High Dimensional Analysis of NK Cells in Smokers and COPD: Associations of NK Cell Phenotype with Exacerbations

Natural killer (NK) cells comprise one subset of the innate lymphoid cell (ILC) family. Despite reported antitumor functions of NK cells, their tangible contribution to tumor control in humans remains controversial. This is due to incomplete understanding of the NK cell states within the tumor microenvironment (TME). Here, we demonstrate that peripheral circulating NK cells differentiate down two divergent pathways within the TME, resulting in different end states. One resembles intraepithelial ILC1s (ieILC1) and possesses potent in vivo antitumor activity. The other expresses genes associated with immune hyporesponsiveness and has poor antitumor functional capacity. Interleukin-15 (IL-15) and direct contact between the tumor cells and NK cells are required for the differentiation into CD49a+CD103+ cells, resembling ieILC1s. These data explain the similarity between ieILC1s and tissue-resident NK cells, provide insight into the origin of ieILC1s, and identify the ieILC1-like cell state within the TME to be the NK cell phenotype with the greatest antitumor activity. Because the proportions of the different ILC states vary between tumors, these findings provide a resource for the clinical study of innate immune responses against tumors and the design of novel therapy.

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FC 129CHANGES IN PERIPHERAL NK CELLS IN KIDNEY TRANSPLANT RECIPIENTS WITH AND WITHOUT HLA DSA

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab148.004 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Laura Llinas ◽

Dolores Redondo Pachon ◽

Dàlia Raïch Regué ◽

Maria Jose Perez-Saez ◽

Sara Sanz ◽

...

Keyword(s):

Nk Cells ◽

Cell Count ◽

Kidney Transplant ◽

Graft Survival ◽

Peripheral Blood ◽

Nk Cell ◽

Cell Phenotype ◽

Transplant Recipients ◽

Kidney Transplant Recipients

Abstract Background and Aims Antibody-mediated rejection (ABMR) is a frequent cause of renal allograft loss. There is increasing evidence of the role of Natural Killer (NK) cells in the establishment of ABMR damage. Our group described that patients with donor-specific antibodies (DSA) and ABMR present higher proportions of NKG2A+ NK cell subset in peripheral blood than those without HLA DSA or HLA antibodies. Method We selected 177 kidney transplant recipients (KT) with renal biopsies 2011-2017: 77 with ABMR (DSA+: 53, DSA-: 24) and 100 without ABMR (DSA+: 15, DSA-: 85). We assessed graft survival with a median time of follow-up since the renal biopsy of 53 months. In 138 KT we evaluated the peripheral blood NK cell immunophenotyping and its value as a prognostic biomarker. Results Graft survival was worse in ABMR-KT at the end of follow-up (p<0.001) independently of DSA detection (p=0.63). Regarding NK cell immunophenotyping, we observed a lower proportion and absolute NK cell count in ABMR+DSA+-KT and ABMR+DSA--KT compared with ABMR-DSA--KT (p=0.027, p=0.017). ABMR+DSA+-KT showed higher proportion of NKG2A+ NK cells compared with ABMR-DSA--KT (p=0.007). All ABMR+ patients, independently of DSA detection, presented lower absolute NKG2A- NK cell count in comparison with ABMR-DSA--KT (p=0.001, p=0.017). Finally, a proportion of NKG2A- <30% was associated with lower graft survival 36 months after graft biopsy with ABMR (p=0.067) (Figure). Conclusion Graft survival is worse in ABMR+ compared with ABMR- KT independently of DSA detection. Kidney transplant recipients with ABMR show reduced peripheral absolute numbers of NK cells and NKG2A- NK cells regardless of undetectable DSA. This NK cell phenotype associated with a worse medium-term graft survival in cases with ABMR.

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Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia

Cancers ◽

10.3390/cancers12082187 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2187

Author(s):

Raffaella Meazza ◽

Michela Falco ◽

Fabrizio Loiacono ◽

Paolo Canevali ◽

Mariella Della Chiesa ◽

...

Keyword(s):

Acute Leukemia ◽

Nk Cells ◽

Nk Cell ◽

Functional Characterization ◽

Cell Subset ◽

Cell Phenotype ◽

Cell Repertoire ◽

Hematopoietic Stem ◽

Γδt Cells ◽

Effector Cells

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIR+NKG2A−CD57+ NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed.

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Abstract 037: A Role for IL-17 to Activate Cytolytic Natural Killer Cells in Response to Placental Ischemia

Hypertension ◽

10.1161/hyp.66.suppl_1.037 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Denise C Cornelius ◽

D'Andrea S Thomas ◽

Jamil Elfarra ◽

Taia R McAfee ◽

Babbette LaMarca

Keyword(s):

Blood Pressure ◽

Nk Cells ◽

Renal Dysfunction ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Blood Pressure Response ◽

Cell Phenotype ◽

Uterine Perfusion ◽

Placental Ischemia

Women with preeclampsia (PE), newly developed hypertension and renal dysfunction during pregnancy, have small-for-gestational-age babies and demonstrate an increase in the inflammatory cytokine IL-17, placental oxidative stress, and cytolytic natural killer (NK) cell activation. The stimulus of the cytolytic NK cell phenotype during PE is currently unknown. Moreover, the specific role of cytolytic NK cells in the pathophysiology of preeclampsia has not been clearly defined. The reduced uterine perfusion pressure (RUPP) model of placental ischemia exhibits many of the characteristics of preeclampsia including hypertension, renal dysfunction, chronic inflammation and intrauterine growth restriction (IUGR). In this study, we tested the hypothesis that placental ischemia results in cytolytic activation of NK cells, and examined a role for the increased IL-17, in response to placental ischemia, to activate cytolytic NK cells. In this study, blood pressure (MAP) and pup weight were measured, and PBMCs and placental lymphocytes were examined via flow cytometry for surface makers of cytolytic NK cell activation. MAP significantly increased in response to placental ischemia from 103±4.1 mmHg in NP (n=6) to 129.1±3.1 mmHg (n=8) in RUPP rats (p<0.001). Neutralization of IL-17 with a soluble receptor attenuated the blood pressure response to 106.3±2.3 mmHg in RUPP+IL-17RC rats (n=3). Pup weight is significantly decreased in RUPP rats (2.52±0.18g in NP vs 2.03±0.05g in RUPP (p<0.05)), which increased to 2.54±0.36g in RUPP+IL-17RC. Cytolytic activation of circulating NK cells was not significantly changed among any of the groups (NP: 2.49±1.1%; RUPP: 7.74±3.2%; RUPP+IL-17RC:5.50±2.8%). However, cytolytic activation of placental NK cells increased in response to placental ischemia (NP: 3.4±1.1% vs RUPP 10.0±3.4%), and was completely attenuated after treatment with the soluble IL-17 receptor (RUPP+IL-17RC: 0.33±0.17%). These results suggest a role for placental ischemia and increased IL-17 to stimulate cytolytic NK cells. Furthermore, this study links the IL-17 pathway with cytolytic NK cell activation and IUGR in response to placental ischemia, potentially identifying new therapeutic targets to improve maternal and fetal outcomes of PE.

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Anti-CD20 Monoclonal Antibody (mAb) with Enhanced Affinity for CD16 Activates NK Cells at Lower Concentrations and More Effectively Than Rituximab ®.

Blood ◽

10.1182/blood.v106.11.348.348 ◽

2005 ◽

Vol 106 (11) ◽

pp. 348-348 ◽

Cited By ~ 4

Author(s):

George J. Weiner ◽

Julie A. Bowles ◽

Brian K. Link ◽

Mary A. Campbell ◽

James E. Wooldridge ◽

...

Keyword(s):

Nk Cells ◽

Mononuclear Cells ◽

Nk Cell ◽

Normal Subjects ◽

Cell Phenotype ◽

P Value ◽

Peripheral Blood Mononuclear ◽

Anti Cd20 ◽

Anti Tumor Activity ◽

Ifng Production

Abstract Growing evidence indicates the affinity of mAb for CD16 (FcgRIII) plays a central role in the ability of the mAb to mediate anti-tumor activity. Both polymorphisms in CD16 and structure of mAb Fc can impact on the affinity between CD16 and mAb. Little is known about how affinity between mAb and CD16 impacts on the ability of mAb to activate NK cells. We evaluated how CD16 polymorphisms, and mAb with modified affinity for CD16, impact on NK cell phenotype and cytokine production. MAb consisted of R, anti-CD20 with enhanced affinity for CD20 (AME-133), and AME-133 with Fc engineered to have enhanced affinity for both CD20 and CD16 (Fc-eng-133). Peripheral blood mononuclear cells were obtained from normal subjects that were homozygous for high affinity CD16 (VV at position 158), homozygous low affinity (FF at 158), and heterozygotes and were mixed with mAb and Raji lymphoma cells at an effector:target ratio of 1:1. Higher concentrations of mAb were needed to induce CD16 modulation, CD54 upregulation, and IFNg production on NK cells from subjects with the lower affinity CD16 polymorphism. The dose of mAb needed to induce NK activation was lower with Fc-eng-133 irrespective of CD16 polymorphism. Measure of activation - % of NK cells CD54 bright: EC50 ng/ml (mean moAb concentration +/− SEM): N=4 per polymorphism R AME-133 Fc-eng-133 p<0.01 - R vs AME-133; R vs Fc-eng-133; AME-133 vs Fc-eng-133 for each polymorphism VV 6.2 +/− 1.0 3.9 +/− 0.5 1.0 +/− 0.3 VF 11.6 +/− 1.7 5.7 +/− 0.6 1.4 +/− 0.4 FF 29.9 +/− 10.6 11.5 +/− 2.5 2.8 +/− 0.3 p value VV vs FF <0.05 <0.05 <0.05 At saturating mAb concentrations, peak NK activation was greater for Fc engineered AME-133 when compared to R mAb for all samples studied Ratio of Fc-eng-133 to R % NK cells CD54 bright 1.35 +/− 0.07 IFNg production 2.64 +/− 0.43 These data indicate tumor cells coated with mAb with enhanced affinity for CD16 are more effective at activating NK cells at both low and saturating mAb concentrations irrespective of CD16 polymorphism, and provide further evidence for the clinical development of such mAb with the goal of improving clinical response to mAb.

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Alterations of NK Cell Phenotype in the Disease Course of Multiple Myeloma

Cancers ◽

10.3390/cancers13020226 ◽

2021 ◽

Vol 13 (2) ◽

pp. 226

Author(s):

Tatiana Pazina ◽

Alexander W. MacFarlane ◽

Luca Bernabei ◽

Essel Dulaimi ◽

Rebecca Kotcher ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Nk Cell ◽

Therapeutic Targets ◽

Progression Free Survival ◽

Cell Phenotype ◽

Healthy Controls ◽

Activating Receptors ◽

And Function ◽

Patient Subgroups

Accumulating evidence demonstrates important roles for natural killer (NK) cells in controlling multiple myeloma (MM). A prospective flow cytometry-based analysis of NK cells in the blood and bone marrow (BM) of MM patient subgroups was performed (smoldering (SMM), newly diagnosed (ND), relapsed/refractory, (RR) and post-stem cell transplantation (pSCT)). Assessments included the biomarker expression and function of NK cells, correlations between the expression of receptors on NK cells with their ligands on myeloma cells, and comparisons between MM patient subgroups and healthy controls. The most striking differences from healthy controls were found in RR and pSCT patients, in which NK cells were less mature and expressed reduced levels of the activating receptors DNAM-1, NKG2D, and CD16. These differences were more pronounced in the BM than in blood, including upregulation of the therapeutic targets TIM3, TIGIT, ICOS, and GITR. Their expression suggests NK cells became exhausted upon chronic encounters with the tumor. A high expression of SLAMF7 on blood NK cells correlated with shorter progression-free survival. This correlation was particularly evident in ND patients, including on mature CD56dim NK cells in the BM. Thus, our NK cell analysis identified possible therapeutic targets in MM and a biomarker with prognostic potential for disease progression.

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710 Differences in the susceptibility of human small cell lung cancer variants to NK cell-mediated lysis can be overcome with the addition of N803 (IL-15 superagonist)

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.710 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A739-A739

Author(s):

Kristen Fousek ◽

Lucas Horn ◽

Haiyan Qin ◽

Bobby Reddy ◽

Lennie Sender ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Nk Cells ◽

Cell Lung Cancer ◽

Nk Cell ◽

Xenograft Model ◽

Immune Checkpoint Blockade ◽

Small Cell ◽

Cell Phenotype ◽

Small Cell Lung

BackgroundSmall cell lung cancer (SCLC) is a highly aggressive tumor with a 5-year survival rate of less than 5%. Traditionally characterized as a neuroendocrine (NE) cancer, several subtypes have now been identified which vary in their phenotypic and transcriptional profiles. Classical NE tumors are molecularly defined as ASCL1+ or NEUROD1+ and exhibit an epithelial phenotype, expressing cytokeratin and E-cadherin (E-Cad). In contrast, non-classical variants express POU2F3 or YAP1 and are enriched in mesenchymal features, such as high levels of vimentin (Vim). Prior studies describe that non-NE variants of SCLC are less susceptible to chemotherapy and may arise via therapeutic selection. With the addition of immune checkpoint blockade to first-line chemotherapy for the treatment of advanced SCLC, understanding whether SCLC variants respond differently to immunotherapy is crucial.MethodsWe utilized a range of pre-clinical models to investigate whether molecular and phenotypic variants of SCLC differ in their susceptibility to immune-mediated lysis. Following extensive characterization at the RNA and protein levels for expression of ASCL1, NEUROD1, POU2F3, YAP1, epithelial E-Cad, mesenchymal Vim, and other markers of cell phenotype, a panel of cells including each variant subtype were selected for further study.ResultsUpon exposure to healthy donor effector NK cells, the more epithelial cells were highly susceptible to NK-mediated cytotoxicity while all mesenchymal SCLC cells remained highly refractory to NK-mediated lysis. This prompted us to investigate immunotherapy approaches such as the addition of N803, a mutant IL-15 superagonist, to improve the activation and proliferation of NK cells. In a xenograft model utilizing the mesenchymal YAP1+ H841 cell line subcutaneously implanted into nude mice devoid of all immune cells except for NK cells, we observed that the weekly administration of N803 resulted in a significant increase in the number of activated NK cells within the spleens of treated mice. Additionally, NK cells from treated mice produced significantly higher levels of IFN-gamma and granzyme B, resulting in a significant decrease in overall tumor burden.ConclusionsOur data indicates that N803-activated NK cells effectively mediate lysis of SCLC across all variant types, including those previously completely refractory to traditional NK cell lysis. These results highlight the potential of N803 as a novel immune-based intervention for the treatment of all variants of SCLC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

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Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma

10.1101/792846 ◽

2019 ◽

Author(s):

Helen J. Close ◽

Lucy F. Stead ◽

Jérémie Nsengimana ◽

Katrina A. Reilly ◽

Alastair Droop ◽

...

Keyword(s):

Nk Cells ◽

Expression Profiling ◽

Neural Progenitor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Single Cells ◽

Tumour Microenvironment ◽

Cell Phenotype ◽

Tumour Immunity ◽

Gene And Protein Expression

AbstractGlioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To better understand this problem we used a combination of NK cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of TGF-β mediated inhibition. This NK cell inhibition is accompanied by expression of mutiple immune checkpoint molecules on T cells. Single cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognised by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more likely benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

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Expansion, persistence and efficacy of donor memory-like NK cells for the treatment of post-transplant relapse

10.1101/2021.08.24.21262547 ◽

2021 ◽

Author(s):

Roman M Shapiro ◽

Grace Birch ◽

Juliana Vergara ◽

Guangan Hu ◽

Sarah Nikiforow ◽

...

Keyword(s):

Nk Cells ◽

Hematopoietic Cell Transplantation ◽

Nk Cell ◽

Phase 1 ◽

Rapid Expansion ◽

Cell Phenotype ◽

In Vivo Evaluation ◽

Post Transplant ◽

Cell Based Therapy

Background. Responses to conventional donor lymphocyte infusion (DLI) for post-allogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse. Methods. We initiated this ongoing phase 1 trial of adoptively transferred cytokine induced memory-like (CIML) NK cells in patients with myeloid malignancies relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5-10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High resolution profiling with mass cytometry and single cell RNA sequencing characterized the expanding and persistent NK cells subpopulations in a longitudinal manner after infusion. In vitro functional studies of infused CIML NK cells were complemented with in vivo evaluation of NK trafficking to disease sites using multiparameter immunofluorescence. Results. In the first 5 patients on the trial, infusion of CIML NK cells led to a rapid 10 to 50-fold in vivo expansion that was sustained over months. The infusion was well-tolerated, with fever and pancytopenia as the most common adverse events. Responses were attained in 4 of 5 patients at day +28. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires. AML relapse after initial response to CIML NK cell therapy was associated with low transcript expression of CD2 ligands, including CD48/SLAMF2 and CD58/LFA3. Conclusion. Given their rapid expansion and long-term persistence, CIML NK cells serve as a promising platform for the treatment of post-transplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T-cells and tumor targets will lead to the development of novel cell-based immunotherapies.

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