There is a body of evidence that CY can induce antitumor effects not only by directly affecting dividing cancer cells, but also indirectly by augmenting T-cell concomitant antitumor immune responses (29
). The immunomodulating effects of CY can be attributed, at least in part, to the ability of CY to eliminate tumor-induced T regulatory/suppressor cells (7
). Several studies have reported the beneficial effects of CY administered prior to adoptive transfer of T cells (30
), dendritic cell vaccines (31
) or activated NK cells (5
). To our knowledge, the results presented here show for the first time that combining CY and immunotherapy can result in enhanced antitumor effects without the involvement of T cells or NK cells.
We have previously shown that αCD40 and CpG, given separately or in combination, can induce T cell-independent antitumor effects involving Mϕ (24
). The results of this study confirm that Mϕ are activated following αCD40/CpG treatment, and show that in vivo
CY treatment did not inhibit the ability of Mϕ to be activated. In contrast to numerous T cell-based approaches, a relatively limited number of approaches aimed at Mϕ activation have been tested for cancer therapy experimentally and in clinical trials. One of them, using muramyl tripeptide phosphatidylethanolamine (MTP-PE), has shown promise in both experimental systems and clinical trials (35
). However, combining MTP-PE with chemotherapy (cisplatin) in mice and dogs did not lead to enhanced antitumor effects (37
). Clinical testing in children with osteosarcoma suggested that the chemotherapy regimen used might influence the potential benefit of MTP-PE treatment and thus improve overall survival (39
). The preclinical data reported here show that αCD40/CpG immunotherapy in combination with CY leads to enhanced antitumor effects. In addition to CY, we have also seen additive or synergistic antitumor effects of αCD40/CpG when combined with the CHO chemotherapeutic regimen consisting of CY, doxorubicin and vincristine (Buhtoiarov et al., in press).
The exact mechanism of the enhanced antitumor activity of the combination of CY and αCD40/CpG is not clear. Our experiments suggest that in vivo
CY treatment may render Mϕ more responsive to the subsequent activation with αCD40/CpG (). This may be due to the increased percentage of Mϕ in the peritoneal cavity, possibly caused by selective reduction of other cells including CD40+
B cells, consequently exposing Mϕ to more αCD40/CpG. However, while some analyses we performed demonstrated enhanced activation of Mϕ with a combination of CY and αCD40/CpG compared to αCD40/CpG without CY, this enhancement was not reproducible (data not shown). It is also possible that CY aids αCD40/CpG therapy by activating cells other than Mϕ. For example, CY has been shown to induce early myeloid cells with the CD11b+
phenotype of myeloid-derived suppressor cells (MDSC), both in mice (40
) and cancer patients (43
). In response to IFNγ and CD40 ligation, CY-induced MDSC produce NO, which has antitumor activity (41
). It was suggested that induction of these cells by CY was dependent on the presence of tumor-specific T cells producing IFNγ (39
). However, we have previously shown that IFNγ can be induced in Mϕ in response to CD40 ligation (24
), suggesting that T cells may not need to be involved. Although adoptive transfer of CY-induced MDSC inhibited T cell-dependent antitumor effects of αCD40 (42
), in our studies the additive effect of CY and immunotherapy was observed in T cell-deficient mice. Our data show an increased percentage of cells with MDSC phenotype (CD11b+
) in PEC of CY, αCD40 and CpG – treated mice, suggesting that they can contribute to the antitumor effects observed in vivo
. The inability of anti-Gr-1 mAb to deplete all Gr-1+
cells or reduce the antitumor effect of CY + αCD40/CpG in our preliminary experiments (data not shown) could be due to the recently described resistance of certain MDSC to anti-Gr-1 mAb – mediated depletion in vivo
). Therefore, the role of MDSC in the observed antitumor effects warrants further investigation.
Our results demonstrate that immunological approaches directed toward activating Mϕ can be combined with chemotherapy to achieve better antitumor effects in immunocompromised mice, and suggest that such strategies might be considered for immunosuppressed cancer patients. This concept might sound counterintuitive, given that tumor-infiltrating Mϕ have been viewed as tumor-promoting rather than antitumor in their effects: their number usually correlates with poor prognosis (46
). However, it also has been reported that chemotherapy combined with immunotherapy (rituximab) resulted in a high content of tumor-associated Mϕ which correlated with favorable outcome in follicular lymphoma patients (47
). Moreover, it has been shown that pro-tumor Mϕ (M2 type) can be converted into tumor-infiltrating antitumor Mϕ (M1 type) by immunotherapy containing CpG (48
). Our data indicate that activation of Mϕ with αCD40 and CpG, especially when combined with chemotherapy, results in antitumor effects in mice, suggesting that this approach should be considered for clinical development and testing.