Bone Marrow Research 3 State of disease Low-dose CYC CD4 tregs NKT cells Window opportunity CD4+CD25+Tregs NKT cells Mq DCs T cells B cells Plasma cells CD4+CD25+Tregs NKT cells Activation of DCs T cells B cells Plasma cells 1 45 90 Injection 135 day CYC CYC CYC CYC Figure 1: Cellular responses to low-dose CYC in mice with MM. maintained only by transfer of BM cells from sick mice to young syngeneic mice. The 5T2MM mouse model resembles the human disease in its main localization to the BM and in the development of hind limb paralysis (due to spinal cord compression) that occurs as an early manifestation of the disease. Further, as the disease progresses, signs of paraplegia and bone lesions take place. Increased accumulation of Tregs is observed in peripheral lymphoid organs, including lymph nodes, BM, and peripheral blood during disease progression [3, 4]. The suppressive functions of Tregs were retained, indicating that the cells were functional in their capacity to regulate immune activity. A correlation between increased Treg levels and disease morbidity was observed. Adoptive transfer of Tregs separated from thymuses of sick 5T2MM mice into 5T2MM-bearing mice serving as Treg recipients, increased severity of MM and enhanced tumor progression. Severe bone destruction, fractures of vertebra, and massive tumor cell inﬁltration into skeletal muscles were observed. The transfer of Treg-free thymocytes did not mediate the tumor-enhancing eﬀects [3]. These results clearly indicated that Tregs can speciﬁcally support in vivo tumor progression. 4. Common Chemotherapeutic Drugs Affecting Treg Depletion A number of chemotherapeutic agents that aﬀect Tregs are used for preventing tumor protection by the immune system. Gemcitabine is a nucleoside analogue that inhibits DNA synthesis. In patients with colon cancer that were treated with gemcitabine, the depletion of Tregs occurred concomitantly with an increase in cytotoxic CD8 T cells [39]. However, when this drug was administered in pancreatic cancer, it did not deplete Tregs although it led to activation of na¨ıve CD4 T cells [40]. Mitoxantrone is an anthracenedione that causes DNA strand breakup and unraveling. Its administration to patients with breast cancer resulted in reduced numbers of Tregs, but this eﬀect was not correlative with the tumor response [41]. Fludarabine is a drug used for treating chronic lymphocytic leukemia (CLL). In vitro eﬀects of this drug were the inhibition of Tregs expansion and sustaining the potency of cytotoxic CD8 T cells [42]. Patients with CLL treated with ﬂudarabine responded with a preferential depletion of Tregs mainly as a result of apoptosis induction [43]. Lenalidomide is a derivative of thalidomide that was approved by the FDA for treating myelodysplastic syndrome and MM. In vitro assays showed that lenalidomide aﬀected Tregs by reducing their expansion and function [44]. CYC is an alkylating agent that is commonly used in various antitumor protocols. In addition to the general cytotoxic eﬀects of CYC, it was reported that doses may inﬂuence selectivity of this drug against Tregs. As a result, the clinical relevance of CYC in oncologic therapy has become stronger. Its use in this regard is discussed below. 5. CYC in Cancer Therapy Therapeutic approaches for breaking tolerance towards tumor cells have been studied extensively. The depletion of Tregs is the most investigated strategy and CYC was found to have speciﬁc eﬀects on T cells with tumor inhibiting proper- ties [45]. In the liver, CYC is converted to the active metabolic aldophosphamide and phosphamide mustards which bind to DNA, thereby inhibiting DNA replication and causing cell death. CYC has a broad antitumor spectrum, a low niche of inducing chemoresistance, and a limited hematopoietic toxicity [46]. High doses of CYC have direct tumoricidal eﬀects, cause immunosuppression by lymphoablation, and is used also in the clinic with the primary aim of damping ongoing immune responses in patients with autoimmune diseases. Administration of low-dose CYC (in the range of 20–150 mg/kg body weight) can paradoxically augment immune responses (demonstrated in many experimental animal models) leading to immunostimulation. Low-dose CYC acts on cycling Tregs and inhibits their suppressor function by enhancing apoptosis and decreasing homeostatic proliferation. The net outcome of CYC immunomodulation depends on the balance between immune suppressive Treg response and non-Treg eﬀector responses. Expression of