Loss of Canonical Notch Signaling Affects Multiple Steps in NK Cell Development in Mice

This paper, published online in The Journal of Immunology in October 2018 by a research team, led by Dr. Ewa Sitnicka out of the division of molecular hematology at the Lund Research Center for Stem Cell Biology and Cell Therapy at Lund University in Sweden. This specific research was spearheaded by Dr. Patricia Chaves and focused on the role of Notch signaling in the development and differentiation of natural killer (NK) cells. NK cells are part of a group of lymphocytes that exist under the umbrella of the innate immune system, but have the capacity to initiate an adaptive immune response (Mak et al., 2014; Fig 1). NK cells are large, non-phagocytotic, lymphoid cells that have the capacity to contain cytotoxic granules that contain the enzymes perforin and granzymes, which have the capacity to rip through cell membranes, destroying the target cell (Fig.1; Mak et al., 2014; de Saint Basile et al., 2010).

Figure 1. A cartoon rendition and an image of a Natural Killer (NK) cell. Created by Mak et al. (2014).

The capacity for an NK cell to be cytotoxic is essential for its primary function within the immune system, to induce cytolysis to destroy tumor cells or virus infected cells (Mak et al., 2014, de Saint Basile et al., 2010, Chaves et al., 2018). Their cytotoxic ability is triggered by a combination of signals received from two competitor signaling pathways; NK activitory signaling and NK inhibitory signaling (Mak et al., 2014). Only if both signals are received and the activitory signal outweighs the inhibitory will the NK cells begin to lyse cells (Mak et al., 2014). NK cells also can travel to any tissue, allowing them to perform their effector functions throughout the host (Mak et al., 2014). In tissue where tumor cells or viral infected cells are present, NK cells can also release cytokines and chemokines (Mak et al., 2014)cells arise from the same precursor cell that T cells arise from, the common lymphoid progenitor cells (CLP), which derive from the hematopoietic stem cells (HSC) developed in the bone marrow (Mak et al., 2014; Chaves et al., 2018; Fig. 2).

Figure 2. The Natural Killer cell lineage pathway. Created by Mak et al. (2014). 

NK cells can then either arise from CPLs present in the bone marrow or from CPLs that have migrated to the thymus (Mak et al., 2014; Chaves et al., 2018; Fig. 2). In either location, the down-regulation of the protein FLT3 and the upregulation of the production of IL-2 and IL-15 on the CLP restricts its differentiation ability and the CLP is renamed to a restricted NK progenitor (rNKP)(Chaves et al., 2018). rNKP cells then upregulate their NK1.1 receptor, distinguishing them now as immature NK cells (iNK). Further differentiation and the changing expression of different surface receptors eventually produce mature NK cells (mNK), which are then capable of carrying out effector functions (Chaves et al., 2018; Fig.3). Early progenitor cells that give rise to NK cells can also follow a differentiation pathway that leads to the production of T cells; an adaptive lymphocyte (Mak et al., 2014; Chaves et al., 2018; Fig. 2). This suggests that T cells and NK cells share similar transcription factors that turn on certain genes because they develop from the same progenitor cell (Chaves et al., 2018). Notch signaling is an essential part of T cell development, as the binding of Notch 1 on cells early in T cell development to Notch promotes further development while blocking all other differentiation pathway (Mak et al., 2014; Chaves et al., 2018). Previous studies to Chaves et al. (2018) indicated that NK cell development could also be promoted by Notch (Kling & Blumenthal, 2016). This connection, as well as the physiological role of Notch signaling in early NK cell development was investigated by Chaves et al. (2018) in the current paper.

Figure 3. The maturation steps of Natural Killer cell in the bone marrow and spleen. Created by Chaves et al. (2018). 

            To observe the role of Notch signaling in the development of NK cells, Chaves et al. (2018) conducted their experiments on mice that contained bone marrow cells that had the Rbp-Jk, a transcriptional effector in Notch signaling, deleted (Fig. 4). Without this transcriptional effector, the signal from the binding of Notch to the NK cell cannot be delivered (Chaves et al., 2018). The NK cell populations studied were derived from the bone marrow, spleen, peripheral blood and thymus of the Rbp-Jk deletion mice and from wild type mice. In these cell populations from each tissues, the researchers used flow cytometry to observe the presence of certain receptors, the production of cytokines, or to sort by cell type (Chaves et al., 2018). RT-PCR was also used to detect certain genes associated with NK cell development to help discern where in the development the role of Notch, if any, comes into play. Lastly, Chaves et al. (2018) implanted the Rbp-Jk deficient bone marrow cells from one mouse into another mouse that had been irradiated to get rid of pre-existing bone marrow cells and then NK cell activity and maturation stage was observed.

Figure 4. The canonical Notch Signaling pathway. The transcriptional effector Rbp-Jk is marked with a gold star. Created by Borggrefe and Oswald (2009). 

            The first experiment in this series assessed the potential involvement of Notch in NK development. The general conclusion reached indicated that the absence of Notch signaling leads to an impairment in hematopoietic development. After exploring the expression of Rbp-Jk and other genes related to the Notch pathway in NK cells from different populations in the host to ensure that knocking out Rbp-Jk in mice would prevent Notch signaling, the research team then compared the make-up of lymphocytes in Rbp-Jk deleted mice to wild type mice. The Rbp-Jk deleted mice decreased significantly in their T cell counts and in their NK cells in the peripheral blood and the thymus while B cells increased, indicating that in the absence of Notch signaling, cells were not differentiating into T cells and NK cells as often as in the presence of Notch signaling (Chaves et al., 2018).

            Further building on the previous result, Chaves et al. (2018) looked next at where the impact Notch signally occurred in NK development. When the cell types present in the bone marrow were compared between Rbp-Jk deleted mice and wild type mice, the Rbp-Jk deleted mice had significantly lower levels of pre-NKP and rNKP cells, while the levels of CLP between the two populations of mice did not change (Chaves et al., 2018). These results indicated that Notch signaling plays an important role in early NK development (Chaves et al., 2018). In addition to this, Chaves et al. (2018) also looked at the population of NK cells in the spleen of the two populations of mice, as NK cells finish their development in the spleen. The levels of iNK cells and an earlier stages of mNK cells were higher in the Rbp-Jk deleted mice than in the wild type, but the final maturation stage (mNK6; Fig. 3) of NK cells in the spleen were lower in the Rbp-Jk deleted mice (Chaves et al., 2018). These results together indicate that Notch signaling plays both a role in early NK development and in the last stages of development (Chaves et al., 2018).

            Chaves et al. (2018) then observed the timing of the expression of key cell surface proteins in the NK cells and the expression of key genes controlling the development of the NK cells. The results of these two sets of experiments revealed that NK cell maturation is delayed and NK regulatory pathways are dysregulated in the absence of Notch signaling (Chaves et al., 2018). Key receptors for NK cell function, such as Ly49, TRAIL, and KLRG1 (see Fig. 3) were expressed in the wrong concentrations at the wrong stage of development in Rbp-Jk deleted mice, indicating that in the absence of Notch the phenotype of the NK cells do not correlate with the phenotype of NK cells in the same stage in the wild type mice, meaning that the NK cells in the Rbp-Jk mice do not appear how they should given their maturation stage and thus their further development could be delayed or impaired because these cells are not expressing the correct concentrations of proteins (Chaves et al., 2018). In addition, as congruent to the results above, the expression levels of key regulatory genes in both early and late stages of development in the Rbp-Jk mice also diverged from the wild type, indicating that in the absence of Notch signaling, the NK development pathway is not correctly regulated and is then impaired in correctly regulating NK development (Chaves et al., 2018).

            While the levels of NK cells in the Rbp-Jk deleted mice was were lower than the wild type mice, the few NK cells that reached maturity in the Rbp-Jk deleted mice exhibited a hyperactive phenotype (Chaves et al., 2018). Chaves et al. (2018) measured this by utilizing the degranulation assay, which included plating spleen cells, identifying the NK cells, stimulating them and then measuring the degree to which they degranulated or produced the inflammatory cytokine IFN-gamma by use of flow cytometry (Chaves et al., 2018). The results of these experiments illustrated that the few mature NK cells in the Rbp-Jk deleted mice exhibited a hyperactive phenotype (Chaves et al., 2018). The NK cells in the Rbp-Jk deleted mice maintained the ability to release cytotoxic granules, as evident by the increase in the cell surface protein LAMP-1, which is an indicator for NK cytotoxic capability because it is up-regulated in NK cells that have become activated and are primed to release granules (Chaves et al., 2018). INF-gamma, the inflammatory cytokine that activates T cells involved in an inflammatory response, was also upregulated in the NK cell population that was present in the Rbp-Jk deleted mice, indicating that the NK cells retained their ability to initiate an adaptive immune response (Mak et al., 2014; Chaves et al., 2018). This hyperactive phenotype in the remaining NK cells can potentially be explained by the increased and prolonged production of the cytokine IL-15 by the NK cells, which in instances of prolonged exposure, has been demonstrated to chronically activate and changes in the phenotype of NK cells (Elpek et al., 2010; Chaves et al., 2018). In summary, while the absence of Notch signaling severely limits the concentration of NK cells, the few NK cells that do mature  develop a hyperactive phenotype, in what seems like an attempt to counterbalance the lack in numbers (Chaves et al., 2018).

            The last set of experiments conducted by Chaves et al. (2018) aimed to answer if the effects of the Rbp-Jk deletion was cell intrinsic, meaning did the effects of Rbp-Jk deletion derive from inside the cell or something in the external environment. To answer this, Chaves et al. (2018) created bone marrow chimeras, which are mice that have their bone marrow irradiated and then replaced with donor lymphoid progenitor cells, in this case the donor cells came from the Rbp-Jk deleted mice. These bone marrow chimeras were unable to reconstruct their hematopoietic cells in addition to having lower levels of early stage NK cells (pre-NKPs and rNKPs) in addition to lower levels of late stage NK cells (mNK6) than control mice (Chaves et al., 2018). This matches the pattern seen in the original Rbp-Jk deleted mice. In addition, the opposite bone marrow chimeras, originally Rbp-Jk deleted mice implanted with wild type bone marrow, were able to produce normal levels of NK cells at all stages (Chaves et al., 2018). These results combined demonstrate that the effects of Notch signaling, or the lack thereof, are cell intrinsic, as the lack of Rbp-Jk led to the same pattern, no matter what mouse these cells were present in (Chaves et al., 2018). The effects of the loss of Notch signaling occur early on in mouse development, as mice in early postnatal stages missing Rbp-Jk had similar NK deficiencies seen in adult mice missing Rbp-Jk (Chaves et al., 2018).
          In conclusion, the loss of Notch signaling effects the development of early stage NK cells as well as late maturation NK cells, both blocking the maturation of existing NK cells as well as severely decreasing the available pool of NK cells in the host. This cell intrinsic effect occurs early in mouse development and persists into adulthood, potentially limits the immune system of the given mouse from protecting itself against tumors and viral infections (Chaves et al., 2018). However, the NK cells that are able to mature exhibit a hyperactive phenotype, a possible attempt by the immune system to offset the limited number of NK cells (Chaves et al., 2018). Chaves et al. (2018) indicated that further research was needed to identify exactly how the loss of Notch signaling impacts NK cells at the molecular level, but another interesting direction further research could take would be to identify how some NK cells are still able to develop despite the lack of Notch signaling, which was demonstrated by Chaves et al. (2018) to be important in their maturation.

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