MCK2-mediated MCMV infection of macrophages and virus dissemination to the salivary gland depends on MHC class I molecules.

Murine cytomegalovirus (MCMV) infection of macrophages relies on MCMV-encoded chemokine 2 (MCK2), while infection of fibroblasts occurs independently of MCK2. Recently, MCMV infection of both cell types was found to be dependent on cell-expressed neuropilin 1. Using a CRISPR screen, we now identify that MCK2-dependent infection requires MHC class Ia/ß-2-microglobulin (B2m) expression. Further analyses reveal that macrophages expressing MHC class Ia haplotypes H-2b and H-2d, but not H-2k, are susceptible to MCK2-dependent infection with MCMV. The importance of MHC class I expression for MCK2-dependent primary infection and viral dissemination is highlighted by experiments with B2m-deficient mice, which lack surface expression of MHC class I molecules. In those mice, intranasally administered MCK2-proficient MCMV mimics infection patterns of MCK2-deficient MCMV in wild-type mice: it does not infect alveolar macrophages and subsequently fails to disseminate into the salivary glands. Together, these data provide essential knowledge for understanding MCMV-induced pathogenesis, tissue targeting, and virus dissemination.

Challenges of CRISPR-Based Gene Editing in Primary T Cells.

Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.

Sorafenib Maintenance After Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia With FLT3-Internal Tandem Duplication Mutation (SORMAIN).

PURPOSE: Despite undergoing allogeneic hematopoietic stem cell transplantation (HCT), patients with acute myeloid leukemia (AML) with internal tandem duplication mutation in the FMS-like tyrosine kinase 3 gene (FLT3-ITD) have a poor prognosis, frequently relapse, and die as a result of AML. It is currently unknown whether a maintenance therapy using FLT3 inhibitors, such as the multitargeted tyrosine kinase inhibitor sorafenib, improves outcome after HCT. PATIENTS AND METHODS: In a randomized, placebo-controlled, double-blind phase II trial (SORMAIN; German Clinical Trials Register: DRKS00000591), 83 adult patients with FLT3-ITD-positive AML in complete hematologic remission after HCT were randomly assigned to receive for 24 months either the multitargeted and FLT3-kinase inhibitor sorafenib (n = 43) or placebo (n = 40 placebo). Relapse-free survival (RFS) was the primary endpoint of this trial. Relapse was defined as relapse or death, whatever occurred first. RESULTS: With a median follow-up of 41.8 months, the hazard ratio (HR) for relapse or death in the sorafenib group versus placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rank P = .013). The 24-month RFS probability was 53.3% (95% CI, 0.36 to 0.68) with placebo versus 85.0% (95% CI, 0.70 to 0.93) with sorafenib (HR, 0.256; 95% CI, 0.10 to 0.65; log-rank P = .002). Exploratory data show that patients with undetectable minimal residual disease (MRD) before HCT and those with detectable MRD after HCT derive the strongest benefit from sorafenib. CONCLUSION: Sorafenib maintenance therapy reduces the risk of relapse and death after HCT for FLT3-ITD-positive AML.

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level BCR-ABL-expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are BCR-ABL-negative in MR, a substantial proportion of BCR-ABL + CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8+ T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML.Significance: CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. Cancer Res; 78(21); 6223-34. ©2018 AACR.

Immunosuppressive capacity of mesenchymal stem cells correlates with metabolic activity and can be enhanced by valproic acid.

BACKGROUND: Mesenchymal stem cells (MSCs) have entered the clinic as an Advanced Therapy Medicinal Product and are currently evaluated in a wide range of studies for tissue regeneration or in autoimmune disorders. Various efforts have been made to standardize and optimize expansion and manufacturing processes, but until now reliable potency assays for the final MSC product are lacking. Because recent findings suggest superior therapeutic efficacy of freshly administered MSCs in comparison with frozen cells, we sought to correlate the T-cell suppressive capacity of MSCs with their metabolic activity. METHODS: Human MSCs were obtained from patients' bone fragments and were employed in coculture with peripheral blood mononuclear cells (PBMCs) in an allogeneic T-cell proliferation assay to measure immunosuppressive function. Metabolic activity of MSCs was measured in real time in terms of aerobic glycolysis quantified by the extracellular acidification rate and mitochondrial respiration quantified by the oxygen consumption rate. RESULTS: We show that MSC-induced suppression of T-cell proliferation was highly dependent on individual healthy donors' lymphocytes. Moreover, coculture with PBMCs increased the glycolytic and respiratory activity of MSCs considerably in a PBMC donor-dependent manner. The twofold to threefold enhancement of cell metabolism was accompanied by higher T-cell suppressive capacities of MSCs. The cryoprotectant dimethyl sulfoxide decreased metabolic and immunosuppressive performances of MSCs while valproic acid (VPA) increased their glycolytic, respiratory and T-cell suppressive capacity. CONCLUSIONS: Functional fitness of MSCs can be determined by measuring metabolic activity and can be enhanced by exposure to VPA. Pretesting the increment of metabolic activity upon interaction of donor MSCs with patient T-cells provides a rational approach for an individualized potency assay prior to MSC therapy.