The body's immune system is composed of many different types of specialized cells that often circulate throughout the body via the vascular system. Upon detection of pathologic insults, such as invading microorganisms or developing tumors, the circulating immune cells stop to react. This process is often initiated by the early, innate immune events in the tissue and secondary lymphoid organs, such as the lymph node and spleen, resulting in the recruitment of still other circulating immune cells to the damaged tissue and stimulation of the adaptive immune response. In contrast to these well-studied circulating immune cells are tissue-resident immune cells, which already reside in selected organs, where they appear poised to respond more rapidly. Although they have been described as counterparts to circulating cells, less is known about their properties.
Conventional natural killer (cNK) cells belong to the innate arm of the immune system and are found in the vascular system. First described on the basis of their inherent capacity to kill tumor cells directly, without prior sensitization, NK cells are now known to develop in the bone marrow and to participate in a wide variety of other immune responses, including responses to viral infections, stem cell transplantation, and pregnancy. In addition, they can respond to proinflammatory cytokines by producing interferon-γ, their signature cytokine, which can have an impact on adaptive immunity. Although classically studied in the mouse spleen, NK cells are found in other organs, such as the liver, where they differ from cNK cells.
Although liver NK cells express surface markers similar to those of cNK cells, their expression of other markers is inconsistent. In particular, a sizable subpopulation can be distinguished from cNK cells because they are DX5– and display high levels of TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), a phenotype similar to immature cNK cells in the bone marrow. These findings initially gave the impression that the liver contains a subpopulation of cNK cells that is not fully differentiated. Our recent studies, however, indicate that these liver NK cells are distinct from immature and mature cNK cells in other ways.
Detailed analysis of liver NK cells revealed that they express either CD49a or DX5; that is, they are CD49a+DX5– or CD49a–DX5+. Following adoptive transfer of CD49a+DX5– liver NK cells into congenically distinct hosts, donor cells were found in the liver but not in the spleen. By contrast, transferred CD49a–DX5+ liver NK cells were found in both the liver and spleen. Based on other markers, the liver CD49a+DX5– were unlike splenic cNK cells, whereas liver CD49a–DX5+ were very similar to splenic cNK cells, suggesting the former are tissue-resident NK (trNK) cells, whereas the latter represent cNK cells circulating in the liver.
We used two approaches to validate this interpretation. One approach was based on the anatomy of the liver vasculature. The liver receives blood from two afferent vessels, the hepatic artery, which supplies oxygenated blood, and the portal vein, which supplies nutrient-rich blood from the intestines and spleen. The blood mixes in the sinusoids, blood vessels that network to form a low-pressure and highly fenestrated vascular system. Selective sampling of the thoracic aorta (which ultimately feeds the hepatic artery), portal vein, and vena cava (which receives blood from the hepatic vein) showed the presence of only CD49a–DX5+ NK cells, consistent with their circulation in the blood vasculature. However, when the three major blood vessels of the liver were clamped before excision and the excised liver was flushed, both CD49a+DX5– and CD49a–DX5+ NK cells were found, similar to single-cell suspensions from homogenized livers. Thus, liver trNK cells selectively reside in the sinusoids.
A second approach utilized parabiotic mice; that is, two mice surgically connected to each other so that they share their blood vasculature within 10 days following surgery. However, the host liver contained CD49a+DX5– NK cells primarily of host origin, whereas CD49a–DX5+ NK cells were derived from both the host and other parabiont. These data strongly suggested that the liver contains two types of NK cells: (1) noncirculating CD49a+DX5– trNK cells in the sinusoids, and (2) CD49a–DX5+ cNK cells circulating through the liver.
When compared to cNK cells, the "immature" phenotype of liver trNK cells suggested that they could be a developmental precursor to cNK cells. Alternatively, the trNK cells could represent a distinct lineage of NK cells. Detailed gene expression profiling showed that the CD49a–DX5+ NK cells in the liver are essentially identical to cNK cells in the spleen, consistent with their circulatory patterns. However, the liver trNK cells did not resemble the cNK cells from either the liver or spleen and were also distinct from immature cNK cells in the bone marrow. Finally, trNK and cNK cells have different functional properties. These data strongly suggested that cNK and trNK cells may be distinct lineages of NK cells.
Indeed, liver trNK and cNK cells have distinct transcription factor requirements. Previous reports indicated an absolute requirement for NF-IL3 (nuclear factor of IL-3 [interleukin-3], also known as E4BP4) for splenic NK cells. In Nfil3-knockout mice, there were no splenic NK cells and our studies indicated that there were no cNK cells in the liver as well. However, liver trNK cells in NF-IL3-deficent mice were present in normal numbers with normal expression of cell surface markers and functional responses. Thus, unlike cNK cells, trNK cells in the liver do not require NF-IL3.
Conversely, we found that liver trNK cells require another transcription factor, T-bet (Tbx21), because Tbx21-knockout mice completely lacked liver trNK cells. In contrast, cNK cells were mostly preserved in the liver and spleen. Thus, liver trNK cells constitute a lineage of NK cells that is distinct from cNK cells, rather than a precursor stage in cNK cell development.
Our studies suggested that other organs may also contain subpopulations of trNK cells as well as circulating cNK cells. Indeed, we discovered that trNK cells in other organs, such as the skin and uterus, express CD49a but not DX5. Skin trNK cells have the same transcription factor requirements as liver trNK cells; that is, the skin trNK cells are dependent on T-bet and independent of NF-IL3, suggesting a developmental relationship between liver and skin trNK cells. However, uterine trNK cells, unlike other NK cells, develop in the absence of T-bet or NF-IL3. Finally, we showed that trNK cells are distinct from previously described thymic NK cells, which are dependent on the GATA-3 transcription factor and express the IL-7 receptor (CD127), whereas none of the trNK cells are dependent on GATA-3 or express IL-7 receptor.
Taken together, our recent studies suggest that NK cells are more diverse than previously recognized because there appear to be at least four distinct lineages of mouse NK cells: (1) cNK cells that circulate, (2) thymic NK cells, (3) our newly described trNK cells in the liver (and skin), and (4) trNK cells in the uterus. Our studies further suggest that these trNK cells should have specialized roles in the host immune response in different organs.
Grants from the National Institutes of Health provide partial support for these projects.
As of April 28, 2016