Abstract The lack of clinically sufficient antitumor immune responses has been attributed to soluble inhibitory factors such as TGF␤1 1,2 or PGE 2 3-5 as well as the induction and expansion of regulatory cells. 6,7 CD4 ϩ CD25 high regulatory T cells (T reg cells) were shown to be expanded in murine tumor models. 8 Moreover, their deletion reinstated an efficient antitumor immune response leading to complete tumor regression. [9] [10] [11] We and others have demonstrated that CD4 ϩ CD25 high FoxP3 ϩ T reg cells are also expanded in patients with solid tumors, [12] [13] [14] [15] [16] [17] [18] [19] Hodgkin lymphoma, 20, 21 or B-cell chronic lymphocytic leukemia (CLL). 22 Both in humans and in animal models, T reg cells have been described as anergic cells exerting strong suppression after T-cell receptor (TCR) stimulation. [23] [24] [25] As demonstrated in murine models, natural T reg cells usually originate from the thymus, 26, 27 although cells with similar characteristics can also be generated in the periphery under appropriate conditions. 28 More recently, the diversity and developmental stage of thymic emigrants with a T reg -cell phenotype as well as CD4 ϩ CD25 ϩ T reg cells within peripheral blood were examined. 29, 30 In healthy individuals, the levels of T-cell receptor excision circles (TRECs) were comparable in both conventional CD4 ϩ CD25 Ϫ and regulatory CD4 ϩ CD25 ϩ thymic populations. However, the number of TRECs was significantly higher in thymic emigrants than in peripheral blood-derived T cells, which strongly suggests thymic development of human CD4 ϩ CD25 high T reg cells. 30 Nevertheless, conventional CD4 ϩ CD25 Ϫ T cells from peripheral blood of healthy donors contained higher TREC numbers than their CD4 ϩ CD25 ϩ counterparts, which is in line with the possibility of extrathymic expansion particularly within the T reg -cell subset. 29 Until recently, CD4 ϩ CD25 high T reg cells have been described to belong to the memory T-cell compartment. 24, [31] [32] [33] Valmori et al, 34 however, identified a T reg -cell population with a naive phenotype (CCR7 ϩ CD45RA ϩ ), which they termed natural naive T reg cells (NnTregs). As expected, the frequency of these NnTregs was relatively low in healthy individuals. NnTregs were shown to vigorously proliferate in response to contact with autologous antigen-presenting cells, suggesting that particularly this subpopulation is enriched in T cells bearing self-reactive T-cell receptors. 34 Most recently, Seddiki et al 35 described the persistence of a population of naive CD45RA ϩ T reg cells in adult life. Abstract Little is known about the differentiation, origin, and mechanisms of expansion of T reg cells in cancer patients. We and others have observed that increase of T reg -cell frequency correlates with disease state, 13,22 which might be explained by an antigendependent mechanism of peripheral expansion in response to tumor progression. However, it is unknown if these T reg cells are also more differentiated toward a central or even effector memory phenotype. M.B. designed research, performed research, wrote the paper, and analyzed data; M.K. designed research and analyzed data; T.G. contributed vital analytic tools and performed research; E.E. contributed vital analytic tools and performed research; M.R.W. performed research; P.A.K. contributed vital analytic tools; S.C. performed research; and J.L.S. designed research, wrote the paper, and analyzed data. Abstract The online version of this article contains a data supplement. Abstract Reprints: Joachim L. Schultze, Molecular Tumor Biology and Tumor Immunology, Clinic I for Internal Medicine, University of Cologne, Joseph-Stelzmann Str 9/Haus 16, 50931 Cologne, Germany; e-mail: mooresusan@example.com. Abstract The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. (MGUS). Whether these cells were coexpressing CD25 was not determined in this study. This is of particular interest since MM has been associated with immune dysfunctions, 37-40 which alternatively might be explained by an increase of regulatory or immune inhibitory mechanisms. 41 MM has been associated with decreased T-cell responses to mitogenic and TCR-mediated stimulations 37-40 that might be-at least in part-due to induction of regulatory T cells. 41, 42 Based on these recent findings, we were interested to answer 2 main questions. First, we wanted to assess frequency and function of CD4 ϩ CD25 high FoxP3 ϩ T reg cells in MM patients using an experimental approach previously applied by us and others in patients with other types of cancer. 12-22 Second, we were interested if naive T reg cells exist in patients with MGUS and MM. Therefore, we have assessed frequency, function, and phenotype of T reg cells in 9 MGUS and 67 MM patients. We demonstrate a significantly increased frequency of CD4 ϩ CD25 high FoxP3 ϩ T reg cells with strong inhibitory function in MGUS as well as untreated or treated MM patients. Unexpectedly, not only T reg cells of central or effector memory phenotype were expanded, but also naive CCR7 ϩ CD45RA ϩ T reg cells. We further demonstrate TREC expression in both MM patients and healthy individuals to be restricted to T cells of a naive phenotype, with the largest TREC numbers expressed by conventional CD4 ϩ CD25 Ϫ T cells, followed by CD4 ϩ CD25 low T cells, and the lowest numbers in CD4 ϩ CD25 high T reg cells. These data strongly suggest that increased frequencies observed in patients with malignant disease are due to peripheral expansion of not only T reg cells of central and effector memory but also of T reg cells of a naive phenotype. Abstract Patients, materials, and methods Abstract Following approval by our institutional review board (University Ethics Committee, Cologne, Germany), peripheral blood (PB) from 42 healthy individuals, 9 MGUS patients, and 67 MM patients as well as bone marrow (BM) from 6 MM patients was taken after informed consent was obtained in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll/Hypaque (Amersham, Uppsala, Sweden) density centrifugation and used immediately or stored in liquid nitrogen until further use, leading to similar phenotypic and functional results. Patients included for phenotypical or functional analysis were either untreated or had not received cytoreductive treatment for a period of at least 1 month prior to investigation. Staging was performed according to the Durie and Salmon classification for MM. Characteristics of the patients studied are summarized in Table S1 , available on the Blood website; see the Supplemental Materials link at the top of the online article. Abstract Phenotype of T cells was defined by flow cytometry using the following antibodies: CD4-FITC, OX40-FITC, cytotoxic T lymphocyte-associated protein 4 (CTLA4)-PE, CD45RA-PE-Cy-5, CD4-APC, CD4-APC-Cy-7 (all from Becton Dickinson PharMingen, Heidelberg, Germany), CD25-PE, CD62L-PE, CD25-PE-Cy7 (all from Becton Dickinson Biosciences, Heidelberg, Germany), CCR7-FITC (R&D Systems, Wiesbaden, Germany), as well as the corresponding isotype control antibodies (BD PharMingen). Intracellular staining was performed with the following antibodies: forkhead box P3 (FoxP3)-PE (eBioscience, San Diego, CA), glucocorticoidinduced TNFR-related protein (GITR)-FITC, IL-10-FITC (both from R&D Systems), TGF␤1-PE (IQ-Products, Groningen, the Netherlands), CTLA4-PE, or with the appropriate isotype controls (all from BD PharMingen). 22 Cells were stained according to the manufacturer's recommendations. Abstract Samples were acquired on a FACSCalibur or FACSCanto and analyzed with FACSDiva software (BD Biosciences) or WinMDI 2.8 (http:// facs.scripps.edu/software.html). CD25 high T cells were gated as previously described. 22 Frequencies of CD4 ϩ CD25 high T cells in PB are shown as percent values of CD4 ϩ T cells. To determine cells positive for T reg -cell markers, we used stringent gating criteria, setting gates at the 1% level of the respective isotype control. Abstract CD4 ϩ T cells were purified from PBMCs using CD4 MACS Beads (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously. 22 To assess polyclonal CD4 ϩ T-cell activation, 1 ϫ 10 5 CD4 ϩ T cells/well were activated in AIM-V (Gibco Invitrogen, Karlsruhe, Germany)/EX-Cell 610 (JRH Biosciences, Lenexa, KS) with pokeweed mitogen (PWM, 10 g/mL), phytohemagglutinin (PHA, 10 g/mL; both from Sigma-Aldrich, Taufkirchen, Germany), or anti-CD3 (0.2 g/mL, OKT-3) and anti-CD28 mAb (0.2 g/mL; kind gift of Dr L. M. Nadler) in 96-well plates. Proliferation of T cells was monitored by measuring BrdU (5-bromo-2Ј-deoxyuridine) incorporation (Roche Diagnostics, Mannheim, Germany). Abstract For functional analysis, CD4 ϩ CD25 high T cells were purified from PBMCs. Briefly, CD4 MACS Multisort Beads (Miltenyi Biotec) were used for isolation of CD4 ϩ T cells. 22 After detaching, cells were washed and CD4 ϩ CD25 high T cells were positively selected using CD25 microbeads (2 L beads/10 7 CD4 ϩ cells). The described technique is optimized for the isolation of human CD4 ϩ CD25 high T cells with high purity. 43 Use of higher concentrations of microbeads for isolation results in better recovery but decreases the purity of the T reg -cell population. The negative fraction of CD4 ϩ CD25 Ϫ T cells from healthy controls was used as effectors to assess T reg -cell function independently of potential defects of conventional CD4 ϩ T cells. Cells were reanalyzed after sorting and routinely showed more than 95% purity. Abstract Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the T reg -cell-associated transcription factor FOXP3. The same primers and conditions were used as previously described. 22 Relative FOXP3 expression in CD4 ϩ CD25 Ϫ and CD4 ϩ CD25 high T cells was normalized to GAPDH expression. Abstract To assess the suppressive activity of CD4 ϩ CD25 high T cells, a modified allogeneic mixed lymphocyte reaction (MLR) was performed as previously described. 22 Briefly, after magnetic separation both highly purified CD4 ϩ CD25 Ϫ and CD4 ϩ CD25 high T cells were incubated for 20 hours with 10 U/mL IL-2 (Proleukin; Chiron, Munich, Germany) and 0.5 g/mL anti-CD3 mAb in X-VIVO 15 (BioWhittakker, Verviers, Belgium). 22, [44] [45] [46] Subsequently, CD4 ϩ CD25 Ϫ T cells (5 ϫ 10 4 /well) were cocultured with irradiated allogeneic PBMCs (2 ϫ 10 5 /well) in X-VIVO 15 supplemented with 10% fetal calf serum, 100 U/mL penicillin/streptomycin, and 2 mM glutamine (all from Gibco Invitrogen). Purified allogeneic CD4 ϩ CD25 high T cells were added at different concentrations as indicated. 34, 47, 48 On day 4, the cells were pulsed with BrdU, and BrdU incorporation was analyzed 20 hours later. There was no influence on the inhibitory effect of CD4 ϩ CD25 high T cells when CD4 ϩ CD25 Ϫ T cells were not preactivated prior to the inhibition assay. Alternatively, 5,6-carboxyfluorescein-diacetatesuccinimidyl-ester (CFSE; Sigma-Aldrich)-stained CD4 ϩ CD25 Ϫ T cells (5 ϫ 10 4 /well) were stimulated with magnetic beads (Dynal Biotech, Oslo, Norway) coated with 5% anti-CD3 (OKT3), 14% anti-CD28 (9.3, kind gift Abstract For personal use only. on July 21, 2017. by guest www.bloodjournal.org From of Drs C. June and J. Riley), and 81% anti-MHC class I (W6/32) at a ratio of 3:1 (cells-beads). PKH-26 (Sigma-Aldrich)-labeled allogeneic CD4 ϩ CD25 high T cells were added at a 1:1 ratio to the culture, and proliferation of CD4 ϩ CD25 Ϫ T cells was determined by assessing CFSE dilution after 4 days of culture. Use of autologous CD4 ϩ CD25 Ϫ T cells yielded comparable results; however, for better comparability of T reg cells from healthy controls, MGUS patients, and MM patients, allogeneic CD4 ϩ CD25 Ϫ T cells were used for most experiments. Abstract The concentration of IFN-␥ in cell culture supernatants was measured using the human Th1/Th2 cytokine kit II (BD PharMingen). In brief, capture beads were mixed with culture supernatants and PE detection reagent and incubated for 3 hours at room temperature. The beads were then washed with wash buffer and analyzed. Abstract Isolation of CD4 ؉ CD25 ؊ , CD4 ؉ CD25 low , and CD4 ؉ CD25 high T cells for DNA isolation and assessment of T-cell receptor excision circles Briefly, CD4 MACS Beads (Miltenyi Biotec) were used for isolation of CD4 ϩ T cells. 22 After staining with CCR7-FITC (R&D Systems), CD25-PE, CD45RA-PE-Cy-5, and CD4-APC (all from BD PharMingen) according to the manufacturer's recommendations, CD4 ϩ C25 Ϫ , CD4 ϩ CD25 low , and CD4 ϩ CD25 high T cells and the respective T-cell subsets, CCR7 ϩ CD45RA ϩ T naive , CCR7 ϩ CD45RA Ϫ T CM , and CCR7 Ϫ CD45RA Ϫ T EM cells, were purified using a FACSDiVa Cell Sorter (BD Biosciences). Abstract DNA was isolated from purified T naive , T CM , and T EM cell subsets of the CD4 ϩ CD25 Ϫ , CD4 ϩ CD25 low , and CD4 ϩ CD25 high T-cell subpopulations using a DNA Isolation Kit (Roche Diagnostics) following the manufacturer's instructions. Abstract Relative TREC levels were determined using real-time PCR with a LightCycler (Roche Diagnostics) based on specific primers and general fluorescence detection with SYBR Green. All PCRs were performed using LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics). All samples were studied in duplicate reactions using the human TREC primer kit (Search-LC, Heidelberg, Germany). The number of TREC molecules in the sample was calculated as number of copies per 10 4 cells (detection limit Ն 10 molecules). Abstract Comparison between paired or unpaired groups was performed using the appropriate Student t test. A P value less than .05 was defined as statistically significant. Due to the explorative nature of this study, no multiplicity adjustment procedures were performed. All statistical analyses were performed using the SPSS statistical software package (SPSS 12.0 for Windows; SPSS, Chicago, IL). Abstract Increased frequencies of CD4 ؉ CD25 high FoxP3 ؉ T reg cells in peripheral blood and bone marrow of patients with MGUS and MM Flow cytometry was used to assess frequencies of CD4 ϩ CD25 high T reg cells in PB of MGUS patients (n ϭ 9), MM patients (n ϭ 67), and healthy controls (n ϭ 26, Figure 1A ). The frequency of T reg cells in controls (4.5% Ϯ 1.1%) was similar to previously published results ( Figure 1B) . 22, 47 In contrast, individuals with MGUS (n ϭ 9, 6.4% Ϯ 2.2%, P Ͻ .05) as well as untreated stage I MM patients (n ϭ 8, 8.0% Ϯ 1.7%, P Ͻ .001), treated stage II MM patients (n ϭ 11, 9.6% Ϯ 3.8%, P Ͻ .005), and treated stage III MM patients (n ϭ 43, 10.6% Ϯ 3.9%, P Ͻ .001) showed significantly increased frequencies of T reg cells compared with healthy individuals ( Figure 1B ). There was a trend toward higher frequencies of T reg cells in untreated MM patients compared with MGUS patients; however, this did not reach statistical significance. In contrast, there was no statistically significant difference of T reg -cell frequencies between treated and untreated MM patients, suggesting that the treatment of MM patients had no selective effect on T-cell subpopulations. However, the absolute number of T cells was generally decreased after therapy of MM patients (data not shown). For 6 patients, we obtained PB and BM at the same time point. Frequencies of T reg cells in BM were comparable with PB (8.8% Ϯ 1.9% PB vs 9.4% Ϯ 2.7% BM, Figure 1C ), indicating that no preferential accumulation in either compartment occurs. Abstract Next, we investigated changes in frequency of T reg cells over time ( Figure 1D ). In 13 patients, we obtained blood samples at 2 or more different time points more than 6 months apart (range, 9 to 30 months). In 3 MM patients treated with conventional chemotherapy (melphalan or bendamustine) no clear pattern emerged ( Figure 1D , Tx), with 1 patient showing similar frequencies at both time points, and 1 patient with lower and 1 patient with higher frequencies after therapy. In 5 MM patients, we obtained blood prior to and after autologous stem cell transplantation ( Figure 1D , SCT). Overall, the frequencies of T reg cells at these 2 time points did not differ significantly. For 5 MM patients who had undergone autologous SCT prior to first T reg -cell analysis, lower frequencies were observed at the second time point of analysis ( Figure 1D , after SCT). Of interest, these patients were at least in partial remission for the observed time period (range, 9 to 19 months). Abstract Among the 67 MM patients, 34 patients had progressive disease at time of analysis, while 33 had stable disease or were in partial or complete remission. When comparing these 2 patient cohorts irrespective of prior treatment, no significant difference in the proportion of T reg cells was observed, while both patient cohorts showed significantly higher frequencies of T reg cells compared with healthy controls (data not shown). Abstract Next, we assessed FOXP3 expression on both mRNA and protein level. For the assessment of FOXP3 mRNA, we purified CD4 ϩ CD25 Ϫ as well as CD4 ϩ CD25 high T cells prior to real-time RT-PCR to avoid potential contamination of other cells expressing FOXP3 and to compare FOXP3 mRNA on a cell-to-cell basis. Increased expression of FOXP3 mRNA was observed in CD4 ϩ CD25 high T cells from MM patients (n ϭ 6) and healthy individuals (n ϭ 7) compared with CD4 ϩ CD25 Ϫ T cells ( Figure 1E ). Although FOXP3 mRNA concentrations in CD4 ϩ CD25 high T cells from MM patients were higher than in healthy individuals, this did not reach statistical significance, suggesting that the amount of FOXP3 mRNA per cell is not increased. To assess the frequency of FoxP3 ϩ CD4 ϩ CD25 high T reg cells in MGUS and MM patients, we assessed FoxP3 protein expression on single cells by intracellular flow cytometry ( Figure 1F ). In MGUS patients (MGUS, n ϭ 8, 3.3% Ϯ 1.4%, P Ͻ .01), untreated MM patients (MM no Tx, n ϭ 7, 3.6% Ϯ 2.1%, P Ͻ .005), as well as treated MM patients (MM Tx, n ϭ 13, 4.0% Ϯ 2.9%, P Ͻ .05), frequencies of FoxP3 ϩ CD4 ϩ CD25 high T reg cells were statistically significantly augmented compared with healthy individuals (control, n ϭ 14, 1.5% Ϯ 0.8%). Abstract Our data indicate that the expansion of CD4 ϩ CD25 high FoxP3 ϩ T reg cells previously described for patients with other types of cancer 13, 14, 18, 22, 49 is also apparent in MM as well as the premalignant precursor MGUS. Increased frequencies of T reg cells might therefore be a general phenomenon in cancer patients. We also assessed previously described surface receptors associated with T reg cells, 25,50-57 including CTLA4, GITR, CD62L, and OX40, on CD4 ϩ CD25 high T cells in MGUS and MM patients as well as healthy individuals. As summarized in Table 1 , the frequencies of T reg cells from PB expressing these surface molecules were always significantly increased in MM patients. This was also true for T reg cells derived from PB of MGUS patients or BM of MM patients, except for OX40. Abstract Intracellular expression of TGF␤1 and IL-10 in PB was assessed since these 2 cytokines have been associated with T reg -cell function. 58, 59 Similar to the T reg -cell-associated receptors, TGF␤1 and IL-10 were significantly increased in CD4 ϩ CD25 high T cells from MM patients (Table 1) . Overall, CD4 ϩ T cells expressing high levels of CD25, FoxP3, CTLA4, CD62L, GITR, OX40, TGF␤1, and IL-10 are significantly increased in MM patients. Abstract In 7 treated MM patients, sufficient numbers of highly purified CD4 ϩ CD25 high T cells were isolated to analyze their inhibitory function in comparison with T reg cells from healthy controls (n ϭ 22). Regulatory function of CD4 ϩ CD25 high T cells was assessed using an allogeneic MLR of CD4 ϩ CD25 Ϫ T cells and allogeneic irradiated PBMCs as stimulators. 22, 25, 34, [46] [47] [48] As we and others have previously shown, the CD4 ϩ CD25 high T-cell population in healthy controls exhibits strong inhibitory function on allogeneic T-cell proliferation. 22, 25, 46 As exemplified in Figure 2A , proliferation of allogeneic conventional CD4 ϩ CD25 Ϫ T cells was significantly inhibited when highly purified CD4 ϩ CD25 high T cells from a healthy donor were added at a 1:1 ratio (P Ͻ .001). This was corroborated by a complete loss of IFN-␥ production by allogeneic conventional T cells in the presence of T reg cells ( Figure 2B ). We next assessed highly purified CD4 ϩ CD25 high T reg cells from treated CD4 ϩ CD25 high T cells (dark gray bars) and CD4 ϩ CD25 Ϫ T cells (light gray bars) were sorted by magnetic activated cell sorting (MACS) from peripheral blood in healthy controls (n ϭ 7) and MM patients (n ϭ 6). Real-time RT-PCR for FOXP3 was performed, and relative expression of FOXP3 in CD4 ϩ CD25 high T cells as well as CD4 ϩ CD25 Ϫ T cells was normalized to GAPDH as described. 22 Error bars represent standard deviation (SD). (F) Flow cytometric analysis of FoxP3 in CD4 ϩ CD25 high Treg cells in MGUS (n ϭ 8) as well as untreated (n ϭ 7) and treated (n ϭ 13) MM patients compared with healthy donors (n ϭ 14). Shown are the median, 75th percentile (box), standard deviation (whiskers), and outliers (dots). *P Ͻ .05, Student t test. Abstract For personal use only. on July 21, 2017. by guest www.bloodjournal.org From MM patients at the same ratios for their inhibitory function on allogeneic T-cell proliferation and cytokine production ( Figure 2A-B) . On a cell-to-cell basis, these T reg cells showed an equally strong inhibitory function on conventional CD4 ϩ CD25 Ϫ T-cell proliferation ( Figure 2A ) and IFN-␥ release ( Figure 2B ). Titration experiments as exemplified in Figure 2C demonstrated that the inhibitory function of T reg cells from healthy individuals and MM patients was comparable also at lower T reg /T conv ratios. When comparing all 7 treated MM patients with the healthy controls, no statistically significant difference of inhibitory capacity of T reg cells was observed ( Figure 2D ). Proliferation of allogeneic or autologous conventional CD4 ϩ CD25 Ϫ T cells stimulated by beads coated with anti-CD3 and anti-CD28 mAbs was used as the readout to assess inhibitory function of CD4 ϩ CD25 high T cells isolated from MGUS patients (n ϭ 3) or untreated MM patients (n ϭ 3) in comparison with T reg cells isolated from healthy individuals (n ϭ 8, Figure 2E ). For better comparison, Figure 2E shows a representative experiment of T reg cells from a healthy individual, an MGUS patient, and an MM patient using the same allogeneic conventional CD4 ϩ CD25 Ϫ T cells as the readout for T-cell It needs to be emphasized that these assays are normalized for the same numbers of T reg cells from healthy controls and MGUS as well as MM patients. Therefore, since MGUS and MM patients have an increased frequency of these cells, the overall inhibitory effect of the whole increased T reg -cell population in MGUS and MM patients will be even more pronounced in vivo. This might also be reflected by an overall reduction of proliferation of CD4 ϩ T cells in response to mitogenic (PWM, PHA) or even supraphysiologic stimulation with anti-CD3 and anti-CD28 mAbs in MM patients compared with healthy controls ( Figure S1 ). Abstract An important question concerning the increase of CD4 ϩ CD25 high FoxP3 ϩ T reg cells in cancer patients including MGUS and MM is the contribution of different T-cell subsets such as naive, central, and effector memory cells. These cells are defined by differential expression of the cell surface receptors CD45RA and CCR7. 60, 61 By multicolor flow cytometry, we determined the frequency of naive, central, and effector memory cells within the CD4 ϩ CD25 high T reg -cell compartment from healthy individuals as well as MGUS and MM patients as previously described by Valmori et al 34 and compared those with the distribution within conventional CD4 ϩ CD25 Ϫ T cells and CD4 ϩ CD25 low T cells (Figure 3) . In healthy individuals (white bars), naive CCR7 ϩ CD45RA ϩ CD4 ϩ CD25 high T reg cells were hardly detectable ( Figure 3A) . T reg cells were almost exclusively of memory phenotype ( Figure 3B-C) with a slightly higher frequency of CD4 ϩ CD25 high T EM cells ( Figure 3C ). In contrast, in PB of MGUS (light gray bars) and MM patients (dark gray bars) a significant expansion of CD4 ϩ CD25 high T reg cells with a CCR7 ϩ CD45RA ϩ naive phenotype was observed ( Figure 3A) . This was further accompanied by an increase of T reg cells with a central memory phenotype in MGUS as well as MM patients ( Figure 3B ), while effector memory T reg cells were augmented in PB of MM patients ( Figure 3C ). CD4 ϩ CD25 low T cells, assumed to be mostly activated T cells, showed an increase of T naive cells ( Figure 3D ) with a concomitant decrease of T EM cells ( Figure 3F ) in PB of MGUS and MM patients, while frequencies of T CM cells remained unchanged ( Figure 3E ). Conversely, among conventional CD4 ϩ CD25 Ϫ T cells, T naive cells ( Figure 3G ) were significantly lower in MM patients than in healthy controls, whereas no statistically significant differences were evident for the T CM ( Figure 3H ) and T EM ( Figure 3J ) subset. In MGUS patients, frequencies of T CM cells were augmented, while T naive and T EM cells were unaffected. Abstract For BM, a preferential T CM phenotype has been described, 62 which we could also observe in BM of patients with MM irrespective of CD25 expression ( Figure 3B ,E,H black bars) with a concomitant decrease of T EM cells ( Figure 3C,F,J) . However, (T CM) , and CCR7 Ϫ CD45RA Ϫ effector memory T cells (TEM) were assessed in peripheral blood (PB, n ϭ 18) and bone marrow (BM, n ϭ 6) of MM patients, MGUS patients (MGUS, n ϭ 8), and healthy individuals (control, n ϭ 30). CD4 ϩ T cells were further subdivided into conventional CD4 ϩ CD25 Ϫ , CD4 ϩ CD25 low , and regulatory CD4 ϩ CD25 high T cells according to their CD25 expression. Significant differences (P Ͻ .05, Student t test) between healthy donors, MGUS patients, and MM patients are marked by an asterisk. For personal use only. on July 21, 2017. by guest www.bloodjournal.org From comparable with PB of MGUS and MM patients, we observed a significant increase of naive CD4 ϩ CD25 high T reg cells in BM of MM patients ( Figure 3A ). This increase of T naive cells, similar to PB, could also be observed in the CD4 ϩ CD25 low T cells ( Figure 3D ), while the decrease of naive conventional CD4 ϩ CD25 Ϫ T cells was also apparent in BM of patients with MM ( Figure 3G ). Abstract To further characterize the increased subset of naive CD4 ϩ CD25 high T reg cells, we analyzed the expression of molecules previously associated with T reg cells, particularly FoxP3. In addition to the expression of CD45RA and CCR7, we found significantly increased levels of FoxP3 on naive CD4 ϩ CD25 high T reg cells in PB of MGUS and MM patients ( Figure 4A ). CD62L was expressed in comparable amounts on naive CD4 ϩ CD25 high T reg cells ( Figure 4B ). Similar to previously published data, expression of intracellular CTLA4 was detectable in naive T reg cells from healthy individuals. 34 Analysis of the increased subset of naive T reg cells in PB and BM from MM patients as well as MGUS patients revealed a significantly higher percentage of CTLA4 ϩ naive T reg cells ( Figure 4C ). Equivalent expression of GITR was observed in naive T reg cells of healthy individuals and in BM of patients with MM, whereas naive T reg cells from PB of MM and MGUS patients showed significantly augmented GITR expression ( Figure 4D ). Abstract As T reg cells with a naive phenotype are increased in patients with MM, we were interested if the increase of T reg cells results from peripheral expansion or possibly thymic generation of CD4 ϩ CD25 high T cells. The level of TRECs has been used as a marker to estimate the developmental vicinity of T cells to the thymus and their division history. 30 We therefore applied TREC analysis to further characterize T reg cells with a naive phenotype in MM patients. PB-derived CD4 ϩ T cells from 2 MM patients and 2 age-matched healthy individuals were first sorted into conventional CD4 ϩ CD25 Ϫ , CD4 ϩ CD25 low , and regulatory CD4 ϩ CD25 high T cells according to their CD25 expression. Subsequently, the cells were separated into the appropriate T-cell subsets, namely T naive , T CM , and T EM , defined by their expression of CCR7 and CD45RA. Abstract TREC values for all 9 highly purified T-cell subsets were assayed by real-time PCR. As expected, the TREC content of conventional CD4 ϩ CD25 Ϫ T naive cells from healthy individuals was significantly higher than that of CD4 ϩ CD25 Ϫ T CM or T EM cells ( Figure 5A ). CD4 ϩ CD25 low T cells ( Figure 5B ), primarily consisting of recently activated T cells, showed lower TREC levels in T naive cells than in conventional CD4 ϩ CD25 Ϫ T cells, and TREC levels were below the detection limit in T CM and T EM cells in CD25 Ϫ T cells ( Figure 5A ) and CD25 low T cells ( Figure 5B ) from healthy individuals. These data are in accordance with the concept of T-cell activation and memory T-cell generation, as CD4 ϩ CD25 low T cells should consist predominantly of activated antigenexperienced memory T cells. CD4 ϩ CD25 high T reg cells showed even lower TREC contents in the naive T-cell population, and TREC levels were below the detection threshold in the T CM and T EM subsets ( Figure 5C ). These observations are in line with the concept of antigen-driven peripheral expansion of T reg cells in healthy individuals rather than recent thymic emigration. Despite significant differences in the frequencies of the distinct T-cell subsets in patients with MM, the TREC content was comparable between MM patients and healthy controls on the single-cell level ( Figure 5D-F) , suggesting that the underlying mechanism of T reg -cell generation and differentiation is the same in healthy individuals and MM patients. This implies that the increased frequency of naive as well as central and effector memory T reg cells in MM patients is due to peripheral expansion of T reg cells rather than newly generated T reg cells within the thymus. For personal use only. on July 21, 2017. by guest www.bloodjournal.org From peripheral expansion of these cells. The assessment of FoxP3 protein expression by CD4 ϩ CD25 high T cells has clearly revealed that there is an increase in frequency of these cells rather than an increase of expression within individual cells. Costaining experiments of intracellular FoxP3 with other previously suggested T reg -cell markers once adequate antibody combinations are available will further elucidate the functional relationship of these proteins on T reg cells, not only in MM patients, but also healthy individuals. Abstract Most recently, Prabhala et al 36 reported lower frequencies of CD4 ϩ FoxP3 ϩ T cells in MM patients. So far, only increased frequencies of CD4 ϩ CD25 ϩ T reg cells with strong inhibitory function were observed in cancer patients, [12] [13] [14] [15] [16] [17] [18] [19] Hodgkin lymphoma, 20,21 and CLL. 22 When assessing FoxP3 expression in the context of CD25 expression, we found increased frequencies of CD4 ϩ CD25 high FoxP3 ϩ T reg cells with normal inhibitory function in MGUS and MM patients. The different results obtained by the previous study are most likely explained by the different experimental approaches chosen. For example, T reg -cell function in the prior study was assessed using bulk PBMCs depleted of CD25 ϩ cells, while we and others used purified cells excluding effects that might be due to other cells within PBMCs. In fact, the significantly lower proliferation of CD25 Ϫ PBMCs in MGUS and MM patients reflects differences that already exist between healthy controls and patients that cannot be contributed to T reg -cell function. It has been previously stated by Baecher-Allan et al 63 that the assessment of human T reg cells is still difficult and the use of different assays sometimes makes it difficult to compare different studies. We and others have previously observed that the analysis of T reg cells in cancer patients can be hampered by concomitant defects in the conventional T-cell compartment. 19, 22 This is particularly true in situations where tumor patients already show a reduced proliferative response of total CD4 ϩ T cells (Figure S1 ), where further inhibitory effects induced by T reg cells are not easily assessed. To circumvent such limitations, we have deployed 2 allogeneic systems where conventional T cells from a healthy donor are used as a readout system to ensure comparability of T reg -cell function of healthy donors and MGUS or MM patients, thereby reducing variables that might have an impact on the outcome of the assessment of T reg -cell function. Abstract An important issue of T reg -cell biology in cancer is to better understand why there is an expansion and how the expansion might be explained. Recent studies have clearly indicated that antigenspecific expansion of T reg cells must occur in cancer, since T reg -cell clones were established and shown to recognize tumor antigen in an MHC class II-restricted fashion. 42 Furthermore, in healthy individuals it was demonstrated that T reg cells exist at all differentiation states, namely naive, central, and effector memory. 30, 34, 35 We therefore addressed whether the increase of T reg cells would be solely driven by an expansion of memory T reg cells. Surprisingly, we did find a significant expansion not only of T reg cells with a central or effector memory phenotype, but also of naive CCR7 ϩ CD45RA ϩ T reg cells. This was not restricted to MM patients but was also observed in MGUS patients, suggesting that the pool of naive T reg cells is also expanded at an early disease state and during tumor progression. Similar to observations in healthy individuals, 34, 35 this opened the question whether naive T reg cells might be recent thymic emigrants or whether peripheral expansion might account for the increased number of these cells. At least for conventional naive T cells, peripheral expansion is well established. 64 The analysis of TREC is prone to answer this critical question. By isolation of 9 T-cell subsets dependent on expression of CD25 and differentiation state, we clearly demonstrate that only T cells of a naive phenotype expressing CCR7 and CD45RA-irrespective of CD25 expression-contain detectable amounts of TRECs. In contrast, TRECs were not detected in central and For personal use only. on July 21, 2017. by guest www.bloodjournal.org From memory effector T cells of healthy individuals and MM patients. Of interest, the number of TRECs differed between CD25 Ϫ , CD25 low , and CD25 high naive T-cell subpopulations. In both MM patients and healthy individuals, we found the highest numbers of TRECs in conventional CD25 Ϫ T cells followed by the CD25 low population and lowest numbers in the CD25 high T reg cells. We conclude from these findings that the increased frequency of naive T reg cells is most likely due to peripheral expansion. Differences in frequencies of T reg cells in different compartments as observed in bone marrow or peripheral blood from MM patients most likely reflect different migration patterns of T reg cells with a naive, central memory, or effector memory phenotype. In murine model systems, it has recently been demonstrated that appropriate localization is crucial for in vivo activity of T reg cells. 65 Our findings open new aspects of T reg -cell biology in cancer patients. It will be an important issue to address whether antigenspecific T reg -cell clones can be established only from the central or effector memory phenotype subpopulations, or whether the T regcell compartment with a naive phenotype already contains such cells. 42 Furthermore, it will also be interesting to see whether T reg -cell expansion in solid tumors is also accompanied by the expansion of naive T reg cells and whether there are differences in different compartments (eg, blood, secondary lymphoid organs, and bone marrow) or at the tumor site. Abstract Taken together, CD4 ϩ CD25 high FoxP3 ϩ T reg cells are increased and fully functional in MGUS as well as early untreated or late-stage-treated MM patients, which might play a crucial role for some of the immune dysfunctions observed in MM patients. By analysis of CCR7 and CD45RA, we demonstrate the expansion of naive T reg cells. According to our TREC analysis, this increase is due to peripheral expansion of naive T reg cells. These findings open new avenues to study T reg -cell biology in the context of tumor development and progression. Introduction The lack of clinically sufficient antitumor immune responses has been attributed to soluble inhibitory factors such as TGF␤1 1,2 or PGE 2 3-5 as well as the induction and expansion of regulatory cells. 6, 7 CD4 ϩ CD25 high regulatory T cells (T reg cells) were shown to be expanded in murine tumor models. 8 Moreover, their deletion reinstated an efficient antitumor immune response leading to complete tumor regression. [9] [10] [11] We and others have demonstrated that CD4 ϩ CD25 high FoxP3 ϩ T reg cells are also expanded in patients with solid tumors, [12] [13] [14] [15] [16] [17] [18] [19] Hodgkin lymphoma, 20, 21 or B-cell chronic lymphocytic leukemia (CLL). 22 Both in humans and in animal models, T reg cells have been described as anergic cells exerting strong suppression after T-cell receptor (TCR) stimulation. [23] [24] [25] As demonstrated in murine models, natural T reg cells usually originate from the thymus, 26, 27 although cells with similar characteristics can also be generated in the periphery under appropriate conditions. 28 More recently, the diversity and developmental stage of thymic emigrants with a T reg -cell phenotype as well as CD4 ϩ CD25 ϩ T reg cells within peripheral blood were examined. 29, 30 In healthy individuals, the levels of T-cell receptor excision circles (TRECs) were comparable in both conventional CD4 ϩ CD25 Ϫ and regulatory CD4 ϩ CD25 ϩ thymic populations. However, the number of TRECs was significantly higher in thymic emigrants than in peripheral blood-derived T cells, which strongly suggests thymic development of human CD4 ϩ CD25 high T reg cells. 30 Nevertheless, conventional CD4 ϩ CD25 Ϫ T cells from peripheral blood of healthy donors contained higher TREC numbers than their CD4 ϩ CD25 ϩ counterparts, which is in line with the possibility of extrathymic expansion particularly within the T reg -cell subset. 29 Until recently, CD4 ϩ CD25 high T reg cells have been described to belong to the memory T-cell compartment. 24, [31] [32] [33] Valmori et al, 34 however, identified a T reg -cell population with a naive phenotype (CCR7 ϩ CD45RA ϩ ), which they termed natural naive T reg cells (NnTregs). As expected, the frequency of these NnTregs was relatively low in healthy individuals. NnTregs were shown to vigorously proliferate in response to contact with autologous antigen-presenting cells, suggesting that particularly this subpopulation is enriched in T cells bearing self-reactive T-cell receptors. 34 Most recently, Seddiki et al 35 described the persistence of a population of naive CD45RA ϩ T reg cells in adult life. Introduction Little is known about the differentiation, origin, and mechanisms of expansion of T reg cells in cancer patients. We and others have observed that increase of T reg -cell frequency correlates with disease state, 13, 22 which might be explained by an antigendependent mechanism of peripheral expansion in response to tumor progression. However, it is unknown if these T reg cells are also more differentiated toward a central or even effector memory phenotype. Introduction Most recently, Prabhala et al 36 published surprising results concerning reduced numbers of CD4 ϩ FoxP3 ϩ T cells in patients with multiple myeloma (MM) as well as in premalignant monoclonal gammopathy of undetermined significance (MGUS). Whether these cells were coexpressing CD25 was not determined in this study. This is of particular interest since MM has been associated with immune dysfunctions, [37] [38] [39] [40] which alternatively might be explained by an increase of regulatory or immune inhibitory mechanisms. 41 MM has been associated with decreased T-cell responses to mitogenic and TCR-mediated stimulations [37] [38] [39] [40] that might be-at least in part-due to induction of regulatory T cells. 41, 42 Based on these recent findings, we were interested to answer 2 main questions. First, we wanted to assess frequency and function of CD4 ϩ CD25 high FoxP3 ϩ T reg cells in MM patients using an experimental approach previously applied by us and others in patients with other types of cancer. [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] Second, we were interested if naive T reg cells exist in patients with MGUS and MM. Therefore, we have assessed frequency, function, and phenotype of T reg cells in 9 MGUS and 67 MM patients. We demonstrate a significantly increased frequency of CD4 ϩ CD25 high FoxP3 ϩ T reg cells with strong inhibitory function in MGUS as well as untreated or treated MM patients. Unexpectedly, not only T reg cells of central or effector memory phenotype were expanded, but also naive CCR7 ϩ CD45RA ϩ T reg cells. We further demonstrate TREC expression in both MM patients and healthy individuals to be restricted to T cells of a naive phenotype, with the largest TREC numbers expressed by conventional CD4 ϩ CD25 Ϫ T cells, followed by CD4 ϩ CD25 low T cells, and the lowest numbers in CD4 ϩ CD25 high T reg cells. These data strongly suggest that increased frequencies observed in patients with malignant disease are due to peripheral expansion of not only T reg cells of central and effector memory but also of T reg cells of a naive phenotype. Patients, materials, and methods Patients and clinical parameters Following approval by our institutional review board (University Ethics Committee, Cologne, Germany), peripheral blood (PB) from 42 healthy individuals, 9 MGUS patients, and 67 MM patients as well as bone marrow (BM) from 6 MM patients was taken after informed consent was obtained in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll/Hypaque (Amersham, Uppsala, Sweden) density centrifugation and used immediately or stored in liquid nitrogen until further use, leading to similar phenotypic and functional results. Patients included for phenotypical or functional analysis were either untreated or had not received cytoreductive treatment for a period of at least 1 month prior to investigation. Staging was performed according to the Durie and Salmon classification for MM. Characteristics of the patients studied are summarized in Table S1 , available on the Blood website; see the Supplemental Materials link at the top of the online article. Antibodies and fluorescence-activated cell sorter (FACS) analysis Phenotype of T cells was defined by flow cytometry using the following antibodies: CD4-FITC, OX40-FITC, cytotoxic T lymphocyte-associated protein 4 (CTLA4)-PE, CD45RA-PE-Cy-5, CD4-APC, CD4-APC-Cy-7 (all from Becton Dickinson PharMingen, Heidelberg, Germany), CD25-PE, CD62L-PE, CD25-PE-Cy7 (all from Becton Dickinson Biosciences, Heidelberg, Germany), CCR7-FITC (R&D Systems, Wiesbaden, Germany), as well as the corresponding isotype control antibodies (BD PharMingen). Intracellular staining was performed with the following antibodies: forkhead box P3 (FoxP3)-PE (eBioscience, San Diego, CA), glucocorticoidinduced TNFR-related protein (GITR)-FITC, IL-10-FITC (both from R&D Systems), TGF␤1-PE (IQ-Products, Groningen, the Netherlands), CTLA4-PE, or with the appropriate isotype controls (all from BD PharMingen). 22 Cells were stained according to the manufacturer's recommendations. Antibodies and fluorescence-activated cell sorter (FACS) analysis Samples were acquired on a FACSCalibur or FACSCanto and analyzed with FACSDiva software (BD Biosciences) or WinMDI 2.8 (http:// facs.scripps.edu/software.html). CD25 high T cells were gated as previously described. 22 Frequencies of CD4 ϩ CD25 high T cells in PB are shown as percent values of CD4 ϩ T cells. To determine cells positive for T reg -cell markers, we used stringent gating criteria, setting gates at the 1% level of the respective isotype control. CD4 ؉ T-cell isolation and culture CD4 ϩ T cells were purified from PBMCs using CD4 MACS Beads (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously. 22 To assess polyclonal CD4 ϩ T-cell activation, 1 ϫ 10 5 CD4 ϩ T cells/well were activated in AIM-V (Gibco Invitrogen, Karlsruhe, Germany)/EX-Cell 610 (JRH Biosciences, Lenexa, KS) with pokeweed mitogen (PWM, 10 g/mL), phytohemagglutinin (PHA, 10 g/mL; both from Sigma-Aldrich, Taufkirchen, Germany), or anti-CD3 (0.2 g/mL, OKT-3) and anti-CD28 mAb (0.2 g/mL; kind gift of Dr L. M. Nadler) in 96-well plates. Proliferation of T cells was monitored by measuring BrdU (5-bromo-2Ј-deoxyuridine) incorporation (Roche Diagnostics, Mannheim, Germany). Isolation of CD4 ؉ CD25 high and CD4 ؉ CD25 ؊ T cells For functional analysis, CD4 ϩ CD25 high T cells were purified from PBMCs. Briefly, CD4 MACS Multisort Beads (Miltenyi Biotec) were used for isolation of CD4 ϩ T cells. 22 After detaching, cells were washed and CD4 ϩ CD25 high T cells were positively selected using CD25 microbeads (2 L beads/10 7 CD4 ϩ cells). The described technique is optimized for the isolation of human CD4 ϩ CD25 high T cells with high purity. 43 Use of higher concentrations of microbeads for isolation results in better recovery but decreases the purity of the T reg -cell population. The negative fraction of CD4 ϩ CD25 Ϫ T cells from healthy controls was used as effectors to assess T reg -cell function independently of potential defects of conventional CD4 ϩ T cells. Cells were reanalyzed after sorting and routinely showed more than 95% purity. RNA extraction and real-time RT-PCR for FOXP3 Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the T reg -cell-associated transcription factor FOXP3. The same primers and conditions were used as previously described. 22 Relative FOXP3 expression in CD4 ϩ CD25 Ϫ and CD4 ϩ CD25 high T cells was normalized to GAPDH expression. Assessment of inhibitory function of CD4 ؉ CD25 high T cells To assess the suppressive activity of CD4 ϩ CD25 high T cells, a modified allogeneic mixed lymphocyte reaction (MLR) was performed as previously described. 22 Briefly, after magnetic separation both highly purified CD4 ϩ CD25 Ϫ and CD4 ϩ CD25 high T cells were incubated for 20 hours with 10 U/mL IL-2 (Proleukin; Chiron, Munich, Germany) and 0.5 g/mL anti-CD3 mAb in X-VIVO 15 (BioWhittakker, Verviers, Belgium). 22, [44] [45] [46] Subsequently, CD4 ϩ CD25 Ϫ T cells (5 ϫ 10 4 /well) were cocultured with irradiated allogeneic PBMCs (2 ϫ 10 5 /well) in X-VIVO 15 supplemented with 10% fetal calf serum, 100 U/mL penicillin/streptomycin, and 2 mM glutamine (all from Gibco Invitrogen). Purified allogeneic CD4 ϩ CD25 high T cells were added at different concentrations as indicated. 34, 47, 48 On day 4, the cells were pulsed with BrdU, and BrdU incorporation was analyzed 20 hours later. There was no influence on the inhibitory effect of CD4 ϩ CD25 high T cells when CD4 ϩ CD25 Ϫ T cells were not preactivated prior to the inhibition assay. Alternatively, 5,6-carboxyfluorescein-diacetatesuccinimidyl-ester (CFSE; Sigma-Aldrich)-stained CD4 ϩ CD25 Ϫ T cells (5 ϫ 10 4 /well) were stimulated with magnetic beads (Dynal Biotech, Oslo, Norway) coated with 5% anti-CD3 (OKT3), 14% anti-CD28 (9.3, kind gift of Drs C. June and J. Riley), and 81% anti-MHC class I (W6/32) at a ratio of 3:1 (cells-beads). PKH-26 (Sigma-Aldrich)-labeled allogeneic CD4 ϩ CD25 high T cells were added at a 1:1 ratio to the culture, and proliferation of CD4 ϩ CD25 Ϫ T cells was determined by assessing CFSE dilution after 4 days of culture. Use of autologous CD4 ϩ CD25 Ϫ T cells yielded comparable results; however, for better comparability of T reg cells from healthy controls, MGUS patients, and MM patients, allogeneic CD4 ϩ CD25 Ϫ T cells were used for most experiments. Cytometric bead array (CBA) for chemokines The concentration of IFN-␥ in cell culture supernatants was measured using the human Th1/Th2 cytokine kit II (BD PharMingen). In brief, capture beads were mixed with culture supernatants and PE detection reagent and incubated for 3 hours at room temperature. The beads were then washed with wash buffer and analyzed. Cytometric bead array (CBA) for chemokines Isolation of CD4 ؉ CD25 ؊ , CD4 ؉ CD25 low , and CD4 ؉ CD25 high T cells for DNA isolation and assessment of T-cell receptor excision circles Briefly, CD4 MACS Beads (Miltenyi Biotec) were used for isolation of CD4 ϩ T cells. 22 After staining with CCR7-FITC (R&D Systems), CD25-PE, CD45RA-PE-Cy-5, and CD4-APC (all from BD PharMingen) according to the manufacturer's recommendations, CD4 ϩ C25 Ϫ ,
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