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Visceral leishmaniasis (VL) has emerged as a clinically important opportunistic infection in HIV-positive patients, as VL/HIV co-infected patients suffer from increased treatment failure, drug toxicity, mortality and VL relapse. Here, I followed cohorts of patients with VL only and co-infected with HIV in Ethiopia and collected detailed clinical and immunological data during 12 months of follow-up, to identify clinical and immunological markers that are associated with VL relapse. By the end of the study, 78.1% of VL/HIV patients, but none of the VL-only patients, had experienced VL relapse. Despite clinical and/or parasitological defined cure at the end of antileishmanial treatment, over time VL/HIV patients maintained high parasite loads, splenomegaly, low BMI, pancytopenia, high levels of programmed cell death protein 1 (PD1) on CD4+ and CD8+ T cells, impaired IFN-γ production capacity in a whole blood assay (WBA) and persistently high level of inflammatory cytokines. In contrast, these parameters were restored over time in patients with VL infection only. Furthermore, during follow-up these parameters were also associated with VL relapse in VL/HIV co-infection. Overall, my results suggest that in VL/HIV patients, VL relapses independent of the HIV viral load and the high rate of VL relapse in VL/HIV patients is at least in part due to their impaired capacity to restore CD4+ T cell counts and antigen-specific IFN-γ production in the whole blood assay; as well as high levels of the inhibitory molecule PD1 on T cells and high levels of pro- and anti-inflammatory cytokines in the plasma. Furthermore, the impaired IFN-γ production and the persistently high levels of PD1 on T cells in VL/HIV patients suggests that these patients might also benefit from immunotherapy with IFN-γ and anti-PD1.

Infection with HIV results in a chronic infection that progressively impairs the immune system. Although depletion of CD4+ T cells is frequently used to explain the deterioration in immune function, the combination of the chronicity of infection and progressive loss of CD4+ T cells are not sufficient to fully account for the immune dysregulation that occurs during the progression to AIDS. Arginase-induced L-arginine deprivation is emerging as a key mechanism for the downregulation of immune responses. In this thesis it is shown that PBMC arginase activity increases with disease severity in HIV seropositive patients. PBMC arginase activity was found to be associated with both CD4+ T cell count and plasma viral load. However, these associations were not found in the cohort of patients that were taking highly active antiretroviral therapy. HIV seropositive patients with high PBMC arginase activity were also found to have decreased levels of CD3! protein by western blot, a marker of T cell dysregulation caused by arginase mediated L-arginine depletion. The arginase1+ cells in the PBMC fraction were identified as CD15+ CD14- granulocytes and therefore named low-density granulocytes (LDGs). The percentage of LDGs was found to be associated with PBMC arginase activity. LDGs were found to be phenotypically different from neutrophils that co-purify with erythrocytes after density gradient centrifugation (normal density granulocytes – NDGs). LDGs have an increased size, increased expression of the activation markers CD63 and CD66b and a decreased expression of arginase 1 compared to NDGs. This suggests that the LDGs may be a population of activated neutrophils that have degranulated releasing arginase 1. As it has been shown that T cells have an absolute requirement for L-arginine in order to proliferate and express CD3! the results presented in this thesis suggest that arginase-induced L-arginine deprivation might contribute to T cell dysfunction in HIV infection.