Human cell reprogramming technologies offer access to live human neurons from patients and provide a new alternative for modeling neurological disorders in vitro. Neural electrical activity is the essence of nervous system function in vivo. Therefore, we examined neuronal activity in media widely used to culture neurons. We found that classic basal media, as well as serum, impair action potential generation and synaptic communication. To overcome this problem, we designed a new neuronal medium (BrainPhys basal + serum-free supplements) in which we adjusted the concentrations of inorganic salts, neuroactive amino acids, and energetic substrates. We then tested that this medium adequately supports neuronal activity and survival of human neurons in culture. Long-term exposure to this physiological medium also improved the proportion of neurons that were synaptically active. The medium was designed to culture human neurons but also proved adequate for rodent neurons. The improvement in BrainPhys basal medium to support neurophysiological activity is an important step toward reducing the gap between brain physiological conditions in vivo and neuronal models in vitro.

Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1+CD14+ population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell–based vaccines, where they suppress CD4+ T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1+CD14+ cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1+CD14+ cell population expressed markers of both BDCA1+ dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332–46. ©2016 AACR.

Background Clinical benefit of cellular immunotherapy has been shown in patients with castration-resistant prostate cancer (CRPC). We investigated the immunological response and clinical outcome of vaccination with blood-derived CD1c⁺ myeloid dendritic cells (mDCs; cDC2) and plasmacytoid DCs (pDCs). Methods In this randomized phase IIa trial, 21 chemo-naive CRPC patients received maximally 9 vaccinations with mature mDCs, pDCs or a combination of mDCs plus pDCs. DCs were stimulated with protamine/mRNA and loaded with tumor-associated antigens NY-ESO-1, MAGE-C2 and MUC1. Primary endpoint was the immunological response after DC vaccination, which was monitored in peripheral blood and in T cell cultures of biopsies of post-treatment delayed-type hypersensitivity-skin tests. Main secondary endpoints were safety, feasibility, radiological PFS (rPFS) and overall survival. Radiological responses were assessed by MRIs and contrast-enhanced ⁶⁸Ga-prostate-specific membrane antigen PET/CT, according to RECIST 1.1, PCWG2 criteria and immune-related response criteria. Results Both tetramer/dextramer-positive (dm⁺) and IFN-γ-producing (IFN-γ⁺) antigen specific T cells were detected more frequently in skin biopsies of patients with radiological non-progressive disease (5/13 patients; 38%) compared to patients with progressive disease (0/8 patients; 0%). In these patients with vaccination enhanced dm⁺ and IFN-γ⁺ antigen-specific T cells median rPFS was 18.8 months (n = 5) vs. 5.1 months (n = 16) in patients without IFN-γ-producing antigen-specific T cells (p = 0.02). The overall median rPFS was 9.5 months. All DC vaccines were well tolerated with grade 1–2 toxicity. Conclusions Immunotherapy with blood-derived DC subsets was feasible and safe and induced functional antigen-specific T cells. The presence of functional antigen-specific T cells correlated with an improved clinical outcome. Trial registration ClinicalTrials.gov identifier NCT02692976, registered 26 February 2016, retrospectively registered.