A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron.

BACKGROUND: Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development. RESULTS: Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community. We used flow cytometry to sort single cells from a pooled library, located mutants within this initial progenitor collection by applying a pooling strategy with barcode sequencing, and re-arrayed specific mutants to create a condensed collection with single-insertion strains covering >2500 genes. To demonstrate the potential of the condensed collection for phenotypic screening, we analyzed growth dynamics and cell morphology. We identified both growth defects and altered cell shape in mutants disrupting sphingolipid synthesis and thiamine scavenging. Finally, we analyzed the process of assembling the B. theta condensed collection to identify inefficiencies that limited coverage. We demonstrate as part of this analysis that the process of assembling an ordered collection can be accurately modeled using barcode sequencing data. CONCLUSION: We expect that utilization of this ordered collection will accelerate research into B. theta physiology and that lessons learned while assembling the collection will inform future efforts to assemble ordered mutant collections for an increasing number of gut microbiota members.

Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry.

Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc.

Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory.

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.

Targeted Delivery of an Anti-inflammatory PDE4 Inhibitor to Immune Cells via an Antibody-drug Conjugate.

Phosphodiesterase 4 (PDE4) inhibitors are approved for the treatment of some moderate to severe inflammatory conditions. However, dose-limiting side effects in the central nervous system and gastrointestinal tract, including nausea, emesis, headache, and diarrhea, have impeded the broader therapeutic application of PDE4 inhibitors. We sought to exploit the wealth of validation surrounding PDE4 inhibition by improving the therapeutic index through generation of an antibody-drug conjugate (ADC) that selectively targets immune cells through the CD11a antigen. The resulting ADC consisted of a human αCD11a antibody (based on efalizumab clone hu1124) conjugated to an analog of the highly potent PDE4 inhibitor GSK256066. Both the human αCD11a ADC and a mouse surrogate αCD11a ADC (based on the M17 clone) rapidly internalized into immune cells and suppressed lipololysaccharide (LPS)-induced TNFα secretion in primary human monocytes and mouse peritoneal cells, respectively. In a carrageenan-induced air pouch inflammation mouse model, treatment with the ADC significantly reduced inflammatory cytokine production in the air pouch exudate. Overall, these results provide compelling evidence for the feasibility of delivering drugs with anti-inflammatory activity selectively to the immune compartment via CD11a and the development of tissue-targeted PDE4 inhibitors as a promising therapeutic modality for treating inflammatory diseases.