Proinflammatory phenotype of iPS cell-derived JAK2 V617F megakaryocytes induces fibrosis in 3D in vitro bone marrow niche.

The myeloproliferative disease polycythemia vera (PV) driven by the JAK2 V617F mutation can transform into myelofibrosis (post-PV-MF). It remains an open question how JAK2 V617F in hematopoietic stem cells induces MF. Megakaryocytes are major players in murine PV models but are difficult to study in the human setting. We generated induced pluripotent stem cells (iPSCs) from JAK2 V617F PV patients and differentiated them into megakaryocytes. In differentiation assays, JAK2 V617F iPSCs recapitulated the pathognomonic skewed megakaryocytic and erythroid differentiation. JAK2 V617F iPSCs had a TPO-independent and increased propensity to differentiate into megakaryocytes. RNA sequencing of JAK2 V617F iPSC-derived megakaryocytes reflected a proinflammatory, profibrotic phenotype and decreased ribosome biogenesis. In three-dimensional (3D) coculture, JAK2 V617F megakaryocytes induced a profibrotic phenotype through direct cell contact, which was reversed by the JAK2 inhibitor ruxolitinib. The 3D coculture system opens the perspective for further disease modeling and drug discovery.

Evolutionary Trajectories of Complex Traits in European Populations of Modern Humans.

Humans have a great diversity in phenotypes, influenced by genetic, environmental, nutritional, cultural, and social factors. Understanding the historical trends of physiological traits can shed light on human physiology, as well as elucidate the factors that influence human diseases. Here we built genome-wide polygenic scores for heritable traits, including height, body mass index, lipoprotein concentrations, cardiovascular disease, and intelligence, using summary statistics of genome-wide association studies in Europeans. Subsequently, we applied these scores to the genomes of ancient European populations. Our results revealed that after the Neolithic, European populations experienced an increase in height and intelligence scores, decreased their skin pigmentation, while the risk for coronary artery disease increased through a genetic trajectory favoring low HDL concentrations. These results are a reflection of the continuous evolutionary processes in humans and highlight the impact that the Neolithic revolution had on our lifestyle and health.

Impact of HBsAg and HBcrAg levels on phenotype and function of HBV-specific T cells in patients with chronic hepatitis B virus infection.

OBJECTIVE: Hepatitis B virus (HBV)-specific T cells are main effector cells in the control of HBV infection and hepatitis B surface antigen (HBsAg) is suggested to be a critical factor in the impaired immune response, a hallmark of chronic HBV infection. In addition to HBsAg, other viral markers such as hepatitis B core-related antigen (HBcrAg) are available, but their potential association with HBV-specific immune responses is not defined yet, which will be important if these markers are used for patient stratification for novel therapies aimed at functional HBV cure. DESIGN: We analysed T cell responses in 92 patients with hepatitis B e antigen negative chronic HBV infection with different HBsAg and HBcrAg levels. Overlapping peptides were used for in vitro response analyses (n=57), and HBV core18-specific and polymerase (pol)455-specific CD8+ T cells were assessed in human leukocyte antigen (HLA)-A*02 patients (n=35). In addition, in vitro responsiveness to anti-programmed cell death-ligand 1 (anti-PD-L1) was investigated. RESULTS: HBV-specific T cell responses were not affected by HBsAg levels, but rather by age and CD4+ T cell responses were highest in patients with low HBcrAg levels. The phenotypes and functionality of HBV core18-specific and pol455-specific CD8+ T cells differed, but HBsAg and HBcrAg levels did not affect their profiles. Blocking with anti-PD-L1 could restore HBV-specific T cells, but the effect was significantly higher in T cells isolated from patients with low HBsAg and in particular low HBcrAg. CONCLUSION: Our data suggest that age and HBcrAg rather than HBsAg, are associated with HBV-specific T cell responses. Finally, very low antigen levels indicated by HBsAg and in particular HBcrAg may influence T cell response to checkpoint inhibition.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes.

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.