Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

Ly49D-mediated ITAM signaling in immature thymocytes impairs development by bypassing the pre-TCR checkpoint.

Activating and inhibitory NK receptors regulate the development and effector functions of NK cells via their ITAM and ITIM motifs, which recruit protein tyrosine kinases and phosphatases, respectively. In the T cell lineage, inhibitory Ly49 receptors are expressed by a subset of activated T cells and by CD1d-restricted NKT cells, but virtually no expression of activating Ly49 receptors is observed. Using mice transgenic for the activating receptor Ly49D and its associated ITAM signaling DAP12 chain, we show in this article that Ly49D-mediated ITAM signaling in immature thymocytes impairs development due to a block in maturation from the double negative (DN) to double positive (DP) stages. A large proportion of Ly49D/DAP12 transgenic thymocytes were able to bypass the pre-TCR checkpoint at the DN3 stage, leading to the appearance of unusual populations of DN4 and DP cells that lacked expression of intracellular (ic) TCRß-chain. High levels of CD5 were expressed on ic TCRß(-) DN and DP thymocytes from Ly49D/DAP12 transgenic mice, further suggesting that Ly49D-mediated ITAM signaling mimics physiological ITAM signaling via the pre-TCR. We also observed unusual ic TCRß(-) single positive thymocytes with an immature CD24(high) phenotype that were not found in the periphery. Importantly, thymocyte development was completely rescued by expression of an Ly49A transgene in Ly49D/DAP12 transgenic mice, indicating that Ly49A-mediated ITIM signaling can fully counteract ITAM signaling via Ly49D/DAP12. Collectively, our data indicate that inappropriate ITAM signaling by activating NK receptors on immature thymocytes can subvert T cell development by bypassing the pre-TCR checkpoint.

In vivo tumor cell rejection induced by NK cell inhibitory receptor blockade: maintained tolerance to normal cells even in the presence of IL-2.

Missing-self-reactivity can be mimicked by blocking self-specific inhibitory receptors on NK cells, leading to increased rejection of syngeneic tumor cells. Using a mouse model, we investigated whether Ab-mediated blocking of inhibitory receptors, to a degree where NK cells rejected syngeneic tumor cells, would still allow self-tolerance toward normal syngeneic cells. Ly49C/I inhibitory receptors on C57BL/6 (H-2(b)) NK cells were blocked with F(ab')(2) fragments of the mAb 5E6. Inhibitory receptor blockade in vivo caused rejection of i.v. inoculated fluorescence-labeled syngeneic lymphoma line cells but not of syngeneic spleen cells, BM cells or lymphoblasts. The selective rejection of tumor cells was NK cell-dependent and specifically induced by Ly49C/I blockade. Moreover, selective tumor rejection was maintained after treatment with 5E6 F(ab')(2) for 9 wk, arguing against the induction of NK cell anergy or autoreactivity during this time. Combination therapy using 5E6 F(ab')(2) together with high dose IL-2 treatment further increased lymphoma cell rejection. In addition, combination therapy reduced growth of melanoma cell line tumors established by s.c. inoculation 3 days before start of treatment. Our results demonstrate that inhibitory receptor blockade does not result in attack on normal cells, despite potent reactivity against MHC class I-expressing tumors.

Spatiotemporal regulation of intracellular trafficking of Toll-like receptor 9 by an inhibitory receptor, Ly49Q.

Toll-like receptor (TLR) 9 recognizes unmethylated microorganismal cytosine guanine dinucleotide (CpG) DNA and elicits innate immune responses. However, the regulatory mechanisms of the TLR signaling remain elusive. We recently reported that Ly49Q, an immunoreceptor tyrosine-based inhibitory motif-bearing inhibitory receptor belonging to the natural killer receptor family, is crucial for TLR9-mediated type I interferon production by plasmacytoid dendritic cells. Ly49Q is expressed in plasmacytoid dendritic cells, macrophages, and neutrophils, but not natural killer cells. In this study, we showed that Ly49Q regulates TLR9 signaling by affecting endosome/lysosome behavior. Ly49Q colocalized with CpG in endosome/lysosome compartments. Cells lacking Ly49Q showed a disturbed redistribution of TLR9 and CpG. In particular, CpG-induced tubular endolysosomal extension was impaired in the absence of Ly49Q. Consistent with these findings, cells lacking Ly49Q showed impaired cytokine production in response to CpG-oligodeoxynucleotide. Our data highlight a novel mechanism by which TLR9 signaling is controlled through the spatiotemporal regulation of membrane trafficking by the immunoreceptor tyrosine-based inhibitory motif-bearing receptor Ly49Q.