taken together, the presence of these spatially distinc t subsets of ecs and scs highlights the uniqueness of the specific microenvironments in which the distinc t mac populations reside. finally, in addition to providing gating strategies for myeloid and cd45 cells, we also wanted to investigate if the cite - seq data would allow us to develop similar strategies for the lymphoid populations. to examine this, we re - clustered the t cells, nk cells, ilcs, b cells, and pdcs from figure 1 b identifying 12 distinc t populations ( figure s5 a ). analysis of the degs highlighted that while b cells, nk cells, ilc1s, and pdcs were distinc t populations, there was considerable overlap between the transcriptomic profiles of the t cells ( figure s5 b; table s5 ). however, by analyzing the deps we were able to define distinc t subsets including naive cd4 and cd8 t cells, t regs, t h 1s, ctls, and t h 17s ( figures s5 a and s5c; table s5 ). moreover, we were able to design a gating strategy to isolate the distinc t populations ( figure s5 d ). to determine the degree of conservation between the mac subsets and their different microenvironmental niches between the mouse and the human liver, we next generated a proteogenomic atlas of the human liver using sc / snrna - seq and cite - seq on 19 liver biopsies ( figures 4 a, 4b, s6 a, and s6b; tables s1 and s5 ). of these, most were histologically healthy with only 5 patients showing > 10 % hepatic steatosis in the absence of any significant fibrosis ( table s6 ). cellular proportions varied according to the isolation technique used, and while there was some variability between patients, this was not linked to the surgery ( figures s6 c – s6e ). further confirming the lack of fibrosis in the steatotic livers, we did not detect any increase in ctls ( figures s5 e – s5i; table s5 ), which have been shown to correlate with non - alcoholic steatohepatitis ( nash ) ( haas et al., 2019 ). a significant increase in ctls was detected in the setting of murine nash induced by feeding a western diet ( wd ) for up to 36 weeks, demonstrating that this is not due a limitation in detecting these differences using cite - seq ( figures s5 j – s5m; table s7 ). as visium reliably located murine hepatic cells, we used this to locate the cells of the human liver in 4 biopsies ( figure 4 c ). however, the visium spots from patients with > 10 % steatosis were found to cluster separately from the healthy samples ( < 10 % steatosis; figures 4 d and 4e ). we therefore used the healthy samples to calculate a baseline zonation and then transferred this trajectory onto the steatotic samples ( figures 4 f and s6 f ). this identified the steatosis to be predominantly present in regions expressing peri - central zonation genes like cyp2e1. this zonation pattern was further validated using molecular cartography ( figure s6 g ). this fits with previous clinical studies demonstrating peri - central steatosis to be most common in non - alcoholic fatty liver disease ( nafld ) patients, especially in early disease ( chalasani et al., 2008; kleiner and makhlouf, 2016 ). however, as these peri - central regions are larger than in the healthy controls, it could also imply that the presence of steatosis alters expression of the zonated hepatocyte genes, but this remains to be tested. notably, the overall cellular distribution was not impacted by the presence of steatosis, although neutrophils and monocytes and monocyte-derived cells were preferentially localized peri - centrally in the steatotic patients correlating with the presence of steatosis ( figure 4 g ). mics 100 - plex protein analysis further validated the cellular distributions predicted by visium and confirmed the increase in neutrophils in the steatotic livers ( figures s6 h and s6i ). to date, no validated markers of bona fide human kcs have been described. explaining the difficulty to ac curate ly define human kcs, we found monocytes and macs formed a single continuum in the human sc / snrna - seq data, preventing a simple definition of human kcs ( figure 4 b ). notably, a similar continuum from monocyte to kcs was also observed in the nash murine liver ( figures s5 j and s6 j – s6n, table s8 ). consistent with our previous report ( remmerie et al., 2020 ), we observed both long - term resident timd4 - expressing kcs and recently recruited timd4 monocyte-derived kcs ( mokcs; figures s6 j – s6n ). as the presence of such a continuum in the human liver suggests that there may also be monocyte contribution to the kc pool in the healthy human liver, we next zoomed in on myeloid cells to examine this, identifying 10 clusters ( figures 4 h, s6 o, and s6p; table s2 ). to define the kcs, we examined expression of the top 25 murine kc genes by these clusters, which identified cluster10 to be the genuine human kcs ( figure s6 q ). unlike in mice, these were preferentially located in the mid zone ( figure s6 r ). cluster9 also expressed many of these genes but lacked timd4 ( figure s6 q ), suggesting that these cells may be recently recruited mokcs. the presence of mokcs in the liver is consistent with reports that host - derived macs are identified in transplanted donor livers ( bittmann et al., 2003; pallett et al., 2020 ) and suggests that the kc population may be a mix of embryonic and monocyte-derived cells. although not the case at mrna level, vsig4 was found to be the best human kc protein marker in the cite - seq data, while folr2, cd163, and cd169 were also identified as useful markers of these cells for flow cytometry and confocal microscopy on frozen and paraffin sections ( figures 4 i and s6 s – s6x ). co - staining human livers for vsig4 protein and kc - specific cd5l mrna and mics 100 - plex protein analysis also confirmed the mid - zonal localization of kcs ( figures 4 j and s6 u ). to assess if kc identity was further conserved in evolution, we profiled macaque, pig, hamster, chicken, and zebrafish livers ( figure 4 k ). we identified the kcs in an unbiased manner by mapping the conserved human - mouse kc signature onto the datasets ( figures 4 k and s7 a – s7c ). we then examined the main features of each kc population identified ( figures s7 d – s7h; table s9 ). a strong overlap in transcriptomes across species was observed likely due to the conserved expression of core kc transcription factors ( figure s7 i ). however, each species also harbored a number of unique kc genes ( figure s7 j; table s9 ). vsig4 protein expression was also conserved in pig and macaque kcs ( figures s7 k – s7m ). similarly, we were also able to identify most of the other hepatic cells across species on the basis of conserved genes ( figures s7 n and s7o ). cdc2s were the main exception to this, as specific cdc2 marker genes were not conserved across all species ( figure s7 o ). alongside kcs, we also identified distinc t clusters of macs in the human myeloid cells ( figure 4 h ). to better understand the nature of these clusters we performed confocal microscopy to examine the specific locations of cd68 vsig4 macs in the human liver ( figure s8 a ). this identified cd68 vsig4 macs in the liver capsule, in close proximity to central and pvs as well as at bile ducts ( bds ) ( figures 5 a – 5c and s8 a ). similar populations were also observed at the pvs and cvs and at the bds in the healthy macaque liver ( figure s7 m ). examination of the scrna - seq data and comparison with murine signatures identified immature and mature lams, with immature lams expressing some monocyte genes ( figures 4 h, 5 d, s8 b, and s8c ). although recently suggested to be specific to fibrotic human livers ( ramachandran et al., 2019 ), we identified lams in all patients profiled with scrna - seq, but there was a trend toward increased proportions of lams in the livers with > 10 % steatosis ( figure s8 d ) consistent with the increased population of lams in murine nafld ( figures s6 j – s6n ).