A comprehensive single-cell map of T cell exhaustion-associated immune environments in human breast cancer

Immune checkpoint therapy in breast cancer remains restricted to triple negative patients, and long-term clinical benefit is rare. The primary aim of immune checkpoint blockade is to prevent or reverse exhausted T cell states, but T cell exhaustion in breast tumors is not well understood. Here, we use single-cell transcriptomics combined with imaging mass cytometry to systematically study immune environments of human breast tumors that either do or do not contain exhausted T cells, with a focus on luminal subtypes. We find that the presence of a PD-1high exhaustion-like T cell phenotype is associated with an inflammatory immune environment with a characteristic cytotoxic profile, increased myeloid cell activation, evidence for elevated immunomodulatory, chemotactic, and cytokine signaling, and accumulation of natural killer T cells. Tumors harboring exhausted-like T cells show increased expression of MHC-I on tumor cells and of CXCL13 on T cells, as well as altered spatial organization with more immature rather than mature tertiary lymphoid structures. Our data reveal fundamental differences between immune environments with and without exhausted T cells within luminal breast cancer, and show that expression of PD-1 and CXCL13 on T cells, and MHC-I - but not PD-L1 - on tumor cells are strong distinguishing features between these environments

Integrated Analysis Of Immunotherapy Treated Clear Cell Renal Cell Carcinomas: An Exploratory Study

Molecular or immunological differences between responders and nonresponders to immune checkpoint inhibitors (ICIs) of clear cell renal cell carcinomas (ccRCCs) remain incompletely understood. To address this question, we performed next-generation sequencing, methylation analysis, genome wide copy number analysis, targeted RNA sequencing and T-cell receptor sequencing, and we studied frequencies of tumor-infiltrating CD8+ T cells, presence of tertiary lymphoid structures (TLS) and PD-L1 expression in 8 treatment-naive ccRCC patients subsequently treated with ICI (3 responders, 5 nonresponders). Unexpectedly, we identified decreased frequencies of CD8+ tumor-infiltrating T cells and TLS, and a decreased expression of PD-L1 in ICI responders when compared with nonresponders. However, neither tumor-specific genetic alterations nor gene expression profiles correlated with response to ICI or the observed immune features. Our results underline the challenge to stratify ccRCC patients for immunotherapy based on routinely available pathologic primary tumor material, even with advanced technologies. Our findings emphasize the analysis of pretreated metastatic tissue in line with recent observations describing treatment effects on the tumor microenvironment. In addition, our data call for further investigation of additional parameters in a larger ccRCC cohort to understand the mechanistic implications of the observed differences in tumor-infiltrating CD8+ T cells, TLS, and PD-L1 expression

On Deciding Local Theory Extensions via E-matching

Satisfiability Modulo Theories (SMT) solvers incorporate decision procedures for theories of data types that commonly occur in software. This makes them important tools for automating verification problems. A limitation frequently encountered is that verification problems are often not fully expressible in the theories supported natively by the solvers. Many solvers allow the specification of application-specific theories as quantified axioms, but their handling is incomplete outside of narrow special cases. In this work, we show how SMT solvers can be used to obtain complete decision procedures for local theory extensions, an important class of theories that are decidable using finite instantiation of axioms. We present an algorithm that uses E-matching to generate instances incrementally during the search, significantly reducing the number of generated instances compared to eager instantiation strategies. We have used two SMT solvers to implement this algorithm and conducted an extensive experimental evaluation on benchmarks derived from verification conditions for heap-manipulating programs. We believe that our results are of interest to both the users of SMT solvers as well as their developers

Quantifier-Free Interpolation of a Theory of Arrays

The use of interpolants in model checking is becoming an enabling technology to allow fast and robust verification of hardware and software. The application of encodings based on the theory of arrays, however, is limited by the impossibility of deriving quantifier- free interpolants in general. In this paper, we show that it is possible to obtain quantifier-free interpolants for a Skolemized version of the extensional theory of arrays. We prove this in two ways: (1) non-constructively, by using the model theoretic notion of amalgamation, which is known to be equivalent to admit quantifier-free interpolation for universal theories; and (2) constructively, by designing an interpolating procedure, based on solving equations between array updates. (Interestingly, rewriting techniques are used in the key steps of the solver and its proof of correctness.) To the best of our knowledge, this is the first successful attempt of computing quantifier- free interpolants for a variant of the theory of arrays with extensionality

A Verification Toolkit for Numerical Transition Systems

This paper presents a publicly available toolkit and a benchmark suite for rigorous verification of Integer Numerical Transition Systems (INTS), which can be viewed as control-flow graphs whose edges are annotated by Presburger arithmetic formulas. We present FLATA and ELDARICA, two verification tools for INTS. The FLATA system is based on precise acceleration of the transition relation, while the ELDARICA system is based on predicate abstraction with interpolation-based counterexample-driven refinement. The ELDARICA verifier uses the PRINCESS theorem prover as a sound and complete interpolating prover for Presburger arithmetic. Both systems can solve several examples for which previous approaches failed, and present a useful baseline for verifying integer programs. The infrastructure is a starting point for rigorous benchmarking, competitions, and standardized communication between tools

Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS

Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP(+) astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS). Our results suggest that the subpopulation of GFAP(+) astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS

Formal mutation testing for Circus

International audienceContext: The demand from industry for more dependable and scalable test-development mechanisms has fostered the use of formal models to guide the generation of tests. Despite many advancements having been obtained with state-based models, such as Finite State Machines (FSMs) and Input/Output Transition Systems (IOTSs), more advanced formalisms are required to specify large, state-rich, concurrent systems. Circus, a state-rich process algebra combining Z, CSP and a refinement calculus, is suitable for this; however, deriving tests from such models is accordingly more challenging. Recently, a testing theory has been stated for Circus, allowing the verification of process refinement based on exhaustive test sets. Objective: We investigate fault-based testing for refinement from Circus specifications using mutation. We seek the benefits of such techniques in test-set quality assertion and fault-based test-case selection. We target results relevant not only for Circus, but to any process algebra for refinement that combines CSP with a data language. Method: We present a formal definition for fault-based test sets, extending the Circus testing theory, and an extensive study of mutation operators for Circus. Using these results, we propose an approach to generate tests to kill mutants. Finally, we explain how prototype tool support can be obtained with the implementation of a mutant generator, a translator from Circus to CSP, and a refinement checker for CSP, and with

Testing robots using CSP

This paper presents a technique for automatic generation of tests for robotic systems based on a domain-specific notation called RoboChart. This is a UML-like diagrammatic notation that embeds a component model suitable for robotic systems, and supports the definition of behavioural models using enriched state machines that can feature time properties. The formal semantics of RoboChart is given using tockCSP, a discrete-time variant of the process algebra CSP. In this paper, we use the example of a simple drone to illustrate an approach to generate tests from RoboChart models using a mutation tool called Wodel. From mutated models, tests are generated using the CSP model checker FDR. The testing theory of CSP justifies the soundness of the tests