High-dose glucocorticoid treatment of near-fatal bocavirus lung infection results in rapid recovery.

Human bocavirus (HBoV) has to be considered a life-threatening pathogen in adults with atypical pneumonia. Pulsed high-dose glucocorticoid treatment may be beneficial in patients suffering from severe pulmonary disease caused by HBoV or other viruses. https://bit.ly/3epiMyO.

Single-cell analysis based dissection of clonality in myelofibrosis.

Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

Efficient Non-Viral T-Cell Engineering by Sleeping Beauty Minicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors.

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-ß upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.

T-cell Receptors for Clinical Therapy: In Vitro Assessment of Toxicity Risk.

Adoptive therapy with T-cell receptor (TCR)-engineered T cells has shown promising results in the treatment of patients with tumors, and the number of TCRs amenable for clinical testing is expanding rapidly. Notably, adoptive therapy with T cells is challenged by treatment-related side effects, which calls for cautious selection of target antigens and TCRs that goes beyond their mere ability to induce high T-cell reactivity. Here, we propose a sequence of in vitro assays to improve selection of TCRs and exemplify risk assessments of on-target as well as off-target toxicities using TCRs directed against cancer germline antigens. The proposed panel of assays covers parameters considered key to safety, such as expression of target antigen in healthy tissues, determination of a TCR's recognition motif toward its cognate peptide, and a TCR's cross-reactivity toward noncognate peptides. Clin Cancer Res; 23(20); 6012-20. ©2017 AACR.

Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.

Identifying T-cell receptors (TCRs) that bind tumor-associated antigens (TAAs) with optimal affinity is a key bottleneck in the development of adoptive T-cell therapy of cancer. TAAs are unmutated self proteins, and T cells bearing high-affinity TCRs specific for such antigens are commonly deleted in the thymus. To identify optimal-affinity TCRs, we generated antigen-negative humanized mice with a diverse human TCR repertoire restricted to the human leukocyte antigen (HLA) A*02:01 (ref. 3). These mice were immunized with human TAAs, for which they are not tolerant, allowing induction of CD8⁺ T cells with optimal-affinity TCRs. We isolate TCRs specific for the cancer/testis (CT) antigen MAGE-A1 (ref. 4) and show that two of them have an anti-tumor effect in vivo. By comparison, human-derived TCRs have lower affinity and do not mediate substantial therapeutic effects. We also identify optimal-affinity TCRs specific for the CT antigen NY-ESO. Our humanized mouse model provides a useful tool for the generation of optimal-affinity TCRs for T-cell therapy.