Programmed death-1 receptor (PD-1) is expressed on T cells following TCR 
activation. Binding of this receptor to its cognate ligands, programmed death 
ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell 
anergy and apoptosis, thus leading to immune suppression. Here, we find that 
using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose 
cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic 
antigen-specific immune responses and reveals novel activities of each agent in 
this combination. This strategy led to complete regression of established tumors 
in a significant percentage of treated animals, with survival prolongation. We 
show for the first time that combining CT-011 and CPM significantly increases 
the number of vaccine-induced tumor-infiltrating CD8(+) T cells, with 
simultaneous decrease in infiltrating Treg cells. Interestingly, we find that 
CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable 
significant synergistic decrease of splenic and tumor-infiltrated Treg cells. 
Surprisingly, we find that the anti-tumor effect elicited by the combination of 
CT-011 and CPM is dependent on both CD8(+) and CD4(+) T-cell responses, although 
the antigen we used is a class I MHC-restricted peptide. Thus, we describe a 
novel and effective therapeutic approach by combining multiple strategies to 
target several tumor-mediated immune inhibitory mechanisms.