together, our findings reveal the complex interplay between tissue, lineage, subset and age in immune homeostasis that is important for defining immune dysfunctions in disease. we isolated mononuclear cells ( mncs ) from blood, multiple lymphoid organs, lungs, airways, intestines and other sites using established protocols from 24 donors ( 10 females and 14 males, aged 20 – 75 years ) ( fig. 1a ). organ donors originated from new york city ( usa ) and cambridge ( uk ) and were free of chronic infection, cancer and overt disease ( supplementary table 1 ). we performed single - cell rna sequencing ( scrna - seq ) on tissues from all donors, including cite - seq with 127 proteins from 22 donors ( supplementary table 2 ). we obtained 1. 28 million immune cell events from 10 sites with > 75, 000 cells per site, including blood, bone marrow ( bm ), spleen, different lymph nodes ( lns ) including lung - associated ln ( lln ), mesenteric ln ( mln ) and inguinal ln ( iln ), lungs, comprising broncho alveol ar lavage ( bal ) and lung parenchyma, and jejunum ( jej ), divided into the intraepithelial layer ( jel ) and lamina propria ( jlp ) ( fig. 1a ). we also purified low numbers of immune cells from liver, skin, colon epithelium and colon lamina propria from 9 donors ( 4 females, aged 25 – 75 years; 5 males, aged 20 – 55 years ). these sites were therefore not included in the annotate d dataset below and are provided as a separate reference ( extended data fig. 1a ). for data integration, we leveraged multi - resolution variational inference ( mrvi ), which is designed for cohort studies. mrvi harmonizes variation between cell states ( for unified annotation of cell states across samples ) and accounts for differences between samples. visualization with uniform manifold approximation and projection ( umap ) showed similar results across us and uk donors, sequencing technologies and other donor covariates such as sex, cytomegalovirus ( cmv ) and epstein – barr virus serostatus ( fig. 1b and extended data fig. 1b – g ). although cells from blood and lymphoid organs ( bm, spleen, lns ) clustered similarly, cells from mucosal sites ( lung, jej ) clustered distinc tly ( fig. 1c ). for annotation, we used multimodal classifier hierarchy ( mmochi ), which leverages both surface protein and gene expression to hierarchically classify cells into predefined categories ( supplementary fig. 1 and supplementary table 3 ). mmochi defined six major immune lineages found across all tissues ( fig. 1d ) comprising 13 t cell, 5 nk / ilc, 6 b cell and 7 myeloid subsets ( extended data fig. 1h ). these results showed broad and consistent representation of major immune lineages in our dataset. we next analyzed the subset composition and heterogeneity for each immune cell lineage across tissues based on the mmochi annotations above. t lymphocytes ( 610, 429 cells ) comprised low - frequency γδ t cells, which develop early in ontogeny, and predominant αβ t cells ( fig. 2a, b and supplementary fig. 2 ). cd4 and cd8 t cells ( αβ tcr ) were subdivided into naive ( t n ), terminal effector ( t emra ) and memory subsets, including effector - memory ( t em ), central memory ( t cm ) and t rm, along with cd4 regulatory t cells ( t reg ) ( fig. 2a, b ). surface proteins were essential for identifying t cell subsets that were not fully resolved by scrna - seq ( fig. 2b and supplementary fig. 3 ), as shown before. for example, surface cd45ra expression was required to disting uish cd45ra t n cells from cd45ra t cm cells and cd45ra t em cells from cd45ra t emra cells, and surface γδ or αβ t cell receptor ( tcr ) expression to ac curate ly identify γδ t cells from cd8 t cells, which can express trdc ( fig. 2b ). in addition, t rm cells were disting uished from t em cells based on surface expression of cd69, cd103 and / or cd49a ( fig. 2b and supplementary fig. 3 ). t cell subsets were differentially distributed across sites; cd4 t n, cd8 t n and cd4 t cm cells were enriched in blood and multiple ln, cd4 t reg cells were enriched in ln, while cd4 and cd8 t rm cells predominated in jej and were present at lower frequencies in lungs, spleen and ln ( fig. 2a, b ). t emra cells were mostly cd8 and enriched in the bm and spleen and, to a lesser extent, in lungs, while t em cells were distributed across most sites ( fig. 2a, b ). mucosal - associated invariant t ( mait ) cells, disting uished by trav1. 2 expression, cd161 and other markers, were predominantly found in the spleen, bm and lungs ( fig. 2a, b ). tcr clonal analysis provided additional correlative evidence for subset delineation and tissue distribution ( for example, with the highest clonality observed in the t emra subset, as previously described ) ( supplementary fig. 2 ). innate lymphocytes ( 130, 414 cells ) were predominantly mature cd56 cd16 nk cells expressing cytolytic markers ( klrf1, gzmb ) and enriched in blood, bm and lungs ( fig. 2c, d ). immature cd56 cd16 nk cells were present at lower frequencies across most tissues ( fig. 2c, d ). we detected low frequencies of ilcs consisting largely of cd16 ncr2 il7r ilc1s with high expression of tissue residency markers ( cd69, cd49a, cd103 ) enriched in jej, and il7r kit rorc ilc3 ( ref. ) found in ln, spleen and jej ( fig. 2c, d ). putative cd127 cd62l tcf7 nk / ilc precursors, resembling cd56 cd16 nk and ilc3s, were enriched in blood but present in all tissues ( fig. 2c, d ). b cells ( 272, 162 cells ) were classified into 6 subsets largely confined to lymphoid organs ( fig. 2e, f ), including igd naive b cells, cd27 memory b cells, germinal center b cells expressing aicda ( encoding activation - induced adenosine deaminase, which mediates somatic mutation and class switch recombination ), plasma cells expressing immunoglobulin genes and sdc1 ( cd138 ) and plasmablasts expressing proliferation markers ( mki67, top2a ) ( fig. 2f ). cd11c memory b cells expressing tbx21 ( encoding the transcription factor t - bet ) resembled ‘ atypical b cells ’ and were found at low frequencies in spleen and bm ( fig. 2e, f ). variable frequencies of memory b cells in ln and spleen expressed cd69 ( fig. 2e, f ), denoting tissue residency. plasma cells expressing iga were enriched in the jlp, while igg plasmablasts were enriched in lymphoid organs ( fig. 2e, f ). b cell receptor ( bcr ) analysis indicated that plasmablasts exhibited the highest clonal expansion across lymphoid sites, while memory b cells and plasma ( but not naive ) cells expressed mutate d bcr ( supplementary fig. 4 ). myeloid lineage cells ( 225, 268 cells ) comprised c1qa ms4a7 macrophages, fcn1 cd14 classical and fcn1 fcgr3a non - classical monocytes and dc subsets, including clec9a dc1, clec10a dc2, ccr7 migratory dcs and cd123 lilra4 plasmacytoid dcs ( pdcs ) ( fig. 2g, h ). classical monocytes were most abundant in blood, while non - classical monocytes were found mainly in bm and lung ( fig. 2g, h ). dcs were found at low frequency across all tissues; pdcs were enriched in the bm, migratory dcs in the lns, while dc2s were found mainly in the lns and jej ( fig. 2g, h ). macrophages were found predominantly in the lung and at low frequencies in the bm, spleen, lns and jej ( fig. 2h ). immune cell subset composition for the lineages above in blood, lymphoid organs and mucosal sites was specific to the site and conserved across donors ( fig. 2i and extended data fig. 2 ). we defined additional subset heterogeneity based on rna expression, identifying proliferating cells across lineages and functional subsets for cd8 t em cells and cd8 t emra cells, also with tissue - specific distribution ( supplementary figs. 5 and 6 ). this comprehensive, annotate d map of immune cells across tissues can serve as a reference for future analysis, and we provided pre - trained models for label transfer of our cell - type annotation in the popv framework ( supplementary fig. 7 ).