Human immunodeficiency virus (HIV) infection is a worldwide epidemic where infected individuals usually develop acquired immunodeficiency syndrome (AIDS). HIV is primarily spread by sexual transmission across mucosal tissue where dendritic cells (DC) reside. DC regulate immune responses through their unique ability to capture antigen, migrate to lymphoid tissue, and activate naive T cells. In this Thesis, we have investigated whether HIV influences the migration of DC, thereby influencing their capacity to regulate immune function and facilitate transport of HIV to T cell rich lymphoid tissue. Transmigration assays demonstrated that the predominant HIV strain during primary infection, R5 HIV-1, was chemotactic for immature DC (iDC). Addition of soluble CD4 enhanced iDC migration to R5 HIV, presumably by binding to R5 HIV and altering the conformation to enhance binding to CCR5. Our results suggested that iDC migrated specifically to R5 and not X4 HIV gp120, through interactions between the extracellular loop-2 (ECL-2) domain of CCR5 with the V3 loop region of R5 gp120. iDC prepared from HIV-infected subjects were shown to have impaired chemotaxis to inflammatory chemokines compared with iDC from healthy individuals. Furthermore, the level of inhibition appeared to be proportional to the severity of disease progression in HIV infected subjects. Interestingly, chemotaxis of iDC from long-term non-progressor individuals was similar to normal individuals, whereas migration of iDC from typical progressors was greatly impaired. These differences did not appear to be related to the level of CCR5 expression or patient viral load. The protease inhibitor Indinavir used in antiretroviral therapy, limited DC trans-endothelial migration to chemokines, reduced DC-SIGN expression and increased CD83 on iDC. The results suggested that Indinavir inhibited proteases necessary for DC migration by adversely affecting interactions between DC-SIGN, VLA-4 and VLA-5 and ligands on the endothelium and underlying fibronectin matrix. A novel method has been successfully developed for amplifying rare HIV-specific CDS cells using DC transfected with HLA antigens matching HIV-infected subjects. This has enabled us to amplify HIV-specific CDS T cells by 10- to 60-fold. This may help us to clone and characterise HIV-specific CDS T cells from highly exposed persistently seronegative (HEPS) individuals. In summary, the results in this Thesis demonstrate that R5 HIV mimics chemokines to subvert the natural trafficking of DC. Indeed, we have shown that DC from typical progressors have severely impaired migration. This may have serious consequences on DC immunoregulation, compromising the immune function of these infected individuals.

Thesis (MScMed)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.