The immune system is the primary interface between an organism and the environment. Interactions with pathogens are constantly changing immune system function across the primate lineage. Host-pathogen interactions represent a major selective pressure for the evolution of primates and as a result immune response genes are among the most rapidly evolving classes of genes. One way that interactions with pathogens can change the function of the immune system is through mutations to cis-regulatory elements that control gene regulation, which can alter gene expression levels and tissue specificity. However, regulatory evolution is not well studied, especially in the context of host-pathogen interactions where most work has focused on changes to protein coding exons. Preliminary data shows that genes encoding transmembrane receptors (which play a central role in host-pathogen interactions) are among the most differentially transcribed genes between human, chimpanzee, and rhesus macaque CD4+ T-cells using Precision run-on and sequencing (PRO-seq). This data supports a broad role for host-pathogen interactions driving regulatory evolution. An excellent example from this data is an 8-fold decrease in ANTXR2 transcription in humans compared with other primate species. ANTXR2 encodes a transmembrane receptor that allows toxins produced by the B. anthracis bacterium to enter cells, leading to anthrax disease and ultimately death of the host. We hypothesize that this change in transcription was caused by selective pressures on humans due to historical human exposure to anthrax. The goal of this work is to understand the impact of human-specific changes in ANTXR2 transcription on sensitivity to anthrax disease. Human and rhesus macaque CD4+ T-cells will be collected, challenged with anthrax toxins, and assayed for viability and cytokine signaling. Expression and genotyping data across human populations and primate species will be analyzed to find signatures of selection and to identify candidate causal alleles. Promising candidate causal alleles will be tested experimentally using reporter assays, viability experiments, and genome editing. Together, the work completed during this proposal will help to understand (1) if certain human populations are more resistant to anthrax disease than non-human primates, (2) how natural selection has shaped the human immune system in response to increasing exposure to anthrax disease, and (3) which DNA sequences are responsible for mediating the changes in ANTXR2 transcription in humans, and how these have contributed to changes in anthrax sensitivity. The proposed work will aid in unraveling the mechanistic basis of regulatory evolution in host-pathogen interactions and potentially provide insight into how disease outcome has been shaped by evolution. This work will be guided by a team of sponsors with expertise in genomics and population genetics in an interdisciplinary environment that fosters collaboration. The proposed training plan will allow the trainee to gain the skills, practical experience, and knowledge needed for a successful research career.