following ligands were not shown: itga9, sema6d, jam3, itgb1 ( stellate cells ); itga9, f8, cd274, hsp90b1 ( lsecs ); c5, f9, f2, fga, tf, ttr, col18a1, col5a3, serpina1, serpinc1 ( hepatocytes ). the depicted target genes are kc - specific in both mouse and human, and a top - predicted target according to the nichenet ligand - target regulatory potential scores. nr1h3 was manually added as a notch2 target based on recent studies ( bonnardel et al., 2019 ). resected human liver was fixed in 4 % formalin for 24 - 48h and subsequently embedded in paraffin. samples were stored for 10 - 15 years at rt before analysis. sections of 6 μm thick were cut using a microm hm360 and mounted on a polarized glass slide. these sections were deparaffinized in xylene and rehydrated in a graded ethanol series. antigen retrieval was performed by immersing the samples for 5 min in ph 8. 3 tris - edta at 98°c. slides were then cooled to rt and washed in pbs. confocal staining was performed as described above. bm was isolated from the tibia and femur of mice by centrifugation. red blood cells were lysed and single cell suspensions were stained with antibod ies for flow cytometry. bm monocytes were sorted as live cd45 + cd11b + ly6g - ly6c + cd115 + cells using a bd facsaria iii. monocytes were resuspended in dmem / f12 media supplemented with 10 % fcs, 30ng / ml csf1, 2mm glutamine and 100u / ml penicillin and streptomycin. 150, 000 monocytes were seeded in each well of an adherent 24 - well plate pre - coated with bovine collagen type i and cultured overnight ( 37c, 5 % co2 ). the following day 0, 25 or 50ng / ml of ac - ldl was added. ac - ldl was kindly provided by sophie janssens, ghent, belgium who received the material from wilfried le goff, paris, france. 14 hours later cells were harvested and live f4 / 80 + cells were facs - purified in rlt plus buffer containing 1 % β - mercaptoethanol. rna isolation, cdna synthesis and qpcr were performed as described above. bone marrow chimeras were generated as described previously ( scott et al., 2016 ). briefly, 6 - 12 week old clec4f - dtr mice ( cd45. 1 ) were anaesthetized by intraperitoneal administration of ketamine ( 150 mg / kg ) and xylazine ( 10 mg / kg ). mice were lethally irradiated with 8 gy, with the livers being protected with a 3 - cm - thick lead cover. once recovered from the anesthesia, mice were reconstituted by intravenous administration of 5 - 10×10 bm cells from cd45. 2 acvrl1 or fcgr1 - crexacvrl1 l mice. 4 weeks after reconstitution mice were administrated a single dose of 500ng dt via intraperitoneal injection to deplete kcs. chimerism was assessed 7 or 13 days later by flow cytometry and compared with chimerism levels in blood ly6c monocytes. clec4f - dtr mice were administered 10mg / kg alk1fc, tgfβriifc or appropriate isotype controls ( higg1 and migg2a; acceleron pharma ) by intraperitoneal injection on days - 1, 2, 3 and 5. on day 0 mice were also administered a single dose of 500ng dt i. p. livers were harvested at day 7 to assess kc development. in all experiments, data are presented as mean ±sem and / or individual data points are presented unless stated otherwise. statistical tests were selected based on appropriate assumptions with respect to data distribution and variance characteristics. details of the precise test used for each analysis can be found in the figure legends. statistical significance was defined as p < 0. 05. sample sizes were chosen according to standard guidelines. number of animals / patients is indicated as ‘ ‘ n ’ ’. the investigators were not blinded to the group allocation, unless otherwise stated. the sc / snrna - sequencing, cite - seq fcs files and spatial transcriptomics datasets will be made available for visualization, analysis and download at www. livercellatlas. org. the datasets generated during this study have been deposited in the gene expression omnibus public database under accession number gse192742. multimodal profiling reveals tissue - directed signatures of human immune cells altered with age source paper: pmc12396968 the immune system comprises multiple cell lineages and subsets maintained in tissues throughout the lifespan, with unknown effects of tissue and age on immune cell function. here we comprehensively profiled rna and surface protein expression of over 1. 25 million immune cells from blood and lymphoid and mucosal tissues from 24 organ donors aged 20 – 75 years. we annotate d major lineages ( t cells, b cells, innate lymphoid cells and myeloid cells ) and corresponding subsets using a multimodal classifier and probabilistic modeling for comparison across tissue sites and age. we identified dominant site - specific effects on immune cell composition and function across lineages; age - associated effects were manifested by site and lineage for macrophages in mucosal sites, b cells in lymphoid organs, and circulating t cells and natural killer cells across blood and tissues. our results reveal tissue - specific signatures of immune homeostasis throughout the body, from which to define immune pathologies across the human lifespan. the immune system leverages a dynamic network of specialized cells spread across the body to defend against infections and cancer, regulate inflammation and repair tissue damage. myeloid cells — macrophages, monocytes and dendritic cells ( dcs ) — initiate innate immunity at mucosal and barrier sites, while adaptive immunity is mediated by antigen-specific t and b lymphocytes in lymphoid organs. immune memory is established following antigen - driven activation and differentiation of t cells and b cells, resulting in heterogeneous subsets of circulating and tissue resident memory t cells ( t rm ) and b cells that persist in diverse tissues. with age, immune memory accumulates, although responses can become dysregulated, increasing susceptibility to infections, cancer and autoimmunity. as human immune cells and the effect of age are mostly studied in blood, we lack a comprehensive understanding of the effect of age on the majority of innate and adaptive immune cells that are maintained in tissues. investigating human tissue immunity across diverse ages has been difficult to achieve. obtaining tissues from organ donors enables the acquisition of blood and multiple tissues from individual donors and the isolation of viable immune cells across lineages for phenotypic, functional and multimodal single - cell profiling. previous studies of lymphocytes from organ donors showed that t lymphocyte, natural killer ( nk ) cell and innate lymphoid cell ( ilc ) subset composition, tissue residence and certain functional attributes are specific to the tissue, indicating that localization has a dominant role in determining the maintenance and functional responses of lymphocytes. whether these tissue - specific effects on human t cells are exhibited by other immune cell lineages, such as b cells and myeloid cells, and whether aging exerts general or tissue - specific effects have not been established. here, we present a comprehensive analysis of human immune cells using cellular indexing of transcriptomes and epitopes ( cite - seq ) to simultaneously profile transcriptomes and > 125 surface proteins of myeloid and lymphoid - lineage cells in 14 tissue sites of 24 organ donors aged 20 – 75 years. we identified a crucial role of tissue on immune cell composition, function, homing and differentiation across myeloid and lymphocyte lineages, including signatures specific for the gut, lungs and lymphoid organs. across age, site - specific immune cell composition was largely maintained, although age - associated changes in function, signaling and metabolism were identified in certain subsets and sites, including macrophages in the lung, b cells in lymphoid organs and cd8 t cells across sites.