CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma

Abstract

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.

Figure 1

SM1 is a weakly immunogenic tumor. Mice were vaccinated s.c. with 1 × 10⁶ irradiated cells of the indicated cell line. Thirty days later, mice were rechallenged with 2 × 10⁵ live SM1 cells and tumor growth was monitored. Incidence of SM1 tumors is indicated in parentheses.

Figure 2

SM1 is not rejected as a consequence of anti-CTLA-4 treatment. Mice were implanted s.c. with SM1 tumors (circles) or B7SM1 tumors (squares) and treated i.p. with 100 μg of either control antibody (solid lines) or anti-CTLA-4 (dashed lines) 4, 7, and 10 days later. Tumor growth was monitored, and incidence is indicated in parentheses.

Figure 3

GM-CSF and anti-CTLA-4 synergize in treatment of SM1 tumors. On day 0, mice were implanted with 2 × 10⁴ (a) or 2 × 10⁵ SM1 (b) cells. (a) On days 0, 3, and 6, mice were injected s.c. on the contralateral flank with the 1 × 10⁶ irradiated cells of the indicated vaccine. On days 4, 7, and 10, mice were injected i.p. with either control antibodies (dashed lines) or anti-CTLA-4 (solid lines). (b) Mice were treated with a combination of anti-CTLA-4 and an irradiated GMSM1 vaccine or anti-CTLA-4 alone as described in a. Growth of the parental SM1 tumor was monitored, and incidence is indicated in parentheses.

Figure 4

Rejection as a consequence of anti-CTLA-4 treatment and a GM-CSF-expressing vaccine results in immunity to rechallenge. Mice were treated as in Fig. 3a. Six weeks after initial challenge with SM1, mice were rechallenged (arrow) on a separate, shaved area of the back with 2 × 10⁵ SM1 cells. Tumor growth was monitored, and incidence is indicated in parentheses.

Figure 5

Both CD4⁺ and CD8⁺ cells are required for regression of SM1 tumors. Six days before SM1 tumor challenge and initiation of treatment, mice were depleted of the indicated cell population as described in Materials and Methods. Mice were implanted with SM1 tumors and treated as described in Fig. 3a, and tumor growth was monitored. In contrast to mice that were mock-depleted (triangles), all mice depleted of either CD4⁺ or CD8⁺ cells (or both populations) grew tumors.