ORIGINAL ARTICLE CD4 þ CD25 þ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge L Elia, L Aurisicchio, A Facciabene, P Giannetti, G Ciliberto, N La Monica and F Palombo Department of Molecular and Cellular Biology, IRBM-Merck Research Laboratory, P. Angeletti, Pomezia, Rome, Italy Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self- antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4 þ CD25 þ regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8 þ , CD4 þ T cells and antibody response. The appearance of a CD4 þ T-cell response correlated with a stronger memory response. The combined CD25 þ inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients. Cancer Gene Therapy (2007) 14, 201–210. doi:10.1038/sj.cgt.7701004; published online 20 October 2006 Keywords: CD25; CEA; adeno FOXP3; tumor; CD8 þ Introduction Immunomodulation has emerged as an important com- ponent in inducing a strong and effective immune response against tumor-associated antigens (TAA). 1 In this respect, inactivation of regulatory T cells (T R ) has proved efficacious against tumor cell lines, although permanent T R depletion is embedded with autoimmune- related side effects such as colitis and gastritis. 2 The induction of a long lasting and effective CD8 þ response is key to most cancer vaccine treatments against TAA. To achieve this result it is also important to induce a CD4 þ helper response that can sustain an effective CD8 þ response over time. 3 However, CD4 þ cells also play a role in regulating the amplitude and duration of the immunological response raised against TAA, mainly through the action of T R . They express high levels of interleukin (IL)-2 receptor (CD25), GITR, 41BB and the transcription factor forkhead FOXP3. 2 T-cell transfer upon T R depletion leads to autoimmune diseases that are suppressed by complementation with T R cells. 4 Thus, an ideal setting in cancer vaccine could be the induction of a transient autoimmune state (T R depletion or inactivation) where antigen-specific immune response could be induced by a genetic vaccination. Many reports have shown depletion of T R in tumor cell vaccination has a positive impact, but only a few have explored the impact of T R on a specific peptide vaccine or xenogeneic vaccine. 5,6 Apparently, the impact of anti-CD25 antibodies on T R is more complex than simple depletion as recently shown by Kohm et al. 7 Anti-CD25 anti- body induces down expression of CD25 receptor, which is associated with functional inactivation, but they remain present, as indicated by FOXP3 expression in CD4 þ cells. The impact of T R depletion or inactivation on self- TAAs vaccination has not been explored extensively. To verify the efficacy of this strategy we explored a transient T R inactivation as a sort of ‘immunization window’ where genetic vaccination against TAA may provoke a stronger immune response that in turn can protect mice against tumor challenge when T R subsequently return to normal level. To test this hypothesis we used a CEA transgenic mouse model where we have recently shown that an adenovirus (Ad) vector expressing the carcinoembryonic antigen (Ad-CEA) succeeded in breaking immunological tolerance. 8 Received 1 April 2006; revised 22 July 2006; accepted 28 August 2006; published online 20 October 2006 Correspondence: Dr F Palombo, Department of Molecular and Cellular Biology, IRBM-Merck Research Laboratory, P. Angeletti, Via Pontina Km 30,600 00040 Pomezia, Rome, Italy. E-mail: Fabio_Palombo@merck.com Cancer Gene Therapy (2007) 14, 201–210 r 2007 Nature Publishing Group All rights reserved 0929-1903/07 $30.00 www.nature.com/cgt