Thymic Natural Killer Cells Research Articles

Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells.

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001.

Emergence of NK-cell progenitors and functionally competent NK-cell lineage subsets in the early mouse embryo

AbstractThe earliest stages of natural killer (NK)–cell development are not well characterized. In this study, we investigated in different fetal hematopoietic tissues how NK-cell progenitors and their mature NK-cell progeny emerge and expand during fetal development. Here we demonstrate, for the first time, that the counterpart of adult BM Lin−CD122+NK1.1−DX5− NK-cell progenitor (NKP) emerges in the fetal liver at E13.5. After NKP expansion, immature NK cells emerge at E14.5 in the liver and E15.5 in the spleen. Thymic NK cells arise at E15.5, whereas functionally competent cytotoxic NK cells were present in the liver and spleen at E16.5 and E17.5, respectively. Fetal NKPs failed to produce B and myeloid cells but sustained combined NK- and T-lineage potential at the single-cell level. NKPs were also found in the fetal blood, spleen, and thymus. These findings show the emergence and expansion of bipotent NK/T-cell progenitor during fetal and adult lymphopoiesis, further supporting that NK/T-lineage restriction is taking place prethymically. Uncovering the earliest NK-cell developmental stages will provide important clues, helping to understand the origin of diverse NK-cell subsets, their progenitors, and key regulators.

Expression of BMPRIA on human thymic NK cell precursors: role of BMP signaling in intrathymic NK cell development

AbstractThe bone morphogenetic protein (BMP) signaling pathway regulates survival, proliferation, and differentiation of several cell types in multiple tissues, including the thymus. Previous reports have shown that BMP signaling negatively regulates T-cell development. Here, we study the subpopulation of early human intrathymic progenitors expressing the type IA BMP receptor (BMPRIA) and provide evidence that CD34+CD1a−BMPRIA+ precursor cells mostly express surface cell markers and transcription factors typically associated with NK cell lineage. These CD34+ cells mostly differentiate into functional CD56+ natural killer (NK) cells when they are cocultured with thymic stromal cells in chimeric human-mouse fetal thymic organ cultures and also in the presence of SCF and IL-15. Moreover, autocrine BMP signaling can promote the differentiation of thymic NK cells by regulating the expression of key transcription factors required for NK cell lineage (eg, Id3 and Nfil3) as well as one of the components of IL-15 receptor, CD122. Subsequently, the resulting population of IL-15–responsive NK cell precursors can be expanded by IL-15, whose action is mediated by BMP signaling during the last steps of thymic NK cell differentiation. Our results strongly suggest that BMPRIA expression identifies human thymic NK cell precursors and that BMP signaling is relevant for NK cell differentiation in the human thymus.

Development of thymic NK cells from double negative 1 thymocyte precursors

AbstractThe differentiation of natural killer (NK) cells and a subpopulation of NK cells which requires an intact thymus, that is, thymic NK cells, is poorly understood. Previous in vitro studies indicate that double negative (CD4−CD8−, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. Sorted DN1 (CD44+CD25−) CD122−NK1.1− thymocytes from Rag1−/− mice were adoptively transferred into Rag1−/−Ly5.1 congenic mice. After intrathymic injection, donor-derived cells phenotypically resembling thymic NK cells were found. To further study their differentiation, we seeded sorted DN1 CD122−NK1.1− thymocytes on irradiated OP9 bone marrow stromal cells with IL-15, IL-7, Flt3L, and stem cell factor. NK1.1+ cells emerged after 7 days. In vitro differentiated NK cells acquired markers associated with immature bone marrow–derived NK cells, but also expressed CD127, which is typically found on thymic NK cells. Furthermore, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated on target exposure. Together, these data indicate that functional thymic NK cells can develop from a DN1 progenitor cell population.

Identification of an NK/T cell–restricted progenitor in adult bone marrow contributing to bone marrow– and thymic-dependent NK cells

AbstractAlthough bone marrow (BM) is the main site of natural killer (NK)–cell development in adult mice, recent studies have identified a distinct thymic-dependent NK pathway, implicating a possible close link between NK- and T-cell development in adult hematopoiesis. To investigate whether a potential NK-/T-lineage restriction of multipotent progenitors might take place already in the BM, we tested the full lineage potentials of NK-cell progenitors in adult BM. Notably, although Lin−CD122+NK1.1−DX5− NK-cell progenitors failed to commit to the B and myeloid lineages, they sustained a combined NK- and T-cell potential in vivo and in vitro at the single-cell level. Whereas T-cell development from NK/T progenitors is Notch-dependent, their contribution to thymic and BM NK cells remains Notch-independent. These findings demonstrate the existence of bipotent NK-/T-cell progenitors in adult BM.

Distal-less homeobox transcription factors regulate development and maturation of natural killer cells

Natural killer (NK) cells constitute a subpopulation of lymphocytes that develop from precursors in the bone marrow (BM), but the transcriptional regulation of their development and maturation is only beginning to be understood, in part due to their relatively rare abundance, especially of developmental subsets. Using a mouse model in which NK cells are arrested at an immature stage of development, and a gene expression profiling approach, we uncovered transient normal NK cell expression of a homeobox transcription factor (TF) family, called Distal-less (Dlx), which had been primarily implicated in murine CNS, craniofacial, limb, and skin development. Our studies demonstrate that Dlx1, Dlx2, and Dlx3 are transiently expressed in immature Mac-1(lo) NK cells within the BM, with Dlx3 being the predominantly expressed member. These genes are expressed in a temporally regulated pattern with overlapping waves of expression, and they display functional redundancy. Expression is extinguished in fully mature splenic NK cells, and persistent expression of Dlx genes leads to functionally immature NK cells arrested at the Mac-1(lo) stage. Whereas conventional splenic NK cells develop but are arrested at an immature stage, there appears to be a complete failure to develop CD127(+) thymic NK cells when Dlx genes are persistently expressed. We also observed that T and B cells fail to develop in the context of persistent Dlx1 expression. Thus, these studies indicate that Dlx TFs play a functional role in lymphocyte development.

10th Meeting of the Society for Natural Immunity, Cambridge, UK, 11–14 April 2007

Natural killer (NK) cells are important members of the innate immune system being involved in the detection and clearance of transformed or virus-infected cells. The 10th meeting of the Society for Natural Immunity was recently held in Cambridge, UK, where leaders from around the globe gathered to discuss the latest developments and understandings in the field of NK cell biology. Among the topics covered in this meeting were NK cell development, the origin of thymic NK cells, contribution of NK cells to viral infections and subsequent tissue damage, crosstalk between NK cells and other immune cells and the role of NK cells at the foetal-maternal interface.

Bone marrow versus thymic pathways of natural killer cell development

Understanding natural killer (NK) cell developmental pathways is crucial for harnessing the potential therapeutic benefits of this specialized lymphocyte subset. The bone marrow (BM) plays a major role in NK cell development, providing the appropriate environmental cues for NK cell commitment and subsequent NK cell differentiation. Nevertheless, the molecular signals provided in this context remain enigmatic. It is widely assumed that BM seeds the periphery with NK cells. However, the precise origins of NK cells found in lymphoid organs and tissues are not defined. Recently, we found that thymic NK cells bear molecular markers and functional attributes that distinguish them from most peripheral NK cells. We find that NK cells are actively exported from the thymus to the periphery, suggesting that thymus-derived NK cells may have unique roles both intrathymically and in secondary lymphoid organs. Here we compare the properties of thymic NK cells with properties of other NK cell subsets that have been identified in the mouse. We propose that heterogeneity in NK cell function can be achieved through distinct thymic and bone marrow pathways of NK cell development.

A thymic pathway of mouse natural killer cell development characterized by expression of GATA-3 and CD127

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.

Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2.

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2-/- mice that exhibit defective NK cell development. In the Id2-/- FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2-/- FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2-/- fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2-/- and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Lineage commitment and differentiation of T and natural killer lymphocytes in the fetal mouse.

T cells and natural killer (NK) cells are presumed to share a common intrathymic precursor. The development of conventional alpha beta T lymphocytes begins within the early fetal thymus, after the colonization of multipotent CD117+ precursors. Irrevocable commitment to the T lineage is marked by thymus-induced expression of CD25. However, the contribution of the fetal thymus to NK lineage commitment and differentiation remains largely unappreciated. Recently, we demonstrated that the development of functional mouse NK cells occurs first in the fetal thymus. Moreover, the appearance of mature fetal thymic NK cells (NK1.1+/CD117-) is preceded by a thymus-induced developmental stage (NK1.1+/CD117+) that marks lineage commitment of multipotent hematopoietic precursors to the T and NK-cell fates. Commitment to the T/NK bipotent stage is induced by fetal thymic stroma, but is not thymus dependent. Recent data indicate that CD90+/CD117lo fetal blood prothymocytes exhibit NK lineage potential and are phenotypically and functionally identical to fetal thymic NK1.1+/CD117+ progenitors. This finding also indicates that full commitment of circulating precursors to the T-cell lineage occurs after thymus colonization. In this review, we discuss recent insights into the cellular and molecular events involved in fetal mouse T and NK lineage commitment and differentiation to unipotent progenitors.

Human natural killer cell committed thymocytes and their relation to the T cell lineage.

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.

Association between the degree of thymic dysplasia and the kinetics of thymic NK cell activity during the graft-versus-host reaction

In this study, by employing different cell doses and parent into F 1 hybrid combinations, we have investigated the relationship between the severity of thymic medullary dysplasia and the kinetics of thymic natural killer (NK) cell activity after the induction of graft-versus-host (GVH) reactions. GVH reactions were induced by injecting different doses (30, 20, 10 × 10 6) of C57BL 6 (B6) of A parental lymphoid cells (PLC) into non-X-irradiated adult B6xAF 1 (B6AF 1) mice. On different days after the induction of GVH reactions, the thymuses were examined histologically and thymocyte NK cell activity was tested by using YAC targets. Our results show that, depending upon the genotype and dose of PLC injected, various degrees of thymic medullary dysplasia (mild, moderate, or severe) can be induced. Furthermore, severe to moderate thymic medullary dysplasia is observed only in those groups of GVH-reactive mice in which thymic NK cell activity occurs early and increases rapidly. In contrast, when mild thymic medullary dysplasia or no thymic alterations was observed, thymic NK cell activity peaked later and was of lower intensity than that of the groups with moderate to severe lesions. These results suggest an association between the degree of thymic medullary dysplasia and the kinetics of NK cell activity in the thymus. Furthermore, the different degrees of thymic medullary dysplasia as described here may serve as a powerful tool to study the role of thymic medullary dysplasia in determining the duration of T-cell immunodeficiency associated with the GVH reactions.

Natural killer cells in murine muscular dystrophy: IV. Characterization of percoll fractionated splenic and thymic natural killer cells and natural killer-sensitive thymocyte targets

The Natural Killer (NK) activity in the thymus and NK-sensitive thymocyte targets of dystrophic mice was investigated. Dystrophic and normal mouse thymocytes or spleen cells were layered on discontinuous Percoll gradients (5 or 10% increments, respectively) between 40 and 70% and centrifuged at 1700g for 30 min. All fractions were tested for either NK activity or used as 51Cr-labeled NK-sensitive targets in a 6-hr 51Cr release assay. The density interface between the 50% (1.060 g/ml) and 60% (1.075 g/ml) Percoll fractions of either dystrophic or normal mouse spleen cells and the 40% (1.050 g/ml) and 50% (1.060 g/ml) Percoll fractions of either dystrophic or normal mouse thymocytes were found to contain the largest proportion of NK activity using YAC-1 lymphoma tumor cells as targets. In addition, the NK activity in dystrophic mouse spleen cells and thymocytes was significantly greater when compared with normal mouse controls. Target binding cell studies revealed that these Percoll fractions of dystrophic mouse spleen cells and thymocytes had greater numbers of conjugate-forming cells when compared with normal control groups. Cell depletion experiments using either anti-Thy 1.2, anti-asialo-GM 1 or anti-NK-1 plus complement treatment revealed that the cell responsible for NK activity in the 50% Percoll fraction interface of dystrophic mouse spleen cells was asialo-GM 1 positive, NK-1 positive, and partially Thy 1.2 positive. However, the cells displaying NK-activity in the thymus of normal or dystrophic mice were found to be highly Thy-1.2 positive and peanut agglutinin (PNA) negative. The density interface between the 60% (1.075 g/ml) and 65% (1.081 g/ml) Percoll fractions of either normal or dystrophic mouse thymocytes contained the largest proportion of NK-sensitive target cells. Interestingly, the 60% Percoll fraction of dystrophic mouse thymocyte targets was significantly more susceptible to NK-mediated lysis than that of the normal mouse thymocyte population. Cell depletion experiments revealed that the NK-sensitive thymocyte population was similar in both mice, that is. Thy-1.2 positive, cortisone sensitive, PNA positive. Dolichos biflorus (DBA) negative and asialo GM-1 negative. The results indicate that there are density differences between splenic and thymic NK cells. In addition, there are density and phenotypic differences between thymic NK cells and thymic NK-sensitive target cells. The findings support the hypothesis that there are different populations of NK cells.