Bortezomib(Velcade®)-Thalidomide-Dexamethasone (VTD) Is Superior to Thalidomide-Dexamethasone (TD) In Patients with Multiple Myeloma (MM) Progressing or Relapsing After Autologous Transplantation

Introduction: The Thalidomide-Dexamethasone (TD) regimen has provided encouraging results in relapsed MM. To improve results, bortezomib (Velcade) has been added to the combination in previous phase II studies, the so called VTD regimen. In January 2006, the European Group for Blood and Marrow Transplantation (EBMT) and the Intergroupe Francophone du Myélome (IFM) initiated a prospective, randomized, parallel-group, open-label phase III, multicenter study, comparing VTD (arm A) with TD (arm B) for MM patients progressing or relapsing after autologous transplantation. Patients and Methods: Inclusion criteria: patients in first progression or relapse after at least one autologous transplantation, including those who had received bortezomib or thalidomide before transplant. Exclusion criteria: subjects with neuropathy above grade 1 or non secretory MM. Primary study end point was time to progression (TTP). Secondary end points included safety, response rate, progression-free survival (PFS) and overall survival (OS). Treatment was scheduled as follows: bortezomib 1.3 mg/m2 was given as an i.v bolus on Days 1, 4, 8 and 11 followed by a 10-Day rest period (days 12 to 21) for 8 cycles (6 months) and then on Days 1, 8, 15, 22 followed by a 20-Day rest period (days 23 to 42) for 4 cycles (6 months). In both arms, thalidomide was scheduled at 200 mg/Day orally for one year and dexamethasone 40 mg/Day orally four days every three weeks for one year. Patients reaching remission could proceed to a new stem cell harvest. However, transplantation, either autologous or allogeneic, could only be performed in patients who completed the planned one year treatment period. Response was assessed by EBMT criteria, with additional category of near complete remission (nCR). Adverse events were graded by the NCI-CTCAE, Version 3.0.The trial was based on a group sequential design, with 4 planned interim analyses and one final analysis that allowed stopping for efficacy as well as futility. The overall alpha and power were set equal to 0.025 and 0.90 respectively. The test for decision making was based on the comparison in terms of the ratio of the cause-specific hazards of relapse/progression, estimated in a Cox model stratified on the number of previous autologous transplantations. Relapse/progression cumulative incidence was estimated using the proper nonparametric estimator, the comparison was done by the Gray test. PFS and OS probabilities were estimated by the Kaplan-Meier curves, the comparison was performed by the Log-Rank test. An interim safety analysis was performed when the first hundred patients had been included. The safety committee recommended to continue the trial. Results: As of 1st July 2010, 269 patients had been enrolled in the study, 139 in France (IFM 2005-04 study), 21 in Italy, 38 in Germany, 19 in Switzerland (a SAKK study), 23 in Belgium, 8 in Austria, 8 in the Czech republic, 11 in Hungary, 1 in the UK and 1 in Israel. One hundred and sixty nine patients were males and 100 females; the median age was 61 yrs (range 29-76). One hundred and thirty six patients were randomized to receive VTD and 133 to receive TD. The current analysis is based on 246 patients (124 in arm A, 122 in arm B) included in the second interim analysis, carried out when 134 events were observed. Following this analysis, the trial was stopped because of significant superiority of VTD over TD. The remaining patients were too premature to contribute to the analysis. The number of previous autologous transplants was one in 63 vs 60 and two or more in 61 vs 62 patients in arm A vs B respectively. The median follow-up was 25 months. The median TTP was 20 months vs 15 months respectively in arm A and B, with cumulative incidence of relapse/progression at 2 years equal to 52% (95% CI: 42%-64%) vs 70% (95% CI: 61%-81%) (p=0.0004, Gray test). The same superiority of arm A was also observed when stratifying on the number of previous autologous transplantations. At 2 years, PFS was 39% (95% CI: 30%-51%) vs 23% (95% CI: 16%-34%) (A vs B, p=0.0006, Log-Rank test). OS in the first two years was comparable in the two groups. Conclusion: VTD resulted in significantly longer TTP and PFS in patients relapsing after ASCT. Analysis of response and safety data are on going and results will be presented at the meeting.

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

Tissue homing and persistence of defined antigen-specific CD8+ tumor-reactive T-cell clones in long-term melanoma survivors.

Tumor antigen-specific cytotoxic T cells (CTLs) play a major role in the adaptive immune response to cancers. This CTL response is often insufficient because of functional impairment, tumor escape mechanisms, or inhibitory tumor microenvironment. However, little is known about the fate of given tumor-specific CTL clones in cancer patients. Studies in patients with favorable outcomes may be very informative. In this longitudinal study, we tracked, quantified, and characterized functionally defined antigen-specific T-cell clones ex vivo, in peripheral blood and at tumor sites, in two long-term melanoma survivors. MAGE-A10-specific CD8+ T-cell clones with high avidity to antigenic peptide and tumor lytic capabilities persisted in peripheral blood over more than 10 years, with quantitative variations correlating with the clinical course. These clones were also found in emerging metastases, and, in one patient, circulating clonal T cells displayed a fully differentiated effector phenotype at the time of relapse. Longevity, tumor homing, differentiation phenotype, and quantitative adaptation to the disease phases suggest the contribution of the tracked tumor-reactive clones in the tumor control of these long-term metastatic survivor patients. Focusing research on patients with favorable outcomes may help to identify parameters that are crucial for an efficient antitumor response and to optimize cancer immunotherapy.

Emerging viral infections after hematopoietic cell transplantation.

This overview summarizes recent data on emerging viruses after hematopoietic cell transplantation (HCT), including adenovirus, BK virus, human metapneumovirus (hMPV), and human herpesvirus (HHV) 6. The increased recognition of these infections is due to improved molecular detection methods, increased surveillance and more profound immunosuppression in the host. Adenovirus can cause serious disease especially in T-cell depleted transplant recipients. Adenovirus viremia is an important risk factor for disease in this setting. BK virus has been associated with hemorrhagic cystitis in HCT recipients. BK viremia is significantly associated with hemorrhagic cystitis. hMPV shows a seasonal distribution and can cause fatal pneumonia in HCT recipients. hMPV may be the etiology of some cases previously categorized as idiopathic pneumonia syndrome. HHV-6 commonly leads to viremia in HCT recipients. HHV-6 has been strongly associated with encephalitis and delayed platelet engraftment. Prospective studies are needed to further examine epidemiology, disease associations, and management strategies for these viruses.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance.

Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

Epithelial Ingrowth After Lasik/altk: An Immunohistological Study Of 4 Cases. Do Epithelial Inclusions Grow From Trapped Corneal Stem-like Cells?

Purpose: To characterize the clinical, morphological and immunohistological features of epithelial ingrowth cells after laser in situ keratomileusis (LASIK) or Automated Lamellar Therapeutic Keratoplasty (ALTK) with specific reference to current markers of corneal stem cells.Methods: Four patients were included in this interventional non-comparative case series. Full ophthalmologic examination was performed. Epithelial ingrowth specimens from 4 patients were removed surgically and immunostained for cytokeratin 3 (CK3), cytokeratin 15 (CK15), cytokeratin 19 (CK19), Muc5AC, p63α, C/EBPδ, Bmi-1, BCRP/ABCG2 and Ki-67.Results: The time interval between LASIK/ALTK and ingrowth surgical removal was, 3, 11, 15 and 36 months. On slit lamp examination, early epithelial ingrowth appeared as whitish pearls and late epithelial ingrowth as confluent whitish opacities. Microscopically, the epithelial ingrowths showed features of a squamous non keratinizing epithelium. No mitotic figure was seen. Ki-67 labelling of 3 cases showed a proliferation index of 3-4%. Superficial squamous cells strongly expressed CK3. Expression of C/EBPδ, BCRP/ABCG2 and p63α was seen in more than 70% of cells and Bmi-1 was positive in up to 30% of cells in the specimens tested. There was no expression of CK19 or CK15.Conclusions: Epithelial ingrowths can persist for up to 3 years following LASIK surgery. They show a capacity for self-renewal and corneal differentiation. Besides, they express p63α, C/EBPδ, Bmi-1, BCRP/ABCG2 which have been proposed as markers of stem cell phenotype. These observations suggest that post-LASIK/ALTK epithelial inclusions could derive from stem-like cells located in the peripheral corneal epithelium.

Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture.

Steady-state hematopoiesis and hematopoietic transplantation rely on the unique potential of stem cells to undergo both self-renewal and multilineage differentiation. Fetal liver (FL) represents a promising alternative source of hematopoietic stem cells (HSCs), but limited by the total cell number obtained in a typical harvest. We reported that human FL nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cells (SRCs) could be expanded under simple stroma-free culture conditions. Here, we sought to further characterize FL HSC/SRCs phenotypically and functionally before and following culture. Unexpanded or cultured FL cell suspensions were separated into various subpopulations. These were tested for long-term culture potential and for in vivo repopulating function following transplantation into NOD/SCID mice. We found that upon culture of human FL cells, a tight association between classical stem cell phenotypes, such as CD34(+) /CD38(-) and/or side population, and NOD/SCID repopulating function was lost, as observed with other sources. Although SRC activity before and following culture consistently correlated with the presence of a CD34(+) cell population, we provide evidence that, contrary to umbilical cord blood and adult sources, stem cells present in both CD34(+) and CD34(-) FL populations can sustain long-term hematopoietic cultures. Furthermore, upon additional culture, CD34-depleted cell suspensions, devoid of SRCs, regenerated a population of CD34(+) cells possessing SRC function. Our studies suggest that compared to neonatal and adult sources, the phenotypical characteristics of putative human FL HSCs may be less strictly defined, and reinforce the accumulated evidence that human FL represents a unique, valuable alternative and highly proliferative source of HSCs for clinical applications.

Mobilization of hemopoietic stem cells with high-dose methotrexate plus granulocyte-colony-stimulating factor in patients with primary central nervous system lymphoma.

BACKGROUND: High-dose therapy with autologous stem cell support after standard dose induction is a promising approach for therapy of primary central nervous system lymphoma (PCNSL). High-dose methotrexate (HD-MTX) is a standard drug for induction of PCNSL; however, data about the capacity of HD-MTX plus granulocyte-colony-stimulating factor (G-CSF) to mobilize hemopoietic progenitors are lacking. STUDY DESIGN AND METHODS: This investigation describes the data from stem cell mobilization and apheresis procedures after one or two cycles of HD-MTX for induction of PCNSL within the East German Study Group for Haematology and Oncology 053 trial. Eligible patients proceeded to high-dose busulfan/thiotepa after induction therapy and mobilization. RESULTS: Data were available from nine patients with a median age of 58 years. The maximal CD34+ cell count per microL of blood after the first course of HD-MTX was 13.89 (median). Determination was repeated in six patients after the second course with a significantly higher median CD34+ cell count of 33.69 per microL. Five patients required two apheresis procedures and in four patients a single procedure was sufficient. The total yield of CD34+ cells per kg of body weight harvested by one or two leukapheresis procedures was 6.60 x 10(6) (median; range, 2.68 x 10(6)-15.80 x 10(6)). The yield of CD34+ cells exceeded the commonly accepted lower threshold of 3 x 10(6) cells per kg of body weight in eight of nine cases. Even in the ninth, hemopoietic recovery after stem cell reinfusion was rapid and safe. CONCLUSION: HD-MTX plus G-CSF is a powerful combination for stem cell mobilization in patients with PCNSL and permits safe conduction of time-condensed and dose-intense protocols with high-dose therapy followed by stem cell reinfusion after HD-MTX induction.

Preemptive treatment approach to cytomegalovirus (CMV) infection in solid organ transplant patients: relationship between compliance with the guidelines and prevention of CMV morbidity.

Cytomegalovirus (CMV) remains a major cause of morbidity in solid organ transplant patients. In order to reduce CMV morbidity, we designed a program of routine virological monitoring that included throat and urine CMV shell vial culture, along with peripheral blood leukocyte (PBL) shell vial quantitative culture for 12 weeks post-transplantation, as well as 8 weeks after treatment for acute rejection. The program also included preemptive ganciclovir treatment for those patients with the highest risk of developing CMV disease, i.e., with either high-level viremia (>10 infectious units [IU]/106 PBL) or low-level viremia (<10 IU/106 PBL) and either D+/R- CMV serostatus or treatment for graft rejection. During 1995-96, 90 solid organ transplant recipients (39 kidneys, 28 livers, and 23 hearts) were followed up. A total of 60 CMV infection episodes occurred in 45 patients. Seventeen episodes were symptomatic. Of 26 episodes managed according to the program, only 4 presented with CMV disease and none died. No patient treated preemptively for asymptomatic infection developed disease. In contrast, among 21 episodes managed in non-compliance with the program (i.e., the monitoring was not performed or preemptive treatment was not initiated despite a high risk of developing CMV disease), 12 episodes turned into symptomatic infection (P=0.0048 compared to patients treated preemptively), and 2 deaths possibly related to CMV were recorded. This difference could not be explained by an increased proportion of D+/R- patients or an increased incidence of rejection among patients with episodes treated in non-compliance with the program. Our data identify compliance with guidelines as an important factor in effectively reducing CMV morbidity through preemptive treatment, and suggest that the complexity of the preemptive approach may represent an important obstacle to the successful prevention of CMV morbidity by this approach in the regular healthcare setting.

Fc block treatment, dead cells exclusion, and cell aggregates discrimination concur to prevent phenotypical artifacts in the analysis of subpopulations of tumor-infiltrating CD11b(+) myelomonocytic cells.

It is well established that cancer cells can recruit CD11b(+) myeloid cells to promote tumor angiogenesis and tumor growth. Increasing interest has emerged on the identification of subpopulations of tumor-infiltrating CD11b(+) myeloid cells using flow cytometry techniques. In the literature, however, discrepancies exist on the phenotype of these cells (Coffelt et al., Am J Pathol 2010;176:1564-1576). Since flow cytometry analysis requires particular precautions for accurate sample preparation and trustable data acquisition, analysis, and interpretation, some discrepancies might be due to technical reasons rather than biological grounds. We used the syngenic orthotopic 4T1 mammary tumor model in immunocompetent BALB/c mice to analyze and compare the phenotype of CD11b(+) myeloid cells isolated from peripheral blood and from tumors, using six-color flow cytometry. We report here that the nonspecific antibody binding through Fc receptors, the presence of dead cells and cell doublets in tumor-derived samples concur to generate artifacts in the phenotype of tumor-infiltrating CD11b(+) subpopulations. We show that the heterogeneity of tumor-infiltrating CD11b(+) subpopulations analyzed without particular precautions was greatly reduced upon Fc block treatment, dead cells, and cell doublets exclusion. Phenotyping of tumor-infiltrating CD11b(+) cells was particularly sensitive to these parameters compared to circulating CD11b(+) cells. Taken together, our results identify Fc block treatment, dead cells, and cell doublets exclusion as simple but crucial steps for the proper analysis of tumor-infiltrating CD11b(+) cell populations.

Novel insulated gamma and lentis retroviral vectors towards safer genetic modification of stem cells

In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. b-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr- MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933

Ochratoxin A at nanomolar concentration perturbs the homeostasis of neural stem cells in highly differentiated but not in immature three-dimensional brain cell cultures.

Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients.

Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4-specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14(++)CD16(-) monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68(+)/CD163(+) macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti-CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.