In drug-drug interactions, there are surprising cases in which the growth inhibition of bacteria by a single antibiotic decreases when a second antibiotic is added. These interactions are termed ...suppressive and have been argued to have the potential to limit the evolution of resistance. Nevertheless, little attention has been given to suppressive interactions because clinical studies typically search for increases in killing efficiency and because suppressive interactions are believed to be rare based on pairwise studies.
Here, we quantify the effects of single-, double-, and triple-drug combinations from a set of 14 antibiotics and 3 bacteria strains, totaling 364 unique three-drug combinations per bacteria strain. We find that increasing the number of drugs can increase the prevalence of suppressive interactions: 17% of three-drug combinations are suppressive compared to 5% of two-drug combinations in this study. Most cases of suppression we find (97%) are "hidden" cases for which the triple-drug bacterial growth is less than the single-drug treatments but exceeds that of a pairwise combination.
We find a surprising number of suppressive interactions in higher-order drug combinations. Without examining lower-order (pairwise) bacterial growth, emergent suppressive effects would be missed, potentially affecting our understanding of evolution of resistance and treatment strategies for resistant pathogens. These findings suggest that careful examination of the full factorial of drug combinations is needed to uncover suppressive interactions in higher-order combinations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer ...gain-of-function activity by converting α-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-γ-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.
During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off ...between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into ...patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse.
During immune surveillance, CD8 T cells scan the surface of antigen-presenting cells using dynamic microvillar palpation and movements as well as by having their receptors preconcentrated into ...patches. Here, we use real-time lattice light-sheet microscopy to demonstrate the independence of microvillar and membrane receptor patch scanning. While T cell receptor (TCR) patches can distribute to microvilli, they do so stochastically and not preferentially as for other receptors such as CD62L. The distinctness of TCR patch movement from microvillar movement extends to many other receptors that form patches that also scan independent of the TCR. An exception to this is the CD8 coreceptor which largely comigrates in patches that overlap with or are closely adjacent to those containing TCRs. Microvilli that assemble into a synapse contain various arrays of the engaged patches, notably of TCRs and the inhibitory receptor PD-1, creating a pastiche of occupancies that vary from microvillar contact to contact. In summary, this work demonstrates that localization of receptor patches within the membrane and on microvillar projections is random prior to antigen detection and that such random variation may play into the generation of many individually composed receptor patch compositions at a single synapse.
A T cell’s ability to efficiently surveil its environment and recognize and respond appropriately to cognate antigen via its antigen receptor is a critical feature of adaptive immunity. Although ...cell-cell interactions are often depicted as two flat surfaces pushing up against each other with molecules moving laterally within a single plane, the reality is that T cells must probe three-dimensionally complex surfaces covered in relatively large, glycosylated molecules as they migrate through lymph nodes and other tissues during immune surveillance. Yet, it is unknown how T cells overcome these barriers to make very close contact with apposing membranes, bringing together the small membrane receptors that initiate T cell signaling. Prior to these studies, whether T cells use their membrane microvilli to aid in detection of antigen was largely unknown. Here, we used lattice light sheet (LLS) microscopy and synaptic contact mapping (SCM) total internal reflection fluorescence (TIRF) imaging to visualize microvilli on T cells with high spatiotemporal resolution prior to and during ligand detection by T cells. We found that T cell microvilli are highly dynamic structures that efficiently probe surfaces in physiologically relevant timescales. Specifically upon binding of antigen receptor to cognate ligand, the underlying microvillus became stabilized, providing a persistent surface for signaling. Of note, ZAP70 signaling and actin polymerization were not required to maintain these close contacts after binding to cognate antigen, indicating that physical pinning of membranes via membrane receptors is a significant contributor to changes in membrane dynamics. Given that natural antigen receptors make use of this cell biology, we then asked whether engineered chimeric antigen receptors (CARs) interact similarly. We found that CARs distributed in the plasma membrane similarly to natural T cell receptors (TCRs) although notably in distinct patches. However, when engaging ligands these induced hyper-stabilization of the underlying microvilli relative to that of the TCR. This hyper-stabilization was dependent on the high affinity and avidity of CAR binding and was associated with altered organization of molecules at the cell-cell interface, decreased effector function, and increased propensity for exhaustion. Thus, although microvillar search is involved in both natural and synthetic ligand detection, microvillar dynamics are differentially altered depending on strength of receptor binding. This work reveals the cell biology underlying T cell ligand detection and its sensitivity to changes in binding dynamics, with likely clinical importance for the development of cell therapies.
T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors ...(TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.
Interactions among drugs play a critical role in the killing efficacy of multi-drug treatments. Recent advances in theory and experiment for three-drug interactions enable the search for emergent ...interactions—ones not predictable from pairwise interactions. Previous work has shown it is easier to detect synergies and antagonisms among pairwise interactions when a rescaling method is applied to the interaction metric. However, no study has carefully examined whether new types of normalization might be needed for emergence. Here, we propose several rescaling methods for enhancing the classification of the higher order drug interactions based on our conceptual framework. To choose the rescaling that best separates synergism, antagonism and additivity, we conducted bacterial growth experiments in the presence of single, pairwise and triple-drug combinations among 14 antibiotics. We found one of our rescaling methods is far better at distinguishing synergistic and antagonistic emergent interactions than any of the other methods. Using our new method, we find around 50% of emergent interactions are additive, much less than previous reports of greater than 90% additivity. We conclude that higher order emergent interactions are much more common than previously believed, and we argue these findings for drugs suggest that appropriate rescaling is crucial to infer higher order interactions.
Gram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We used Escherichia coli mutants that are partially ...sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-type E. coli at concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enter E. coli but normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489-494, 2006, http://dx.doi.org/10.1038/ng1755).
Generation of tumor-infiltrating lymphocytes (TILs) begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated amongst the ...large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DC and are then transferred amongst DC subsets. A synapse is formed between interacting DC and vesicle transfer takes place in the absence of free exosomes. DC containing vesicles can uniquely activate T cells whereas DC lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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