Cyclophosphamide Induced CD11b⁺ Cells Can Inhibit Anti-CD40 Monoclonal Antibody (mAb) Therapy of B-Cell Lymphoma.

Immunotherapy with mAb is emerging as an important component of treatment for B-cell lymphoma. Furthermore, anti-tumor effects may be enhanced by combining mAb with a range of conventional cancer treatments and recently impressive results have been acheived with mAb in combination with chemotherapy. Here, we have assessed the efficacy of combining an agonistic anti-CD40 mAb with cyclophosphamide (CY). We have previously shown that large repeated doses of anti-CD40 mAb alone may stimulate cytotoxic T-cell lymphocyte (CTL) responses and provide robust protection in syngeneic murine tumors, such as BCL₁ lymphoma. In this study we therefore used single smaller doses of the mAb that alone fail to provide long-term clearance thus allowing us to look for additive or synergistic effects of anti-CD40 and CY. Interestingly, the timing of CY relative to mAb administration appeared critical to therapeutic outcome. Cohorts given CY 7 days (d7 CY) prior to anti-CD40 show reduced survival compared to those receiving mAb alone. In contrast treatment with CY given 1 day before, or on the same day as mAb resulted in greatly increased tumor free survival. In vivo tracking experiments revealed that there is a potent CTL response that clears tumor around 5 days after anti-CD40 treatment. However, when CY was given d7 prior to anti-CD40 mAb, CTL expansion was reduced compared with other treatments, and instead resulted in increased numbers of splenic CD11b⁺ cells. These CD11b⁺ cells have an immature myeloid precursor phenotype and were able to produce nitric oxide (NO). Co-culture of CD11b⁺ cells with murine splenocytes resulted in greatly reduced cytotoxic CTL proliferation, but no apoptosis, which was reversed in the presence of NO inhibitors. Similarly, splenic CTL proliferation in vivo is inhibited by this increased population of CD11b⁺ cells. Importantly adoptive transfer of CY-induced CD11b⁺ into non-CY-treated mice inhibited anti-CD40 mAb tumor protection in BCL₁ lymphoma. This data indicate that combining CY with immunotherapy can enhance therapeutic responses, but the timing of CY relative to immunotherapy is critical to the therapeutic outcome.