The targeting of NK and NKT cells within the liver microenvironment represents an important goal for immunotherapy of liver disease (). Many combination immunotherapies have been described that augment NK cell numbers and activity within the liver microenvironment. One tactic has been the use of pathogens, such as attenuated Listeria monocytogenes,
that specifically target NK cells. Bahjat, et al showed that this approach resulted in the migration and activation of NK cells within the liver and the concomitant NK-dependent destruction of hepatic tumors [47
]. This study demonstrates that microbial stimuli are capable of potent immune activation resulting in the establishment of tumor-specific immune responses. Immune modulating cytokines comprise another, major approach for the manipulation of NK cells. Smyth, et al showed that IL-2 and IL-12 each resulted in the suppression of tumor metastases through an NKG2D-dependent pathway that involved perforin-mediated cytolysis [48
]. These two cytokines were more effective against tumors expressing NKG2D ligands. In contrast, IL-18 was found to mediate the NKG2D-independent, Fas ligand-mediated rejection of tumors [48
]. The implications of this important study are that the Fas ligand-sensitivity and expression of NKG2D ligands on tumors needs to be monitored as this may reflect the tumor responsiveness to a particular immunotherapy. In our own studies, we utilized plasmid DNA encoding cytokine genes with the rationale that these may serve as useful adjuvants for cancer vaccines and might also be potentially efficacious in combination with other immunomodulatory agents. We reported that the intradermal injection of plasmid DNA encoding murine IL-12 elicited the systemic expression of IL-12 as well as IFNγ and IFNγ-inducible chemokines within 24 hours [49
]. The expressed cytokine was functional in that NK cell activity was augmented even in mice deficient in endogenous IL-12 p40 expression. In another study, we showed similarly that hydrodynamically delivered IL-2 cDNA caused a sustained increase in NK cell numbers and NK-mediated cytolytic activity in liver and spleen leukocytes [50
]. Furthermore, the treatment of mice bearing established lung and liver metastases showed that IL-2 plasmid DNA was an effective treatment against liver metastasis and had moderate effectiveness against lung metastasis as well.
Early and ongoing studies from our laboratory have characterized the mechanisms that regulate the recruitment of NK and NKT cells to the liver in response to proinflammatory cytokines. We showed that a variety of exogenously added cytokines resulted in the recruitment and activation of hepatic NK cells. For example, systemic IL-2 administration resulted in the rapid and sustained recruitment of NK cells in the liver[50
]. IL-12 also induced NK recruitment to the livers of treated mice through an IFN-γ dependent pathway [7
]. Less is known about the recruitment of NKT cells to the liver following activation, however the chemokine receptor CXCR6 plays a crucial role in NKT cell homeostasis and for patrolling the liver sinusoid [10
]. Human NKT cells were examined for chemokine receptor profiles and were found to express receptors associated with inflammatory chemokines [51
]. In contrast to conventional T cells, only a low percentage of NKT express CCR7, a chemokine receptor found on naïve or memory T-cells. This chemokine receptor profile suggests NKT cells intrinsically have an activated/primed phenotype permitting quick mobilization to sites of inflammation. Certain viral infections similarly augment NK cell number and/or activity within the liver, and this is frequently associated with the production of proinflammatory cytokines. The mechanisms whereby this occurs include the induction of chemokines, such as macrophage inflammatory protein (MIP) 1-α that mediates the CCR5-dependent recruitment of NK cells into the liver [8
]. Our lab and others have shown an important role for cytokines such as TNFα in the recruitment of NK cells into the liver[52
]. Based on the well established ability of TNFαto upregulate adhesion molecule expression on endothelial cells, we also demonstrated the critical role for vascular cell adhesion molecule-1 / very late activation antigen-4 interactions in mediating NK recruitment and subsequent activation within the livers of poly-ICLC and IL-2 treated mice[7
]. Although the complex milieu of cytokines produced in vivo
by resident hepatocytes, Kupffer cells, endothelial cells as well as infiltrating leukocytes makes it challenging to assign the relative importance of a single cytokine for the recruitment of NK cells into the liver, it is evident that a more complete understanding of the molecular mechanisms regulating NK recruitment and activity within the liver microenvironment is essential for the design of improved immunotherapy to treat liver diseases.
Clearly, much more work needs to be done in order to define the contributions of NKT cells as cytolytic cells. NKT cells were first shown as anti-tumor effector cells when it was found that the potent anti-tumor ceramide αGalCer, activated NKT cells in a CD40/CD40L and B7 dependent manner [53
]. Subsequent studies examining the inflammatory response of αGalCer-activated NKT cells found a coordinated interaction between NKT cells and APCs expressing αGalCer that leads to activation of NK cells. Kitamura found αGalCer activated NKT cells express CD40L that engages CD40 on APCs triggering IL-12 production that stimulated NKT cells to produce IFNγ [54
]. In turn, NKT-derived IFNγ activates NK cells causing them to become more cytotoxic and induce the activation of CD8+
cytotoxic T lymphocytes [55
]. Smyth, et al expanded these findings by demonstrating that αGalCer activated NKT cells stimulate the proliferation and cytolytic activity of NK cells and that this newly-expanded NK cell population could be made to be even more cytolytic by the subsequent administration of systemic IL-21 [56
]. Crosstalk cascades have been found with dendritic cells for both NK and NKT cells. Adam, et al showed that primary rejection of lymphoma and the establishment of long-term T cell memory was dependent upon reciprocal cellular receptor- and cytokine-mediated interactions between NK cells and DC [57
]. αGalCer-pulsed DC also result in NK-dependent tumor rejection in a number of hepatic metastasis models [29
]. These crosstalk studies further confirmed a critical role for IL-12 production by the APC and the ensuing IFNγ production by NK and NKT cells. Because IL-12 is a central player in the αGalCer-induced immune cascade, other therapies using IL-12 have demonstrated remarkably similar anti-tumor activities and a dependence on NKT and NK cells [26
]. However, a study examining B16.F10 melanoma in the liver found that IL-12 had therapeutic effects in the absence of NKT cells and found NK cells were the essential cells for tumor rejection [59
]. Thus, contrasting roles for hepatic NK and NKT cells may exist and may possibly be dependent upon the cellular microenvironment. The unique cellular interactions between immune cells are likely to play an important role in the priming and regulation of both innate and adaptive immune responses against liver tumors. It is critical that we now define the molecules involved in these cell-cell interactions, so that improved therapies can be developed.
Intriguingly, the use of cytokine combinations to treat tumor-bearing mice has suggested that the liver microenvironment, but not that of other organs may be particularly sensitive to immune surveillance by NK cells. Although synergistic anti-tumor responses against orthotopic kidney tumors have been achieved using IL-2/IL-12 or IL-2/IL-18 combinations, these anti-tumor effects were maintained in mice depleted of NK cells [25
]. Although, for a variety of transplantable and chemically-induced murine tumors, the depletion of NK cells (using anti NK1.1 antibody) resulted in the growth and metastatic spread of several transplantable and chemically-induced murine tumors [60
], a caveat of these studies however, is that anti-NK1.1 depletes not only NK cells, but also NKT cells. The development of mice deficient in NK cells, but not NKT or T cells, would therefore be optimally suited for the refinement of NK cell contributions towards anti-tumor responses. For tumors arising in the liver, however, we believe that the augmented NK cytolytic activity within this particular microenvironment endows them with a critical role for the control of hepatic tumors. We showed recently that NK cells were an important component for tumor regression in a murine liver tumor model () [20
]. In this study, the combined administration of IL-18 and IL-12 augmented the numbers of and production of IFNγ by hepatic NK cells concomitantly with the removal of immunosuppressive NKT cells, which resulted in significantly reduced tumor nodules in the livers of treated mice (). Ongoing experiments in our laboratory are examining whether the IL-12/IL-18 regimen uniquely primes NK cells for enhanced anti-tumor responses. Taken together, these data suggest that although NK cells may not be principal mediators of the anti-tumor response in certain tumor models, they are critical components of effective anti-tumor responses in the liver microenvironment and possibly other sites.
Although this work focuses on tumor immunotherapy, it is equally clear that there are significant roles for NK, NKT cells and of course innate immunity in the regulation of the normal immune response as well as in autoimmunity. We will not review this literature but will cite recent work [61
]. We believe it likely that the most effective immunotherapeutic approaches will be those that take advantage of the unique cellular microenvironment of the liver for robust NK cell activation and the coordinated engagement of NKT cells.