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We recently reported proliferating cell nuclear antigen (PCNA) as a ligand for the NK cell activating receptor, NKp44, which unexpectedly triggers inhibition. The recognition of nuclear proteins such as PCNA, by related NK cell receptors has been reported. Widespread upregulation of PCNA in tumor cells may therefore promote immune evasion.
Natural killer (NK) cells provide primary innate immune defense against cancer, and natural cytotoxic receptors (NCRs) are key activating receptors for tumor recognition. The purpose of our recent study was to identify and characterize tumor associated ligands for the NCR, NKp44.1 It is currently believed that tumors that survive immune surveillance have developed mechanisms to evade the immune system.2 Proliferating cell nuclear antigen (PCNA) is commonly overexpressed in cancer cells, where it contributes to cellular proliferation and transformation.3 We recently showed that the interaction of NKp44 with target cell-expressed PCNA paradoxically inhibits NK cell function. We further demonstrated that PCNA promotes tumor survival by suppressing NK activation through the immunoreceptor tyrosine-based inhibitory motif (ITIM) on the NKp44 cytoplasmic domain. Finally, we observed that nuclear/cytoplasmic PCNA from target cells is recruited to the NK immunological synapse (NKIS) when NKp44 is expressed on the NK cells.
Using recombinant soluble chimeric NK cell receptors fused to the human IgG Fc domain, we showed that PCNA protein manifests binding to NKp44 but not to other NK cell receptors, including LIR1, NKG2D, KIR2DL4, NKp30 and NKp46. Immunoprecipitation studies also confirmed that NKp44 interacts with soluble PCNA and native PCNA from cell lysates, while LIR1 does not. Using surface plasmon resonance analysis, NKp44 displayed a characteristic receptor-ligand binding affinity to PCNA (KD = 3.4 × 10−9 M).
We next expressed PCNA-GFP in HeLa target cells and showed that PCNA overexpression suppressed IFNγ secretion and lysis by NKp44-expressing NK cells. In addition, incubation of NKp44-expressing NK cells (NK92-44) with blocking anti-NKp44 antibodies abolished the inhibition of lysis.
We further investigated the effect of endogenous PCNA-downregulation using an siRNA approach. Downregulation of PCNA in PANC-1 (pancreas), MCF-7 (breast), DU145 (prostate), HeLa (cervix), and U251 (brain) tumor cells resulted in enhanced lysis by NK92-44 cells. Downregulation of endogenous PCNA in target cancer cells also enhanced IFNγ secretion by NKp44-expressing NK cells, whereas responses by NKp44null NKL cells were unaffected.
We further tested whether this PCNA-mediated inhibition might involve the ITIM located in the cytoplasmic tail of NKp44. We employed NK-92 cells transduced to express a form of NKp44 in which the cytoplasmic tail is either truncated prior to the ITIM or mutated at the tyrosine site within the ITIM. In this setting, the overexpression of PCNA resulted in significantly reduced functions of NK92 cells expressing wild type, as compared with the mutated forms of NKp44. Thus, the integrity of the ITIM on the NKp44 cytoplasmic domain is essential for the PCNA-mediated inhibitory effect on NK cells.
We also studied whether soluble PCNA, target cell-conditioned medium, or exosomal fractions could mediate PCNA inhibition of NK cell function. None of these conditions could mimic inhibition of NK cell function by PCNA-expressing target cells. On the contrary, a direct interaction between the NK cell and the target cell was found to be imperative for PCNA-mediated effects, both in vitro and in vivo. Furthermore, using live imaging confocal microscopy, we showed that PCNA in the tumor target cell can be recruited to the NKIS, and that this recruitment correlates with a high surface density of NKp44 on the conjugated NK cell.
The overexpression of PCNA by tumor cells strongly correlates with cancer virulence, since PCNA contributes to many survival processes, including DNA replication, DNA repair, and cell-cycle progression.3 Our data suggest that PCNA may additionally promote tumor survival by promoting immune evasion through NKp44-mediated inhibition of NK cell attack. Although PCNA is primarily a nuclear/cytoplasmic factor, other cytoplasmic or nuclear proteins have been shown to serve as ligands to membrane-associated NCRs, notably the NKp30 ligands, CMV-pp65 and nuclear BAT3,4,5 and the NKp46 ligand, vimentin.6 Collectively, these observations suggest that the localization of NCR ligands need not be primarily restricted to the membrane.
We further hypothesize that the PCNA-NKp44 interaction may also contribute to fetal protection from maternal NK cells during pregnancy. Interestingly, PCNA is overexpressed in decidual tissue, and maternal-derived decidual NK cells constitutively express NKp44.7 During the first trimester of pregnancy, these decidual NK cells comprise 50% to 90% of the lymphoid cells in the decidua, and they are tolerized toward the fetal tissue.7 Interestingly, this NK cell tolerance correlates with PCNA overexpression in fetal-derived trophoblast cells during the first trimester.8 Therefore, PCNA-induced inhibition through NKp44 may also contribute to the immune tolerance of maternal NK cells during pregnancy.
Although PCNA has not been previously reported to localize at the plasma membrane, we observed its accumulation at the contact interface with NKp44-expressing NK cells, which is commonly called the NKIS. PCNA was recently shown to interact with the membrane-associated protein, Annexin A2 (ANXA2),9 which was also recently identified as a potential cell surface tumor antigen.10 Although yet untested, we speculate that ANXA2 may play a role in recruiting PCNA to the NKIS upon interaction of a tumor cell with NKp44-expressing NK cells (Fig. 1).
To summarize, this work identifies a new ligand for NKp44, demonstrates a novel inhibitory signaling function for NKp44, reveals a previously unrecognized mechanism of immune evasion, and re-emphasizes the role of nuclear proteins as important ligands for NCRs.
Previously published online: www.landesbioscience.com/journals/oncoimmunology/article/19366