We show that the two-stage activation process that was previously described only in vitro26 can adequately explain the situation in vivo. However, tumor escape seems to be more complex than might be suggested by definitions in terms of type 1 or type 2 resistance. λ-myc transgenic mice express the myc oncogene under the control of Ig-λ chain regulatory sequences and spontaneously develop tumors of the B-cell lineage that share multiple features of human Burkitt lymphoma 29. Animals with lymphadenopathy were sacrificed and NK cells from spleens and lymph nodes were phenotypically analyzed. The absolute number of NK cells was strongly increased in tumor lymph nodes.
The highest numbers were found in cervical and mandibular lymph nodes, Metabolism inhibitor the primary site of lymphoma growth (Fig. 1A). Inguinal and axillary lymph nodes and other lymphoid organs are infiltrated by tumor cells later during disease progression. Obviously, there is either an active migration of NK cells into the developing lymphomas or an enhanced proliferation in the tumor lymph nodes.
Most activating receptors including NKG2D and the inhibitory receptors tested were diminished, and expression of typical activation markers, such as CD45R and CD69, was enhanced (Fig. 1B). We assume that interaction of NK cells with tumor cells gave rise to NK-cell activation entailing up- or down-regulation of several surface receptors. A correlation between NK-receptor levels and NK/tumor-cell ratios in the different compartments was not seen in mice with visible tumor burdens suggesting strong activating signals as soon as visible tumor FK228 price growth has started. To obtain more information on NK-cell activation in vivo, we also analyzed transgenic mice prior to macroscopic
tumor manifestation. NK cells from these animals already showed slight alterations of the surface molecules (data not shown), which might be due to incipient, yet undetectable lymphomas. To investigate effector functions, NK cells were tested for cytotoxicity by chromium release assay and for IFN-γ expression by RT-PCR and protein staining. In contrast to normal NK cells, highly enriched NK cells from tumor-bearing animals did not exert any cytotoxicity against the NK-sensitive Molecular motor YAC-1 target (Fig. 2A). Lytic activity of NK cells from clinically unapparent λ-myc transgenic mice (before manifestation of visible tumors) was also impaired but its decrease was often less pronounced than in tumor-bearing mice. For IFN-γ mRNA expression, a clear hierarchy was observed in NK cells derived from WT, clinically unapparent λ-myc transgenic and lymphoma-bearing animals, respectively (Fig. 2B). These differences were confirmed at the protein level by IFN-γ capture assays and intracellular IFN-γ staining (Fig. 2C). As in T lymphocytes activation-induced anergy may be overcome by stimulation of TLR, we treated freshly isolated NK cells with CpG-oligonucleotide (CpG-ODN) 1668, a stimulatory TLR9 ligand.