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Immune rejection is one of the most serious challenges in allogeneic transplantation, including allogeneic induced pluripotent stem cell (allo-iPSC)-derived cell therapy. Beta-2-Microglobulin gene-knockout, human leukocyte antigen (HLA) class I-deficient iPSCs can evade immune rejection by host T cells, which occurs due to HLA mismatches. However, natural killer (NK) cells recognize HLA class Ⅰ-deficient cells and reject them, which is known as the missing-self response. Introducing chimeric HLA-E protein to HLA class Ⅰ-deficient iPSCs suppresses the missing-self response of NK cells expressing the inhibitory receptor NKG2A; however, technology to suppress NKG2A-negative NK cells is still required. Here, we developed novel agonists for the other inhibitory receptor, killer immunoglobulin receptor (KIR), on NK cells. We found that antibodies that bind to activating KIR enhance NK cell activation and developed selective agonists for inhibitory KIRs (KIR2DL1, KIR2DL2/3, and KIR3DL1). Introducing these selective inhibitory KIR agonists on T cells and HLA class Ⅰ-deficient iPSCs allowed them to evade immune rejection by NK cells. Additionally, we identified an NKG2A-selective agonist as an alternative to chimeric HLA-E, which stimulates the activating receptor NKG2C. This technology enhances immune tolerance in allo-iPSCs and facilitates the development of various iPSC-derived regenerative medicines.

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.

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

The effect of natural killer (NK) cell alloreactivity on the outcome of unrelated hematopoietic SCT (HSCT) remains a topic of debate. NK cell alloreactivity after allogeneic HSCT is regulated by killer-cell Ig-like receptors (KIRs). To investigate the influence of KIRs on outcome after unrelated HSCT, we retrospectively analyzed the HLA and KIR genotypes of 116 donor–recipient pairs. We found that missing KIR ligands in recipients were significantly associated with a decreased leukemic relapse risk ( P =0.019, HR=0.329), mainly in myeloid disease ( P =0.003, HR=0.193). This beneficial effect was seen in AML/myelodysplastic syndrome and also in chronic myeloid leukemia. In myeloid disease, missing KIR ligands also improved 5-year OS ( P =0.034, HR=0.430) and disease-free survival (DFS) ( P =0.024, HR=0.445). Meanwhile, the presence of donor-activating KIR2DS3 gene was associated with increased relapse risk ( P =0.003, HR=5.046), decreased OS ( P =0.004, HR=3.181) and DFS ( P =0.003, HR=2.919) in myeloid disease. No effect was seen in patients with lymphoid disease. Our study indicated that, in unrelated HSCT for myeloid leukemia, missing KIR ligands in recipients offered a lower relapse risk and a long-term survival advantage. The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.

Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT. The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into 'discovery' (135 pairs) and 'validation' (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50-5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33-5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42-4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11-2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10-0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17-0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants. The present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.

Group I metabotropic glutamate receptor (mGluR I) activation exerts a slow postsynaptic excitatory effect in the CNS. Here, the issues of whether and how this receptor is involved in regulating retinal ganglion cell (RGC) excitability were investigated in retinal slices using patch-clamp techniques. Under physiological conditions, RGCs displayed spontaneous firing. Extracellular application of LY367385 (10 µM)/MPEP (10 µM), selective mGluR1 and mGluR5 antagonists, respectively, significantly reduced the firing frequency, suggesting that glutamate endogenously released from bipolar cells constantly modulates RGC firing. DHPG (10 µM), an mGluR I agonist, significantly increased the firing and caused depolarization of the cells, which were reversed by LY367385, but not by MPEP, suggesting the involvement of the mGluR1 subtype. Intracellular Ca -dependent PI-PLC/PKC and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways mediated the DHPG-induced effects. In the presence of cocktail synaptic blockers (CNQX, D-AP5, bicuculline, and strychnine), which terminated the spontaneous firing in both ON and OFF RGCs, DHPG still induced depolarization and triggered the cells to fire. The DHPG-induced depolarization could not be blocked by TTX. In contrast, Ba , an inwardly rectifying potassium channel (Kir) blocker, and Cs and ZD7288, hyperpolarization-activated cation channel (I ) blockers, mimicked the effect of DHPG. Furthermore, in the presence of Ba /ZD7288, DHPG did not show further effects. Moreover, Kir and I currents could be recorded in RGCs, and extracellular application of DHPG indeed suppressed these currents. Our results suggest that activation of mGluR I regulates the excitability of rat RGCs by inhibiting Kir and I .

Alloreactivity of natural killer (NK) cells contributes to the GVL reaction after allogeneic hematopoietic SCT (allo-HSCT). However, various procedure-related factors may affect NK cell maturation and their ability to recognize and kill leukemic cells. In this study, we prospectively evaluated expression of NK cell inhibitory receptors in 83 adults treated with myeloablative, killer cell Ig-like receptor (KIR)-ligand-matched allo-HSCT. NK cell maturation was evaluated by comparing the phenotypic patterns after allo-HSCT with the donor ones. The frequencies of KIR3DL1 were comparable to the donor ones on day +28, while they decreased significantly starting from day +100. The expression of KIR2DL2/3 was significantly lower in patients compared with donors up to day +100. The expression of KIR2DL1, despite continues growth, remained significantly decreased for 1 year after allo-HSCT. NKG2A was over-expressed up to day +180. Within 1 year after allo-HSCT, the NK cell phenotypic pattern tended to recapitulate the donor type. The process was disturbed by the use of steroids with significant differences observed on days +56 ( P =0.01) and +100 ( P =0.04). Up to day +100, reconstitution of NK cell receptor repertoire correlated with the absolute numbers of circulating CD3 + , CD3 + CD4 + and CD3 + CD8 + cells. Our observations should be taken into account when trying to predict potential benefit from NK cell alloreactivity.

Dopaminergic (DA) periglomerular (PG) neurons are critically placed at the entry of the bulbar circuitry, directly in contact with both the terminals of olfactory sensory neurons and the apical dendrites of projection neurons; they are autorhythmic and are the target of numerous terminals releasing a variety of neurotransmitters. Despite the centrality of their position, suggesting a critical role in the sensory processing, their properties -and consequently their function- remain elusive. The current mediated by inward rectifier potassium (Kir) channels in DA-PG cells was recorded by adopting the perforated-patch configuration in thin slices; IKir could be distinguished from the hyperpolarization-activated current (I h ) by showing full activation in <10 ms, no inactivation, suppression by Ba(2+) in a typical voltage-dependent manner (IC50 208 μM) and reversal potential nearly coincident with EK. Ba(2+) (2 mM) induces a large depolarization of DA-PG cells, paralleled by an increase of the input resistance, leading to a block of the spontaneous activity, but the Kir current is not an essential component of the pacemaker machinery. The Kir current is negatively modulated by intracellular cAMP, as shown by a decrease of its amplitude induced by forskolin or 8Br-cAMP. We have also tested the neuromodulatory effects of the activation of several metabotropic receptors known to be present on these cells, showing that the current can be modulated by a multiplicity of pathways, whose activation in some case increases the amplitude of the current, as can be observed with agonists of D2, muscarinic, and GABAA receptors, whereas in other cases has the opposite effect, as it can be observed with agonists of α1 noradrenergic, 5-HT and histamine receptors. These characteristics of the Kir currents provide the basis for an unexpected plasticity of DA-PG cell function, making them potentially capable to reconfigure the bulbar network to allow a better flexibility.

An inwardly rectifying K + current is present in atrial cardiac myocytes that is activated by acetylcholine (I KACh ). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gβγ G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPγS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.