The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor

Abstract

Some stimulatory receptors of the innate immune system, such as the NKG2D receptor expressed by NK cells and activated CD8 + T cells, recognize self-molecules that are upregulated in diseased cells by poorly understood mechanisms 1 . Here we show that mouse and human NKG2D ligands are upregulated in non-tumor cell lines by genotoxic stress and stalled DNA replication, conditions known to activate a major DNA damage checkpoint pathway initiated by ATM (Ataxia telangiectasia, mutated) or ATR (ATM-and Rad3-related) protein kinases 2 . Ligand upregulation was prevented by pharmacological or genetic inhibition of ATR, ATM or Chk1, the latter a downstream transducer kinase in the pathway. Furthermore, constitutive ligand expression by a tumor cell line was inhibited by ATM siRNA, suggesting that ligand expression in established tumor cells, which often harbor genomic irregularities, may be due to chronic activation of the DNA damage response pathway. Thus, the DNA damage response, previously shown to arrest the cell cycle and enhance DNA repair functions or to trigger apoptosis, may also participate in alerting the immune system to the presence potentially dangerous cells.To investigate mechanisms leading to NKG2D ligand upregulation, we examined two transformed ovarian epithelial cell lines from p53 −/− mice 3 . The C1 cell line had been transduced with K-ras and c-myc, whereas the C2 cell line had been transduced with AKT and c-myc (Fig. 1A). Both transformed cell lines grew well in cell culture but did not express appreciable levels of mouse NKG2D ligands as detected by staining with a tetrameric NKG2D reagent that binds to all mouse NKG2D ligands (Rae1, MULT1 and H60 1 ) (Fig. 1B). Ligand upregulation failed to occur when C1 and C2 cells were transfected or super-transduced with numerous other oncogenes, including E6, E7, E1A, or Ras V12 (data not shown), some of which interfere with expression of the retinoblastoma tumor suppressor gene. When injected into the ovaries of nude mice, both cell lines generated ovarian epithelial tumors, which were established as cell lines T1 and T2 3 . Both T1 and T2 exhibited significant upregulation of NKG2D ligands (Fig. 1B) including Rae1 (see below). These findings suggested that ligand upregulation was not associated with transformation per se.Ligand expression by C1 or C2 cells was not upregulated by numerous cell stress conditions, including heat shock, hyperoxia, hypoxia, inhibition of the cell cycle (by roscovitine), exposure to inflammatory cytokines such as TNF, interferon or IL-6, or incubation in medium of pH6 or pH8.5 ( Fig. 2A, S1, data not shown). In contrast, NKG2D ligands were upregulated in C1 or C2 cells exposed to high doses of ionizing radiation, inhibitors of DNA replication such as mitomycin C, hydroxyurea (HU), 5-fluorouracil (5-FU) and the DNA polymerase inhibitor aphidicolin, or chromatin modifying treatments such as trichostatin A, chloroquine and

Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer

“…112 Platinum derivatives and dacarbazine were shown to stimulate the expression of ligands for NKG2D, an NK cellactivating receptor, resulting in augmented NK-cell cytotoxicity and IFN-g production. 113 Numerous agents promote functional downregulation of the inhibitory NK ligand Clr-b and upregulation of stimulatory NKG2D ligand on tumor cells, thus enhancing the susceptibility of target cells to NK cellmediated lysis. 114 Interestingly, gemcitabine has been shown to increase the expression of HLA on malignant cells, 115 and to enhance the cross-presentation of tumor antigens to CD8 T-cells.…”

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

“…On the other hand, the possibility that this T lymphocyte subset may also expand in response to different antigens, including other microbial pathogens, is not ruled out. Regardless of their primary antigenic specificity, NKG2D would enhance the response of potentially autoreactive CD4 + T cells against noninfected tissues, where expression of its ligands may be either constitutive or inducible by a variety of stimuli [26,55,56].…”

PGE2 inhibits natural killer and γδ T cell cytotoxicity triggered by NKR and TCR through a cAMP-mediated PKA type I-dependent signaling

“…Indeed, genotoxic stress that activates DNA damage responses has been shown to induce the expression of NKG2D ligands (MICA, MICB, and ULBPs) and most of established tumor cell lines constitutively express one or more of these NKG2D ligands [43]. NK and  T cells express high levels of NKG2D which has been shown to directly promote their cytotoxic responses against target cells [5,44].…”