integrating single - cell and spatial transcriptomics data, we have dissected the molecular and cellular basis of the histological changes in cd, including villus epithelial changes, crypt hyperplasia and intraepithelial lymphocytosis. this cellular, spatial transcriptomics description builds on the marsh – oberhuber histological description, with complex, highly localized, mucosal cell communities, including focal lymphoid organization where specific cell types, including gluten - specific t fh - like cd4 t cells, b cells and t reg cells, are co - located. overall, our study of the mucosal cellular and spatial landscape in cd provides a detailed foundation from which to explore potential therapeutic targets, and highlights the need to explore the clinical implications of the prolonged epithelial – immune scar in tcd. study participants with cd, and hcs, were identified via oxford university hospitals nhs trust cd clinic and endosc opy service ( oxford, uk ). blood and intestinal biopsy samples were taken at endosc opy with informed consent under the oxford gastrointestinal illnesses biobank study ( rec: 21 / yh / 0206 ). study participant demographics and study inclusion / exclusion criteria are summarized in supplementary table 1. participants were not compensated financially. peripheral blood mononuclear cells were extracted from whole blood or leukocyte cones via density gra dien t centrifugation. briefly, peripheral blood was diluted at a 1: 1 ratio with dulbecco ’ s phosphate buffered saline without calcium or magnesium ( pbs ) and layered over lymphoprep ( axis - shield ) before centrifugation ( 973 g for 30 min at 20 °c without brake ). the mononuclear layer was retrieved and washed twice in pbs or r10 culture media ( rpmi - 1640 ( sigma - aldrich ), 10 % fcs, 1 % penicillin / streptomycin, 1 % l - glutamine ). viable mononuclear cells stained with trypan blue were counted manually by microscopy using a hemocytometer before downstream applications. if remnant red blood cells were present, they were lysed with ammonium – chloride – potassium solution for 2 – 3 min, then washed again in r10. samples were cryopreserved in freezing medium ( 90 % fcs ( sigma - aldrich ), 10 % dimethylsulfoxide ( sigma - aldrich ) ). when needed, samples were thawed rapidly in a water bath ( 37 °c ), then washed twice in r10 before downstream use. intestinal biopsy samples were collected at endosc opy from duodenum. biopsy samples for intestinal lymphocyte extraction were immediately placed in sterile r10 medium ( as above ) or macs tissue storage solution ( miltenyi biotec ) on ice for transportation, before cryopreservation in cryostor cs10 ( stemcell technologies ). this approach preserves immune cell viability and surface marker expression. when required, samples were rapidly thawed in a 37 °c water bath and washed in 20 ml r10 before tissue dissociation. for immune cell isolation from duodenal biopsy samples for scrna - seq ( 10x genomics, dataset 1 ), biopsy samples were incubated in r10 medium with 1 mg ml collagenase d ( roche ) and 100 mg ml dnase ( thermo fisher scientific ) for 1 h in a shaking incubator at 37 °c. biopsy samples were then dissociated by vigorous agitation using a gentlemacs dissociator ( miltenyi biotec ), then strained through a 70 - μm filter. the mononuclear cells were isolated on a discontinuous 70 % and 35 % percoll gra dien t ( ge healthcare ) by centrifugation at 700 g for 20 min without brake. mononuclear cells were collected from the interface and washed in r10. cells were washed with r10 medium before antibod y staining and downstream applications. the complete protocol with all steps is available at https: / / www. protocols. io / view / freezing - and - processing - intestinal - biopsies - for - th - dm6gp8745lzp / v1 / ( oxford hca, 2019 ). the method for ec isolation from duodenal biopsy samples for scrna - seq ( 10x genomics, dataset 1 ) was adapted from ref.. biopsy samples were washed in wash medium ( hpga, 1 mm edta, 1 mm dithioth reitol ), then incubated in chelation medium ( hpga, 1 mm edta ) at 37 °c for 40 min with agitation. the supernatant, which was removed and replaced every 10 min and contained epithelial crypts, was digested into a single - cell suspension by dissociation in a shaking incubator with tryple express and dnase ( 50 µg ml ) for 60 min at 37 °c. the epithelial single - cell suspension was washed with pbs and passed through a 30 - µm filter. cell counts and viability were confirmed with a manual hemocytometer before further processing. the complete protocol with all steps is available at https: / / www. protocols. io / view / isolation - of - cells - from - the - epithelial - layer - of - fr - e6nvw9e6dgmk / v1 / ( oxford hca, 2019 ). for immune cell isolation from duodenal biopsy samples for scrna - seq and proteomics ( bd rhapsody, dataset 2 ), samples were thawed, then diluted with warm x - vivo ( lonza ) + 1 % ab serum ( sigma - aldrich ). biopsy samples underwent enzymatic and mechanical digestion using 0. 042 mg ml liberase tl ( roche ) and 1 mg ml dnase i ( thermo fisher ). samples were placed horizontally in a shaking incubator for 20 min at 37 °c and homogenized using a gentlemacs dissociator ( miltenyi biotech ). dissociated cells were passed through a 70 - μm strainer. lymphocytes were enriched by percoll density centrifugation as above. duodenal biopsy samples for flow cytometry were processed as for scrna - seq of immune cells ( see above ). cells were washed with r10 medium before antibod y staining and downstream applications. duodenal biopsy samples for fluorescence - activated cell sorting ( facs ) of iels for rna - seq and tcr repertoire sequencing were placed in 10 ml hbss with 1 mm edta and 1 mm dithioth reitol ( both sigma - aldrich ) and placed in a shaking incubator ( 200 rpm, 37 °c ) for 15 min. iels were strained through a 70 - μm filter and washed two to three times with r10 before downstream applications. for spatial transcriptomics, single intestinal biopsy samples were embedded in oct cryo - embedding matrix ( thermo fisher scientific ) then frozen in isopentane ( sigma - aldrich ) suspended over liquid nitrogen or dry ice, and stored at −80 °c until use. for surface marker staining, cells were stained in 50 ml of facs buffer ( pbs + 1 mm edta + 0. 05 % bsa ) for 30 min at 4 °c. surface antibod ies and clones used are listed in supplementary table 3. antibod ies were purchased from biolegend, bd biosciences, miltenyi biotec or thermo fisher scientific. after staining, cells were stored at 4 °c protected from light until data acquisition. flow cytometry data were acquired on a bd lsr ii flow cytometer ( bd biosciences ). facs samples were surface stained as above, with sytox green ( thermo fisher scientific ) used as a viability dye. facs was performed on an aria iii ( bd biosciences; 70 - mm nozzle ). for sorting by facs for scrna - seq of intestinal immune populations, cells were stained with epcam - pe, cd27 - bv421 and cd45 - apc - cy7. live cd45 or cd27 cells were sorted to include all mucosal immune cell populations, including long - lived cd27 plasma cells, which can downregulate surface cd45 expression. for sorting by facs for scrna - seq of intestinal epithelial populations, cells were stained with epcam - pe and cd45 - af700, with live epcam cells sorted. for sorting by facs for bulk rna - seq or tcr - seq of cd8 iel populations, cells were stained with cd45 - bv785, cd3 - bv711, αβtcr - apc, γδtcr - pe, cd4 - bv650 and cd8a - af700, with live cd45 cd3 αβtcr cd8 cd4 cells sorted. libraries were generated using 10x genomics chromium single cell v ( d ) j reagents kits ( v1 chemistry ) per the manufacturer ’ s instructions. sorted cells suspended in pbs ( plus 0. 04 % bsa ) at a concentration of 1, 000 cells per microliter were loaded into one lane of a chromium controller.