4 GITR and Foxp3 which are involved in the suppressive activity of Tregs is downregulated by CYC administration [47]. Studies have shown the presence of a large number of Foxp3+ Tregs in a variety of tumors and the enhancement of natural as well as vaccine-induced antitumor T cell responses by systematically or locally removing Foxp3+ Tregs [48]. A single low-dose CYC causing Treg depletion was shown to be curative by potentiating tumor-speciﬁc immunotherapy against established tumors [49]. High doses of CYC were less eﬀective in rejecting tumor growth [50]. Treatment of neu-N mice with vaccine and low-dose CYC chemotherapy rejected tumor challenge due to the recruitment of latent pools of CD8+ cells to the antitumor immune response [51]. Mice bearing poorly immunogenic B16 melanoma did not evoke immunity; however, the depletion of CD4+CD25+Tregs resulted in CD8+ T-cell mediated rejection of secondary challenged B16 tumors [52]. CYC was shown to induce IFN- α production, thereby augmenting lymphocyte proliferation including CD44high memory T-cell phenotype that might account for the increased antibody responses and the persistence of memory cells [53]. Low-dose CYC, in addition to its detrimental eﬀects on Tregs, was shown to augment the antitumor immunogenicity of DC vaccine tested in B16 melanoma or C26 colon cancer [54]. Increased Tregs in the peripheral blood and tumor microenvironment were observed in many human malig- nancies. However, the current clinical application of the latter in cancer chemoimmunotherapy is limited. Oral adminis- tration of metronomic low-dose CYC regimen (100 mg at regular intervals) in advanced cancer patients induced a profound and selective reduction of Tregs and restoration of T-cell proliferation and innate killing activity by NK eﬀector functions. An increased dose of CYC (200 mg taken daily) failed to deplete Tregs or to restore immune functions [55]. These studies validated CYC ability to induce transient Treg depletion in humans. The combination of chemotherapy and immunotherapy was tested in advanced pancreatic cancer patients. The vaccination was induced using allogeneic GM-CSF secreting cell lines alone or in sequence with CYC (250 mg/m2) before vaccination. Further, patients with metastatic pancreatic cancer treated with CYC followed by GM-CSF had CD8+ T cell responses to HLA class I-restricted mesothelium epitope, and the patients’ survival was prolonged [56]. A phase I clinical trial in patients with metastatic carci- noma and a high Treg level in their peripheral blood tested the eﬀect of a single intravenous infusion of CYC in three dif- ferent doses (e.g. 250, 500, or 700 mg/m2) when administered concomitantly with a nonspeciﬁc immunotherapy using intratumoral Bacilli Calmette-Guerin (BCG). It was found in this trial that CYC at the doses tested did not modulate signiﬁcantly Treg numbers and function, and the authors concluded that CYC may not represent an optimal therapy for eliminating Tregs [57]. Nevertheless, it has been reported (in many animal models) that only CYC at low doses (20– 150 mg/kg body weight) could enhance immunostimulation due to the selective killing of Tregs [4, 46, 47, 51]. Hence, it should not come as a surprise that high doses of 500 and 700 mg/m2 in the reported trial failed to reduce the Bone Marrow Research frequency of Tregs or to modulate their function; in fact, the best reduction of Tregs in this trial was observed in patients who were treated with the lowest dose of three tested, for instance, 250 mg/m2. In the metronomic regimen studies [55], it also was stressed that only the low daily oral dose of 100 mg (in contrast to the nonactive 200 mg dose) selectively depleted Tregs cells. Another clinical trial that followed after the frequency and function of Tregs in patients with advanced hepatocellular carcinoma who were treated with CYC at doses 150, 250, or 350 mg/m2 on day 1 and day 29 found that only the lower doses of CYC (150 and 250 mg/m2) could impair Treg number and function and unmask α-fetoprotein-speciﬁc T cell responses. In fact, a better and more prolonged suppression of Treg function was observed in patients treated with 250 mg/m2. In contrast, the reduction of Tregs in response to the high CYC dose (350 mg/m2) was not signiﬁcant [58]. There is limited information about the kinetics and function of Tregs following CYC induced transient Treg depletion (including recovery to pretreatment levels) in patients with cancer. The duration of the blocked renewal of Tregs following CYC treatment might be important when considering repeated administrations of low-dose CYC at longer intervals using the window opportunity [4, 59]. 5.1. Eﬀects of a Single Low- or High-Dose CYC on 5T2MM Progression. High doses of CYC are eﬀective in tumor therapy because of their direct cytotoxic eﬀect and inhibitory activity against cycling cells, thus killing tumor cells but along with the depletion of immune cells. In contrast, low doses of the drug have been shown to selectively reduce the number and function of Tregs and induce antitumor- mediated eﬀects. Within 24 hours after CYC administration to 5T2MM-bearing mice at the clinical phase of the disease (60–70 days after tumor cell challenge), both low- and high-dose CYC (100 and 200 mg/kg body weight, resp.) resulted in normalization of serum paraprotein level. In the BM cavity, plasma tumor cells were replaced with normal cell populations, in association with prolonged survival. However, a higher MM incidence (80–85%) was observed in those treated with the high-dose in comparison with the low-dose CYC (40–50%) [4]. Further, mice treated not with low-dose but with high-dose CYC still had residual 5T2MM cells, because adoptive transfer of BM cells from grossly appearing mice 170 days after initiation of treatment into young syngeneic recipients resulted in the development of disease in the BM of the latter 3-4 months afterwards [59]. Inhibitory eﬀects of CYC on Tregs have been shown in previous studies [46, 47, 51]. Mechanistic pathways to explain the susceptibility of Tregs to CYC includes downregulated expression of the survival molecule Bcl-xL and of costimulatory CTLA-4 in CD4+CD25high cells, and a signiﬁcant decreased production of IL-2 by CD4 eﬀector cells [4]. The inhibitory eﬀects of low-dose CYC on Tregs are essential for accomplishing antimyeloma activities, because coadministration of Tregs 24 hours following the injection of low-dose CYC (when the cytotoxic eﬀects of the drug were substantially diminished [46]), to 5T2MM-bearing mice, abrogated the beneﬁcial eﬀects of CYC on MM [4]. Selective