Taken together, a moderate bonus effect on direct tumor killing may be achieved by using the combination approach. Since trAbs not only mediate direct tumor cell killing, but also elicit a vaccination effect , we then asked whether the immunologic memory induced by trAbs is affected by the combination with anti-CTLA-4 treatment. These experiments were only done using the B78-D14 melanoma because the B16-EpCAM model is less stringent due to its higher inherent immunogenicity (our unpublished data). Since effective long-lasting tumor protection is dependent on T-cell responses of the Th1 type [27, 28], we first compared the levels of Th1 and Th2 cytokines in sera of mice that had been vaccinated with trAb Surek or HB304 or the antibody combination together with replication-incompetent tumor cells as an antigen source. After Surek immunization, high concentrations of Th1 cytokines were detected (Figure 4A). The data confirmed our previous results, which indicated a clear Th1 bias involving IL-12 expression by DCs and IFN-γ production by T cells . Accordingly, an upregulation of Th2 cytokines was also observed, because this is a prerequisite for inducing Th1 responses (see Discussion). In contrast, HB304 alone did not induce any cytokine response. Furthermore, the cytokine levels that were induced by Surek could not be further enhanced by combining trAb treatment with HB304 (Figure 4A). Likewise, when T cells from vaccinated mice were restimulated with melanoma-derived peptides in vitro, the combination group neither showed enhanced IFN-γ release as compared to animals vaccinated with Surek alone (not shown).