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Competing Interests: The authors declare no conflict of interest. Given Roberto Bei’s role as the Editorial Board Member, he had no involvement in the peer-review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Graham Pawelec.

Human cytomegalovirus (CMV) is a common herpesvirus causing lifelong latent infection in most people and is a primary cause of congenital infection worldwide. Given the role of NK cells in the materno-fetal barrier, we investigated peripheral blood NK cell behavior in the context of CMV infection acquired during pregnancy. We analyzed the NK phenotype and CD107a surface mobilization on PBMCs from CMV-transmitting and non-transmitting mothers and newborns with or without congenital infection. NK cells from non-transmitting mothers showed the typical phenotype of CMV-adaptive NK cells, characterized by higher levels of NKG2C, CD57, and KIRs, with reduced NKG2A, compared to transmitting ones. A significantly higher percentage of DNAM-1+, PD-1+, and KIR+NKG2A-CD57+PD-1+ CD56dim cells was found in the non-transmitting group. Accordingly, NK cells from congenital-CMV (cCMV)-infected newborns expressed higher levels of NKG2C and CD57, with reduced NKG2A, compared to non-congenital ones. Furthermore, they showed a significant expansion of CD56dim cells co-expressing NKG2C and CD57 or with a memory-like (KIR+NKG2A-CD57+NKG2C+) phenotype, as well as a significant reduction of the CD57-NKG2C- population. Degranulation assays showed a slightly higher CD107a geomean ratio in NK cells of mothers who were non-transmitting compared to those transmitting the virus. Our findings demonstrate that both CMV-transmitting mothers and cCMV newborns show a specific NK profile. These data can guide studies on predicting virus transmission from mothers and congenital infection in infants.

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cifaldi, Melaiu, Giovannoni, Benvenuto, Focaccetti, Nardozi, Barillari and Bei.)

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 D’Amico, D’Alicandro, Compagnone, Tempora, Guida, Romania, Lucarini, Melaiu, Falco, Algeri, Pende, Cifaldi and Fruci.)

Immune escape mechanisms employed by neuroblastoma (NB) cells include secretion of immunosuppressive factors disrupting effective antitumor immunity. The use of cellular therapy to treat solid tumors needs to be implemented. Killing activity of anti-GD2 Chimeric Antigen Receptor (CAR) T or natural killer (NK) cells against target NB cells was assessed through coculture experiments and quantified by FACS analysis. ELISA assay was used to quantify interferon-γ (IFNγ) secreted by NK and CAR T cells. Real Time PCR and Western Blot were performed to analyze gene and protein levels modifications. Transcriptional study was performed by chromatin immunoprecipitation and luciferase reporter assays on experiments of mutagenesis on the promoter sequence. NB tissue sample were analyzed by IHC and Real Time PCR to perform correlation study. We demonstrate that Indoleamine-pyrrole 2,3-dioxygenase1 (IDO1), due to its ability to convert tryptophan into kynurenines, is involved in NB resistance to activity of immune cells. In NB, IDO1 is able to inhibit the anti-tumor effect displayed by of both anti-GD2 CAR (GD2.CAR) T-cell and NK cells, mainly by impairing their IFNγ production. Furthermore, inhibition of MYCN expression in NB results into accumulation of IDO1 and consequently of kynurenines, which negatively affect the immune surveillance. Inverse correlation between IDO1 and MYCN expression has been observed in a wide cohort of NB samples. This finding was supported by the identification of a transcriptional repressive role of MYCN on IDO1 promoter. The evidence of IDO1 involvement in NB immune escape and its ability to impair NK and GD2.CAR T-cell activity contribute to clarify one of the possible mechanisms responsible for the limited efficacy of these immunotherapeutic approaches. A combined therapy of NK or GD2.CAR T-cells with IDO1 inhibitors, a class of compounds already in phase I/II clinical studies, could represent a new and still unexplored strategy capable to improve long-term efficacy of these immunotherapeutic approaches., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma., (©2020 American Association for Cancer Research.)

Tumor-infiltrating lymphocytes play an essential role in improving clinical outcome of neuroblastoma (NB) patients, but their relationship with other tumor-infiltrating immune cells in the T cell-inflamed tumors remains poorly investigated. Here we show that dendritic cells (DCs) and natural killer (NK) cells are positively correlated with T-cell infiltration in human NB, both at transcriptional and protein levels, and associate with a favorable prognosis. Multiplex imaging displays DC/NK/T cell conjugates in the tumor microenvironment of low-risk NB. Remarkably, this connection is further strengthened by the identification of gene signatures related to DCs and NK cells able to predict survival of NB patients and strongly correlate with the expression of PD-1 and PD-L1. In summary, our findings unveil a key prognostic role of DCs and NK cells and indicate their related gene signatures as promising tools for the identification of clinical biomarkers to better define risk stratification and survival of NB patients.

Natural killer (NK) cells are a population of innate lymphoid cells playing a pivotal role in host immune responses against infection and tumor growth. These cells have a powerful cytotoxic activity orchestrated by an intricate network of inhibitory and activating signals. The importance of NK cells in controlling tumor growth and in mediating a robust anti-metastatic effect has been demonstrated in different experimental mouse cancer models. Consistently, high density of tumor-infiltrating NK cells has been linked with a good prognosis in multiple human solid tumors. However, there are also tumors that appear to be refractory to NK cell-mediated killing for the presence of an immunosuppressive microenvironment affecting NK cell function. Immunotherapeutic strategies aimed at restoring and increasing the cytotoxic activity of NK cells in solid tumors, including the adoptive transfer of NK and CAR-NK cells, are currently employed in preclinical and clinical studies. In this review, we outline recent advances supporting the direct role of NK cells in controlling expansion of solid tumors and their prognostic value in human cancers. We summarize the mechanisms adopted by cancer cells and the tumor microenvironment to affect NK cell function, and finally we evaluate current strategies to augment the antitumor function of NK cells for the treatment of solid tumors., (Copyright © 2020 Melaiu, Lucarini, Cifaldi and Fruci.)

Natural Killer (NK) cells play a critical role in host defense against viral infections. The mechanisms of recognition and killing of virus-infected cells mediated by NK cells are still only partially defined. Several viruses induce, on the surface of target cells, the expression of molecules that are specifically recognized by NK cell-activating receptors. The main NK cell-activating receptors involved in the recognition and killing of virus-infected cells are NKG2D and DNAM-1. In particular, ligands for DNAM-1 are nectin/nectin-like molecules involved also in mechanisms allowing viral infection. Viruses adopt several immune evasion strategies, including those affecting NK cell-mediated immune surveillance, causing persistent viral infection and the development of virus-associated diseases. The virus's immune evasion efficacy depends on molecules differently expressed during the various phases of infection. In this review, we overview the molecular strategies adopted by viruses, specifically cytomegalovirus (CMV), human immunodeficiency virus (HIV-1), herpes virus (HSV), Epstein-Barr virus (EBV) and hepatitis C virus (HCV), aiming to evade NK cell-mediated surveillance, with a special focus on the modulation of DNAM-1 activating receptor and its ligands in various phases of the viral life cycle. The increasing understanding of mechanisms involved in the modulation of activating ligands, together with those mediating the viral immune evasion strategies, would provide critical tools leading to design novel NK cell-based immunotherapies aiming at viral infection control, thus improving cure strategies of virus-associated diseases.

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, causes death in almost 15% of children affected by cancer. Treatment of neuroblastoma is based on the combination of chemotherapy with other therapeutic interventions such as surgery, radiotherapy, use of differentiating agents, and immunotherapy. In particular, adoptive NK cell transfer is a new immune-therapeutic approach whose efficacy may be boosted by several anticancer agents able to induce the expression of ligands for NK cell-activating receptors, thus rendering cancer cells more susceptible to NK cell-mediated lysis. Here, we show that chemotherapeutic drugs commonly used for the treatment of NB such as cisplatin, topotecan, irinotecan, and etoposide are unable to induce the expression of activating ligands in a panel of NB cell lines. Consistently, cisplatin-treated NB cell lines were not more susceptible to NK cells than untreated cells. The refractoriness of NB cell lines to these drugs has been partially associated with the abnormal status of genes for ATM, ATR, Chk1, and Chk2, the major transducers of the DNA damage response (DDR), triggered by several anticancer agents and promoting different antitumor mechanisms including the expression of ligands for NK cell-activating receptors. Moreover, both the impaired production of reactive oxygen species (ROS) in some NB cell lines and the transient p53 stabilization in response to our genotoxic drugs under our experimental conditions could contribute to inefficient induction of activating ligands. These data suggest that further investigations, exploiting molecular strategies aimed to potentiate the NK cell-mediated immunotherapy of NB, are warranted.

Type 1 diabetes is an autoimmune disease, in which pancreatic β cells are destroyed by autoreactive T cells in genetically predisposed individuals. Serum beta cell autoantibody specificities have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives. In recent years, approaches were attempted to solve the difficult issue of detecting rare antigen-specific autoreactive T cells and their significance to etiopathogenesis such as the use of the MHC multimer technology. This tool allowed the specific detection of increased percentages of GAD65 autoreactive T cells by means of HLA A*02:01 GAD65 AA 114-122 pentamers in newly diagnosed diabetics. Here we provide evidence that GAD65 AA 114-122 pentamers can depict a GAD65 AA114-122 peptide expandable population of functionally and phenotypically skewed, preliminary characterized CD3-CD8dullCD56+ 'memory-like' NK cells in PBMC of newly diagnosed diabetics. Our data suggest that the NK cell subset could bind the HLA class I GAD65 AA 114-122 pentamer through ILT2 inhibitory receptor. CD107a expression revealed increased degranulation of CD3-CD8dullCD56+ NK cells in GAD65 AA 114-122 and FLU peptide expanded peripheral blood mononuclear cells of diabetics following GAD65 AA 114-122 peptide HLA A*02:01 presentation in respect to the unpulsed condition. CD107a expression was enriched in ILT2 positive NK cells. As opposite to basal conditions where similar percentages of CD3-CD56+ILT2+ cells were detected in diabetics and controls, CD3-CD56+CD107a+ and CD3-CD56+ILT2+CD107a+ cells were significantly increased in T1D PBMC either GAD65 AA 114-122 or FLU peptides stimulated after co-culture with GAD65 AA 114-122 pulsed APCs. As control, healthy donor NK cells showed similar degranulation against both GAD65 AA 114-122 pulsed and unpulsed APCs. The pathogenetic significance of the CD3-CD8dullCD56+ 'memory-like NK cell subset' with increased response upon secondary challenge in diabetics remains to be elucidated.

Natural killer (NK) cells efficiently recognize and kill tumor cells through several mechanisms including the expression of ligands for NK cell-activating receptors on target cells. Different clinical trials indicate that NK cell-based immunotherapy represents a promising antitumor treatment. However, tumors develop immune-evasion strategies, including downregulation of ligands for NK cell-activating receptors, that can negatively affect antitumor activity of NK cells, which either reside endogenously, or are adoptively transferred. Thus, restoration of the expression of NK cell-activating ligands on tumor cells represents a strategic therapeutic goal. As discussed here, various anticancer drugs can fulfill this task via different mechanisms. We envision that the combination of selected chemotherapeutic agents with NK cell adoptive transfer may represent a novel strategy for cancer immunotherapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

NK cell cytotoxicity in Chédiak-Higashi syndrome (CHS) is strongly impaired as lytic granules are not released upon NK-target cell contact, contributing to several defects typical of this severe immunodeficiency. Correction of NK cell defects in CHS should improve the outcome of hematopoietic stem-cell transplantation, proposed as therapy. We investigated NK cell functions in a CHS patient before and after cord-blood transplantation, and the ability of in vitro IL-2 treatment to restore them. Before the transplant, the strong defect in NK cell-mediated natural and antibody-dependent cytotoxicity, as well as in IFN-γ production, could be restored up to normal levels by in vitro IL-2 treatment. This cytokine also caused the appearance of smaller lysosomal granules and their orientation towards the NK-target cell contact area, thus suggesting that IL-2 had a more general capacity to restore NK cell effector functions. Moreover after the transplant, although the successful engraftment, NK cell cytotoxicity resulted still partially impaired at one year, almost normal at ten years and, anyhow, fully recovered by in vitro IL-2 treatment. Taken together, our results indicate that IL-2 had a wide capacity to restore NK cell effector functions, being able to reverse the altered cytotoxic activity, lytic granule pattern, and cytokine production observed in the CHS patient., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Objective: Systemic juvenile idiopathic arthritis (JIA) is associated with high levels of interleukin-6 (IL-6) in the serum and synovial fluid, and impairment of natural killer (NK) cell function is often observed. This study was undertaken to evaluate a possible link between these 2 biologic findings and whether they may be associated with the development of macrophage activation syndrome, a condition frequently observed in systemic JIA., Methods: Splenocytes from wild-type (WT) or IL-6-transgenic (Tg) mice were evaluated for NK cell cytotoxicity using a (51) Cr-release assay. Numbers of NK cells and expression of perforin, granzyme B, CD69, and CD107a were evaluated by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were treated with IL-6 and cultured in the presence or absence of tocilizumab (TCZ), an IL-6 receptor blocker. Human polyclonal NK cells from healthy donor PBMCs were evaluated for cell cytotoxicity and expression of perforin, granzyme B, and CD107a. PBMCs harvested from patients with systemic JIA during periods of active or inactive disease were left untreated or treated with IL-6 in combination with soluble IL-6 receptor and analyzed for the expression of perforin and granzyme B., Results: Splenic NK cell cytotoxicity was reduced in IL-6-Tg mice compared to WT mice. Levels of CD69 and CD107a showed no significant differences, whereas expression of perforin and granzyme B was impaired in NK cells from IL-6-Tg mice. Exposure of human peripheral blood NK cells to IL-6 led to reduced expression of perforin and granzyme B. Culturing human polyclonal NK cells in the presence of TCZ significantly increased cell cytotoxicity, and also increased expression of perforin and granzyme B. In patients with systemic JIA, a reduction in IL-6 plasma levels during disease remission correlated with the rescue of perforin and granzyme B expression in NK cells from these patients., Conclusion: In both mice and humans, IL-6 down-modulated the cytotoxic activity of NK cells. This decrease was associated with reduced perforin and granzyme B levels in the absence of altered granule exocytosis., (© 2015, American College of Rheumatology.)

NK cells expressing TIM-3 show a marked increase in IFNγ production in response to acute myeloid leukemia (AML) blast cells that endogenously express Gal-9. Herein, we demonstrate that NK cell-mediated production of IFNγ, induced by TIM-3/Gal-9 interaction and released in bone marrow microenvironment, is responsible for IDO1 expression in AML blasts. IDO1-expressing AML blasts consequently down-regulate NK cell degranulation activity, by sustaining leukemia immune escape. Furthermore, the blocking of TIM-3/Gal-9 interaction strongly down-regulates IFNγ-dependent IDO1 activity. Thus, the inhibition of TIM-3/Gal-9 immune check point, which affects NK cell-dependent IFNγ production and the consequent IDO1 activation, could usefully integrate current chemotherapeutic approaches.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy., (©2015 American Association for Cancer Research.)

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy., (©2011 AACR.)

Allergic contact dermatitis is a common disease caused by an exaggerated T cell-mediated immune response to skin-applied haptens. We show in this study that NK cells affect skin immune responses to haptens by releasing type 1 cytokines and inducing keratinocytes apoptosis. Immunohistochemical stainings demonstrated that NK lymphocytes constitute approximately 10% of the inflammatory infiltrate mostly distributed in the superficial dermis and in the epidermis at the site of intense spongiotic changes. More than 90% of NK cells isolated from allergic contact dermatitis skin showed a CD3-CD56(high)CD16- phenotype by FACS analysis. In addition, they uniformly expressed NKG2A, intermediate to high levels of perforin, and the activating receptors, NKG2D, NKp44, and NKp46, but lacked NKp30 and killer Ig-related receptors. Skin NK lymphocytes displayed a CXCR3+CCR6+CCR5+ chemokine receptor asset for homing into inflamed skin, but not CD62L and CCR7 for lymph node homing. When NK cells from nickel-allergic donors were exposed in vitro to the metal, they failed to proliferate, to upregulate CD69, and to release IFN-gamma, thus indicating that NK lymphocytes do not exhibit memory-like properties to haptens. However, IL-2 released by hapten-driven T lymphocytes rapidly induced the release of IFN-gamma by NK cells and promoted the NK-mediated apoptosis of autologous keratinocytes in a hapten-independent manner. Our findings underline the importance of the interaction between innate and adaptive immune mechanisms for amplification of skin allergic responses to haptens and full expression of allergic contact dermatitis.

In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or beta2-integrin engagement on NK cells.

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.