John B. Yates

Inhibition of NF-κB and Oxidative Pathways in Human Dendritic Cells by Antioxidative Vitamins Generates Regulatory T Cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and α-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2α, NF-κB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4+CD45RO, CD4+CD45RA, and CD4+CD25− subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-β. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.

Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis

CD4+ CD25+ regulatory T cells have been shown to be a vital component of the mechanisms that prevent autoreactivity in mice and also in humans. Previous studies have examined CD4+ CD25hi regulatory T cell frequency and function in patients with systemic lupus erythematosus (SLE) with mixed results. We investigated frequency, phenotype and function in 21 patients with SLE and six with inactive disease. We found no reduction in frequency of the CD25hi subset, although active disease was associated with an increased proportion of CD4+ CD25+ T cells. When examining function, in the majority of individuals suppression was comparable with controls, although cells isolated from one patient with active disease failed to suppress proliferation. On testing the effect of CD25hi depletion on the responses of whole peripheral blood mononuclear cells to nucleosomes we found that, where a response was detectable from patients, depletion augmented interferon‐γ secretion, demonstrating intact suppression of responses implicated in the pathogenesis of SLE. Our results did not confirm an association of failure in CD4+ CD25hi regulatory T cell function or a reduction in their frequency with active disease. Instead, perturbations in the CD4+ CD25hi regulatory T cell population may play a role in disease in only a minority of the patients afflicted by the diverse syndromes of SLE.

Location of major histocompatibility complex class II molecules in rafts on dendritic cells enhances the efficiency of T-cell activation and proliferation.

The existence of major histocompatibility complex (MHC) class II molecules in lipid rafts has been described in dendritic cells (DC); however, the importance of rafts in T‐cell activation has not been clarified. In this study, the distribution of the lipid raft components (CD59 and GM1 ganglioside) in human monocyte‐derived DC was investigated. DC had an even distribution of these components at the cell surface. In addition, raft‐associated GM1 ganglioside colocalized with cross‐linked MHC class II. This implies coaggregation of raft components with these MHC molecules, which may be important in the interaction between T cells and antigen‐presenting cells. In studies carried out to investigate the effect of the DC : T‐cell interaction on raft distribution, we found a clustering of the lipid raft component CD59 on DC at the synaptic interface, with associated activation of the interacting T cell. In an antigen‐specific response between DC and CD4+ T‐cell clones, disruption of lipid rafts resulted in inhibition of both CD59 clustering and T‐cell activation. This was most pronounced when limiting amounts of cognate peptide were used. Together, these data demonstrate the association of MHC class II with lipid rafts during DC : T‐cell interaction and suggest an important role for DC lipid rafts in T‐cell activation.

CREATION OF TOLEROGENIC ANTIGEN PRESENTING CELLS VIA INTRACELLULAR CTLA4: A NOVEL STRATEGY WITH POTENTIAL CLINICAL UTILITY IN TRANSPLANTATION.

Aims: Activation of T lymphocytes requires the recognition of peptide–MHC complexes and co-stimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T cell activation without co-stimulation can lead to T cell anergy, a state of T cell hyporesponsiveness. In this study we developed a novel strategy to inhibit expression of B7 molecules by transfecting APCs with a gene construct encoding a modified CTLA4 molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). Our approach is to express within the cells CTLA4-KDEL. This will bind to the newly synthesised B7 molecules in the ER and prevent them from reaching the cell surface. Methods: The CTLA4-KDEL gene was constructed using standard technology. It was then transfected into a range of APCs. In the case of DCs transfection was performed using a new non-viral gene therapy system. The APCs were then tested in their ability to activate peptide specific T cell clones and primary allospecific T cells. OVA TG model was used to assess in vivo tolerance. Results: Our in vitro and in vivo data using CTLA4-KDEL in various APCs show that this strategy does indeed block B7 expression by APC and that antigen-reactive (both allospecific and peptide-specific) T cells not only are hyporesponsive to antigen presented by these cells but also are rendered anergic. Conclusions: This gene-based strategy to inhibit expression of key surface receptors in order to generate ‘B7deficient’ APCs has significant advantages over the currently described approaches for treatment of transplant rejection. Transplant 6328