Co-transducing B7H3 CAR-NK cells with the DNR preserves their cytolytic function against GBM in the presence of exogenous TGF-β

Cord blood (CB)-derived natural killer (NK) cells that are genetically engineered to express a chimeric antigen receptor (CAR) are an attractive off-the-shelf therapy for the treatment of cancer, demonstrating a robust safety profile in vivo. For poor prognosis brain tumors such as glioblastoma multiforme (GBM), novel therapies are urgently needed. Although CAR-T cells demonstrate efficacy in preclinical GBM models, an off-the-shelf product may exhibit unwanted side effects like graft-versus-host disease. Hence, we developed an off-the-shelf CAR-NK cell approach using a B7H3 CAR and showed that CAR-transduced NK cells have robust cytolytic activity against GBM cells in vitro. However, transforming growth factor (TGF)-β within the tumor microenvironment has devastating effects on the cytolytic activity of both unmodified and CAR-transduced NK cells. To overcome this potent immune suppression, we demonstrated that co-transducing NK cells with a B7H3 CAR and a TGF-β dominant negative receptor (DNR) preserves cytolytic function in the presence of exogenous TGF-β. This study demonstrates that a novel DNR and CAR co-expression strategy may be a promising therapeutic for recalcitrant CNS tumors like GBM

To automate or not to automate: this is the question

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected

Direct Visualization of Peptide/MHC Complexes at the Surface and in the Intracellular Compartments of Cells Infected In Vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay.

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, we designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96 well plate to fully characterize T cell viability, function, growth, and differentiation. Peripheral blood mononuclear cells (PBMC) from six consenting donors were seeded at 100,000 cells/well with pools of CMV peptides from IE-1 and pp65, and combinations of IL15, IL6, IL21, IFNα, IL12, IL18, IL4, and IL7. Ten-day cultures were tested by 13 color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers, and IFNγ and TNFα expression. Combinations of IL15/IL6 and IL4/IL7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold respectively compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL15/IL6, and 9-fold in IL4/IL7 cultures (p< 0.0001). CD4+ T cells expanded 27-fold in IL4/IL7 and 15-fold in IL15/IL6 (p< 0.0001). CD45RO+ CCR7- effector memory T cells were the preponderant cells (76.8% and 72.3% in IL15/IL6 and IL15/IL7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL15/IL6 producing IFNγ (7.6% producing both TNFα and IFNγ), and 18.5% of CD3+ cells grown in IL4/IL7 (9% producing both TNFα and IFNγ). This study shows the utility of this single plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10(7) PBMC

Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B

Altres ajuts: This work is supported by a MDA Career Development Award (MDA477331) to M.W.H. Additional financial support was provided by NIAMS (R01AR055686) and MDA (MDA277389) to J.K.J. and NIH (K26OD011171; R24HD050846, P50AR060836) grants to K.N. The authors acknowledge Carsten Bönnemann's contribution to this study. Microscopy imaging was performed at the CRI Cellular Imaging Core, which is supported by funds from CRI and NICHD (U54HD090257).Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients