Evidence That Novel NK-NK Cell Subset Regulation Exists With Regard To Effects In Tumor and Viral Models

Natural killer (NK) cells are lymphocytes of the innate immune system and are classically associated with cytotoxic responses to both virally infected as well as neoplastic cells. Activation of NK cells to exhibit their cytotoxicity is dependent on signaling through a number of activating and inhibitory receptors. In mice, one such family of inhibitory receptors is the C-type lectin-like Ly49 family. In humans, the killer immunoglobulin-like receptors (KIRs) serve as the primary family of inhibitory receptors and are functional analogs of the Ly49s. Despite markedly different structures, the Ly49s and KIRs display similar binding capabilities and bind primarily to distinct MHC class I haplotypes, which plays an important role in regulating NK cell function. NK cells that express inhibitory receptors that are specific for the MHC class I haplotype of the individual are termed “licensed” and have been shown to have increased functionality in terms of cytotoxicity and cytokine production. In contrast, NK cells that express inhibitory receptors that are unable to bind to the MHC class I haplotype of the individual are termed “unlicensed” and have been shown to be hyporesponsive.

We have recently reported on the role of NK licensing on the immune response to viral infections such as MCMV. In addition, we have previously described how regulatory T cells can regulate NK cell activity in vivo. However, there are limited data examining the interaction and regulation between the different NK subsets based on differences in licensing. We hypothesized that different NK cell subsets, based on licensing, can regulate each other in the context of anti-tumor and anti-viral responses.

Here we first provide in vitro data providing evidence to support the hypothesis of NK-NK regulation based on licensing. In vitro killing assays using MCMV infected fibroblasts, or C1498 (murine acute myeloid leukemia) cells as targets and using different combinations of murine NK Ly49 subsets as effectors were used to assess this NK-NK regulation. To further test our hypothesis, in vivo experiments were also performed using a mouse leukemia model as well as an MCMV model. Mice were injected with C1498 cells and then given hematopoietic stem cell transplantation (HSCT). The mice were then depleted of all NK cells or either licensed or unlicensed subsets by antibody depletion once a week, and monitored for survival. Mice that were depleted of the unlicensed population survived significantly longer compared with the other depleted groups, suggesting a negative regulation of the anti-tumor response by the unlicensed population resulting in greater tumor burden and death in the presence of the unlicensed population. This negative regulation by the unlicensed population is further supported by another experiment where mice were infected with MCMC following total NK or subset depletion and monitored for ten days throughout the course of the immune response to MCMV. Mice that were depleted of their unlicensed population displayed a significantly larger expansion of the licensed population of NK cells, without reciprocal greater expansion of the unlicensed population upon licensed NK cell depletion. More specifically, depletion of the unlicensed population resulted in an expansion of the Ly49H⁺NK cells which have previously been shown to be the primary effector population during MCMV infection. Thus, the unlicensed NK cells are playing a role in down-regulating the anti-viral response by limiting the expansion of the effector licensed population.

Our data highlight a role for the murine NK subsets to negatively regulate the immune response of the effector licensed NK population in the context of anti-tumor and anti-viral responses. This new insight into the regulatory role of NK cells may have clinical benefit for patients receiving bone marrow transplants during cancer treatment to enhance graft vs. tumor effects, and to combat opportunistic viral infections that may manifest in the immune compromised environment of the BMT patient.