While the concept of immunologic memory has been well described in T and B adaptive immune lymphocytes, several recent studies suggest that NK cells can also exhibit properties of memory, both specific and non-specific (10
). O’Leary et al
) demonstrated that in the absence of T and B cells, NK cells can mediate a hapten-specific contact hypersensitivity (CHS) response, a classic example of adaptive immune-mediated delayed type hypersensitivity reaction. A CHS response was present in recombination-activating gene 2 (Rag2)-deficient and sever combined immunodeficient (SCID) mice lacking T and B cells but possessing NK cells, whereas the response was absent in Rag2-deficient, γc-deficient mice which lack T, B, and NK cells. Moreover, adoptive transfer of NK cells from hapten-sensitized mice into naive mice resulted in a CHS reaction when recipients were challenged with the original hapten (10
). This observation is consistent with a memory-like response mediated by NK cells and suggests that NK cells are sufficient for CHS. It is unclear whether there was direct recognition of haptenated protein by NK cells; however, the response was specifically seen after the transfer of liver NK cells expressing the Ly49C/I+
). This receptor specificity suggests two possibilities. The first is that NK cell licensing via inhibitory receptors for self-MHC (Ly49C recognition of H2Kb
in this case) is a pre-requisite for development of NK cells with memory-like functions. A second possibility is that there are alterations in NK cell recognition of haptenated cells specific to Ly49C-positive NK cells. While the exact mechanism remains unclear, these studies suggested that NK cells are capable of mediating a specific recall response that was traditionally a hallmark property of adaptive immunity.
In our own studies of NK cell ‘memory’, we explored the possibility that previously stimulated NK cells would display a component of classical immunological memory, i.e. an enhanced response upon secondary challenge. Moreover, of the two primary and complementary mechanisms to activate NK cells, cytokines and NKRs, we chose to investigate the functional consequences of cytokine stimulation of NK cells. We utilized an in vivo
adoptive transfer system to address the question of whether stimulation of NK cells via cytokines alone renders a memory-like effect (11
). In this system, freshly isolated splenic NK cells were activated with IL-12 and IL-18 and cultured overnight in the presence of a low-dose of IL-15 as a survival factor. NK cells were then labeled with carboxyfluorescein diacetate succinamidyl ester (CFSE) and adoptively transferred into naive hosts in parallel with control-treated NK cells, which received only IL-15. The combination of IL-12 and IL-18 induced >90% of cells to produce IFN-γ, ensuring that nearly all transferred cells were secreting IFN-γ at the time of transfer. However, by one week after transfer, previously activated NK cells returned to a resting state and did not constitutively produce IFN-γ (11
). At that timepoint, preactivated NK cells did not exhibit a distinct phenotype and expressed similar levels of multiple activation and cytokine receptors as control-transferred cells. One notable difference between preactivated and control cells is that cells stimulated with cytokines went on to divide in vivo
within 72 h of transfer (11
, authors’ unpublished observations). Transferred NK cells were seen in multiple organs, and similar proliferation was observed in the spleen, liver, and lymph nodes. These data suggest that following a response to local inflammation, NK cells may have the capacity to traffic to distant sites where they can then proliferate and reside.
While preactivated NK cells did not continuously produce IFN-γ at one week following adoptive transfer, they demonstrated a significantly more robust response to re-activation in vitro
than control-transferred or endogenous host NK cells (11
). This was observed when cells were re-stimulated ex vivo
with cytokines (such as IL-12 + IL-15), or via engagement of the activating NKRs NK1.1 and Ly49H, activation signals to which they had never been previously exposed. This effect was NK-intrinsic and not related to alteration of the host environment, since co-transfer of preactivated and control-treated congenic NK cells into the same host resulted in similar findings (authors’ unpublished observations) (). Enhanced response to NK cell re-stimulation by preactivated cells persisted greater than four weeks (authors’ unpublished observations) (), a relatively long time for NK cells considering that their half-life had been estimated to be approximately one to two weeks (68
). These findings suggest that based on a prior experience, NK cells fundamentally change the way that they respond to later activation, a central property of immunologic memory. This property is best described as ‘memory-like’, since it represents a non-specific activation of a long-term memory type response.
Enhanced IFN-γ production by memory-like NK cells is maintained for at least 4 weeks
One potential explanation for gain of memory-like responses by NK cells in this system is that proliferation in vivo
might allow for changes in daughter NK cells enabling them to produce IFN-γ. However, this was not the case, and identification of the original parental NK cells and daughter generations by CFSE dilution clearly showed that all preactivated NK cell generations had a similar memory-like phenotype (11
). These data suggest that NK memory-like responses are heritable and passed on to daughter cells that were never previously activated.
NK cell memory-like responses were also recently evaluated in the context of receptor activation (12
). While the overall NK cell receptor repertoire is limited, some NKRs have been found to specifically bind pathogen-encoded ligands, akin to foreign antigens as discussed. The most well-described of these interactions is the activating Ly49H receptor, which is expressed on approximately 50% of NK cells from certain mouse strains, including C57BL/6 mice, and is specific for the MCMV-encoded ligand m157 (54
). We previously demonstrated that Ly49H+
NK cells specifically undergo a rapid proliferative phase with significant expansion followed by a contraction phase two weeks after infection (71
). Expansion of Ly49H+
NK cells is driven by signals delivered directly through Ly49H and observed even in the absence of cytokines such as type I interferons (72
). Using a model where splenic NK cells were adoptively transferred into DAP12-deficient mice, Sun et al
) recently found that following MCMV infection, previously activated Ly49H+
NK cells persist greater than two months. Interestingly, these NK cells were more responsive to activation in vitro
70 days after initial MCMV infection and expressed a more ‘mature’ phenotype with low levels of CD27 and higher levels of Ly6C, KLRG1, and CD43 (12
). In addition, adoptive transfer of Ly49H+
NK cells that were previously activated during MCMV infection provided superior protection compared to naïve NK cells against MCMV challenge of DAP12−/−
newborn mice which, for the most part, lack functional Ly49H (12
). These authors suggest that Ly49H NK cells differentiate into antigen-specific ‘memory’ NK cells with characteristics more typical of memory T cells than cytokine-induced memory-like NK cells (12
). It will be interesting to learn whether the protective effect of Ly49H+
MCMV-induced memory NK cells extends to other infections or is specific to m157-expressing MCMV, as would be predicted based on adaptive immune models of memory. Moreover, it will be of interest to determine if memory Ly49H+
NK cells control reactivation of MCMV, which undergoes latency. However, MCMV can mutate such that m157-deficient viral clones can emerge as early as 3 weeks after infection (75
), suggesting that MCMV could escape from antigen-specific memory NK cells.
These studies point toward a previously unrecognized attribute of NK cells and innate immunity, the capacity to exhibit memory-like immune responses. It appears that memory-like NK cells can be generated via activation with cytokines or through engagement of activating receptors. The relationship between these two mechanisms of activation and relevance in vivo remains to be seen, although it is clear that NK cells acquire the ability to mediate enhanced responses based on prior activation.