Purpose: Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma. Experimental Design: Autologous DC were pulsed with MART-1 26-35 peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point. Results: Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response. The CTL-associated antigen 4 (CTLA4) is a main negative regulator of the immune system, which inhibits costimulatory signaling provided by dendritic cells (DC) to activate T lymphocytes. Antibodies to CTLA4 block this negative signaling, which allows a dominant positive signaling provided by costimulatory molecules on DCs recognized by CD28 on T cells (1). In animal models, administration of CTLA4-blocking antibodies alone induced rejection of established tumors, provided that these were immunogenic tumor models (1, 2). However, in poorly immunogenic tumor models, which may more closely resemble human disease, prior immunization with tumor vaccines, including DC-based vaccines, was required for CTLA4 blockade to exert robust antitumor effects (1).Tremelimumab 206) is a fully human CTLA4-blocking monoclonal antibody being developed for the treatment of cancer (3). In phase I testing, plasma levels of antibody of 30 μg/mL, which corresponds to levels predicted to result in continuous CTLA4 blockade in vitro, were achieved for at least 1 month at doses beyond 6 mg/kg (4). Cumulative clinical data suggests that single agent tremelimumab has antitumor activity in ∼10% of patients with advanced melanoma, and these responses tend to be long

Background CTLA4 blocking monoclonal antibodies provide durable clinical benefit in a subset of patients with advanced melanoma mediated by intratumoral lymphocytic infiltrates. A key question is defining if the intratumoral infiltration is a differentiating factor between patients with and without tumor responses. Methods Paired baseline and post-dosing tumor biopsies from 19 subjects, including three patients with an objective tumor response, were prospectively collected from patients with metastatic melanoma receiving the anti-CTLA4 antibody tremelimumab within a clinical trial with primary endpoint of quantitating CD8+ cytotoxic T lymphocyte (CTL) infiltration in tumors. Samples were analyzed for cell density using automated imaging capture, and further characterized for functional lymphocyte properties by assessing the cell activation markers HLA-DR and CD45RO, the cell proliferation marker Ki67 and the T regulatory cell marker FOXP3. Results There was a highly significant increase in intratumoral infiltration by CD8+ cells in biopsies taken after tremelimumab treatment. This included increases between 1-fold and 100-fold changes in 14 out of 18 evaluable cases regardless of clinical tumor response or progression. There was no difference between the absolute number, location or cell density of infiltrating cells between clinical responders and patients with non-responding lesions that showed acquired intratumoral infiltrates. There were similar levels of expression of T cell activation markers (CD45RO, HLA-DR) in both groups, and no difference in markers for cell replication (Ki67) or the suppressor cell marker FOXP3. Conclusion CTLA4 blockade induces frequent increases in intratumoral T cell infiltration despite which only a minority of patients have objective tumor responses.

Background: Th17 cells are CD4+ cells that produce interleukin 17 (IL-17) and are potent inducers of tissue inflammation and autoimmunity. We studied the levels of this T cell subset in peripheral blood of patients treated with the anti-CTLA4 antibody tremelimumab since its major dose limiting toxicities are inflammatory and autoimmune in nature.

Purpose: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity. Experimental Design: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). Results: Clinically responding lesions had diffuse intratumoral infiltrates of CD8 + Tcells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8 + intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8 + cells with or without a concomitant increase in CD4 + cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3

Purpose: PLX4032 (RG7204), an oncogenic BRAF kinase inhibitor undergoing clinical evaluation, has high response rates in early clinical trials in patients with advanced BRAF V600E mutant melanoma.Combining PLX4032 with immunotherapy may allow expanding the durability of responses. The effects of PLX4032 on immune cells were studied to explore the feasibility of future combinatorial approaches with immunotherapy for melanoma. Experimental Design: Peripheral blood mononuclear cells (PBMC) and BRAF V600E mutant melanoma cells were exposed to increasing concentrations of PLX4032 and the cell viability, proliferation, cell cycle, apoptosis, and phosphorylation of signaling proteins were analyzed. Effects of PLX4032 on antigenspecific T-cell function were analyzed by specific cytokine release and cytotoxicity activity.Results: The 50% inhibition concentration (IC 50 ) of PLX4032 for resting human PBMC was between 50 and 150 mmol/L compared with an IC 50 below 1 mmol/L for sensitive BRAF V600E mutant melanoma cell lines. Activated lymphocytes were even more resistant with no growth inhibition up to concentrations of 250 mmol/L. PLX4032 had a marginal effect on cell-cycle arrest, apoptotic cell changes or alteration of phosphorylated signaling molecules in lymphocytes. Functional analysis of specific antigen recognition showed preserved T-cell function up to 10-mmol/L concentration of PLX4032, whereas the cytotoxic activity of PLX4032 was maintained up to high concentrations of 50 mmol/L. Conclusions:The preserved viability and function of lymphocytes exposed to high concentrations of PLX4032 suggest that this agent could be a potential candidate for combining with immunotherapy strategies for the treatment of patients with BRAF V600E mutant melanoma.

Background: CTLA4-blocking antibodies induce tumor regression in a subset of patients with melanoma. Analysis of immune parameters in peripheral blood may help define how responses are mediated.

Emerging scientific evidence indicates that frequent digital technology use has a significant impact—both negative and positive—on brain function and behavior. Potential harmful effects of extensive screen time and technology use include heightened attention-deficit symptoms, impaired emotional and social intelligence, technology addiction, social isolation, impaired brain development, and disrupted sleep. However, various apps, videogames, and other online tools may benefit brain health. Functional imaging scans show that internet-naive older adults who learn to search online show significant increases in brain neural activity during simulated internet searches. Certain computer programs and videogames may improve memory, multitasking skills, fluid intelligence, and other cognitive abilities. Some apps and digital tools offer mental health interventions providing self-management, monitoring, skills training, and other interventions that may improve mood and behavior. Additional research on the positive and negative brain health effects of technology is needed to elucidate mechanisms and underlying causal relationships.

BackgroundThe effects on cell signalling networks upon blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA4) using the monoclonal antibody tremelimumab were studied in peripheral blood mononuclear cell (PBMC) samples from patients with metastatic melanoma.Methodology/PrincipalFindings Intracellular flow cytometry was used to detect phosphorylated (p) signaling molecules downstream of the T cell receptor (TCR) and cytokine receptors. PBMC from tremelimumab-treated patients were characterized by increase in pp38, pSTAT1 and pSTAT3, and decrease in pLck, pERK1/2 and pSTAT5 levels. These changes were noted in CD4 and CD8 T lymphocytes but also in CD14 monocytes. A divergent pattern of phosphorylation of Zap70, LAT, Akt and STAT6 was noted in patients with or without an objective tumor response.Conclusions/SignificanceThe administration of the CTLA4-blocking antibody tremelimumab to patients with metastatic melanoma influences signaling networks downstream of the TCR and cytokine receptors both in T cells and monocytes. The strong modulation of signaling networks in monocytes suggests that this cell subset may be involved in clinical responses to CTLA4 blockade.Clinical Trial Registrationclinicaltrials.gov; Registration numbers NCT00090896 and NCT00471887