BRAFV600E accelerates disease progression and enhances immune suppression in a mouse model of B-cell leukemia.

Mutated mitogen-activated protein kinase (MAPK) pathway components promote tumor survival, proliferation, and immune evasion in solid tumors. MAPK mutations occur in hematologic cancers as well, but their role is less clear and few models are available to study this. We developed an in vivo model of disseminated BRAFV600E B-cell leukemia to determine the effects of this mutation on tumor development and immune evasion. Mice with B-cell-restricted BRAFV600E expression crossed with the Eµ-TCL1 model of chronic lymphocytic leukemia (CLL) developed leukemia significantly earlier (median, 4.9 vs 8.1 months; P < .001) and had significantly shorter lifespan (median, 7.3 vs 12.1 months; P < .001) versus BRAF wild-type counterparts. BRAFV600E expression did not affect B-cell proliferation but reduced spontaneous apoptosis. BRAFV600E-mutant leukemia produced greater T-cell effects, evidenced by exhaustion immunophenotype and CD44+ T-cell percentage, as well as increased expression of PD-L1 on CD11b+ cells. Results were confirmed in syngeneic mice engrafted with BRAFV600E leukemia cells. Furthermore, a BRAFV600E-expressing CLL cell line more strongly inhibited anti-CD3/CD28-induced T-cell proliferation, which was reversed by BRAFV600E inhibition. These results demonstrate the immune-suppressive impact of BRAFV600E in B-cell leukemias and introduce a new model to develop rational combination strategies targeting both tumor cells and tumor-mediated immune evasion.

Visualization of extracellular matrix components within sectioned Salmonella biofilms on the surface of human gallstones.

Chronic carriage of Salmonella Typhi is mediated primarily through the formation of bacterial biofilms on the surface of cholesterol gallstones. Biofilms, by definition, involve the formation of a bacterial community encased within a protective macromolecular matrix. Previous work has demonstrated the composition of the biofilm matrix to be complex and highly variable in response to altered environmental conditions. Although known to play an important role in bacterial persistence in a variety of contexts, the Salmonella biofilm matrix remains largely uncharacterized under physiological conditions. Initial attempts to study matrix components and architecture of the biofilm matrix on gallstone surfaces were hindered by the auto-fluorescence of cholesterol. In this work we describe a method for sectioning and direct visualization of extracellular matrix components of the Salmonella biofilm on the surface of human cholesterol gallstones and provide a description of the major matrix components observed therein. Confocal micrographs revealed robust biofilm formation, characterized by abundant but highly heterogeneous expression of polysaccharides such as LPS, Vi and O-antigen capsule. CsgA was not observed in the biofilm matrix and flagellar expression was tightly restricted to the biofilm-cholesterol interface. Images also revealed the presence of preexisting Enterobacteriaceae encased within the structure of the gallstone. These results demonstrate the use and feasibility of this method while highlighting the importance of studying the native architecture of the gallstone biofilm. A better understanding of the contribution of individual matrix components to the overall biofilm structure will facilitate the development of more effective and specific methods to disrupt these bacterial communities.