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Tumor cells develop numerous mechanisms to escape from the control exerted by the immune system. One of these strategies is the accumulation of regulatory T cells (Treg) within the tumor, which keep effector T cells (Teff) and dendritic cells (DC) in an inactive state. An efficient approach to overcome the inhibitory potential of Treg focuses on OX40, a costimulatory molecule constitutively expressed by Treg and induced in activated Teff. The treatment of mouse transplantable tumor models with the mAb OX86, the agonist of OX40, induces tumor rejection by acting on both these T cell subsets. In this study we investigated the fine cellular mechanisms at the basis of this process, dissecting the effects of OX86 on Treg and on CD4+Foxp3•CD44highCD62L,owOX40+ effector memory T cells (Tem), which represent the most abundant Teff subset in the tumor. Upon OX40 stimulation, Treg are "contra-suppressed" and down-modulate the expression of the transcription factor interferon regulatory factor 1 (IRFl), thus reducing the secretion of IL- 10. Conversely OX86 provides activating stimuli to Tern, which up-regulate CD40L and in turn promote the maturation of DC. OX86 shifts the tumoral milieu from tolerogenic to immunogenic, favoring the activation and migration of DC from the tumor to the draining lymph node (dLN) and the subsequent new CTL induction. The relevance of OX40 in Treg biology goes beyond the modulation of their suppressive abilities. OX40 increases the sensitivity of Treg to IL-2, facilitating the phosphorylation of STAT5 through high level of the mirl55 and low level of SOCS 1. The overexpression of miR155 endowed Treg of higher suppressive functions, further enhancing tumor growth. These data clearly remark the key roles exerted by OX40 in influencing Treg and Teff behavior. Understanding how to manipulate OX40 signaling will provide great advantage in the development of efficient therapy for both tumors and autoimmune diseases.

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In the present study, the effects of two caspase inhibitors, z-VAD-FMK and z-IETD-FMK, on T cell activation and proliferation were compared to the negative control, z-FA-FMK. All three compounds inhibited T cell proliferation, blast formation, CD25 expression, IL-2 signalling and nuclear translocation of the NF-kappaB subunit, p65. However, unlike z-VAD-FMK and z-IETD-FMK, z-FA-FMK inhibited IL-2 and IFN-gamma secretion and blocked cell cycle entry. Furthermore, although both z-VAD-FMK and z-IETD-FMK inhibited Fas-induced caspase activation, they had no effect on caspase-8 and -3 processing during T cell activation. In contrast, z-FA-FMK, which did not inhibit caspase processing during apoptosis, blocked caspase-8 and -3 processing in activated T cells. This suggests that peptidyl-fluoromethylketones have pleiotropic immunosuppressive properties which may not involve the processing of caspase-8 or -3. In addition, these data suggest that the processing of caspases is regulated differently during T cell activation compared to apoptosis. To further characterise the role of caspase-8 during T cell activation, an activation-induced cell death (AICD) model using caspase-8-/- Jurkat T cells was examined. In addition, the involvement of FADD, the docking protein for caspase-8, was also investigated. Caspase-8-/- Jurkat T cells were resistant to AICD induced by PMA and ionomycin, whereas FADD-/- Jurkat T cells underwent TNF-alpha-mediated necrosis. These data suggest that caspase-8 but not FADD is required for the T cell activation signalling pathway and also implicate TNF-alpha in AICD in the absence of the Fas-signalling pathway. In conclusion, the results suggest that non-specific effects of caspase inhibitors are involved in the inhibition of T cell activation and proliferation and that the regulation of caspases during T cell activation is different from that during apoptosis.

The presence of inflammatory cells within the central nervous system (CNS) including the retina is characteristic of disease states such as human uveitis and multiple sclerosis, both CD4+ T cell mediated immune inflammatory diseases. Normal CNS is devoid of inflammatory cells and is maintained by the specialized nature of the isolated microenvironments. The blood brain and blood retinal barriers (BBB, BRB) composed of highly specialized endothelial cells physically protect the CNS by regulating immune cell entry. The CNS is actively protected by immune privilege in which the local environment is able to suppress potentially devastating immune reactions, through the local expression of FasL and production of cytokines such as TGF-p. Additionally a role for a group of compounds called Statins has been described in the prevention of leukocyte migration from blood vessels to the sites of atherosclerotic lesions. This study aims to determine potential mechanisms by which microvascular endothelial cells (EC) of the CNS are able to regulate T lymphocyte function, and whether these mechanisms are implicated in disease. The effects of statins on an inflammatory disease of the CNS were also investigated. An in vitro model of the BBB was used to investigate the immunomodulatory effects of EC upon T cells. A transwell insert system was employed to investigate the effects on T cells of co-culture with EC whilst preventing cell-to-cell contact and also, the effect of transmigration through EC monolayers. Studies were also undertaken to investigate potential mechanisms by which this regulation may occur. The effect of statins was investigated using two models of Experimental Autoimmune Uveoretinitis (EAU) as a model of human posterior Uveitis. EAU was induced using SAg peptide or IRBP peptide and the effects of daily administration of statin were assessed using Fluorescein Angiography. Retinal sections were also examined by classical histology. The effects of statins were also investigated using an in vitro model of the BRB, time lapse microscopy and flow cytometry. The effects of statins on T cell function were assessed ex vivo by splenocyte proliferation assays.

The human T-cell Lymphotropic Virus type 1 (HTLV-1) infection generates a strong cytotoxic T lymphocyte (CTL) response which controls the pro viral load. The aim of this work was to characterize functional mechanisms involved in the efficiency of killing of infected cells by the CD8⁺-cells.

PD-l , a costimulatory receptor expressed by T -cells, B-cells, NKT cells and monocytes, has emerged as one of the most potent inhibitory molecules in the immune system, and a very promising therapeutic target. The biophysical properties of the interactions of PD-i with its two ligands PD-Lt and PD-L2, however, are incompletely characterized. The question of why there are two ligands for PD-l is also unanswered. An unexpected interaction between PD-Lt and B7- 1, which also binds to other costimulatory receptors, has further complicated the interpretation of the functions of these molecules. In this thesis , experiments were undertaken to more fully characterize the interactions of PD-l within the wider context of costimulatory interactions involving T cells. Monomeric forms of PD-l, PD-Lt, PD-L2 and B7-1 were produced in order to re-analyze their interactions using surface plasmon resonance-based assays. The three- to four-fold greater affinity of PD-L2 versus PD-Ll for PO-l in humans (PD-IIPO-Ll- 7.8 μM, PO-IIPO-L2 -2.2μM) was principally due to the three-fold smaller dissociation rate for PO-L2 binding. The affinity of PO-Ll with B7-1 was much weaker than previously reported, and the interactions of PO-l with its ligands in the murine system had similar affinities, albeit ones considerably weaker than the respective human interactions. The thermodynamic properties of the PD-l system were characterized using two different approaches, van't Hoff analysis and isothermal calorimetry, and this showed that the PO-IIPO-Ll interaction is driven entropically, whereas for PD-I and PD-L2 binding enthalpy is dominant. The ΔCp values for both interactions were similar, with a slightly more negative value obtained for PO-IIPO-Ll than for PO-IIPD-L2, and consistent with binding involving the burial of relatively large hydrophobic surfaces. Comparison of the binding surface residues perturbed during ligand binding using nuclear magnetic resonance (NMR)-based analyses showed that the binding foot-print of PD-Ll is larger than that of PD-L2, even though PD-L2 binds PD-l more strongly. Comparison of the NMR data with the published crystal structures of murine PD- I complexed with human PD-Ll and mouse PD-L2 suggested that the human and mouse interactions differ in detail. Mathematical simulations based on the affinity and kinetic data, and on rigorous expression data revealed an unexpectedly limited contribution of PD-L2 to PD-I ligation during interactions of activated T-cells with mature dendritic cells (mDCs): PD-I engaged more than four-fold fewer PD-L2 than PD-Ll molecules due to the low expression of PD-L2 on mDCs. These observations suggest that the function of PD-L2 is not to enhance human PD-l engagement but to provide qualitatively different signals. The simulations also implied that the B7-IIPD-Ll interaction might have limited impact in the presence of CTLA-4, CD28 and PD-I. Finally, a novel method for identifying new receptor ligands suggested that there are unlikely to be high affinity ligands for several B7-, PD-Ll- and PD-L2-related proteins, raising the possibility that these proteins have ligand-independent functions. These findings provide a new, more rigorous structural and biophysical framework for interpreting the functions of PD-I.

Immature dendritic cells (DCs) can be instrumental in the induction of peripheral tolerance promoting the generation of regulatory T cells. Since NF-kB is central to DC maturation, rat bone marrow-derived DCs were transfected with an adenoviral vector encoding for a kinase-defective dominant negative form of IKK2, namely AdVdnIKK2, to block NF-kB activation and inhibit DC maturation. DnIKK2-DCs had an immature phenotype, as shown by the low expression of MHCII, CD86 and IL-12. Moreover, they had an impaired allostimulatory capacity and generated CD4+ T cells with regulatory function (CD4+ dnIKK2-Treg). I have investigated the potency, the phenotype, and the mechanism of action of CD4+dnIKK2 Treg. The results revealed that CD4+dnIKK2-Treg were CD4+CD25-/dim and expressed Foxp3, IL-10, TGF-P, IL-2, and inducible nitric oxide synthase (iNOS). CD4+ dnIKK2-Treg had an extremely potent donor-specific regulatory activity, in fact when cocultured with responder T cells in a primary MLR, suppressed T-cell proliferation to alloantigens until at up to 1:10¹⁵ ratio.