Macrophages from mice vaccinated with 10 μg LPG and re-stimulated in vitro with 1 μg LPG, showed diminished expression of PD-L2 whereas vaccination with 100 μg LPG tended to increase the expression of PD-L2 in macrophages after receiving secondary stimuli with LPG ( Fig. 5A). Mice infected with 1 × 104 or 1 × 105 parasites down-regulated PD-L2 expression by 50% (Fig. 5B). Re-stimulation of macrophages from mice infected with 1 × 104 parasites Small molecule library in vivo with LPG always showed diminished expressions of this inhibitory

marker, whereas those from mice infected with 1 × 105 parasites slightly increase their PD-L2 expression, albeit never reaching the levels expressed in cells of non-infected mice (Fig. 5B). Together, these data show that Leishmania infections reduce PD-L2 expression in spleen macrophages and that this down-regulation persists despite secondary in vitro stimulation

with LPG. Our data shed new light on the cause of enhanced disease progression after immunization with Leishmania LPG that has also been reported in the literature [16]. In an attempt to understand the underlying cause of this unsuccessful vaccination with LPG, we immunized mice with different concentrations of LPG and thereafter stimulated CB-839 their spleen cells with various doses of LPG in vitro in an attempt to simulate a secondary exposure to LPG antigen, as would occur during a natural infection. isothipendyl Additionally, we infected mice with different L. mexicana numbers and also re-exposed their lymphocytes to a secondary challenge with LPG. We here show that immunization of BALB/c mice with LPG or infections with L. mexicana promastigotes enhances the expression of the inhibitory receptor PD-1 in CD8+, whereas CD4+ T cells remain unaltered. The increase of these inhibitory molecules in CD8+ T cells acts in concert with their reduction of the activating molecule CD137, when these cells are

confronted with a new challenge of LPG. These changes vary according to the amount of the LPG used for the vaccination and the parasite load during infection and they also vary according to the amount of parasite antigen (LPG) encountered by these cells after renewed exposure. The combination of these events possibly leads to a severe down-regulation of the functional capacity of CD8+ T cells in controlling the parasite infection. The response of CD4+ T cells was less clear. PD-1 (programmed-death 1) receptor is related to CD28 and CTLA-4. It is inducible after T cell activation and down-regulates activated T cells [11]. Its ligands, PD-L1 and PD-L2, are up-regulated in APCs following activation [8]. PD-1 and PD-L2 may have distinctive roles in regulating Th-1 and Th-2 responses and reducing T cell proliferation by arresting the cell cycle [17] and [18].