scholarly journals Donor and Host Coexpressing KIR Ligands Promote NK Education Post Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4519-4519
Author(s):  
Xingxing Yu ◽  
Xiang-Yu Zhao ◽  
Zhengli Xu ◽  
Xunhong Cao ◽  
Mingrui Huo ◽  
...  
Keyword(s):  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Hla Typing ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Hla Ligands ◽  
Ifn Γ ◽  
Hla Genotype

Background:Educated NK cells prevent autoreactive behavior but also permit cytotoxicity against target cells that have down-regulated HLA class I expression. When and how the process of education occurs has not been clearly discerned. Several groups reported that both the donor and host MHC could influence NK cell education in mouse models. In humans, Dulphy et al demonstrated that NK-cell education is shaped by donor HLA genotype. Moreover, our previous study found NK-cell education was shaped by host HLA genotype post allo-HSCT. However, due to the lack of single-KIR+ NK cells, functional analysis limited the full evaluation of the interaction between donor/host HLA and donor inhibitory KIR, so the contribution of donor HLA could not be excluded. Aims: In this research, we have investigated the relative contributions of donor and recipient HLA to NK cells education, the interplay between functional reconstitution and the involvement of donor/host HLA interaction in NK cell control of leukemia cells. Methods: Two cohorts of patients were enrolled in this study. We first prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore NK cell phenotypes and functional reconstitution. From June 2012 to April 2016, 276 AML/MDS patients that underwent haploidentical transplantation were enrolled in the second cohort to analyze the effect of donor-host KIR-HLA combinations on relapse post transplantation. Molecular HLA typing and KIR genotyping were performed according to the manufacturer's instructions (One Lambda, Canoga Park, CA, USA). Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry. The cytotoxicity and cytokine secretion of NK cells was determined using CD107a expression and IFN-γ production against the K562 cell line. Single-KIR+ NK cells were grouped into the following groups: (A) nsKIR: where both hosts and donors lacked HLA ligands for one donor KIR; (B) d-rsKIR, where donors and hosts, encoded HLA ligands for donor KIRs; (C) dsKIR, where donors, but not hosts, encoded HLA ligands for donor KIR; and (D) rsKIR, where hosts, but not donors, encoded HLA ligands for donor KIR. Results: 1. Donor KIR ligated by both donor and host HLA is associated with better single-KIR+ NK cell education among the same patients. KIR2DL2/L3 single+ NK cell exhibited higher reactivity compared to KIR2DL1 single+ NK cell in pairs of donors C1C1 or C1C2 and host C1C1. KIR2DL2/L3 single+ NK cell exhibited higher reactivity than KIR3DL1 single+ NK cell in pairs of donors Bw4C1Cx and host C1Cx. KIR2DL1 single+ NK cell exhibited comparable reactivity with KIR2DL2/L3 single+ NK cell in pairs of donor Bw4C1C2 and host Bw4C1C2. 2. Donor KIR ligated by both donor and host HLA contribute to better single-KIR+ NK cell education among the same single-KIR+ NK cells. KIR2DL2/L3 single+ NK cell in the group of d-rsKIR (C1Cx-C1Cx) exhibit higher reactivity compared with other groups (dsKIR (C1Cx-C2C2), rsKIR (C2C2- C1Cx)). KIR2DL1 single+ NK cells in group of d-rsKIR (C2Cx-C2Cx) exhibited higher reactivity compared with other groups (nsKIR (C1C1-C1C1), dsKIR (C2Cx-C1C1), rsKIR (C1C1-C2Cx)). KIR3DL1 single+ NK cells in groups of d-rsKIR (Bw4Bwx-Bw4Bwx) exhibited higher reactivity compared with other groups (nsKIR (Bw6Bw6-Bw6Bw6), dsKIR (Bw4Bwx-Bw6Bw6), rsKIR (Bw6Bw6-Bw4Bwx)). 3. Both donor and host HLA must coexist for maximum education of NK cells given donor 3 inhibitory KIRs. When both of donor and host presenting all HLA (Bw4C1C2), we showed a remarkable hierarchy of responses among NK populations. NK cells with two inhibitory KIRs for self-HLA exhibited higher NK responsiveness (CD107α and IFN-γ) compared with single KIR+ NK cells. NK cells with 3 inhibitory KIRs for self-HLA exhibited maximum responsiveness. 4. Both donor and host exhibiting all HLA (Bw4C1C2) for donor 3 inhibitory KIRs contributes to least relapse following haploidentical allo-HSCT. In the second cohort, the lowest relapse rate was found in d-rsKIR group (n=31, 0%) compared with rsKIR group (n=55 ,0% vs. 10.0±4.9%, P=0.115), dsKIR group (n=33, 0% vs 14.9%±7.0%, P=0.039), or nsKIR group (n=156, 0% vs. 18%±3.5%, P=0.022). Summary: This study demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients. Disclosures No relevant conflicts of interest to declare.

GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2707-2707 ◽  
Author(s):  
Britnie Spaunhorst ◽  
George J Weiner
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Autologous Serum ◽  
Target Cells ◽  
Type I ◽  
Raji Cells ◽  
Anti Cd20

Abstract Abstract 2707 Poster Board II-683 Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC. GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present. Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors. We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Antineoplastic and anti-inflammatory effects of bortezomib on systemic chronic active EBV infection

2021 ◽  
Vol 5 (7) ◽  
pp. 1805-1815
Author(s):  
Mayumi Yoshimori ◽  
Haruna Shibayama ◽  
Ken-Ichi Imadome ◽  
Fuyuko Kawano ◽  
Ayaka Ohashi ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Messenger Rna ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Intraperitoneal Administration ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Infected Cells ◽  
Ifn Γ

Abstract Systemic chronic active Epstein-Barr virus (EBV; sCAEBV) infection, T- and natural killer (NK)-cell type (sCAEBV), is a fatal disorder accompanied by persisting inflammation harboring clonal proliferation of EBV-infected T or NK cells. Today’s chemotherapy is insufficient to resolve disease activity and to rid infected cells of sCAEBV. The currently established treatment strategy for eradicating infected cells is allogeneic hematopoietic stem cell transplantation. In this study, we focused on the effects of proteasome inhibitor bortezomib on the disease. Bortezomib suppressed survival and induced apoptosis of EBV+ T- or NK-cell lines and peripheral mononuclear cells containing EBV-infected T or NK cells of sCAEBV patients. Bortezomib enhanced binding immunoglobulin protein/78-kDa glucose-regulated protein (Bip/GRP78) expression induced by endoplasmic reticulum stress and activated apoptosis-promoting molecules JNK and p38 in the cell lines. Bortezomib suppressed the activation of survival-promoting molecule NF-κB, which was constitutively activated in EBV+ T- or NK-cell lines. Furthermore, quantitative reverse transcription–polymerase chain reaction demonstrated that bortezomib suppressed messenger RNA expression of proinflammatory cytokines tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in EBV+ T or NK cells from the patients. Finally, we examined the effects of bortezomib using xenograft models of sCAEBV generated by IV injection of patients’ cells. The intraperitoneal administration of bortezomib significantly reduced EBV-DNA load in peripheral blood and the infiltration of EBV-infected cells in the models’ livers. Moreover, the serum concentration of TNF-α and IFN-γ decreased after bortezomib treatment to the models. Our findings will be translated into the treatment of sCAEBV not only to reduce the number of tumor cells but also to suppress inflammation.

JAK1 and JAK2 Modulate Myeloma Cell Susceptibility to NK Cells Through the Interferon Gamma (IFN-γ) Pathway,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3960-3960
Author(s):  
Roberto Bellucci ◽  
Allison Martin ◽  
Marc Buren ◽  
Hong-Nam Nguyen ◽  
Davide Bommarito ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Specific Antigen ◽  
Innate Immune ◽  
Target Cells ◽  
Jak Inhibitor ◽  
Stat Proteins ◽  
Ifn Γ

Abstract Abstract 3960 Multiple myeloma (MM) is a B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite the use of new drugs such as lenalidomide and bortezomib, MM remains an incurable disease. Successful treatment of MM with allogeneic stem cell transplantation suggests that MM is susceptible to immunologic approaches. NK cells are the primary effectors of the innate immune response against infectious pathogens and malignant transformation. Unlike T and B cells, NK cells do not recognize antigens in the context of classical major histocompatibility complex (MHC) but lyse target cells without specific antigen recognition. Nevertheless, MM cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not well understood. To identify novel pathways that modulate MM cell resistance to the immune system, we previously developed a genetic screen to detect cell-cell interactions using a large lentiviral shRNA library containing a total of 6,144 shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 results in significantly increased MM cell susceptibility to NK cell lysis. This effect was not noted when JAK3 and TYK2 were targeted. JAK1, JAK2 JAK3 and TYK2 are members of a family of tyrosine kinases that are constitutively associated with many membrane cytokine receptors. After activation, JAK proteins regulate phosphorylation/activation of STAT proteins, which subsequently initiate gene transcription. To understand JAK1 and JAK2 involvement in MM resistance to NK cells, we undertook a series of experiments to analyze the JAK signaling pathway in MM cells. We first analyzed the activation status of STAT proteins in a series of MM cell lines (IM-9, KM12BM, RPMI 8226, U266) in which JAK1 and JAK2 expression was reduced by specific shRNAs. Constitutive activation of STAT proteins was not affected by JAK1 or JAK2 gene silencing suggesting that these kinases were not activated in the absence of cytokine receptor-mediated signaling. Since JAK1 and JAK2 are associated with the IFN-γ receptor and we previously showed that JAK1 and JAK2 silencing induces increased secretion of IFN-γ from NK cells, we pre incubated MM cell lines with NK activated supernatant or recombinant IFN-γ and tested them for STAT activation. 15 min incubation was sufficient to initiate phosphorylation of STAT1 but no other STATs were activated. Silencing of JAK1 or JAK2 with specific shRNAs prevented STAT1 activation. To validate this finding, we tested primary MM cells treated with different concentrations of Jak inhibitor 1 (0 nM, 10 nM, 30 nM and 40 nM). These cells had a similar STAT profile at their basal level when compared with the previously tested MM cell lines. Pre-incubation with NK activated supernatant or IFN-γ also induced rapid activation of STAT1, which was completely inhibited when cells were pre-treated with Jak inhibitor 1. Treatment of MM cells with 10, 30 and 40 nM of Jak inhibitor enhanced killing by NK cells by 46.6%, 51% and 53%, compared to untreated cells (p=0.0036, p=0.0011 and p=0.0010 respectively). These findings demonstrate that IFN-γ signals rapidly enhance resistance of MM cells to NK cells but inhibition of this pathway at the level of JAK1 and JAK2 reverses this effect and induces susceptibility to NK cell mediated lysis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aberrant anti-viral response of natural killer cells in severe asthma

2020 ◽  
Vol 55 (5) ◽  
pp. 1802422
Author(s):  
Justine Devulder ◽  
Cécile Chenivesse ◽  
Valérie Ledroit ◽  
Stéphanie Fry ◽  
Pierre-Emmanuel Lobert ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Severe Asthma ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Asthma Patients ◽  
Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

CX3CR1 expression defines 2 KLRG1+ mouse NK-cell subsets with distinct functional properties and positioning in the bone marrow

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4467-4475 ◽  
Author(s):  
Giuseppe Sciumè ◽  
Giulia De Angelis ◽  
Giorgia Benigni ◽  
Andrea Ponzetta ◽  
Stefania Morrone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Cell Population ◽  
Nk Cell ◽  
Target Cells ◽  
Functional Features ◽  
Ifn Γ ◽  
Cell Positioning ◽  
Cytokine Stimulation ◽  
Cx3cr1 Expression

Abstract During development in the bone marrow (BM), NK-cell positioning within specific niches can be influenced by expression of chemokine or adhesion receptors. We previously demonstrated that the maintenance in the BM of selected NK-cell subsets is regulated by the CXCR4/CXCL12 axis. In the present study, we showed that CX3CR1 is prevalently expressed on KLRG1+ NK cells, a subset considered terminally differentiated. Two KLRG1+ NK-cell populations endowed with distinct homing and functional features were defined according to CX3CR1 expression. In the BM, KLRG1+/CX3CR1− NK cells were mainly positioned into parenchyma, while KLRG1+/CX3CR1+ NK cells exhibited reduced CXCR4 expression and were preferentially localized in the sinusoids. We also showed that α4 integrin plays a pivotal role in the maintenance of NK cells in the BM sinusoids and that α4 neutralization leads to strong reduction of BM KLRG1+/CX3CR1+ NK cells. Moreover, we found that KLRG1+/CX3CR1+ cells originate from KLRG1+/CX3CR1− NK-cell population and display impaired capability to produce IFN-γ and to lyse YAC-1 target cells on cytokine stimulation. Altogether, our findings show that CX3CR1 represents a marker of a KLRG1+ NK-cell population with unique properties that can irreversibly differentiate from the KLRG1+/CX3CR1− NK cells during steady state conditions.

scholarly journals Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 753 ◽  
Author(s):  
Loris Zamai ◽  
Genny Del Zotto ◽  
Flavia Buccella ◽  
Sara Gabrielli ◽  
Barbara Canonico ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Response ◽  
Cell Activation ◽  
Nk Cell ◽  
High Sensitivity ◽  
Leukemic Cells ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Oncologic Patients ◽  
The Way

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

scholarly journals Chlamydia psittaci-Infected Dendritic Cells Communicate with NK Cells via Exosomes To Activate Antibacterial Immunity

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Nadine Radomski ◽  
Axel Karger ◽  
Kati Franzke ◽  
Elisabeth Liebler-Tenorio ◽  
Rico Jahnke ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Early Response ◽  
Chlamydia Psittaci ◽  
Target Cells ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Host Cell Death ◽  
Ifn Γ

ABSTRACT Dendritic cells (DCs) and natural killer (NK) cells are critically involved in the early response against various bacterial microbes. Functional activation of infected DCs and NK cell-mediated gamma interferon (IFN-γ) secretion essentially contribute to the protective immunity against Chlamydia. How DCs and NK cells cooperate during the antichlamydial response is not fully understood. Therefore, in the present study, we investigated the functional interplay between Chlamydia-infected DCs and NK cells. Our biochemical and cell biological experiments show that Chlamydia psittaci-infected DCs display enhanced exosome release. We find that such extracellular vesicles (referred to as dexosomes) do not contain infectious bacterial material but strongly induce IFN-γ production by NK cells. This directly affects C. psittaci growth in infected target cells. Furthermore, NK cell-released IFN-γ in cooperation with tumor necrosis factor alpha (TNF-α) and/or dexosomes augments apoptosis of both noninfected and infected epithelial cells. Thus, the combined effect of dexosomes and proinflammatory cytokines restricts C. psittaci growth and attenuates bacterial subversion of apoptotic host cell death. In conclusion, this provides new insights into the functional cooperation between DCs, dexosomes, and NK cells in the early steps of antichlamydial defense.

CD69 Is Required for Activated NK Cell-Mediated Killing of Resistant Targets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3322-3322 ◽  
Author(s):  
Chloe M. Marden ◽  
Janet North ◽  
Robert Anderson ◽  
Ismail A. Bakhsh ◽  
Elena Addison ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Nk Cells ◽  
Myeloid Leukaemia ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Autologous Bone Marrow Transplantation ◽  
Target Cells ◽  
Monomeric Form ◽  
Normal Peripheral Blood ◽  
Raji Cells

Abstract The C-type lectin receptor CD69 is expressed on a range of haematopoietic cells following activation. On natural killer (NK) cells CD69 may play a direct role in mediating cytotoxicity of tumour targets. We have previously shown that remission following chemotherapy or autologous bone marrow transplantation (BMT) for acute myeloid leukaemia (AML) is dependent on NK cell cytotoxicity and have observed CD69 capping the immune synapse between autologous NK cells and conjugated AML cells (Lowdell et al, Br J Haematol, 2002; 117(4):821–7). Tumour cells which are resistant to NK-mediated lysis are often susceptible to lysis by activated NK cells (e.g. after stimulation with IL-2); thus we hypothesized that the interaction between CD69 on activated NK cells and its unidentified ligand (CD69L) on target cells is required for target cell lysis. Here we use soluble recombinant CD69 (rCD69) to investigate the role of CD69-CD69L interaction in mediating activated-NK cytotoxicity of NK-resistant and NK-sensitive target cells. The extracellular domain of CD69 (amino acids 65–199) fused with an N-terminal biotinylation sequence (Avidity) was expressed in Escherichia coli and a multivalent rCD69 reagent was created by binding biotinylated CD69 protein to avidin coated fluorescent beads (Spherotech Inc). Binding of rCD69 to NK-resistant Raji and Daudi Burkitt’s lymphoma cell line targets was determined by flow cytometry (11.9%, 12.4% positive respectively) and confocal microscopy; rCD69 did not bind to 293 kidney epithelial cells, K562 chronic myeloid leukaemia (an NK-sensitive target) or normal peripheral blood mononuclear cells. rCD69 was used to block the interaction between activated-NK cells and target cells; pre-incubation of Raji target cells with rCD69 reduced specific cytotoxicity to the level of unactivated NK cells (31.2 +/−1.6% to 8.0 +/−0.7%, Figure 1). Furthermore, activation of the intracellular tyrosine kinase Syk, which is selectively phosphorylated following CD69 signalling on activated NK cells (Pisegna et al, JI, 2002; 169: 68–74), was abrogated by rCD69 pre-incubation as determined by confocal microscopy. These data show that rCD69 binds NK-resistant target cells and blocks the killing of these cells by activated NK cells. We conclude that CD69 is required for activated NK-cell-mediated killing of resistant targets and that CD69L may be a tumour-restricted marker. Screening of primary tumours for CD69L is ongoing. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only. Figure 1. rCD69 fractions block lysis of Raji cells by activated NK cells. Killing of Raji target cells by activated NK cells (aNK) is reduced to that of unactivated NK cells by pre-incubating Raji with HPLC purified fractions of rCD69. F3 contains rCD69 in dimeric and monomeric form, F2 in monomeric form only.

Human Natural Killer Cells Achieve Tolerance by Preferentially Endowing Functional Competence to Inhibitory Killer Ig-Like Receptors Specific for Self-HLA Class I.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 919-919
Author(s):  
Junli Yu ◽  
Clara Pinto-Agnello ◽  
Joseph Chewning ◽  
Katharine C. Hsu
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Class I ◽  
Target Cells ◽  
Inhibitory Receptors ◽  
Hla Ligand ◽  
Self Tolerance ◽  
Hla Ligands ◽  
Ifn Γ ◽  
Haplotype A

Abstract Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p<0.001). All 3 inhibitory KIR (KIR2DL3, 2DL1, and 3DL1) demonstrated capacity for tolerance, with predictable response patterns based on the HLA background of the individual. NK cells lacking all inhibitory NK receptors (KIR−CD94/NKG2A−ILT2−) were identifiable and were markedly hyporesponsive. KIR−CD94/NKG2A+ILT2+ NK cells were also minimally responsive, comparable to the NK subset expressing inhibitory KIR for non-self HLA. These results demonstrate NK tolerance in humans, consistent with the licensing model proposed in mice: NK cells expressing inhibitory KIR to self-HLA are significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA, and are rendered tolerant to self through inhibition upon binding to self-HLA ligands. NK cells expressing inhibitory KIR for non-self HLA are hyporesponsive and therefore rendered tolerant and incapable of autoreactivity when ligand is not engaged.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

Sign in / Sign up

Export Citation Format

Share Document