Tissue samples (spleen, bursa of Fabricius, jejunum, caecum, and colon) were derived from an in vivo study carried out at the infectiology platform PFIE (INRA, Val de Loire) in accordance with the national and international regulations specific to the research facility as part of the Development of Immune Function and Avian Gut Health (DIFAGH) consortium. One group of PA12 white leghorn chickens (n=5) was reared under germ-free conditions, whilst another group (n=5) was reared under conventional specific pathogen-free (SPF) conditions. For the germ-free birds, the eggs were collected immediately after laying and surface sterilized by immersion in 1.5% Divosan Plus VT53 (Johnson Diversey, France) for 5 minutes at room temperature. Subsequently, these eggs were transferred into HEPA-filtered incubator. After 18 days at 37°C, the surface of the eggs was sterilized in 1.25% Divosan for 4 min at 37 °C. After hatching, the temperature of the isolator was maintained at 37.5 °C for 7 days, then reduced by 1 °C per day until reaching a stable temperature of 25 °C. Chickens were offered X ray-irradiated starter diet (Special Diets Services; Dietex, Argenteuil, France) and sterilized water ad libitum. The sterility of chickens was confirmed weekly by incubating fresh faecal droppings in 10 mL of sterile brain–heart infusion broth under both aerobic and anaerobic conditions, which allows for bacterial, yeast, and fungal growth. The absence of non-culturable bacteria in faecal and caecal samples of germ-free chickens was confirmed by quantitative PCR of a conserved region of the bacterial ribosomal 16S gene. Individuals from both groups were culled at day 55 post-hatch, when tissue samples were harvested and preserved in RNAlater (Thermo Fisher Scientific) according to manufacturer's instructions. Samples were stored at –800C prior to processing. Tissue samples were weighed and 15 mg of each sample was placed in 600 µl of RLT lysis buffer (Qiagen) together with 100 µl of 0.2 mm silica beads (Thistle Scientific), and subjected to five cycles of 1-minute homogenisation in a Mini-Beadbeater-24 (BioSpec) and 30 seconds of cooling on ice. RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol. For all samples, on-column genomic DNA digestion was performed, using the RNase-Free DNase Set (Qiagen) in accordance with the manufacturer’s instructions. The resulting RNA was eluted in 40 µl of nuclease-free water. The quality and integrity of several samples was tested using an RNA ScreenTape (Agilent Technologies) on the 4200 TapeStation (Agilent Technologies). The RNA samples were stored at –80 °C until further processing. 5’RACE-ready cDNA was generated using the SMARTer kit (Takara), following the manufacturer’s instructions. Subsequently, for each cDNA sample, specific 7-bp barcoded forward primers (5’-NNNNNNNGAAAAGATGACCACATCTGGTTC-3’) for the chicken TCRβ were used for the 5’RACE PCRs. Universal SMARTer kit reverse primers were used for all samples, specific to the common 5’ adapter that was added during 5’RACE cDNA synthesis. Briefly, for each 25 µl reaction, 5 µl of Phusion 5X Buffer (New England Biolabs), 0.5 µl of 10 mM dNTP, 0.5 µl of 10 µM UPA-short primer, 0.5 µl of 2 µM UPA-long and 0.25 µl Phusion Hot Start Flex DNA Polymerase (New England Biolabs) were added to 15.25 µl nuclease-free water, for a total of 22 µl volume. To this, 0.5 µl of the 10µM gene-specific 7 bp-barcoded primer and 2.5 µl of cDNA were added. The individual 25 µl volume 5’RACE PCR reactions were then carried out in 96-well plates using the thermocycler program recommended by the 5’RACE kit (Takara), with 35 cycles of gene-specific amplification with an annealing temperature of 60 °C. After PCR amplification, barcoded samples were pooled and subjected to electrophoresis on a 1.4% agarose in Tris/Borate/EDTA (TBE) buffer gel containing 1:10,000 SYBR green (Sigma-Aldrich) at 120 V for 35 minutes. The bands of the expected lengths were gel extracted and purified using the QIAquick Gel Extraction Kit (Qiagen). DNA libraries of the pooled barcoded PCR samples were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s instructions. Briefly, this involved the ligation of library adapters, excision, PCR enrichment, and clean-up using AMPure XP beads. The quantity and quality of the DNA libraries were analysed using the NEBNext Library Quant Kit for Illumina (New England Biolabs) and a D1000 DNA tape (Agilent Technologies) on the 4200 TapeStation (Agilent Technologies). The libraries were sequenced using an Illumina MiSeq platform at the Department of Biology, University of Oxford.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRβ repertoire and the influences of microbial colonisation on tissue-resident TCRβ+ cells. The results reveal that the microbiome is an important driver of TCRβ diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRβ sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRβ clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide insight into the avian adaptive immune system and the influence of the microbiota on the TCRβ repertoire.

This is a *.csv file and can be opened with programs such as Microsoft Office Excel. For processing and analysis, we recommend the use of a program such as R studio, as was done during the data analysis described in the publication.