The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

Defining the role of IL-15 trans-presentation by distinct cell-types during the development and homeostasis of Natural Killer and invariant Natural Killer T cells

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK cells, I revealed the non-hematopoietic compartment provides IL-15 to NK cells in the early stages of development while hematopoietic cells were crucial for the generation and maintenance of mature NK cells. Regarding iNKT cells in the thymus, IL-15 trans-presentation by non-hematopoietic cells was crucial for the survival of mature iNKT cells. In the liver, both hematopoietic and non-hematopoietic compartments provided IL-15 to both immature and mature iNKT cells. This IL-15 signal helped mediate the survival and proliferation of both NK and iNKT cells as well as induce the functional maturation of mature iNKT cells via enhanced T-bet expression. In conclusion, my work illustrates an important notion that the immunological niche of NK and iNKT cells is tightly regulated and that this regulation is meticulously influenced by the tissue microenvironment.

Mechanism of surgical stress impairment of murine natural killer cell cytotoxicity

Natural killer cells may provide an important first line of defense against metastatic implantation of solid tumors. This antitumor function occurs during the intravascular and visceral lodgment phase of cancer dissemination, as demonstrated in small animal metastasis models. The role of the NK cell in controlling human tumor dissemination is more difficult to confirm, at least partially because of ethical restraints on experimental design. Nonetheless, a large number of solid tumor patient studies have demonstrated NK cell cytolysis of both autologous and allogeneic tumors.^ Of the major cancer therapeutic modalities, successful surgery in conjunction with other treatments offers the best possibility of cure. However, small animal experiments have demonstrated that surgical stress can lead to increased rates of primary tumor take, and increased incidence, size, and rapidity of metastasis development. Because the physiologic impact of surgical stress can also markedly impair perioperative antitumor immune function in humans, we examined the effect of surgical stress on perioperative NK cell cytolytic function in a murine preclinical model. Our studies demonstrated that hindlimb amputation led to a marked impairment of postoperative NK cell cytotoxicity. The mechanism underlying this process is complex and involves the postsurgical generation of splenic erythroblasts that successfully compete with NK cells for tumor target binding sites; NK cell-directed suppressor cell populations; and a direct impairment of NK cell recycling capacity. The observed postoperative NK cell suppression could be prevented by in vivo administration of pyrimidinone biologic response modifiers or by short term in vitro exposure of effector cells to recombinant Interleukin-2. It is hoped that insights gained from this research may help in the future development of NK cell specific perioperative immunotherapy relevant to the solid tumor patients undergoing cancer resection. ^

ANALYSIS OF THE CORYNEBACTERIUM PARVUM-INDUCED DECLINE OF MURINE NATURAL KILLER CELL-MEDIATED CYTOTOXICITY

Treatment of mice with the immunomodulating agent, Corynebacterium parvum (C. parvum), was shown to result in a severe and long-lasting depression of splenic natural killer (NK) cell-mediated cytotoxicity 5-21 days post-inoculation. Because NK cells have been implicated in immunosurveillance against malignancy (due to their spontaneous occurrence and rapid reactivity to a variety of histological types of tumors), as well as in resistance to established tumors, this decreased activity was of particular concern, since this effect is contrary to that which would be considered therapeutically desirable in cancer treatment (i.e. a potentiation of antitumor effector functions, including NK cell activity, would be expected to lead to a more effective destruction of malignant cells). Therefore, an analysis of the mechanism of this decline of splenic NK cell activity in C.parvum treated mice was undertaken.^ From in vitro co-culturing experiments, it was found that low NK-responsive C. parvum splenocytes were capable of reducing the normally high-reactivity of cells from untreated syngeneic mice to YAC-1 lymphoma, suggesting the presence of NK-directed suppressor cells in C. parvum treated animals. This was further supported by the demonstration of normal levels of cytotoxicity in C. parvum splenocyte preparations following Ficoll-Hypaque separation, which coincided with removal of the NK-suppressive capabilities of these cells. The T cell nature of these regulatory cells was indicated by (1) the failure of C. parvum to cause a reduction of NK cell activity, or the generation of NK-directed suppressor cells in T cell-deficient athymic mice, (2) the removal of C. parvum-induced suppression by T cell-depleting fractionation procedures or treatments, and (3) demonstration of suppression of NK cell activity by T cell-enriched C. parvum splenocytes. These studies suggest, therefore, that the eventual reduction of suppression by T cell elimination and/or inhibition, may result in a promotion of the antitumor effectiveness of C. parvum due to the contribution of "freed" NK effector cell activity.^ However, the temporary suppression of NK cell activity induced by C. parvum (reactivity of treated mice returns to normal levels within 28 days after C. parvum injection), may in fact be favorable in some situations, e.g. in bone marrow transplantation cases, since NK cells have been suggested to play a role also in the process of bone marrow graft rejection.^ Therefore, the discriminate use of agents such as C. parvum may allow for the controlled regulation of NK cell activity suggested to be necessary for the optimalization of therapeutic regimens. ^

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

Ascorbic acid effects on the immune system and type -1 /type -2 cytokine balance in humans

Vitamin C (ascorbic acid--AA) can have a substantial impact on human health by reducing the incidence and/or severity of coryza. Studies also suggest it has immunomodulatory functions in humans. Immune function is controlled by cytokines, such as type-1 cytokines (IFNγ) that promote antiviral immunity and type-2 cytokines (IL-4, IL-10) that promote humoral immunity. Knowing the mechanisms responsible for both antiviral immunity and type-1/type-2 cytokine balance, we sought to identify AA-induced alterations of human peripheral blood mononuclear cells (PBMC) in vivo and in vitro . We hypothesized that AA modulates the immune system, altering both number and function of PBMC. We first described the effect of 14 days of oral (1 gram) AA in healthy subjects. AA increased circulating natural killer (NK) cells, CD25+ and HLA-DR+ T cells, and PMA/ionomycin-stimulated intracellular IFNγ. We subsequently developed models for in vitro use. We determined that AA was toxic in vitro to T cells when used at doses found intracellularly but doses found in plasma from individuals taking 1gm/day AA were nontoxic. The model that most fully reproduced our in vivo intracellular cytokine findings used dehydroascorbic acid and buffers to deliver AA intracellularly. This model generated the largest increase in IFNγ at physiologic plasma concentrations. Previous studies demonstrate that chronic psychological stress is associated with a type-2 cytokine response. We hypothesized that vitamin C could prevent the type-2 cytokine shift associated with stress. In a study of medical students taking 1 g AA or placebo, a significant increase in IFNγ was seen intracellularly in CD4+ and CD8+ cells and in tetanus-stimulated cultures in the AA group only. We also observed increases in IFNγ/IL-4 and IFNγ/IL-10 ratios with AA supplementation, indicating a type-1 shift. Furthermore, we noted increased numbers of NK cells and activated T cells in the peripheral blood in the AA treated group only. Lastly, we investigated the role of the CD40L/CD40 and CD28/B7 costimulatory pathway in these cytokine alterations. AA did not have any effect on either pathway studied. Thus costimulatory pathways are not contributing to AA induced modulation of the type-1/type-2 immune balance. ^