Frank Stegelmann

Coordinate Expression of CC Chemokine Ligand 5, Granulysin, and Perforin in CD8 + T Cells Provides a Host Defense Mechanism against Mycobacterium tuberculosis

The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.

Abelson Tyrosine Kinase Controls Phagosomal Acidification Required for Killing of Mycobacterium tuberculosis in Human Macrophages

The mechanisms that regulate the acidification of intracellular compartments are key to host defense against pathogens. In this paper, we demonstrate that Abl tyrosine kinase, a master switch for cell growth and trafficking of intracellular organelles, controls the acidification of lysosomes in human macrophages. Pharmacological inhibition by imatinib and gene silencing of Abelson (Abl) tyrosine kinase reduced the lysosomal pH in human macrophages by increasing the transcription and expression of the proton pumping enzyme vacuolar-type H+-adenosine triphosphatase. Because lysosomal acidification is required for antimicrobial activity against intracellular bacteria, we determined the effect of imatinib on the growth of the major human pathogen Mycobacterium tuberculosis. Imatinib limited the multiplication of M. tuberculosis, and growth restriction was dependent on acidification of the mycobacterial compartment. The effects of imatinib were also active in vivo because circulating monocytes from imatinib-treated leukemia patients were more acidic than monocytes from control donors. Importantly, sera from imatinib-treated patients triggered acidification and growth restriction of M. tuberculosis in macrophages. In summary, our results identify the control of phagosomal acidification as a novel function of Abl tyrosine kinase and provide evidence that the regulation occurs on the level of the vacuolar-type H+-adenosine triphosphatase. Given the efficacy of imatinib in a mouse model of tuberculosis and our finding that orally administered imatinib increased the ability of human serum to trigger growth reduction of intracellular M. tuberculosis, clinical evaluation of imatinib as a complementary therapy of tuberculosis, in particular multidrug or extremely drug-resistant disease, is warranted.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.