All results were analyzed by using FlowJo software, version 9.6.4. IHC Staining and Evaluation. the averages SEM of triplicate means from three or four independent experiments in three different healthy donors and four different patients with melanoma (*** 0.001 for pairwise comparisons between CD25bright vs. CD25int or CD25neg T cells targeted by CD14+CD16++ monocytes blocked or not with anti-CD16 for all those E:T ratios tested; value not significant for pairwise comparisons between CD25bright vs. CD25int or CD25neg T cells targeted by CD14++CD16? monocytes blocked or not with anti-CD16 for all those E:T ratios tested; ANOVA). (and 0.01 and *** 0.001, unpaired two-tailed Student test). Baseline Enrichment of CD68+CD16+ Macrophages in the TME of Patients Responding to Ipilimumab. To investigate the TME, we analyzed matched pre- and postipilimumab metastatic lesions from 13 patients with melanoma and assessed by immunohistochemistry (IHC) the presence of CD8+ T cells, CD56+ NK cells, Foxp3+ Forsythoside A Tregs, and CD68+ or CD163+ macrophages in the tumor nests. Tissue from melanoma metastases was obtained according to the study protocol at baseline and 3C4 wk after the last ipilimumab dose from shrinking lesions from responding patients or progressing lesions from nonresponding patients, respectively. Whereas, at baseline, Foxp3+ tumor-infiltrating Treg counts were comparable in responding vs. nonresponding patients (Fig. 3and and 0.01, paired two-tailed Student test). At baseline (pre), infiltration with Foxp3+ Tregs was comparable in responders vs. nonresponders (= 0.074, not significant, paired two-tailed Student test). ( 0.001, paired two-tailed Student test). ( 0.01 and * 0.05 for CD16+CD68+ or CD16+CD163+ cellular Forsythoside A density Forsythoside A in responders vs. nonresponders, respectively, paired two-tailed Student test). (for plasma preservation, followed by PBMC preparation by gradient centrifugation using Lymphoprep (Ficoll comparative; Axis-Shield). All cells were used new or after cryopreservation. Viable cell recovery was Forsythoside A consistently 85C100%. Patients in this study were diagnosed with metastatic melanoma and received a maximum of four cycles of 3 mg/kg ipilimumab i.v. every 3 wk upon disease progression with at least one prior treatment. Blood samples were withdrawn at baseline, during treatment, 20 d after treatment, and then monthly for as long as 14 mo after the last ipilimumab dose. Cell Sorting. CD3+CD4+ T cells were enriched by using Dynabeads FlowComp Human CD4 kit (Invitrogen/Molecular Probes). The following antibodies were used to stain cells for subsequent FACS sorting: antiCCD3-APC-H7 (isotype IgG1; BD Biosciences), antiCCD4-ECD (isotype IgG1; Beckman Coulter), and antiCCD25-PE (isotype IgG2a; BD Biosciences), and AmCyan was used as a live/lifeless marker (Invitrogen-Molecular Probes). CD3+CD4+ T cells with high (CD25bright, i.e., Tregs), intermediate (CD25int), and low (CD25neg) levels of CD25 expression were sorted by using a BD FASCAria cell sorter. The portion purity was 97% on average. Purification of CD16+ and CD14+ Monocytes. Adherent cells, after 1 h of PBMC incubation in a tissue culture dish with a 20-mm grid (Plasma 150 20 Style; Becton Dickinson) in RPMI 1640 with 2% (vol/vol) FCS, 1% penicillin/streptomycin, and 2 mM AAG (Arg, Asp, Glu), PI4KA were gently trypsinized. CD16+ and CD14+ monocytes were separated by magnetic-activated cell sorting (human CD16 and CD14 microbeads; Miltenyi Biotec) according to manufacturer instructions. The purity of these cell subsets was checked with the following antibodies: antiCHLA-DR-APC (isotype IgG2a; BD Biosciences), antiCCD14-FITC (isotype IgG2a; Beckman Coulter), and antiCCD16-ECD (isotype IgG1; Beckman Coulter), and Vivid-Red (Invitrogen-Molecular Probes) was used as a live/lifeless marker. A Gallios circulation cytometer was utilized for the measurement. Cell purity was 90% for CD14++CD16? and 80% for CD14+CD16++ subsets. ADCC Assay. CD3+CD4+ T cells with different expression levels of CD25 (CD25bright, CD25int, CD25neg) obtained and sorted from healthy donors or patients with melanoma were cocultured with autologous CD14++CD16? or CD14+CD16++ monocytes at the effector:target cell (E:T) ratios 40:1, 10:1, 5:1, and 1:2..