this subset analysis showed consistent, tissue - specific composition between sites. to understand the influence of tissue localization on gene expression, we performed a two - step differential expression ( de ) analysis. first, we compared the major immune lineages ( cd8 t cells, cd4 t cells, γδ / μαιτ cells, myeloid cells, nk / ilc and b cells ) within each site ( for example, blood, bm, spleen, ln, lung, jej ) versus the other sites using pseudobulked linear mixed modeling ( lmm ), controlling for covariates ( for example, sex, cmv serostatus ) ( fig. 3a and supplementary tables 4 and 5 ). we identified 13 clusters from significantly differentially expressed genes ( degs ) ( c1 – 13 ) grouped by lineage and / or tissue ( fig. 3b and supplementary table 6 ). we then conducted a similar across - tissue de analysis for each subset and evaluated whether these signatures were expressed by individual subsets within a lineage using gene set enrichment analysis ( gsea ) ( fig. 3a ). to visualize effect sizes, determine the contribution of compositional differences and identify tissue - specific signatures across lineages, we integrated this analysis with subset frequencies within a tissue, fold changes ( fc ) compared to the other sites and the average expression of gene clusters by subset ( fig. 3c – h, extended data fig. 3, and supplementary tables 7 and 8 ). myeloid cells from all tissues exhibited transcriptional profiles not expressed by other immune lineages ( clusters c1 – c4 ), which also varied by tissue ( fig. 3b ). c1 ( chemok ine, complement, lipid transport ) and c2 ( ppar signaling associated with alveol ar macrophages ) were enriched in the jej and lung, respectively, particularly in macrophages ( fig. 3c, d ). by contrast, c4 ( anti - microbial peptide production and cell signaling ) enrichment was explained by increased frequencies of bm and spleen monocytes relative to macrophages rather than transcriptional changes within and across subsets ( extended data fig. 3a, b ). tissue - associated genes within clusters c5 – c9 were expressed primarily by t cells and innate lymphocytes ( fig. 3b ). c5 genes encoding stem - like transcription factors and markers ( tcf7, lef1, itga6 ) and ln homing receptors ( ccr7, sell ) were enriched in cd4 and cd8 t rm cells and t em cells in ln ( fig. 3e, f ). conversely, c6 genes encoding molecules for mucosal residency ( itga1, cxcr6, itgae ) and gut homing ( ccr9 ) were enriched in cd4 and cd8 t rm cells, γδ t cells, mait cells and nk cells in the jej ( fig. 3e, f ). cd8 t emra cells, γδ cells, mait cells and nk / ilcs in the jej showed reduced expression of c9 genes associated with cytolytic effector function ( gzmb, prf1, ifng, nkg 7 ) compared to other sites, while c9 was enriched in the lung ( fig. 3b and extended data fig. 3c, d ). c7 genes associated with protein metabolism were enriched in nk / ilcs and innate t cells in the jej, while c8 genes encoding chemok ines ( ccl4, xcl1 ) and innate lymphocyte functions ( klrc1, ncr1 ) were expressed by t cell and nk / ilc subsets across all sites ( fig. 3b and extended data fig. 3e, f ). therefore, this analysis identified shared gene expression profiles across t and innate lymphocyte subsets that varied by site. tissue - enriched signatures for b cells ( c10 – c13 ) were mostly associated with specific subsets ( fig. 3b, g, h and extended data fig. 3g, h ). c10 included cell cycle genes and was enriched in b cells and plasma cells in the lungs ( fig. 3g, h ), possibly because of higher plasmablast abundance. jej b cells showed increased expression of c12 ( b cell differentiation and protein transport ) and c13 ( b cell activation ) genes ( fig. 3b ), which were derived from plasma cells comprising the majority of the b cell lineage in intestinal sites ( fig. 3g, h ). we also applied consensus single - cell hierarchical poisson factorization ( schpf ) to identify gene co - expression patterns common to different immune lineages or sites ( considering the jej, lung and ln ) ( extended data fig. 4a and supplementary table 9 ). we found a proliferation module enriched in the lungs, a lymphoid - specific module in the lns, an intestine - specific residency module and modules associated with effector and cytolytic functions prominent in the lungs but not jej or lns ( extended data fig. 4a – d ). the lymphoid module included genes associated with stemness ( klf7, lef1, sox4 ) and lymphoid homing ( ccr7, sell, itga6 ) markers expressed mostly in t cells, some nk cells and ilcs, and not in b cells or myeloid cells ( extended data fig. 4b – e and supplementary table 10 ). the jej residency module included intestinal tissue residency genes ( ccr9, itgae, cd101, cd160 ) enriched in t cells, nk cells and ilcs ( extended data fig. 4f – h ). these signatures were also reflected at the surface protein level ( extended data fig. 4e, i and supplementary table 11 ). thus, tissue - specific gene expression modules spanned multiple cell types, suggesting shared tissue adaptations. the tissue environment poses unique requirements for resident immune populations such as t rm cells, plasma cells and macrophages for maintaining homeostasis. we integrated the de analysis shown in fig. 3 with surface protein expression to define site - specific signatures for resident immune cells ( fig. 4 and supplementary table 12 ). cd4 and cd8 t rm cells expressed genes and / or surface proteins for tissue residency ( cd103, cd101, cd49a ), gut homing and localization ( ccr9, ccr5 for cd4 t rm cells ) and reduced pd - 1 ( for cd8 t rm cells ) in intestines relative to lungs and lymphoid organs ( fig. 4a – f ). lung t rm cells showed increased expression of effector or cytotoxicity ( ifng, gzmh, gzma, prf1 ) and regulatory ( ctla4 ) genes relative to the jej and lymphoid organs; t rm cells in ln, spleen and bm had higher expression of stem - like markers ( tcf7, klf2 ) and certain integrins and costimulatory markers ( itgb2, cd27, cd28, icos ) relative to the jej and lungs ( fig. 4a – f ). these site - specific profiles for t rm cells showed adaptations related to migration, localization and function. tissue plasma cells and macrophages also exhibited distinc t tissue signatures. for plasma cells, we found several jej - enriched genes, including igha2 ( consistent with predominant iga plasma cells in the gut ), the non - classical hla molecule cd1d, plasma cell transcription factors ( runx2, id3 ) and the tissue residency marker cd69 ( fig. 4g – i ). plasma cells in lungs, and to a lesser extent, ln and spleen, expressed higher levels of integrins ( cd11c, cd18 ) ( fig. 4g – i ). jej macrophages had increased expression of integrins ( icam1, itga4 ), chemok ines ( ccl24, ccl3l1 ) and regulators of macrophage activation ( for example, slamf7 ), whereas lung macrophages had higher expression of cd11c ( itgax ), markers of efferocytosis ( mrc1, marco ) and lipid metabolism ( pparg, trem2, fabp4 ) ( fig. 4j – l ), consistent with alveol ar macrophages. lastly, macrophages in the spleen expressed markers of red pulp macrophages ( spic, vcam1 ) ( fig. 4j ). these observations revealed site - specific signatures for activation, migration, metabolism and cell – cell interactions involved in tissue residency. we investigated age - associated effects across immune lineages and tissues. a global analysis of transcriptional variance within each major lineage revealed that tissue site explained the majority of variance, while age accounted for a much smaller fraction ( fig. 5a and supplementary table 13 ). most of the top age - dependent genes in each lineage ( fig. 5b ) also exhibited tissue - specific variation.