CRISPR-Mediated Editing of the B Cell Receptor in Primary Human B Cells.

Vaccination approaches have generally focused on the antigen rather than the resultant antibodies generated, which differ greatly in quality and function between individuals. The ability to replace the variable regions of the native B cell receptor (BCR) heavy and light chain loci with defined recombined sequences of a preferred monoclonal antibody could enable curative adoptive cell transfer. We report CRISPR-mediated homologous recombination (HR) into the BCR of primary human B cells. Ribonucleoprotein delivery enabled editing at the model CXCR4 locus, as demonstrated by T7E1 assay, flow cytometry, and TIDE analysis. Insertion via HR was confirmed by sequencing, cross-boundary PCR, and restriction digest. Optimized conditions were used to achieve HR at the BCR variable heavy and light chains. Insertion was confirmed at the DNA level, and transgene expression from the native BCR promoters was observed. Reprogramming the specificity of antibodies in the genomes of B cells could have clinical importance.

Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice.

Cancer cells from a primary tumor can disseminate to other tissues, remaining dormant and clinically undetectable for many years. Little is known about the cues that cause these dormant cells to awaken, resume proliferating, and develop into metastases. Studying mouse models, we found that sustained lung inflammation caused by tobacco smoke exposure or nasal instillation of lipopolysaccharide converted disseminated, dormant cancer cells to aggressively growing metastases. Sustained inflammation induced the formation of neutrophil extracellular traps (NETs), and these were required for awakening dormant cancer. Mechanistic analysis revealed that two NET-associated proteases, neutrophil elastase and matrix metalloproteinase 9, sequentially cleaved laminin. The proteolytically remodeled laminin induced proliferation of dormant cancer cells by activating integrin α3ß1 signaling. Antibodies against NET-remodeled laminin prevented awakening of dormant cells. Therapies aimed at preventing dormant cell awakening could potentially prolong the survival of cancer patients.

Extended release of perioperative immunotherapy prevents tumor recurrence and eliminates metastases.

Cancer immunotherapy can confer durable benefit, but the percentage of patients who respond to this approach remains modest. The ability to concentrate immunostimulatory compounds at the site of disease can overcome local immune tolerance and reduce systemic toxicity. Surgical resection of tumors may improve the efficacy of immunotherapy by removing the concentrated immunosuppressive microenvironment; however, it also removes tumor-specific leukocytes as well as tumor antigens that may be important to establishing antitumor immunity. Moreover, surgery produces a transient immunosuppressive state associated with wound healing that has been correlated with increased metastasis. Using multiple models of spontaneous metastasis, we show that extended release of agonists of innate immunity-including agonists of Toll-like receptor 7/8 (TLR7/8) or stimulator of interferon genes (STING)-from a biodegradable hydrogel placed in the tumor resection site cured a much higher percentage of animals than systemic or local administration of the same therapy in solution. Depletion and neutralization experiments confirmed that the observed prevention of local tumor recurrence and eradication of existing metastases require both the innate and adaptive arms of the immune system. The localized therapy increased the numbers of activated natural killer (NK) cells, dendritic cells, and T cells and induced production of large amounts of type I interferons, thereby converting an immunosuppressive post-resection microenvironment into an immunostimulatory one. The results suggest that the perioperative setting may prove to be a useful context for immunotherapy, particularly when the release of the therapy is extended locally.

Deficiency of the myeloid differentiation primary response molecule MyD88 leads to an early and rapid development of Helicobacter-induced gastric malignancy.

Approximately 50% of the world's population is infected with Helicobacter pylori, leading to chronic inflammation, which increases the risk for gastric adenocarcinoma. MyD88 is a key adaptor molecule in inflammatory pathways involved in interleukin 1 (IL-1)/IL-18/Toll-like receptor signaling and has been shown to have divergent effects in carcinogenesis. The role of MyD88 in Helicobacter-induced gastric malignancy is unknown. Using a mouse model of Helicobacter-induced gastric cancer, we assessed the role of MyD88 in cancer development by evaluating gastric histopathology, apoptosis, proliferation, and cytokine expression. Infection of MyD88-deficient (Myd88(-/-)) mice with Helicobacter resulted in early and rapid advancement to gastric dysplasia as early as 25 weeks postinfection. The progression of Helicobacter-induced disease to precancerous and cancerous lesions in the absence of MyD88 signaling was accompanied by increased gastric epithelial apoptosis and proliferation. In addition, inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-6, and IL-1ß were highly expressed in association with the development of gastric dysplasia. These data suggest that MyD88 signaling retards development and progression to cancer during Helicobacter infection. This is the first study to show evidence of MyD88 protection in an infection-driven inflammation-associated cancer model.

Bacterial Pili exploit integrin machinery to promote immune activation and efficient blood-brain barrier penetration.

Group B Streptococcus (GBS) is the leading cause of meningitis in newborn infants. Bacterial cell surface appendages, known as pili, have been recently described in streptococcal pathogens, including GBS. The pilus tip adhesin, PilA, contributes to GBS adherence to blood-brain barrier (BBB) endothelium; however, the host receptor and the contribution of PilA in central nervous system (CNS) disease pathogenesis are unknown. Here we show that PilA binds collagen, which promotes GBS interaction with the α2ß1 integrin resulting in activation of host chemokine expression and neutrophil recruitment during infection. Mice infected with the PilA-deficient mutant exhibit delayed mortality, a decrease in neutrophil infiltration and bacterial CNS dissemination. We find that PilA-mediated virulence is dependent on neutrophil influx as neutrophil depletion results in a decrease in BBB permeability and GBS-BBB penetration. Our results suggest that the bacterial pilus, specifically the PilA adhesin, has a dual role in immune activation and bacterial entry into the CNS.