Generation and quality control of mature monocyte-derived dendritic cells for immunotherapy.

Dendritic cell vaccination is a form of active immunotherapy that aims to exploit the crucial role of DC in the initiation of T-cell responses. Numerous vaccination trials have been conducted targeting various tumor entities, including glioblastoma, the most frequent and aggressive malignant brain tumor in adults. They have demonstrated feasibility and safety and suggest improved survival, associated with induction of anti-tumoral immunity. Here, we describe in detail a large-scale 2-step protocol for successive GMP-compliant generation of immature and mature dendritic cells, yielding a highly homogenous population of CD83+ mature DC expressing CD40, CD80, CD86 and HLA-DR at high density, lacking activity of the immunosuppressive enzyme indoleamine-2,3-dioxygenase, migrating towards the chemokine CCL19 and showing highly potent T-cell stimulatory activity. Loaded with autologous tumor lysate, these cells are currently being evaluated in a phase II controlled randomized clinical trial (GlioVax) in glioblastoma patients.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.