29The relative contributions of genetic and environmental factors to variation in immune 30 responses are still poorly understood. Here, we performed a deep phenotypic analysis of 31 immunological parameters of laboratory mice released into an outdoor enclosure, carrying 32 susceptibility genes (Nod2 and Atg16l1) implicated in the development of inflammatory 33 bowel diseases. Variations of immune cell populations were largely driven by environment, 34whereas cytokine production in response to stimulation was affected more by genetic 35 mutations. Multi-omic models identified transcriptional signatures associated with differences 36 in T cell populations. Subnetworks associated with responses against Clostridium perfringens, 37 Candida albicans and Bacteroides vulgatus were also coupled with rewilding. Hence, 38 exposing laboratory mice carrying different genetic mutations to a natural environment 39 uncovered important contributors to immune variation.40 41 One sentence summary 42 Natural environment exposure in laboratory mice primarily promotes variation in population 43 frequencies of immune cells, whereas cytokine responses to stimulation are affected more by 44 genetic susceptibility to inflammatory bowel disease. 45 46 47 93 in wild type animals, we reasoned that they could be an interesting model system to 94 investigate gene-environment interactions in the outdoor enclosure.95We hypothesized that by introducing laboratory mice carrying IBD susceptibility 96 mutations into the outdoor enclosure, the drastic change in environment may trigger 97 alterations in the immune response and the microbiota, which would have greater adverse 98 effects on mutant mice than wildtype mice. Hence, to study the immunological consequences 99 of rewilding, we released Atg16l1 T316A/T316A , Atg16l1 T316A/+ , Nod2 -/mice in addition to 100 C57BL/6J wild type (WT) mice to determine whether mutations in these genes alter the 101 immune response to microbial exposure in a natural environment. Here we present a detailed 102 systems immunology profile of both rewilded and laboratory mice to further investigate 103 variation in immune response and the relationship with environment and genetic mutations. 105 RESULTS 106Study design for immune profiling of rewilded and laboratory mice 107 From 116 mice released into the outdoor enclosure for 6-7 weeks we recovered 104 mice 108 (25 WT, 28 Nod2 -/-, 27 Atg16l1 T316A/+ , and 24 Atg16l1 T316A/T316A ) in time for analysis and 109 compared them to 80 matched controls (19 WT, 19 Nod2 -/-, 20 Atg16l1 T316A/+ , 22 110 Atg16l1 T316A/T316A ) maintained under specific pathogen free (SPF) conditions (herein referred 111 to as lab mice) ( Figure 1A). Blood samples were collected at the time of sacrifice and 112 analyzed by flow cytometry with a lymphocyte panel (Table S1). Additionally, cytokine 113 production in the plasma was assayed ( Figure 1A). Fecal samples and cecal contents were 114 collected for microbial profiling, and for reconstitution experiments (Companion study, 115 Yeung et. al.). ...