Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. Xudong Fan. Includes bibliographical references.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Committee Chair: Zhu, Cheng; Committee Member: Babensee, Julia; Committee Member: Dustin, Michael; Committee Member: Garcia, Andres; Committee Member: Jo, Hanjoong; Committee Member: van der Merwe, Anton. Part of the SMARTech Electronic Thesis and Dissertation Collection. Non-Latin script record

Liver transplantation is the best therapy for patients with end-stage liver diseases and unresectable early hepatocellular carcinoma (HCC). Living donor liver transplantation (LDLT) has been successfully implemented as an alternative to deceased donor liver transplantation (DDLT) and likewise offers comparable excellent survival rate. However, the inferior post-transplant oncological outcomes are found in LDLT recipients with HCC. The liver grafts used in LDLT are usually small-for-size and less effective in coping with shear stress from transient portal hypertension, which results in small-for-size liver graft injury. Acute phase small-for-size liver graft injury may promote late phase tumor recurrence, whereas the underlying mechanism remains unclear. CXCL10, an inflammatory chemokine, initiates liver inflammatory response during hepatic ischemia-reperfusion (IR) injury and may link acute phase small-for-size liver graft injury and late phase tumor recurrence, yet the precise mechanisms remain elusive. Endothelial progenitor cells (EPCs) participate in tissue repair for graft recovery and also provide an angiogenic environment for tumor growth. Myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) can suppress the activation of the immune system and play a critical role in graft rejection and cancer development. We here established the rat orthotopic liver transplantation with whole graft or small-for-size graft model to study the impact of acute phase small-for-size liver graft injury on the mobilization of EPCs, MDSCs and Tregs, and intragraft CXCL10 and its receptor, CXCR3,gene expressions. We further subjected CXCL10-/-mice and CXCR3-/-mice to hepatic IR injury and major hepatectomy to study the role of CXCL10/CXCR3 signaling on the mobilization of EPCs, MDSCs and Tregs. We also investigated the effect of CXCL10 on EPC migration and tube formation in vitroas well as intratumoral microvessel density (MVD) in the rat liver transplantation with tumor growth model and EPCs on tumor growth in nude mice. Key findings: 1. Liver transplantation with small-for-size graft resulted in severe intragraft vascular injury and higher CXCL10 andCXCR3 gene expressions as well as more EPC, MDSC and Treg cell mobilizationin circulation than whole graft. 2. CXCL10-/-mice and CXCR3-/-mice had less circulating EPCs, MDSCs and Tregs than WT mice after hepatic IR injury and major hepatectomy. 3. CXCL10 recruited EPCs in dose-dependent and CXCR3-dependent manners and promoted EPC tube formation in vitro. 4. Higher intratumoral MVD was observed in small-for-size graft than in whole graft in liver transplantation with tumor growth model. 5. Tumor grew more quickly by combining EPC infusionin nude mouse orthotopic liver tumor model. In conclusion, acute phase small-for-size liver graft injury significantly mobilizes EPCs, MDSCs and Tregs after transplantation through CXCL10/CXCR3 signaling. More EPC mobilization and intragraft differentiation after transplantation with small-for-size liver graft may be related to higher intratumoral MVD in small-for-size liver graft after transplantation with tumor development. Therefore, targeting at post-transplant CXCL10/CXCR3 signaling may not only attenuate early phase liver graft injury but also prevent late phase tumor recurrence. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Allergic asthma is a chronic inflammatory airway disease that is dominated by Th2 immune responses, with accumulation of eosinophils, IgE and IgG1 production, and airway hyperresponsiveness. We reported previously that treatment of OVA-asthmatic mice with allergen-presenting IL-10-differentiated dendritic cells (DC) (DC10) leads to progressive and long-lasting full-spectrum asthma tolerance. However, little has been done in investigating a role for antigen-specific B cells in DC10-induced tolerance. In this study, we characterized the surface markers of DC10 and found that these cells expressed lower levels of CD40, CD80, MHC II, PD-L1 and PD-L2 relative to immunostimulatory LPS-differentiated DCs (DCLPS). Co-culturing DC10 or DC10-induced regulatory T cells (iTreg) with CD4+ Th2 effector T cells from asthmatic mice led to a marked suppression of DCLPS-induced T effector cell proliferation. Moreover, DC10 treatment of asthma phenotype mice down-regulated airway eosinophilic inflammation as determined 48 h after a recall allergen challenge, and reduced pulmonary parenchymal tissue OVA-specific IgG1-secreting (OVA-IgG1) plasma cell numbers. The number of lung OVA-specific IgG1 plasma cells decreased by 46.7% over a 2 week period in the absence of repeated allergen challenge, while the numbers of bone marrow OVA-specific IgG1 plasma cells stayed relatively stable over a 6 week period, as determined 48 h after a single allergen challenge of asthmatic mice. DC10 treatment had a significant impact on the serum of IgG1/IgE response. To address the question of how DC10 influence OVA-IgG1 plasma cells responses, we co-cultured enzymatically-dispersed lung total cells from asthmatic mice with or without DC10, and found that the DC10 significantly suppressed OVA-IgG1 plasma cell antibody production. To determine whether DC10 required input from T cells to accomplish this, we co-cultured CD4 T cell-depleted, B cell-enriched populations from the lungs of asthmatic mice with or without DC10, and found that DC10 strongly (65.4+/-3.5%) suppressed OVA-IgG1 plasma cells in CD4 T cell-depleted lung cell cultures. To assess whether DC10-induced Treg also suppress IgG1-secretion, we co-cultured lung CD4+ T cells from untreated or DC10-tolerized asthmatic mice with total lung cells from asthmatic donors, and found that the DC10-induced Tregs effectively (52.2+/-8.7%) suppressed OVA-IgG1 plasma cell responses. In summary, DC10 treatment strongly down-regulate OVA-specific IgG1 plasma cell responses of asthmatic mice, both in vivo and in vitro by at least two mechanisms: directly via DC10 as well as indirectly through DC10-induced Tregs.

There is an urgent need for the development of an effective therapeutic vaccine against cancer caused by human papilloma virus (HPV). We focused on HPV-induced malignancies because of their high worldwide prevalence (e.g., cervical carcinoma and head & neck cancer). A successful therapeutic vaccine could prevent the 250 000 deaths/year worldwide and the 2.25 billion dollars that are expended in related care in the US. We used an HPV-induced mouse cancer model to test vaccines composed of a CD8 T cell peptide epitope administered with potent adjuvants designed to generate vast numbers of high avidity cytotoxic T lymphocytes specific for the HPV16-E7 antigen. One vaccination strategy (TriVax) consists of intravenous administration of synthetic peptide HPV16-E749-57 administered together with a Poly-IC (a TLR3 agonist) and anti-CD40 monoclonal antibody(αCD40 mAb) while the second more simple strategy (BiVax) comprises solely of peptide plus Poly-IC. We used an E7 peptide as antigen in the vaccination strategies because expression of the viral E6 and E7 proteins is required to maintain oncogenic phenotype and because normal cells do not express E6/E7, therefore a therapeutic vaccine targeting these proteins has several advantages: a) a strong immune response can be induced since immune tolerance to these foreign antigens does not exist and b) the strong immune response should not inflict damage to normal cells. TriVax and BiVax generate a high number of antigen specific CD8 T cells capable clear subcutaneous tumors and prevent recurrences, both vaccines are efficient through the i.v. and i.m. route. TriVax (prime-boost) clears tumor in 100% of mice while BiVax clears tumor in 50% of mice, this differential effect is due to the number of antigen specific CD8 T cells and increasing the number of booster shot makes BiVax as immunogenic and efficient in clearing tumors. In the absence of type-I IFN signaling (in IFNαΒR KO mice), TriVax is less effective in generating sufficient numbers of CD8 T cells that could be necessary for total disease eradication. We observed a significant anti-tumor effect of TriVax in the absence of interferon gamma, however the cytokine may play some role in the overall effectiveness of TriVax to completely reject the tumors. Immune responses produced by BiVax are highly dependent on the simultaneous administration of peptide and Poly-IC, on the peptide composition, vaccine formulation and route of administration. The magnitude of the response is dependent on the expression of the Poly-IC receptors TLR3 and melanoma differentiation-associated protein 5 (MDA5). Interestingly, the magnitude and duration of the CD8 T cell responses generated by peptide and Poly-IC mixtures does not rely on the presence of CD4 T cells, scavenger receptor-A (SR-A) or type-I IFN signals and was minimally affected by the absence of CD40 signaling. The present findings could facilitate the development of simple and effective subunit vaccines for diseases where CD8 T cells may hold a therapeutic benefit.

<p>Liver cancer is a devastating disease that is the 5th most common cancer in men, 7th most common cancer in women, and the 3rd leading cause of cancer-related mortality. This disease arises from multiple etiological factors, including hepatitis viruses, environmental toxins, alcohol abuse, and metabolic syndrome, which induce a state of chronic inflammation. This diseased liver tissue background is a drastically different microenvironment from the healthy liver, especially with regards to immune cell prevalence and presence of mediators of immune function. It has been well-established that this altered tissue background contributes significantly to the tumorigenic process, yet its effects on the progression of the disease are more poorly understood. </p><p>To better understand the consequences of liver disease on tumor growth and the interplay with its microenvironment, we first utilized two standard methods of fibrosis induction and orthotopic implantation of tumors into the inflamed and fibrotic liver to mimic the liver condition in human HCC patients, and examined the immune infiltrate. Compared to non-diseased controls, tumor growth is significantly enhanced under fibrotic conditions. The immune cells that infiltrated the tumors are also drastically different, with decreased proportions of natural killer cells but greatly increased numbers of immune-suppressive CD11b+ Gr1hi myeloid cells in both models of fibrosis. In addition, there are model-specific differences: increased proportions of CD11b+ myeloid cells and CD4+ CD25+ T-cells are found in tumors in the bile duct ligation model but not in the carbon tetrachloride model. Importantly, the skewed immune infiltration into the tumor, while having some commonalities with the non-tumor tissue, had several distinct, tumor-specific populations. Induction of fibrosis also alters the cytokine production of implanted tumor cells, which could have far-reaching consequences on the immune infiltrate and its functionality. Taken together, this work demonstrates that the combination of fibrosis induction with orthotopic tumor implantation results in a markedly different tumor microenvironment and tumor growth kinetics. </p><p>Appreciating that the altered immune microenvironment dramatically shifts tumor progression, we sought to further explore the effects of individual inflammatory mediators on the development of the disease. Interleukin 18 (IL-18) is an inflammatory cytokine that is markedly increased in the circulation of patients with HCC correlated with poor prognosis. However, the precise role for IL-18 in HCC remains unclear, with reports presenting both pro- and anti-tumorigenic activities. To answer this question definitively, we interrogated in more detail the expression profiles of IL-18 in tissue specimens from HCC patients and conducted experimentation using multiple clinically relevant mouse models to explore the functional role of this cytokine in the context of HCC. Our results indicate that IL-18 exerts a tumor-suppressive effect mediated in large part by alterations in survival and functionality of T-lymphocytes which infiltrated the tumor microenvironment. This tumor-suppressive effect is however dependent upon the inflammatory milieu: In the absence of an inflammatory environment, whether from a chemical carcinogenesis model or a fibrosis induction model, loss of IL-18 signaling does not affect tumor growth. This effect is also stage-dependent. Taken together, our findings establish a tumor-suppressive role for IL-18 in established HCC and provide a mechanistic explanation for the complex relationship between its expression pattern and HCC prognosis. </p><p>In summary, this work demonstrates a dramatic shift in the microenvironment of developing HCC tumors in the presence of chronic inflammatory stimuli. This microenvironment, which more accurately models the situation in which tumors develop and progress in patients, alters the presence and functionality of many immune mediators. In particular, IL-18 signaling is a powerful mediator of tumor progression, however observation of its functionality is dependent on an inflammatory context. This work provides new insight into the complex processes underlying HCC tumor progression, and emphasizes the necessity for more accurate modeling of HCC progression in mice which takes into account the drastic changes in the tissue caused by chronic liver disease.</p> / Dissertation

CD8+ T cells responses are critical in the immune defense against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection. A major challenge for vaccine development is that HIV/SIV can rapidly mutate to escape containment by the CD8+ T cell response. Therefore, optimal virus control by a vaccine will likely require clonally diverse CD8+ T cells capable of recognizing mutant viruses. Mucosal tissues play a fundamental role in early HIV/SIV pathogenesis by serving as the site for viral entry, CD4+ T cell depletion, and a reservoir for viral replication. Vaccine strategies that induce effective mucosal immunity will likely be critical for protection against HIV/SIV. We examined the SIV Gag p11C-specific CD8+ T cell responses in peripheral blood, gastrointestinal (GI) mucosal and lung mucosal tissues of rhesus monkeys expressing the MHC class I molecule Mamu-A*01. We first investigated the clonal composition of this cell population during the acute and chronic phases of SIVmac infection. We showed that there is a narrowing of the clonal repertoire from acute to chronic infection and the same clonal populations of virus-specific CD8+ T cells are present in the systemic and mucosal compartments of chronically SIV-infected animals. These data indicated that virus-specific CD8+ T cells establish broadly distributed immune responses. Next, we examined the clonal diversity of systemic and mucosal p11C-specific CD8+ T cells induced by prime-boost vaccination. We found that systemic prime-boost vaccination induced clonally diverse p11C-specific populations in mucosal tissues. There were high levels of clonal sharing between systemic and mucosal compartments soon after vaccination. However, later following vaccination there was decreased clonal sharing between the GI mucosa and the systemic circulation. We showed that this was due to limited trafficking of p11C-specific CD8+ T cells to the GI mucosa following vaccination. Overall, these studies indicate that following SIV infection and systemic vaccination the same p11C-specific clones are present in mucosal and systemic compartments. Moreover, the apparent immune compartmentalization is a consequence of differences in cell trafficking between systemic and mucosal CD8+ T cells. These observations have important implications for the design of HIV vaccines that generate effective mucosal immunity.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Irregular screening to identify infected individuals and a lack of sterilizing immunity to C. trachomatis has led to a dramatic increase in the number of reported C. trachomatis infections over the last twenty years. Repeated infections with C. trachomatis lead to serious sequelae such as pelvic inflammatory disease and ectopic pregnancy, which can result in infertility.

The ultimate goal of antitumor vaccines is to develop memory CD8+ cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. Previous work in our lab demonstrated that the ovalbumin (OVA)-specific CD4+ T cell-based (OVA-TEXO) vaccine generated using OVA-pulsed dendritic cell (DCOVA)-released exosomes (EXOOVA) stimulates CTL responses via interleukin (IL)-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4+ OVA-TEXO cells or OVA-TEXO cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/OVA257–264 tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10OVA. We demonstrate that CD4+ OVA-TEXO cells form immunological synapses with cognate CD8+ T cells in vitro. By assessment of the pattern of ex vivo interactions between OTI CD8+ T cells and OVA-TEXO or (Kb-/-)TEXO cells lacking peptide/major histocompatibitity complex (pMHC)-I expression, we provide the first visible evidence on the critical role of exosomal pMHC-I in targeting OVA-TEXO to cognate CD8+ T cells using two-photon microscopy. By assessing primary and recall CTL responses in mice immunized with OVA-TEXO cells or with OVA-TEXO cells lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TEXO cells. Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10OVA tumors. Overall, this work suggests that a novel CD4+ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination. Breast cancer is the most common cancer among women in the western world. Approximately 20-30% of invasive breast carcinomas are proto-oncogene human epidermal growth factor receptor (HER)-2 positive and associated with increased metastatic potential and poor prognosis. The survival benefit of anti-HER2 driven therapies demonstrated in clinical trials indicates that HER2 is one of the most promising molecules for targeted therapy to date. Above results prompt us to assess whether CD4+ T-cell-based vaccine can stimulate efficient HER2-specific CD8+ CTL responses and antitumor immunity in transgenic mice with HER2-specific self-immune tolerance. We prepared HER2-specific HER2-TEXO using ConA-stimulated CD4+ T cells with uptake of exosomes released from HER2-expressing AdVHER2-transfected DCs. We found that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2/HER2-expressing B16 melanoma BL6-10HLA-A2/HER2 in 2/8 double transgenic HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2+ breast cancer. In contrast to CD4+CD25+ regulatory T cells (Tregs), mechanisms of CD8+CD25+ Treg-mediated immunosuppression are not well understood. In this study, we purified polyclonal CD8+CD25+ Tregs from C57BL/6 mouse splenocytes and expanded them in culture medium containing CD3/CD28 microbeads. By using these amplified CD8+CD25+ Tregs, we demonstrated that CD8+CD25+ Tregs inhibit naive CD4+ T-cell proliferation and induce naive T-cell anergy by up-regulating T-cell anergy-associated early growth response 2 (EGR2), and by decreasing T-cell proliferation and IL-2-secretion upon stimulation. They also impact the expression of perforin on effector CTLs and directly induce perforin-mediated CTL apoptosis. CD8+CD25+ Tregs, when pulsed with OVA323-339 peptide, exert an enhanced inhibition. Interestingly, CD8+CD25+ Tregs, when pulsed with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, become capable of inhibiting MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). Two-photon microscopic observations suggest that OVA323-339-pulsed (armed) CD8+CD25+ Tregs reduce the interactions between DCs and cognate CD4+ T cells ex vivo by increasing velocities of T cells in mouse lymph nodes. Therefore, redirecting antigen-specificity to nonspecific CD8+CD25+ Tregs can be achieved for enhanced immunosuppression through their arming with the antigen-specific pMHC-II complexes. This approach may have great impact on improvement of endogenous polyclonal Treg-mediated immunotherapy for autoimmune diseases. Taken together, our studies demonstrate that nonspecific polyclonal CD4+ T cells and CD8+CD25+ Tregs, when armed with HER2 and MOG antigen-specific pMHC-I and -II complexes, become capable of stimulating enhanced HER2-specific CTL responses and antitumor immunity in double transgenic HLA-A2/HER2 mice and inducing enhanced MOG-specific immunosuppression in MOG-induced EAE mice, respectively. Therefore, redirecting antigen specificity to nonspecific CD4+ T and CD8+CD25+ Tregs by pMHC complex arming may have great impact in development of novel T cell-based vaccines for treatment of cancer and autoimmune diseases.

Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.