Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumour cell intrinsic and microenvironmental features underpinning CPI sensitization. Here we collated whole-exome and transcriptomic data for >1000 CPI-treated patients across eight tumor-types, utilizing standardized bioinformatics-workflows and clinical outcome-criteria to validate multivariate predictors of CPI-sensitization. Clonal-TMB was the strongest predictor of CPI response, followed by TMB and CXCL9 expression. Subclonal-TMB, somatic copy alteration burden and HLA-evolutionary divergence failed to attain significance. Discovery analysis identified two additional determinants of CPI-response supported by prior functional evidence: 9q34.3 (TRAF2) loss and CCND1 amplification, both independently validated in >1600 CPI-treated patients. We find evidence for collateral sensitivity, likely mediated through selection for CDKN2A-loss, with 9q34.3 loss as a passenger event leading to CPI-sensitization. Finally, scRNA sequencing of clonal neoantigen-reactive CD8-TILs, combined with bulk RNAseq analysis of CPI responding tumors, identified CCR5 and CXCL13 as T cell-intrinsic mediators of CPI-sensitisation.

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SummaryCarbon dioxide (CO 2 ) is the most commonly used agent for euthanasia of laboratory rodents, used on an estimated tens of millions of laboratory rodents per year worldwide, yet there is a growing body of evidence indicating that exposure to CO 2 causes more than momentary pain and distress in these and other animals. We reviewed the available literature on the use of CO 2 for euthanasia (as well as anaesthesia) and also informally canvassed laboratory animal personnel for their opinions regarding this topic. Our review addresses key issues such as CO 2 flow rate and final concentration, presence of oxygen, and prefilled chambers (the animal is added to the chamber once a predetermined concentration and flow rate have been reached) versus gradual induction (the animal is put into an empty chamber and the gas agent(s) is gradually introduced at a fixed rate). Internationally, animal research standards specify that any procedure that would cause pain or distress in humans should be assumed to do so in non-human animals as well (Public Health Service 1986, US Department of Agriculture 1997, Organization for Economic Cooperation and Development 2000). European Union guidelines, however, specify a certain threshold of pain or distress, such as 'skilled insertion of a hypodermic needle', as the starting point at which regulation of the use of animals in experimental or other scientific procedures begins (Biotechnology Regulatory Atlas n.d.). There is clear evidence in the human literature that CO 2 exposure is painful and distressful, while the non-human literature is equivocal. However, the fact that a number of studies do conclude that CO 2 causes pain and distress in animals indicates a need for careful reconsideration of its use. Finally, this review offers recommendations for alternatives to the use of CO 2 as a euthanasia agent.Keywords Carbon dioxide; euthanasia; pain; distress; anaesthesia; welfare; rodents Carbon dioxide (CO 2 ) is commonly used for euthanasia and anaesthesia of laboratory rodents, largely because of its ease of use, relative safety, and low cost, as well as its capacity to euthanize large numbers of animals in a short time span (Ambrose et al. 2000). In large institutions and those with significant rodent-breeding programmes, large numbers of rodents are often euthanized in a short time (Kline et al. 1963) and an appropriate gas agent is often the best way to approach such a challenge. However, CO 2 is not physiologically inert and the published evidence on whether or not CO 2 administration causes pain or distress in animals raises questions about its routine use. This paper reviews this published evidence and includes information on the effects of CO 2 in both humans and nonhumans. Methodological details are included when possible in order to provide a clear

Simple SummaryThe pet overpopulation problem in the United States has changed significantly since the 1970s. The purpose of this review is to document these changes and propose factors that have been and are currently driving the dog population dynamics in the US. In the 1960s, about one quarter of the dog population was still roaming the streets (whether owned or not) and 10 to 20-fold more dogs were euthanized in shelters compared to the present. We present data from across the United States which support the idea that, along with increased responsible pet ownership behaviors, sterilization efforts in shelters and private veterinary hospitals have played a role driving and sustaining the decline in unwanted animals entering shelters (and being euthanized). Additionally, data shows that adoption numbers are rising slowly across the US and have become an additional driver of declining euthanasia numbers in the last decade. We conclude that the cultural shift in how society and pet owners relate to dogs has produced positive shelter trends beyond the decline in intake. The increased level of control and care dog owners provide to their dogs, as well as the increasing perception of dogs as family members, are all indicators of the changing human-dog relationship in the US.AbstractDog management in the United States has evolved considerably over the last 40 years. This review analyzes available data from the last 30 to 40 years to identify national and local trends. In 1973, The Humane Society of the US (The HSUS) estimated that about 13.5 million animals (64 dogs and cats per 1000 people) were euthanized in the US (about 20% of the pet population) and about 25% of the dog population was still roaming the streets. Intake and euthanasia numbers (national and state level) declined rapidly in the 1970s due to a number of factors, including the implementation of shelter sterilization policies, changes in sterilization practices by private veterinarians and the passage of local ordinances implementing differential licensing fees for intact and sterilized pets. By the mid-1980s, shelter intake had declined by about 50% (The HSUS estimated 7.6–10 million animals euthanized in 1985). Data collected by PetPoint over the past eight years indicate that adoptions increased in the last decade and may have become an additional driver affecting recent euthanasia declines across the US. We suspect that sterilizations, now part of the standard veterinary care, and the level of control of pet dogs exercised by pet owners (roaming dogs are now mostly absent in many US communities) played an important part in the cultural shift in the US, in which a larger proportion of families now regard their pet dogs as “family members”.

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