently capture antigens, express high levels of costimulatory molecules, and produce the combination of cytokines required to create a powerful immune response. They are also considered to be important in initiating autoimmune disease by efficiently presenting autoantigens to self-reactive T cells that, in this case, will mount a pathogenic autoimmune reaction. Triggering T cells is not a simple on–off procedure, as T cell receptor responds to minor changes in ligand with gradations of T cell activation and effector functions. These “misfit” peptides have been called Altered Peptide Ligands, and have been shown to have important biological significance. Here, we show that fully capable dendritic cells may present, upon natural antigen processing, a self-epitope with Altered Peptide Ligands features that can unexpectedly induce anergy in a human autoreactive T cell clone. These results indicate that presentation of a self-epitope by immunologically competent dendritic cells does not always mean “danger” and show a mechanism involved in the fine balance between activation and tolerance induction in humans.

B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.