hla class ii gene and protein expression, specifically hla - dq, was highest in cd27 and cycling b cells ( extended data fig. 4g, h ). intestinal myeloid cell populations are impacted by cd and may be involved in antigen presentation and oral tolerance. myeloid cells ( dataset 2 ) formed 11 transcriptionally distinc t clusters, including macrophages, conventional dendritic cells and plasmacytoid dendritic cells ( supplementary fig. 2a – c ). hla - dq expression was highest on macrophage populations, particularly cd163 cells. in contrast to prior studies, cd163 macrophages were reduced in acd, with expansion of a conventional dendritic cell 2 population, which showed increased il - 1b expression ( supplementary fig. 2d, e ). intestinal cd8 t cells showed considerable heterogeneity in transcriptional states, with multiple tissue - resident memory cd8 t ( t rm ) cells, including an itgae il7r population, a ccl4 cd69 itgae population and two subsets of itgae t rm cells ( fig. 4, extended data fig. 5a and supplementary table 7 ). these aligned with gene signatures defining subsets of bona fide human t rm cells. fgfbp2 effectors aligned with previously described itgb2 itgae t rm cells, while t rm ( 1 ), t rm ( 2 ) and cycling subsets aligned with cd103 t rm cells ( extended data fig. 5b ). ccl4 and il7r populations likely represent intermediate states in t rm cell development. small natural iel and cycling mki67 populations were seen ( extended data fig. 5b, c ). analogous cd8 t cell subsets were seen in dataset 2 ( fig. 4d, e and extended data fig. 5a ), with additional resolution for tissue - resident γδ t cells, and innate - like t cells ( mucosal - associated invariant t cells and vδ2vγ9 cells ). we analyzed subsets relevant to cd, including natural killer ( nk ) - receptor expressing iels and killer - cell immunoglobulin - like receptor ( kir ) - positive cd8 t cells. klrc1 ( nkg2a ) was expressed by ccl4 cells, while klrc2 ( nkg2c ) was expressed by resident il7r, t rm ( 1 ) and t rm ( 2 ) subsets ( extended data fig. 5c, d ). inhibitory kir molecule expression was confined to a small fgfbp2 effector population. t rm ( 2 ) and cycling populations were enriched in acd, but not t rm ( 1 ) cells ( fig. 4f, g ). t rm ( 2 ) cells were rare in health, but increased to form 20 – 40 % of cd8 t cells in acd, which persisted in tcd ( fig. 4h ). natural iels were reduced in acd ( extended data fig. 5e ). cycling cd8 t cells increased to form 2 – 4 % of cells in acd ( fig. 4i ). most cycling cells showed a t rm ( 2 ) phenotype ( extended data fig. 5f ). as t rm ( 2 ) cd8 t cells were increased in proportion and proliferating in acd, we profiled them in depth ( fig. 5a – e and extended data fig. 6 ). t rm ( 2 ) cd8 t cells showed a cd103 tissue - resident phenotype, high gzma and absent gzmk expression, along with high expression of cxcr6, activation markers ( hla - dr ) and genes expressing co - stimulatory and co - inhibitory molecules ( tigit, tnfrsf9 ( 4 - 1bb ), entpd1 ( cd39 ) and layn ( fig. 4b, c ). comparison of t rm ( 2 ) cells in acd versus tcd showed increased expression of activation markers and increased effector function with ifng, gzmb and il32 expression ( extended data fig. 5g ). we examined pseudotime trajectories of tissue - resident clusters with tcr repertoire clonal sharing ( t rm ( 1 ), t rm ( 2 ), il7r and ccl4 effectors ) to infer putative differentiation pathways ( fig. 5a, b, e ). the pseudotime trajectory showed two branches, formed predominantly of t rm ( 1 ) cells in branch 1 and t rm ( 2 ) cells in branch 2, developing from il7r and ccl4 populations ( fig. 5a ). while branch 1 cells were seen in both controls and cd, strikingly, branch 2 was almost totally restricted to acd and tcd ( fig. 5b ). we examined cytokine, chemok ine and tf expression by cd8 t cell subsets. the predominant cd8 sources of ifng were cd8 t rm ( 2 ) and cycling clusters ( fig. 5c, d and extended data fig. 5g – i ). these populations also expressed the chemok ine ccl5, cd70 and faslg. natural iels ( reduced in acd ), produced ccl2, cxcl2, cxcl3, il12, il18 and type i interferon. t rm ( 1 ) and t rm ( 2 ) cd8 subsets showed distinc t tf and regulon profiles; t rm ( 2 ) cells were associated with the tf regulons bach1, cebpz, crem, irf4 and nr3c1 and tf expression of rora, prdm and foxo1 ( extended data fig. 5j – l ). cd8 t cell - induced epithelial damage is thought to be mediated via tcr - independent mechanisms. we hypothesized that cd8 t cell tcr repertoires would be similar in health and disease. single - cell tcr sequences were examined, which showed expected clonal overlap between tissue - resident populations ( fig. 5e and extended data fig. 6a ). cluster trbv gene usage was examined between health and cd. several high - frequency trbv segments ( > 1 % total clones ) were overrepresented in cd ( fig. 5f and extended data fig. 6b ). however, statistical power was limited due to low clonotype numbers. consequently, we sorted intraepithelial cd8 t cells from 12 adults with and without cd ( dataset 3 ) and performed bulk rna - seq. this showed significant enrichment of one trbv segment, trbv28, enriched in acd and tcd, but not controls ( fig. 5g ). trbv28 was the high - frequency v segment with the highest fold change for enrichment in cd within the t rm ( 2 ) population ( fig. 5f ). we validated this by performing bulk tcr repertoire sequencing on 1, 068, 814 mucosal cd8 t cells from 20 donors with and without cd ( dataset 4 ). again, trbv28 was highly upregulated in cd, forming 10 % of unique cdr3 sequences in acd and tcd, versus 2 % in controls ( fig. 5h ). trbv28 was also enriched within the top 100 most expanded clonotypes. no association with trav usage was seen. clonotypes containing trbv28 in cd paired with multiple trbj segments, and showed altered cdr3 amino acid usage, with enrichment of germline - encoded and non - germline - encoded leucine residues ( extended data fig. 6c – e ). we examined bulk tcr repertoires of intestinal cd8 t cells of colonic and small intestinal biopsy samples from three separate studies examining non - cd inflammatory gastrointestinal conditions. there was no signal for enrichment of trbv28 gene usage in these disease settings ( extended data fig. 6f – h ). we hypothesized that differences in mucosal cd8 tcr repertoire / phenotype may be mirrored within gut - homing cd8 t cells in the circulation, as seen following gluten challenge. we examined trbv28 usage by circulating cd8 t cells using flow cytometry ( dataset 9 ). using tcr sequencing ( tcr - seq ), we validated the specificity of the trbv28 - specific antibod y clone ( jovi. 3; extended data fig. 7a – c ). as expected, there was no difference in the fraction of trbv28 cells in total peripheral cd3 or cd8 t cell compartments in participants with and without cd. however, within cd8 t cell populations expressing gut - specific chemok ines ( ccr9 ) or integrins ( cd103 / β - integrin ), the fraction of trbv28 cells was increased in acd and tcd ( extended data fig. 7d – h ). intraepithelial duodenal γδ t cells are increased in cd, although their role is unclear. we analyzed a further dataset of 5, 552 sorted intestinal cd8 αβ and γδ t cells ( dataset 8; extended data fig. 8 ). clustering of cell transcriptional states recapitulated the key populations described above ( extended data fig. 8a, b ). as previously, the t rm ( 2 ) population ( in this case split into ifng and ikzf2 subpopulations ) was increased in acd, along with cycling t cells ( extended data fig. 8c ). γδ t cells showed overlapping transcriptional profiles with mucosal cd8 αβ t cells, albeit with enrichment within specific clusters ( extended data fig. 8d, e ). γδ t cells were most enriched within a natural iel phenotype cluster and the gzmk / fgfbp2 effector populations, and were also present in the ccl4 effector and ikzf2 t rm ( 2 ) population.