Liver-resident NK cells confer adaptive immunity in skin-contact inflammation (original) (raw)
Hepatic DX5– NK cells confer CHS responses. Since hepatic NK cells, but not splenic NK cells, can elicit hapten-specific CHS responses (16, 17), we hypothesized that the phenomenon was due to a distinct NK cell subset present in the liver and not in the spleen. One clue to this subset was the description of DX5– NK cells enriched in the liver that were speculated to be an immature population similar to one identified in the BM (6). Here, we examined the expression of DX5 and NK1.1 on CD122+CD3–CD19– cells from different organs in B6 mice (Figure 1, A and B). Consistent with previous findings, DX5–NK1.1– cells resembling NKPs were detectable in the BM, liver, spleen, and lymph nodes (12). The DX5+NK1.1+ subset, defined as conventional NK cells, was abundant in all examined organs. Surprisingly, however, nearly half of hepatic NK1.1+ NK cells were DX5–, whereas approximately 5%–8% of NK1.1+ NK cells in other organs were DX5–.
Hepatic DX5– NK cells confer memory responses in CHS. (A) Expression of DX5 versus NK1.1 was analyzed on CD122+CD3–CD19– cells from liver, spleen, peripheral blood, BM, and iLNs of WT B6 mice. Representative FACS plots are shown. (B) The percentages of DX5– NK cells among NK (CD122+NK1.1+CD3–CD19–) cells in different organs are shown (n = 7). (C) Ear swelling in naive B6 mice that received 8 × 104 DX5+ (n = 6) or DX5– (n = 4) NK (NK1.1+ CD3– CD19–) cells from OXA-sensitized _Rag1_–/– donor liver. Recipients were challenged 1 month later with OXA on 1 ear and solvent on the other. (D) Ear swelling in naive B6 mice that received 8 × 104 hepatic NK cell subsets (n = 3 per group) or splenic NK cells (n = 6) from OXA-sensitized WT mice and were challenged 1 month later. (C and D) Data are from 1 experiment representative of at least 2 independent experiments. **P < 0.01; ***P < 0.001. Unpaired Student’s t test (C) or ANOVA (D). Means ± SEM are shown in B–D.
To test the potential role of the DX5– subset in CHS, we sensitized _Rag1_–/– or WT mice with oxazolone (OXA) then adoptively transferred their DX5– or DX5+ liver NK cells into naive WT recipients. One month later, recipients were challenged with OXA and ear swelling was measured. Only recipients that received sensitized hepatic DX5– NK cells displayed marked ear inflammation, whereas recipients of sensitized hepatic DX5+ NK cells or sensitized splenic NK cells were unresponsive (Figure 1, C and D). Thus, the immunological memory capacity of liver NK cells in CHS models can be attributed to hepatic DX5– NK cells, suggesting that hepatic DX5– NK cells are functionally distinct from hepatic DX5+ and splenic NK cells, potentially as a unique subset.
“Memory” NK cells remain DX5– during hapten priming in the liver. To verify whether the sensitized “memory” NK cells are naive hepatic DX5– NK cells, but not differentiated from other NK cell subsets including both non-liver NK cells and DX5+ NK cells, we transferred naive hepatic DX5– or DX5+ NK cells sorted from GFP transgenic mice into lethally irradiated recipients followed by sensitization with OXA at days 1 and 2. The liver mononuclear cells (MNCs) from the lethally irradiated recipients were isolated at day 5 (before irradiation-induced death), and adoptively transferred by intravenous injection into secondary naive recipients, which was followed by challenge 1 month later (Figure 2A). Before secondary transfer, donor GFP+DX5– NK cells in irradiated recipients remained DX5– following sensitization with OXA, and donor GFP+DX5+ NK cells consistently maintained expression of DX5 (Figure 2B), indicating that hapten sensitization did not affect DX5 expression on NK cells. Importantly, we found that only secondary recipients that received liver MNCs from the first sensitized recipients, which in turn had received naive hepatic DX5– NK cells, could mount hapten-specific CHS responses (Figure 2C). These results demonstrate that only naive hepatic DX5– NK cells, not other NK cell subsets, have memory potential.
Hepatic NK cells with memory capacity remain DX5– during hapten sensitization. (A) DX5– or DX5+ NK (NK1.1+CD3–CD19–) cells (105) from naive GFP transgenic mice were adoptively transferred into lethally irradiated recipients, which were sensitized with 5% OXA on days 1 and 2. On day 5, liver MNCs from recipients or control irradiated mice that did not receive any cells were adoptively transferred into secondary recipients that were challenged 1 month later. (B) DX5 expression on GFP+ NK cells in the irradiated recipients that received GFP+DX5– (top panel) or GFP+DX5+ (bottom panel) NK cells was analyzed on day 5 before transfer. (C) Ear swelling of secondary recipients described in A was measured after OXA challenge (n = 5 per group). ***P < 0.001, ANOVA. Means ± SEM are shown. Hepatocytes (D) or F4/80+ Kupffer cells (E) in the liver of sensitized (blue) or control (gray shaded) WT mice were respectively isolated by a 2-step collagenase perfusion method, then gated and analyzed for FITC expression; the statistical results show FITC MFI on sensitized (n = 6) or control (n = 2) hepatocytes. *P < 0.05, unpaired Student’s t test. Means ± SEM are shown. (F) FITC+ cells in _Rag1_–/– mice 24 hours after sensitization of FITC on 2 consecutive days. _Rag1_–/– mice without sensitization were set as controls. FITC+ cells among liver MNCs from sensitized mice were analyzed for the expression of indicated markers. (B–F) Data are representative of 2 or 3 independent experiments. SSC, side scatter.
Taken together with the rarity of naive DX5– NK cells in peripheral tissues except the liver, these findings raise the possibility that the liver may be a site where naive NK cells are primed, in which case haptens would need to be delivered to the liver to elicit CHS responses. To examine this hypothesis, we sensitized naive WT mice with the hapten FITC and found that FITC fluorescence on hepatocytes of sensitized mice was detected as opposed to normal autofluorescence of hepatocytes (Figure 2D), implying that hepatocytes may absorb haptens after sensitization and consistent with the liver as a possible site for hapten sensitization. However, Kupffer cells, the resident macrophages in the liver, seemed less likely to be involved in this process, as FITC+ expression was only very rarely detectable in the Kupffer cell population (Figure 2E). Accordingly, FITC-laden cells infiltrated the liver, but there were no FITC+ cells in the spleen in FITC-sensitized _Rag1_–/– mice (Figure 2F). Phenotypic assessment of these liver FITC+ cells revealed that they were NK1.1loLy6Glo cells, with some cells expressing CD11c and CD11b. Further analysis showed that double-positive CD11b+CD11c+ cells existed among FITC+ cells, suggestive of the involvement of dendritic cells in NK cell priming. Also, CD11b+F4/80+Gr-1+ cells with monocyte phenotypes were observed, indicating that circulating macrophages may also function as APCs. Thus, the retention of circulating cells with antigen-presenting ability in the liver makes it possible that DX5– NK cells are directly primed in the liver.
Preferential trafficking of DX5– NK cells to the liver without further differentiation. In our initial studies to investigate whether DX5– NK cells selectively reside in the liver, we adoptively transferred unseparated liver MNCs from CD45.1+ mice into sublethally irradiated CD45.2+ recipients via intravenous injection. One day after transfer, we found that donor-derived DX5– NK cells accumulated only in the liver, whereas donor-derived DX5+ NK cells showed distribution to other sites (Figure 3A), suggestive of distinct migratory tendencies between these 2 subsets. However, it is possible that this result is due to rapid conversion from DX5– to DX5+ NK cells in various tissues except the liver, and that there is no intrinsic difference between them in migratory capacity. To further determine whether they differ in tissue migration, we adoptively transferred sorted DX5– and DX5+ liver NK cells and analyzed recipient mice; we consistently found that donor-derived DX5– NK cells were only detected in the host liver, whereas donor-derived DX5+ NK cells were found in both the host liver and spleen (Figure 3B). To further confirm the preferential migration of DX5– NK cells to the liver, we used another marker of donor NK cells on the purified DX5– or DX5+ liver NK cells, i.e., the stable expression of GFP, and a different route of transfer. Following adoptive transfer into sublethally irradiated recipients via intraperitoneal injection, donor GFP+DX5– NK cells were only found in the liver (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI66381DS1). Moreover, transferred DX5– NK cells remained DX5– in recipient liver (Figure 3B), suggesting that hepatic DX5– NK cells are phenotypically stable and do not differentiate into DX5+ NK cells in the steady state. Thus, compared with nondiscriminatory migration of the DX5+ subset to different tissues, hepatic DX5– NK cells preferentially traffic to the liver and have a low capacity to repopulate other tissues after transfer.
Hepatic DX5– NK cells preferentially traffic to the liver and maintain their phenotypic features after adoptive transfer. (A) Liver MNCs (106) from CD45.1+ mice were adoptively transferred intravenously into sublethally irradiated (IR) CD45.2+ B6 mice. Twenty-four hours after transfer, CD45.1+ NK (NK1.1+CD3–CD19–) cells were analyzed for DX5 expression in recipient liver, spleen, and BM. (B) DX5– or DX5+ liver NK cells (105) were sorted from CD45.1+ mice and intravenously transferred into sublethally irradiated CD45.2+ B6 mice. DX5 expression of donor NK cells before transfer is shown pre- and post-sort. Seven days after transfer, CD45.1+ NK cells were analyzed for DX5 expression in recipient liver and spleen. All data in this figure are representative of 2 independent experiments.
The transcriptional, phenotypic, and functional features of hepatic DX5– NK cells. We next sought to determine other differences between DX5– and DX5+ NK cells in transcriptional signature, phenotype, and functional capacity. DX5–NK1.1+ and DX5+NK1.1+ liver NK cells were respectively sorted for gene expression microarray analysis (GEO accession number GSE43339). With nearly 22,000 genes tested, 11,122 expressed genes showed no change in DX5– versus DX5+ NK cells, while 1,507 expressed genes showed a fold change of 2 or greater, among which 566 genes were overexpressed in DX5– NK cells (Figure 4A). The differentially expressed genes included those encoding inhibitory and activating receptors, cytokines, chemokines, adhesion molecules, cytokine receptors, cytotoxic effectors, and transcription factors (Figure 4B). We confirmed several of these findings by flow cytometry (Supplemental Table 1). Molecules associated with adhesion, such as CD49a and CD51, and chemokine receptors, CXCR3 and CXCR6, were upregulated on DX5– NK cells at both transcript and protein levels. We consistently found that CD69 was expressed at higher levels on DX5– NK cells, but not on S1PR1, which is regulated by CD69 (24). Some adhesion molecules, such as CD62L (Sell), CD11b (Itgam), CD29 (Itgb1), and CCR9 (Ccr9), were significantly downregulated on DX5– NK cells. In addition, we observed that Il21r, Rora, and Ahr, which are related to IL-17 production and Th-17 cell development (25), were also relatively higher in DX5– NK cells. Eomes, which is implicated in inducing IFN-γ production (26) and NK cell maturation (22), was expressed at much higher levels in DX5+ NK cells. These differentially expressed transcription factors may reflect the distinct patterns of cytokines released by those 2 NK cell subsets. Genes encoding LAG-3, Helios, and Egr-2, which are preferentially expressed by Treg cells (27–29), were also found to be overexpressed by DX5– NK cells, raising the possibility that DX5– NK cells may exert negative regulatory actions within the liver. Interestingly, C_d3g_, which has been previously reported to be expressed in fetal NK cells (30) was highly expressed in DX5– NK cells, supporting the possibility of common origins between NK and T cells. In brief, we found that genes associated with immune tolerance, negative regulation, and activation were enhanced in DX5– NK cells, while DX5+ NK cells were relatively enriched in genes associated with migration, immune response, proliferation, and cell maturation (Figure 4C and Supplemental Table 2).
Hepatic DX5– NK cells are distinct from other NK cell populations. (A) Comparative transcriptome analysis between DX5– and DX5+ liver NK (NK1.1+CD3–) cells was performed by Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Genes that were expressed greater than or equal to 2-fold higher or lower than DX5+ NK cells are highlighted in black. The number of genes upregulated or downregulated for each comparison is indicated. (B) Differentially expressed genes in DX5– and DX5+ NK cells were selected and classified into different groups. The listed genes had greater than or equal to 2-fold differences and are shown with their signal values. (C) Distribution by functional category of upregulated genes in hepatic DX5+ and DX5– NK cell subsets. Genes with greater than or equal to 2-fold differences were included. (D) Expression of CD107a and IFN-γ is shown for stimulated DX5– and DX5+ liver NK (NK1.1+CD3–CD19–) cells. Liver MNCs were stimulated with IL-2 and IL-12 (for IFN-γ analysis) or with YAC-1 cells (for CD107a analysis). Blue lines represent staining of the indicated molecules, and gray-shaded curves represent isotype controls. (E) Statistical results of percentages of CD107a+ (n = 6) or IFN-γ+ (n = 11) cells are shown. *P < 0.05 and **P < 0.01, paired Student’s t test. Means ± SEM are shown.
Regarding function, effector molecules such as TRAIL (Tnfsf10), granzyme c (Gzmc), and granzyme f (Gzmf) were overexpressed in DX5– NK cells, implying that DX5– NK cells are functional. However, stimulation assays showed that DX5– NK cells expressed markedly lower levels of CD107a, a degranulation marker of cytotoxic NK cells, and produced slightly lower levels of IFN-γ than DX5+ NK cells after activation (Figure 4, D and E). These data suggest that the 2 NK cell subsets are functionally different. Further analysis revealed that DX5+ NK cells of the liver phenotypically resembled those of the spleen (Supplemental Figure 2). Hence, previous findings that bulk liver NK cells are phenotypically different from NK cells in other organs (6, 11, 31) may be due to the unique DX5– subset of NK cells in the liver.
As the primary site for NK cell development (4, 5), the BM also contains a very small subpopulation of DX5– NK cells which were considered to be immature NK cells (6, 7). Indeed, the low expression of Ly49 receptors and the high expression of NKG2A on liver DX5– NK cells are consistent with a phenotypically immature population (6, 31). However, TRAIL was not expressed on BM DX5– NK cells, in contrast to highly expressed TRAIL on hepatic DX5– NK cells (ref. 11 and Supplemental Figure 3). Meanwhile, hepatic DX5– NK cells expressed much higher CD11c than did BM DX5– NK cells, indicating that hepatic DX5– NK cells are phenotypically different from those of the BM that are considered to be immature NK cells (7). Taken together, the results indicate that hepatic DX5– NK cells are distinct from DX5+ NK cells and DX5– NK cells at other sites in transcriptional, phenotypic, and functional aspects.
CD49a+DX5– NK cells are liver resident. DX5 is widely used to define NK cells in mouse strains that are NK1.1– (12), and identification of DX5– would not be useful as a marker of hepatic NK cell subsets in such strains, indicating that a “positive marker” to define liver NK cell subsets is necessary. By transcriptome analysis, we found that Cd49a, also known as Itga1, was the most highly expressed gene (20-fold higher) among the top 20 most upregulated genes in DX5– NK cells (Supplemental Figure 4). Selective expression was further verified by flow cytometry, showing that CD49a was constitutively and highly expressed by hepatic DX5– NK cells, but not by hepatic DX5+ NK cells or NK cells from other tissues (Figure 5A). In addition, NKp46, a pan-NK cell marker (32), was expressed at high levels on both DX5– and DX5+ NK cells. Though CD51 was expressed by the majority of DX5– NK cells, a proportion of DX5+ NK cells were also CD51+ regardless of tissue origin. The expression of CD27 and Thy1.2 on NK cells has been reported to be associated with immune memory by hepatic NK cells (16, 20, 33), but their expression was detected on NK cells from each examined tissue. CXCR6 was found to be required for NK cell–mediated CHS responses (17), but NK cells from other organs besides liver also expressed CXCR6, and a fraction of hepatic DX5– NK cells did not express CXCR6 (Figure 5A), suggesting that it is not an exclusive marker of liver NK cells. After adoptive transfer, hepatic DX5– NK cells maintained stable expression of CD49a in recipients, while hepatic DX5+ NK cells were still CD49a– (Figure 5B). Furthermore, hepatic CD49a+ NK cells were identified in BALB/c and _Rag1_–/– mice as well as in B6 mice (Figure 5C), and they were selectively accompanied by high expression of TRAIL, CD51, CXCR3, LAG3, CD44, and CD2 (Supplemental Figure 5), similar to what was observed in B6 mice (Supplemental Table 1). Thus, these results strongly suggest that CD49a+DX5– NK cells represent an NK cell subset that preferentially resides in the liver.
CD49a is a specific surface marker of liver-resident DX5– NK cells. (A) Expression of CD49a, NKp46, CD51, CD27, CXCR6, and Thy1.2 versus DX5 was analyzed on NK1.1+CD3–CD19– cells or NK1.1+CD3– cells from the indicated organs of WT or CXCR6+/– (for CXCR6 detection) mice. Data are representative of 4 to 6 individual mice. (B) DX5– or DX5+ liver NK (NK1.1+ CD3– CD19–) cells (105) were sorted from CD45.1+ mice and intravenously transferred into sublethally irradiated CD45.2+ B6 mice. Seven days later, NK cells from recipient liver were analyzed for the expression of CD45.1 and CD49a. Data are representative of 2 independent experiments. (C) Expression of CD49a was analyzed on NK cells from unmanipulated BALB/c and _Rag1_–/– mice. Numbers indicate the percentages of cells expressing CD49a among NKp46+CD3–CD19– (BALB/c) or NK1.1+ (_Rag1_–/–) cells. Blue lines represent staining of the indicated molecules, and gray-shaded curves represent isotype controls. Data are representative of 4 mice per group. (D) As described in Methods, blood was collected from the unfractionated liver and other indicated sites from WT B6 mice, and then MNCs were isolated. Expression of CD49a versus DX5 was analyzed on NK1.1+CD3–CD19– cells. Plots are representative of at least 5 individual mice. (E) Immunofluorescence histology of frozen sections of mouse liver stained with anti-NKp46 (green), anti-CD31 (blue), anti-CD49a (red), or anti-CD49b (DX5; red). Original magnification, ×250. (F) Histological analysis of _Rag1_–/– mouse liver stained with anti-CD49a (brown) and NK1.1 (purple). Original magnification, ×320. (E and F) Data are representative of at least 2 independent experiments.
Importantly, we observed that the CD49a+DX5– NK cells resided neither in afferent (arterial or portal venous) nor in efferent (postcaval venous) blood of the liver as opposed to CD49a–DX5+ NK cells (Figure 5D). Instead, CD49a+DX5– NK cells were present in the liver sinusoidal blood. Immunofluorescence and immunohistochemistry further confirmed the localization of CD49a+ NK cells in the liver sinusoids, albeit somewhat rare on tissue sections (Figure 5, E and F). Moreover, CD49a+DX5– NK cells were seldom present in the spleen, BM, lung, and lymph nodes (Figure 5A and data not shown).
Furthermore, we studied unmanipulated CD49a+DX5– and CD49a–DX5+ NK cells in unirradiated CD45 congenic mice that were surgically joined by parabiosis. At the time when substantial chimerism of splenic leukocytes (Supplemental Figure 6) was taking place, the CD49a–DX5+ NK cells from each parabiont were found in the spleens and livers of both parabionts (Figure 6, A and B). However, CD49a+DX5– NK cells expressing CD45.1+ were essentially confined to the CD45.1+ livers which contained few, if any, CD49a+DX5– NK cells from the CD45.2+ parabiont, and vice versa. Taken together with the samplings of the liver afferent, efferent, and sinusoidal vasculature, the preferential migration of transferred, purified hepatic DX5– NK cells to the liver (Figure 3B) and the phenotypic similarities of the DX5+ NK cells in the liver and spleen (Supplemental Figure 2) indicate that CD49a+DX5– NK cells stably reside in the liver sinusoidal space, whereas CD49a–DX5+ NK cells are circulating NK cells passing through the liver under noninflammatory homeostatic conditions.
Minimal exchange of liver CD49a+DX5– NK cells in parabiotic mice. WT B6 (CD45.2) mice were parabiosed to congenic B6-CD45.1 mice. At day 14 postsurgery, the spleen and liver were harvested and flow cytometry was performed. (A) Representative dot plot of the liver and spleen gated on live CD3–NK1.1+ cells followed by a CD45.1 gate (left panels) and CD45.2 gate (right panels) for each parabiont as indicated. The percentages of CD49a+DX5– and CD49a–DX5+ cells are shown in the dot plots. (B) The percentages of CD49a+DX5– and CD49a–DX5+ cells in the liver and spleen from A are depicted in the stacked bar graph which represents 7 parabiotic pairs. The CD49a+DX5– NK cells are indicated by the solid shading, and the CD49a–DX5+ NK cells are indicated by the open box, with each column representing the indicated parabiont, according to the CD45.1+ or CD45.2+ gate.
CD49a+ NK cells confer hapten-specific CHS responses. The exclusive and constitutive expression of CD49a by hepatic DX5– NK cells in the steady state prompted us to examine the function of sensitized CD49a+ NK cells in CHS. The recipients of sensitized hepatic CD49a+ NK cells displayed vigorous CHS responses after challenge, whereas recipients of the CD49a– subset failed to respond (Figure 7A). Moreover, the magnitude of ear swelling showed a nonstatistically significant positive trend for the number of transferred hepatic CD49a+ NK cells (Figure 7B). To confirm that the recall responses conferred by hepatic CD49a+ NK cells were hapten specific, we adoptively transferred hepatic CD49a+ NK cells from OXA- or FITC-primed mice into WT recipients. Only when challenged with the sensitizing hapten were the recipients able to mount hapten-specific CHS responses; otherwise, the recipients were unresponsive (Figure 7, C and D). These data indicate that the hepatic CD49a+ NK cells confer hapten-specific CHS responses.
CD49a+ NK cells have the capacity to confer hapten-specific CHS responses. (A) Naive B6 mice received 8 × 104 CD49a+ or CD49a– liver NK cells from OXA-sensitized WT mice and were challenged 1 month later (n = 4 per group). (B) Naive B6 mice received the indicated number of CD49a+ liver NK cells from OXA-sensitized WT mice and were challenged 1 month later (n = 3 per group). (C and D) Naive B6 mice received 8 × 104 CD49a+ or CD49a– liver NK cells from OXA–-sensitized (C), or FITC–-sensitized (D) WT mice and were challenged with OXA or FITC 1 month later as indicated (n = 4 for the FITC plus OXA group; n = 5 for the other groups). All data are from 1 experiment representative of 2 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired Student’s t test (A, C, and D), or ANOVA (B). Means ± SEM of ear swelling are shown in all panels.
During the memory phase, however, we found that CD49a–DX5+ NK cells dramatically increased in the liver and were predominant at the ears after challenge (data not shown). Surprisingly, CD49a+DX5– NK cells were still absent in the blood, and NK cells at the site of ear swelling after challenge were predominantly CD49a–DX5+ by flow cytometric analysis (Supplemental Figure 7 and data not shown), which raise the possibility that liver-resident CD49a+DX5– NK cells may change their phenotype into CD49a–DX5+ NK cells upon exiting the liver, or at the site of inflammation, or they may alternatively cooperate with other cells in the effector phase of CHS.
Liver-resident HPCs differentiate into CD49a+DX5– NK cells. Since NK cells are commonly considered to be derived from BM hematopoietic stem cells (BM HSCs) throughout adult life, syngeneic BM transplantation (syn-BMT) was performed to investigate the developmental pathway of hepatic DX5– NK cells. In contrast to the enrichment of DX5– NK cells in the livers (nearly 50%) of normal mice, very few DX5– NK cells (less than 15%) were found in the livers of syn-BM transplanted mice even 3 months after BMT (Figure 8A), suggesting that DX5– NK cells are not predominantly derived from BM. We then transplanted liver MNCs into lethally irradiated mice, and surprisingly, liver MNCs as well as BM HSCs, supported the survival of irradiated mice (Figure 8B), suggestive of the existence of HSCs in the liver. To further address whether the liver contains precursors for this unique NK subset, we adoptively transferred a mixture of BM cells containing heterozygous CD45.1+CD45.2+ double-positive (DP) BM cells and homozygous CD45.1+ single-positive (SP) liver MNCs at a 1:1 ratio into lethally irradiated homozygous CD45.2+ mice (Figure 8C). DP and SP cells in recipients were analyzed 1 month after BMT. Interestingly, liver MNC–derived SP cells were preferentially found in the liver rather than in other organs, but BM-derived DP NK cells were found mainly in the non-liver organs (Supplemental Figure 8). In contrast to the predominance of the DX5+ subset among DP NK cells differentiated from BM-HSCs, SP NK cells derived from liver MNCs were primarily DX5– (Figure 8D), further suggesting that the HPCs in hepatic liver MNCs are critical for the generation of hepatic DX5– NK cells. In order to exclude the influence of homeostatic proliferation by donor NK cells, we also transferred purified hepatic CD45.1+ non-T, non-B, non-NK precursors (CD3–CD19–NK1.1– cells) together with supporting Nfil3–/– BM cells (e.g., NK cell–deficient BM) to lethally irradiated recipients. CD49a+DX5– NK cells were generated in the recipient liver 1 month after transfer (Figure 8E), demonstrating that there are unique direct precursors of the hepatic DX5– NK cell subset in the liver, and that the hepatic microenvironment supports their further development. Moreover, the presence of CD49a–DX5+ NK cells originating from donor hepatic precursors in the liver of recipients indicates that the liver also contains HPCs for conventional NK cell development. Thus, these results suggest that the liver harbors NK1.1–CD3–CD19– HPCs, which are capable of generating liver-resident CD49a+DX5– NK cells, whereas BM cells are deficient in giving rise to this NK subset.
The liver retains HPCs capable of generating CD49a+DX5– NK cells. (A) Lethally irradiated CD45.2+ B6 mice that received 2 × 106 BM cells from CD45.1+ B6 mice were analyzed for the expression of DX5 on hepatic CD45.1+ NK (NK1.1+CD3–CD19–) cells. Means ± SEM are shown (n = 3 for each time point). (B) Survival of lethally irradiated B6 mice that received 3 × 106 BM cells (BMT), liver MNCs (LT), or splenocytes (ST). Lethally irradiated mice without transplantation were set as the control group. Means ± SEM are shown (n = 5 per group). (C and D) Lethally irradiated CD45.2+ B6 mice received 106 CD45.1+CD45.2+ (DP) BM cells mixed with 106 CD45.1+CD45.2– (SP) liver MNCs. One month after transfer, DP and SP cells in the recipient livers were respectively gated to analyze the expression of DX5 on NK cells. Means ± SEM are shown (n = 3 per group). (E) BM cells (2 × 106) from Nfil3–/– mice mixed with 105 hepatic NK1.1–CD3–CD19– cells from CD45.1+ B6 mice were transferred into lethally irradiated CD45.2+ B6 mice. One month after transfer, CD45.1+ NK cells in the recipient livers were analyzed for the expression of DX5 and CD49a. All data are representative of 2 independent experiments.