scholarly journals CAR-NK Cells Targeting Sars-Cov-2 Glycosites As COVID-19 Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2803-2803
Author(s):  
Ilias Christodoulou ◽  
Ruyan Rahnama ◽  
Wesley J. Ravich ◽  
Jaesung Seo ◽  
Sergey Zolov ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Single Amino Acid ◽  
Banana Fruit ◽  
S Protein ◽  
Target Ratio ◽  
Infected Cells ◽  
High Mannose

Abstract Introduction: Banana Lectin (BanLec) is a glycoprotein-binding lectin derived from banana fruit that has antiviral activity. BanLec binds high mannose glycans expressed on the viral envelopes of HIV, Ebola, influenza, and coronaviruses. BanLec mitogenicity can be divorced from antiviral activity via a single amino acid change (H84T). The SARS-CoV-2 spike (S) protein is decorated with high mannose N-glycosites that are in close proximity to the viral receptor binding domain (RBD). Our goal was to use the H84T-BanLec as the extracellular targeting domain of a chimeric antigen receptor (CAR). We hypothesized that engineering NK cells to express an H84T-BanLec CAR would specifically direct antiviral cytotoxicity against SARS-CoV-2. Methods: H84T-BanLec was synthesized and added to a 4-1BB.ζ CAR by subcloning into an existing retroviral vector. To modify primary human NK cells, CD3-depleted peripheral blood mononuclear cells were first activated with lethally irradiated feeder cells (K562.mbIL15.4-1BBL), then transduced with transiently produced replication incompetent γ-retrovirus carrying the H84T-BanLec.4-1BB.ζ CAR construct. Vector Copy Number (VCN) per cell was measured and CAR protein expression detected with Western blotting. 293T cells were engineered to express human ACE2 (hACE2.293T), the binding receptor for SARS-CoV-2. CAR expression on NK cells and SARS-CoV-2 S-protein binding to hACE2.293T were measured using FACS. S-protein pseudotyped lentivirus carrying a firefly Luciferase (ffLuc) reporter was produced. Viral infectivity was measured using bioluminescence (BL) detection in virally transduced cells. H84T-BanLec CAR NK cells were added to our S-protein pseudotyped lentiviral infectivity assay and degree of inhibited transduction was measured. NK cell activation was assessed with detection of IFNγ and TNFα secretion using ELISA. Results: A median of 4.5 integrated H84T-BanLec CAR copies per cell was measured (range 3.5-7.45, n=4). The CAR was detected by Western blot in NK cell lysates using antibodies to TCRζ and H84T-BanLec. Surface expression of the CAR on primary NK cells was recorded on day 4 after transduction (median [range], 67.5% CAR-positive [64.7-75%], n=6; Fig. 1). CAR expression was maintained on NK cells in culture for 14 days (58.9% CAR-positive [43.6-66.7%], n=6; Fig. 1). ACE2 expression and binding of recombinant S-proteins to hACE2 on hACE2.293T but not parental 293Ts was verified. S-protein pseudotyped lentiviral transduction of hACE2.293T was confirmed with increase in BL from baseline across diminishing viral titer (n=3; Fig. 2). Control 293T cells without hACE2 expression were not transduced, confirming specificity of viral binding and entry dependent on hACE2 (n=3; Fig. 2). S-protein pseudoviral infectivity of hACE2.293T cells was inhibited by both H84T-BanLec CAR-NK and unmodified NK cells, with enhanced inhibition observed in the CAR-NK condition (mean % pseudovirus infectivity +/- SEM of hACE2.293T in co-cultures with unmodified NK vs. H84T-BanLec CAR-NK; 65 +/-11% vs 35%+/- 6% for 1:1 effector-to-target ratio, p=0.05; 78 +/-3% vs 68%+/- 3% for 1:2.5 effector-to-target ratio, p=0.03; n=6; Fig.3). Both unmodified and H84T-BanLec CAR-NK cells were stimulated to secrete inflammatory mediators when co-cultured with pseudoviral particles and virally infected cells. CAR-NK cells showed overall higher cytokine secretion both at baseline and with viral stimulation. Conclusions: A glycoprotein binding H84T-BanLec CAR was stably expressed on the surface of NK cells. CAR-NK cells are activated by SARS-CoV-2 S-pseudovirus and virally infected cells. Viral entry into hACE2 expressing cells was inhibited by H84T-BanLec CAR-NK cells. Translation of H84T-BanLec CAR-NK cells to the clinic may have promise as an effective cellular therapy for SARS-CoV-2 infection. Figure 1 Figure 1. Disclosures Markovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct. Bonifant: Merck, Sharpe, Dohme: Research Funding; BMS: Research Funding; Kiadis Pharma: Research Funding.

scholarly journals Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Ilias Christodoulou ◽  
Ruyan Rahnama ◽  
Jonas W. Ravich ◽  
Jaesung Seo ◽  
Sergey N. Zolov ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
S Protein ◽  
Effector Cells ◽  
Lentiviral Infection ◽  
Infected Cells ◽  
High Mannose ◽  
Viral Envelopes ◽  
Innate Effector

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.

A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2517-2517 ◽  
Author(s):  
Zachary B. Davis ◽  
Todd Lenvik ◽  
Louis Hansen ◽  
Martin Felices ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Mhc I ◽  
Variable Heavy ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv Envelope

Abstract Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

scholarly journals Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 557
Author(s):  
Xuewen Deng ◽  
Hiroshi Terunuma ◽  
Mie Nieda
Keyword(s):  
Nk Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Healthy Lifestyle ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Effector Cells ◽  
Cell Dysfunction ◽  
Forest Bathing ◽  
Infected Cells

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

scholarly journals NK Cell Regulation in Cervical Cancer and Strategies for Immunotherapy

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3104
Author(s):  
Adriana Gutiérrez-Hoya ◽  
Isabel Soto-Cruz
Keyword(s):  
Cervical Cancer ◽  
Nk Cells ◽  
Nk Cell ◽  
Hpv Infection ◽  
Cytolytic Activity ◽  
Cellular Immunotherapy ◽  
Antigen Receptors ◽  
Cytokines And Chemokines ◽  
Hpv Status ◽  
Infected Cells

Cervical cancer is one of the most prevalent gynaecological malignancies worldwide and is related to human papillomavirus (HPV) infection, viral persistence, progression, and invasion. Therefore, the immune response is linked to HPV status. Natural killer (NK) cells play a central role against virus-infected cells and tumours through a delicate balance between activating and inhibitory receptors and secretion of cytokines and chemokines. These cells also play a crucial role in tumour immunosurveillance. For these reasons, there is growing interest in harnessing NK cells as an immunotherapy for cervical cancer. These studies are diverse and include many strategies such as transferring activated autologous or allogeneic NK cells, improving the activation and cytolytic activity of NK cells using cytokines or analogues and modifying chimeric antigen receptors to increase specificity and targeting NK cells. However, research regarding the application of NK cells in immunotherapy is limited. This article focuses on recent discoveries about using NK cells to prevent and treat cervical cancer and the possibility of cellular immunotherapy becoming one of the best strategies to exploit the immune system to fight tumours.

scholarly journals Natural Killer Cells in the Malignant Niche of Multiple Myeloma

2022 ◽  
Vol 12 ◽  
Author(s):  
Ondrej Venglar ◽  
Julio Rodriguez Bago ◽  
Benjamin Motais ◽  
Roman Hajek ◽  
Tomas Jelinek
Keyword(s):  
Multiple Myeloma ◽  
Nk Cells ◽  
Natural Killer ◽  
Defense Mechanisms ◽  
Hematological Malignancies ◽  
Cell Function ◽  
Nk Cell ◽  
Current Knowledge ◽  
Suppressor Activity ◽  
Infected Cells

Natural killer (NK) cells represent a subset of CD3- CD7+ CD56+/dim lymphocytes with cytotoxic and suppressor activity against virus-infected cells and cancer cells. The overall potential of NK cells has brought them to the spotlight of targeted immunotherapy in solid and hematological malignancies, including multiple myeloma (MM). Nonetheless, NK cells are subjected to a variety of cancer defense mechanisms, leading to impaired maturation, chemotaxis, target recognition, and killing. This review aims to summarize the available and most current knowledge about cancer-related impairment of NK cell function occurring in MM.

scholarly journals BMT CTN 1803: Haploidentical Natural Killer Cells (CSTD002) to Prevent Post-Transplant Relapse in AML and MDS (NK-REALM)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1955-1955
Author(s):  
Sumithira Vasu ◽  
Nelli Bejanyan ◽  
Steven Devine ◽  
Elizabeth Krakow ◽  
Elizabeth Krakow ◽  
...  
Keyword(s):  
High Risk ◽  
Nk Cells ◽  
Cumulative Incidence ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Marrow Transplant ◽  
Free Survival ◽  
Blood And Marrow Transplant ◽  
Relapse Rates

Background and Rationale: Relapse remains the leading cause of treatment failure for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) undergoing allogeneic blood or marrow transplantation (BMT). Although relapse rates vary based on patient population, age, and conditioning intensity, relapse is experienced in at least 30-50% after conventional BMT in high-risk AML/MDS. Initial safety and post-BMT relapse risk reduction results are reported by investigators at MD Anderson Cancer Center in a phase I study of ex vivo-expanded, donor-derived, haploidentical natural killer (NK)-cell infusion in conjunction with haploBMT. Of 13 patients with high-risk myeloid malignancies treated with NK cells, no infusion reactions or dose-limiting toxicities occurred and only 1 patient, treated at the lowest dose of 1×105 cells/kg, relapsed (Ciurea, Blood 2017). This experience supports investigation of CSTD002, a product derived from haploidentical donor NK cells and expanded ex vivo using plasma membrane (PM21) nanoparticles bearing membrane-bound IL-21 and 4-1BBL. This study represents a public-private partnership between the sponsor (Kiadis Pharma) and the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), leveraging existing National Institutes of Health-supported clinical trials infrastructure to conduct a complex cellular immunotherapy trial. We used contemporary, unpublished data from the Center for International Blood and Marrow Transplant Research registry to determine baseline relapse rates that informed the statistical design. Doses of NK cells expanded by a novel method and exceeding those previously achieved in most published studies will be given in the peri-transplant period to test the hypothesis that haploidentical NK cells can mediate an effective anti-leukemia response. Trial Design and Methods: BMT CTN 1803 is a phase II, single-arm, open-label, multicenter trial designed to investigate the safety and efficacy of CSTD002 for the treatment of patients with high-risk AML or MDS undergoing haploBMT. An initial safety run-in phase will precede enrollment into the full study of approximately 60 patients. Major inclusion criteria of patients and donors are listed in the Table. Peripheral blood will be drawn from the donor to start the NK-cell expansion approximately 5 weeks before the planned haploBMT. Patients will receive intravenous (IV) melphalan 140 mg/m2 (100 mg/m2 for patients ≥60 years old) on Day -7; fludarabine 40 mg/m2 IV on Days -7, -6, -5, and -4; and 2 Gy of total body irradiation on Day -3. Donor bone marrow will be harvested and given on Day 0. Three doses of CSTD002 will be administered IV on Days -2, +7, and +28, relative to the haploBMT. The recommended dose of CSTD002 for administration will be formulated at 1×108 NK cells/kg of recipient body weight. Graft-versus-host disease (GVHD) prophylaxis is post-transplantation cyclophosphamide with tacrolimus and mycophenolate mofetil. The primary endpoint is cumulative incidence of relapse at 1 year post haploBMT in patients receiving at least 1 infusion of CSTD002. Secondary endpoints are safety and tolerability of CSTD002; overall survival; non-relapse mortality; relapse-free survival; GVHD-free survival; cumulative incidence of acute GVHD and chronic GVHD; hematologic recovery; donor-cell engraftment; primary and secondary graft failure; overall incidence of toxicity; and cumulative incidence of infections including cytomegalovirus re-activation and symptomatic BK virus hemorrhagic cystitis. Exploratory endpoints are systemic immunosuppression-free survival; immune reconstitution at Days 28, 100, and 365 post haploBMT; proportion of patients with detectable minimal residual disease at Days 28 and 100 post haploBMT; feasibility of administering the planned CSTD002 doses; and impact of NK-cell alloreactivity on relapse and survival. Disclosures Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: Clinical trial support. Bejanyan:Kiadis Pharma: Other: advisory board. Devine:Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta; Bristol Myers: Other: Grant for monitoring support & travel support. Krakow:Bellicum Pharmaceuticals: Research Funding; Highpass Bio: Research Funding; Magnolia Innovations: Other: Personal fees. Logan:Eisai: Other: Personal fees; Astellas: Other: Grant; Kiadis (formerly Cytosen): Other: Grant; Novartis: Other: Personal fees; Kite: Other: Grant. Luznik:Merck: Research Funding, Speakers Bureau; Genentech: Research Funding; AbbVie: Consultancy; WindMiL Therapeutics: Patents & Royalties: Patent holder. Barrett:Kiadis Pharma (formerly Cytosen): Other: Personal fees; Biologics Consulting Company: Other: Personal fees. Shan:Kiadis Pharma (formerly Cytosen): Employment. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Sanofi-Genzyme: Research Funding.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

scholarly journals IL-17 constrains natural killer cell activity by restraining IL-15–driven cell maturation via SOCS3

2019 ◽  
Vol 116 (35) ◽  
pp. 17409-17418 ◽  
Author(s):  
Xuefu Wang ◽  
Rui Sun ◽  
Xiaolei Hao ◽  
Zhe-Xiong Lian ◽  
Haiming Wei ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Natural Killer Cell Activity ◽  
Killer Cell ◽  
Cell Activity ◽  
Cell Maturation ◽  
Dependent Manner ◽  
Deficient Mice

Increasing evidence demonstrates that IL-17A promotes tumorigenesis, metastasis, and viral infection. Natural killer (NK) cells are critical for defending against tumors and infections. However, the roles and mechanisms of IL-17A in regulating NK cell activity remain elusive. Herein, our study demonstrated that IL-17A constrained NK cell antitumor and antiviral activity by restraining NK cell maturation. It was observed that the development and metastasis of tumors were suppressed in IL-17A–deficient mice in the NK cell-dependent manner. In addition, the antiviral activity of NK cells was also improved in IL-17A–deficient mice. Mechanistically, ablation of IL-17A signaling promoted generation of terminally mature CD27−CD11b+ NK cells, whereas constitutive IL-17A signaling reduced terminally mature NK cells. Parabiosis or mixed bone marrow chimeras from Il17a−/−and wild-type (WT) mice could inhibit excessive generation of terminally mature NK cells induced by IL-17A deficiency. Furthermore, IL-17A desensitized NK cell responses to IL-15 and suppressed IL-15–induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) via up-regulation of SOCS3, leading to down-regulation of Blimp-1. Therefore, IL-17A acts as the checkpoint during NK cell terminal maturation, which highlights potential interventions to defend against tumors and viral infections.

scholarly journals Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

2009 ◽  
Vol 206 (3) ◽  
pp. 515-523 ◽  
Author(s):  
Agnieszka Kielczewska ◽  
Michal Pyzik ◽  
Tianhe Sun ◽  
Astrid Krmpotic ◽  
Melissa B. Lodoen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Deletion Mutant ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Receptor ◽  
Major Histocompatability Complex ◽  
Wild Type ◽  
Mcmv Infection ◽  
Host Protection ◽  
Infected Cells

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-Dk and the MCMV m04 protein. Using H2 chimeras between H2-Db and -Dk, we demonstrate that the H2-Dk peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-Dk, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor.

Mono/Oligoclonal T and NK Cells Are Common in Philadelphia Chromosome Positive (Ph+) Leukemia Patients at Diagnosis and Expand During Successful Tyrosine Kinase Inhibitor Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 856-856
Author(s):  
Anna Kreutzman ◽  
Vesa Juvonen ◽  
Veli Kairisto ◽  
Marja Ekblom ◽  
Leif Stenke ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Gene Rearrangements ◽  
Imatinib Treatment ◽  
Healthy Controls ◽  
Clonal Rearrangement

Abstract Abstract 856 Introduction. Central to current treatment of Ph+ leukemia patients are tyrosine kinase inhibitors (TKIs), which predominantly target the BCR-ABL1 kinase in malignant cells. However, broader-spectrum 2nd generation TKIs, such as bosutinib, dasatinib and nilotinib, also inhibit off-target kinases with important physiological functions. Several in vitro studies have implied that TKIs may have immunosuppressive effects by suppressing activation and proliferation of effector lymphocytes. In contrast, we recently observed immunostimulation during dasatinib therapy in the form of marked expansion of clonal cytotoxic lymphocytes (T- and NK cells) resulting in chronic LGL-type lymphocytosis in peripheral blood (PB). The prevalence, detailed molecular background and clinical implications of clonal lymphocytes during TKI therapy are currently unknown. The aim of this study was to comprehensively analyze clonality and evolution of lymphocyte clones during TKI therapy. Patients and methods. The study population included patients with Ph+ leukemia, both CML (n=28) and ALL patients (n=4) on dasatinib (n=23) and imatinib (n=9) therapies. In addition, samples from 12 healthy controls and diagnostic samples from the nine imatinib treated patients were analyzed. Lymphocyte clonality was determined by analysis of PB mononuclear cells (MNC) for clonal T cell receptor (TCR) γ and δ gene rearrangements by 18 primer pairs covering most known clonal TCR γ and δ rearrangements. Upon positive reaction in heteroduplex analysis, the purified PCR products were sequenced. If clonal rearrangement was observed, allele-spesific PCR primers were designed to allow for quantitative follow-up of lymphocyte clones in each patient. Results. Sequencing-confirmed clonal TCR γ rearrangement was observed only in 1 of 12 healthy controls and no TCR δ gene rearrangements were found in this group. Surprisingly, 7 of 9 (78%) CML patients showed clonal TCR rearrangements at diagnosis. In 3 patients the clonal rearrangement was detected in the TCR δ genes, in 7 patients in the TCR γ genes and 3 patients had rearrangemens both in TCR δ and γ genes. After one year of imatinib treatment the same clones could be detected in 5 of the 7 patients (71%). Although clonal cells were observed, none of the imatinib patients had signs of a concomitant lymphoproliferative disorder and the distribution of lymphocyte subclasses was normal. Next, 23 patients treated with dasatinib were studied, 10 without (LGLneg) and 13 with PB LGL lymphocytosis (LGLpos) including T- or NK-cell expansions. In all LGLpos dasatinib patients (including patients with a CD3neg NK-cell expansion) clonal TCR γ or δ rearrangements were found. In LGLneg dasatinib patients the prevalence of TCR rearrangements was 80%. LGLpos patients had more often clonal rearrangements in TCR δ genes (62%) than LGLneg patients (10%). No differences in clonal rearranged TCR γ genes (77% vs. 80%) were detected. Most patients displayed more than one clonal TCR rearrangement. Quantitative follow-up of LGLpos patients revealed that the expansion of a single predominant lymphocyte clone accounted for LGL lymphocytosis. Intriguingly, quantitative follow-up of lymphocyte clones by PCR showed that the observed clones existed at low levels already before start of dasatinib therapy during imatinib treatment, but no lymphocyte expansions were then seen. Sorting of lymphocytes showed that clonal cells resided in the CD8+ and CD4+ T-cell populations and, strikingly, also among CD16/56+CD3neg NK cells. All dasatinib patients with NK cell expansions (n=3) showed TCR δ rearrangements in their NK cells. Conclusions. Clonal lymphocytes could rarely be found in healthy controls. In contrast, they were frequently present in CML patients at diagnosis and persisted during TKI therapy. In a distinct subgroup of dasatinib treated patients, clonal cells massively expanded during successful therapy. Clonal TCR rearrangements were detected in CD4+, CD8+ and, unexpectedly, also in NK cells. The epitopes and function of clonal, CML-associated lymphocytes are under investigation. Previous studies showed that clonal expansions during dasatinib were associated with excellent, long-lasting therapy responses in advanced leukemia. We therefore hypothesize, that the clonal lymphocytes present at CML diagnosis may be anergic anti-leukemic cells and part of the immune escape mechanisms inherent to leukemogenesis and that dasatinib therapy can reverse this anergy. Disclosures: Ekblom: BMS: Honoraria. Seggewiss:BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:BMS: Honoraria.

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