Multiple myeloma (MM) is a B-cell malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow. Recently, we showed a correlation between increased ratios of functional regulatory T cells (Treg) and disease progression in a unique mouse model that mimics the human disease. Cyclophosphamide (CYC) is a cytotoxic alkylating agent widely used in chemotherapeutic regimens. Low-dose CYC was previously reported to selectively reduce Treg levels and to contribute to immunostimulation. Our objectives were (a) to determine whether treatment using a low-dose CYC could reduce MM progression and (b) to further characterize the modes of action underlying these effects. We found that both low-and high-dose CYC given to sick mice with hind limb paralysis resulted in the disappearance of the paralysis, the replacement of plasma tumor cells in the bone marrow by normal cell populations, and a significant prolongation of survival. However, only low-dose CYC treatment decreased the incidence of MM. Low-dose CYC rendered Tregs susceptible to apoptosis because of the downregulation of Bcl-xL and CTLA-4 in these cells, and a decreased production of interleukin 2 by effector CD4 cells. Moreover, using this treatment, we noted the recovery of IFN-γ-producing natural killer T cells and maturation of dendritic cells. Treatment of tumor-bearing mice with repeated administrations of low-dose CYC at longer time intervals (coinciding with the blocked renewal of Tregs) resulted in reduced tumor load, and the prevention or delay of disease recurrence, thereby breaking immune tolerance against MM tumor cells. Cancer Prev Res; 3(10); 1265-76. ©2010 AACR.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a central role in cancer tolerance. However, mechanisms leading to their accumulation in cancer remain unknown. Although the thymus is the main site of Treg development, thymic contribution to Treg expansion in cancer has not been directly examined. Herein, we used two murine models of multiple myeloma (MM), 5T2 MM and 5T33 MM, to examine Treg accumulation in peripheral lymphoid organs, including spleen, lymph nodes, bone marrow, and blood, and to explore thymic Treg development during malignancy. We found that peripheral ratios of suppressive-functional Tregs increased in both models of MM-inflicted mice. We found that thymic ratios of Treg development in MM increased, in strong association with thymus atrophy and altered developmental processes in the thymus. The CD4+CD8+ double-positive population, normally the largest thymocyte subset, is significantly decreased, whereas the CD4−CD8− double-negative population is increased. Administration of thymocytes from MM-inflicted mice compared with control thymocytes resulted in increased progression of the disease, and this effect was shown to be mediated by Tregs in the thymus of MM-inflicted mice. Our data suggest that increased ratios of Treg development in the thymus may contribute to disease progression in MM-inflicted mice.