Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fc␥ receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fc␥ receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.antibody-dependent cell-mediated cytotoxicity ͉ Fc␥R ͉ protein engineering ͉ cancer

The lower urinary tract’s virtually inevitable exposure to external microbial pathogens warrants efficient tissue-specialized defenses to maintain sterility. The observation that the bladder can become chronically infected in combination with clinical observations that antibody responses following bladder infections are not detectable, suggest defects in the formation of adaptive immunity and immunological memory. We have identified a broadly immunosuppressive transcriptional program specific to the bladder, but not the kidney, during infection of the urinary tract that is dependent on tissue-resident mast cells (MCs). This involves localized production of interleukin-10 and results in suppressed humoral and cell mediated responses and bacterial persistence. Therefore, in addition to the previously described role of MCs orchestrating the early innate immunity during bladder infection, they subsequently play a tissue-specific immunosuppressive role. These findings may explain the prevalent recurrence of bladder infections and suggest the bladder as a site exhibiting an intrinsic degree of MC-maintained immune privilege.

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.

Mast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that during E. coli infection, the primary humoral response in MC-deficient mice is significantly diminished, and was found to be less protective in a urinary tract infection (UTI) model compared to the response from MC-sufficient counterparts. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) over a prolonged time-course. This pattern of trafficking was facilitated by MC generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs, and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

The vigorous cytokine response of immune cells to Gram-negative bacteria is primarily mediated by a recognition molecule, Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide (LPS) and initiates a series of intracellular NF-κB–associated signaling events. Recently, bladder epithelial cells (BECs) were reported to express TLR4 and to evoke a vigorous cytokine response upon exposure to LPS. We examined intracellular signaling events in human BECs leading to the production of IL-6, a major urinary cytokine, following activation by Escherichia coli and isolated LPS. We observed that in addition to the classical NF-κB–associated pathway, TLR4 triggers a distinct and more rapid signaling response involving, sequentially, Ca2+, adenylyl cyclase 3–generated cAMP, and a transcriptional factor, cAMP response element–binding protein. This capacity of BECs to mobilize secondary messengers and evoke a more rapid IL-6 response might be critical in their role as first responders to microbial challenge in the urinary tract.

Summary Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1β (IL-1β) secreted by BECs following inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 (mMCPT4)), BECs underwent caspase-1 associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.

Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

Cells adapt to stress by altering gene expression at multiple levels. Here, we propose a new mechanism regulating stress-dependent gene expression at the level of translation, with coordinated interplay between the tRNA epitranscriptome and biased codon usage in families of stress-response genes. In this model, auxiliary genetic information contained in synonymous codon usage enables regulation of codon-biased and functionally related transcripts by dynamic changes in the tRNA epitranscriptome. This model partly explains the association between synchronous stress-dependent epitranscriptomic marks and significant multi-codon usage skewing in families of translationally regulated transcripts. The model also predicts translational adaptation during viral infections.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1611-1) contains supplementary material, which is available to authorized users.