Enriched Natural Killer Cells Research Articles

P078 Inflammatory transcriptomic signatures and cell type compositions in colonic mucosa of ulcerative colitis

Abstract Background Previous studies have explored transcriptomic profiles associated with active inflammation in patients with IBD patients, but consistent characteristics of gene expression and enriched pathways, particularly in Asian patients with UC, remain unconfirmed. Here, we aimed to assess distinctive transcriptomic changes associated with persistent active mucosal inflammation in Asian patients with UC despite of medical treatment. Methods We obtained colonic mucosa biopsies of inflamed and noninflamed tissue from 15 patients with UC and 15 healthy controls and performed RNA sequencing analysis. Utilizing publicly available single cell RNA sequencing (scRNAseq) data, we also identified cell types expressing differentially expressed genes from RNAseq data and performed data deconvolution using CIBERSORTx. Results Transcriptomics profiling revealed that the inflammatory transcriptomic signature was highly enriched in the colonic mucosa of UC-active compared to UC-inactive and controls. By incorporating scRNAseq data, the genes upregulated in UC-active were highly expressed in M cells, inflammatory monocytes, and inflammatory fibroblasts, while downregulated genes were prominent in BEST4+ enterocytes and WNT5B+ fibroblasts. Notably, a sub-cluster of enterocytes associated with SAA1, SLC6A14, and DUOXA2 genes exhibited high expression in UC-active. Deconvolution analysis using CIBERSORTx identified significant enrichment of natural killer cells, inflammatory monocytes, Tuft cells, inflammatory fibroblast, WNT2B+Fos-lo1, and pericytes in UC-active. Conclusion Our RNAseq analysis not only identified significant gene expression changes but also revealed shifts in cell type proportions associated with persistent inflammation. These findings offer valuable insights for developing novel treatment strategies for UC.

Exploration of the prognostic effect of costimulatory genes in bladder cancer.

A prognostic model of bladder cancer was constructed based on costimulatory molecules, and its stability and accuracy were verified in different datasets. The expression profile of bladder cancer RNA and the corresponding clinical data in The Cancer Genome Atlas (TCGA) database were analyzed employing computational biology, and a prognostic model was constructed for costimulating molecule-related genes. The model was applied in GSE160693, GSE176307, Xiangya_Cohort, GSE13507, GSE19423, GSE31684, GSE32894, GSE48075, GSE69795 and GSE70691 in TCGA dataset and Gene Expression Omnibus database. The role of costimulating molecules in bladder cancer tumor subtypes was also explored. By consistent cluster analysis, bladder cancer in the TCGA dataset was categorized into two subtypes: C1 and C2. The C1 subtype exhibited a poor prognosis, high levels of immune cell infiltration and significant enrichment of natural killer cells, T cells and dendritic cells in the C1 subtype. In addition, the ImmuneScore calculated by the ESTIMATE algorithm differed greatly between the two subtypes, and the ImmuneScore of the C1 subtype was greater than the C2 subtype in a significant manner. This study also assessed the relationship between costimulating molecules and immunotherapy response. The high-risk group responded poorly to immunotherapy, with significant differences in the amount of most immune cells between the two groups. Further, three indices of the ESTIMATE algorithm and 22 immune cells of the CIBERSORT algorithm were significantly correlated with risk values. These findings suggest the potential value of costimulating molecules in predicting immunotherapy response. A costimulatory molecule-based prognostic model for bladder cancer was established and validated across multiple datasets. This model introduces a novel mode for tailoring treatments to each individual with bladder cancer, and offers valuable insights for informed clinical choices. Simultaneously, this research also delved into the significance of costimulating molecules within distinct bladder cancer subtypes, shedding novel insights into improving immunotherapy strategies for the treatment of bladder cancer.

Multiomic Characterization Reveals a Distinct Molecular Landscape in Young-Onset Pancreatic Cancer.

Using a real-world database with matched genomic-transcriptomic molecular data, we sought to characterize the distinct molecular correlates underlying clinical differences between patients with young-onset pancreatic cancer (YOPC; younger than 50 years) and patients with average-onset pancreatic cancer (AOPC; 70 years and older). We analyzed matched whole-transcriptome and DNA sequencing data from 2,430 patient samples (YOPC, n = 292; AOPC, n = 2,138) from the Caris Life Sciences database (Phoenix, AZ). Immune deconvolution was performed using the quanTIseq pipeline. Overall survival (OS) data were obtained from insurance claims (n = 4,928); Kaplan-Meier estimates were calculated for age- and molecularly defined cohorts. Significance was determined as FDR-corrected P values (Q) < .05. Patients with YOPC had higher proportions of mismatch repair-deficient/microsatellite instability-high, BRCA2-mutant, and PALB2-mutant tumors compared with patients with AOPC, but fewer SMAD4-, RNF43-, CDKN2A-, and SF3B1-mutant tumors. Notably, patients with YOPC demonstrated significantly lower incidence of KRAS mutations compared with patients with AOPC (81.3% v 90.9%; Q = .004). In the KRAS wild-type subset (n = 227), YOPC tumors demonstrated fewer TP53 mutations and were more likely driven by NRG1 and MET fusions, whereas BRAF fusions were exclusively observed in patients with AOPC. Immune deconvolution revealed significant enrichment of natural killer cells, CD8+ T cells, monocytes, and M2 macrophages in patients with YOPC relative to patients with AOPC, which corresponded with lower rates of HLA-DPA1 homozygosity. There was an association with improved OS in patients with YOPC compared with patients with AOPC with KRAS wild-type tumors (median, 16.2 [YOPC-KRASWT] v 10.6 [AOPC-KRASWT] months; P = .008) but not KRAS-mutant tumors (P = .084). In this large, real-world multiomic characterization of age-stratified molecular differences in pancreatic ductal adenocarcinoma, YOPC is associated with a distinct molecular landscape that has prognostic and therapeutic implications.

Single-cell profiling reveals the heterogeneity of NK cells during anti-PD-1 therapy in non-small-cell lung cancer

BackgroundThe efficacy of immune checkpoint inhibitors remains limited in non-small cell lung cancer (NSCLC). Natural killer (NK) cells serve as the key element of innate immunity and play an important role in anti-tumor immunity, the impact of NK cells on efficacy of anti-PD-1 therapy in NSCLC is worth exploring. MethodsWe analyzed single-cell transcriptome data derived from biopsies of NSCLC patients receiving anti-PD-1 treatment. Immune cell subtypes were identified and further cell–cell communication were analyzed and verified. ResultsWe observed totally 6 distinct NK cells clusters in NSCLC infiltrating immune cells. It’s worth noting that enrichment of immature NK cells was found in responsive group. A series of marker genes of immature NK cells were associated with anti-PD-1 response and related to immune regulation processes such as antigen processing, Th1, Th17 cells activation. Moreover, effector CD8+ T cells were significantly enriched in responsive group and showed similar trajectories with immature NK cells. Cell-cell communication analysis showed that immature NK cells showed strong interactions with Th17 cells and effector CD8+ T cells. Furthermore, when validating the expression of immature NK cells marker genes, we found that CXCR4 was associated with enriched infiltration of CD8+ T cells. ConclusionsIn conclusion, immature NK cells may facilitate the efficacy of anti-PD-1 therapy by interacting with Th1 cells, Th17 cells and enhancing infiltration of effector CD8+ T cells. Our data suggested that NK cells could be a promising target to improve the prognosis of NSCLC patients.

Abstract 364: Multi-omics approaches revealed the involvement of immune cells in skeletal muscles of lung cancer cachexia mouse model

Abstract Cancer-associated cachexia (CAC) is a multifactorial syndrome characterized by loss of body weight and skeletal muscle mass that cannot be fully recovered by nutritional support. It accounts for approximately 20 % of cancer-related deaths. However, there are no approved treatments for CAC yet. In order to better understand CAC, we wanted to apply the multi-omics approaches to simultaneously assess tumors and skeletal muscles in CAC mouse models. First, we established a lung cancer cachexia mouse model by orthotopic transplantation of KRAS-mutant lung cancer cells. Lung tumor-bearing mice developed cachexia at 7 weeks post injection, showing over 20 % of body weight loss and wasting of skeletal muscles and fats. The histological and immunoblotting analyses revealed degradation of gastrocnemius (GA) and tibialis anterior (TA) muscles in CAC group. Then, we performed the bulk RNA sequencing to assess transcriptomic changes in lung tumors and GA muscles of CAC mice. In lung tumors, genes associated to ATP metabolic process was upregulated. However, skeletal muscles of CAC mice showed upregulation of genes involved in protein degradation and immune responses, while genes related to energy metabolism were downregulated. Notably, the immunohistochemical staining revealed the increased numbers of infiltrating macrophages and CD8+ T cells in skeletal muscles of CAC mice. These data suggest the involvement of immune cells in lung cancer-associated cachexia. To further identify the distinct immune cell populations involved in CAC, the single cell RNA sequencing was conducted using CD45+ immune cells infiltrated into GA muscles of CAC mice. The enrichment of natural killer cells, dendritic cells and T cells was detected in GA of CAC mice. Moreover, we confirmed the roles of the infiltrating immune cells by using the neutralizing antibodies in CAC mice. Taken together, these data suggest the involvement of the immune cells in the skeletal muscle wasting of CAC mice. These multi-omics approaches will shed light on development of therapeutics for cancer-induced cachexia. Citation Format: Se-Young Park, Bomi Kim, Myunggyo Lee, Haeseung Lee, Na-Young Song. Multi-omics approaches revealed the involvement of immune cells in skeletal muscles of lung cancer cachexia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 364.

Small Interfering RNA Delivery Into Primary Human Natural Killer Cells for Functional Gene Analyses

Studying gene functions in human natural killer (NK) cells is key for advancing the understanding of NK cell biology and holds promise to pave the way for improving NK cell therapies against cancer. However, NK cells are challenging to manipulate, and investigation of gene functions in NK cells is hampered by variable delivery efficiencies and impaired viability upon electroporation, lipofection, or viral transduction. Here, we report a simple workflow for delivery of commercially available small interfering RNA molecules into primary human NK cells to enable functional gene analyses. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enrichment of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Preparation of small interfering RNA Basic Protocol 3: Delivery of small interfering RNA into natural killer cells Support Protocol 1: Isolation of human peripheral blood mononuclear cells from buffy coats Support Protocol 2: Thawing and recovery of cryopreserved peripheral blood mononuclear cells Support Protocol 3: Evaluation of natural killer cell purity following magnetic enrichment Support Protocol 4: Exemplary assessment of knockdown efficiency.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

Identification of nafamostat mesylate as a selective stimulator of NK cell IFN-γ production via metabolism-related compound library screening

Natural killer (NK) cells play important roles in controlling virus-infected and malignant cells. The identification of new molecules that can activate NK cells may effectively improve the antiviral and antitumour activities of these cells. In this study, by using a commercially available metabolism-related compound library, we initially screened the capacity of compounds to activate NK cells by determining the ratio of interferon-gamma (IFN-γ)+ NK cells by flow cytometry after the incubation of peripheral blood mononuclear cells (PBMCs) with IL-12 or IL-15 for 18 h. Our data showed that eight compounds (nafamostat mesylate (NM), loganin, fluvastatin sodium, atorvastatin calcium, lovastatin, simvastatin, rosuvastatin calcium, and pitavastatin calcium) and three compounds (NM, elesclomol, and simvastatin) increased the proportions of NK cells and CD3+ T cells that expressed IFN-γ among PBMCs cultured with IL-12 and IL-15, respectively. When incubated with enriched NK cells (purity ≥ 80.0%), only NM enhanced NK cell IFN-γ production in the presence of IL-12 or IL-15. When incubated with purified NK cells (purity ≥ 99.0%), NM promoted NK cell IFN-γ secretion in the presence or absence of IL-18. However, NM showed no effect on NK cell cytotoxicity. Collectively, our study identifies NM as a selective stimulator of IFN-γ production by NK cells, providing a new strategy for the prevention and treatment of infection or cancer in select populations.

Multidimensional profiling depicts infiltrating immune cell heterogeneity in the tumor microenvironment of stage IA non-small cell lung cancer.

BackgroundEmerging evidence has underscored infiltrating immune cells in the tumor microenvironment (TME) of non‐small cell lung cancer (NSCLC). Owing to screening programs, the prevalence of early‐stage NSCLC is growing, but its high recurrence risk and poor survival impose an increasing demand for further understanding the TME.MethodsTissue and plasma samples from 33 resectable stage IA NSCLC patients were collected from the surgery and subject to histological and genomic analyses. The distribution of CD8+ T cells, tumor‐associated macrophages (TAMs, M1 polarization and M2 polarization), and natural killer (NK) cells (CD56dim and CD56bright) was analyzed. The impact of clinical characteristics and immunotherapy‐related biomarkers on immune cell infiltration were also investigated.ResultsUsing multiplex immunohistochemistry (mIHC), we found a significantly higher M1 polarization proportion of total TAMs in tumor parenchyma than in other tissues, while other immune cells remained stable. Patients under 50 showed higher infiltrating CD8+ T cell density and M1 ratio in tumor tissues. Tumors carrying RAS‐MAPK mutations were associated with significantly increased infiltration of CD8+ T cells. We also identified significantly higher infiltration of CD8+ T cells and enrichment of CD56bright NK cells in high tumor mutation burden (TMB) and high programmed cell death ligand 1 (PD‐L1) samples, respectively.ConclusionsOur study highlighted the heterogeneity and dynamics of infiltrating immune cells in stage IA NSCLC TME, featured by M1 TAM enrichment in tumor parenchyma. Age, driver mutation type, TMB, and PD‐L1 level were found to associate with immune cell infiltration in the TME, shedding light on immunotherapy development.

A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

IntroductionAs the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells.MethodsNK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use.ResultsOverall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016).ConclusionsOur data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. [Display omitted] DisclosuresLee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

Manufacturing A Donor Lymphocyte Product Depleted Of TCR-αβ T Cells And CD19+ B Cells For Prophylactic Infusion Following Allogeneic Stem Cell Transplantation

Background & Aim Background In allogeneic stem cell transplantation (HSCT), relapse of hematologic malignancies is a leading cause of post-transplant morbidity. In the early post-transplant period, when immune reconstitution is pending, residual leukemic clones can proliferate, resulting in disease relapse with bleak prognosis and limited treatment options. Donor lymphocyte infusions (DLI) are often used as treatment of post-transplant leukemia relapse but this practice is limited by graft versus host disease (GVHD) as a complication. Adoptive transfer of selected lymphocyte subsets that effect tumor cytotoxicity without inducing GVHD, such as natural killer (NK) cells and TCRγδ T cells, may provide early donor-derived surveillance for residual or early relapse disease after HSCT. With this background, a Phase I clinical trial (CASE1Z19) was designed to explore the prophylactic administration of donor lymphocytes depleted of TCR-αβ T cells and CD19+ B cells early in the post-transplant course. A maximum threshold of Methods, Results & Conclusion Methods Three qualification runs were conducted using the Miltenyi CliniMACS Plus system to deplete TCR-αβ T and CD19+ B cells from non-mobilized, peripheral blood mononuclear cells (PBMC) in leukapheresis products sourced by a commercial vendor. Each product was analyzed for lymphocyte subset composition and stability post-processing. Results The mean starting total white blood cell count was 6.4 × 109 cells from which TCR-αβ T cells and CD19+ B cells were maximally depleted by >99.8% with mean Log10 reductions of 3.10 and 3.06 respectively (Table1). The TCR-αβ T cell content in products generated from each run were 0.2-0.5 × 105/kg, based on an average 60kg adult. The yield of NK cells and TCRγδ T cells from each manufacturing ranged from 50-97% and 86-100% respectively. All products were stable post-processing, at room temperature for up to 6 hours, with a mean cell viability of 95.1%. Conclusion A donor lymphocyte product sufficiently purified of TCRαβ T and CD19+ B cells with subsequent enrichment of NK cells and TCR-γδ T cells can consistently be generated for prophylactic adoptive transfer in the post-transplant setting. Additional analysis characterizing NK and TCR-γδ T cell subsets for function and activation is underway.

Unique natural killer cell subpopulations are associated with exacerbation risk in chronic obstructive pulmonary disease

Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death worldwide. COPD is frequently punctuated by acute exacerbations that are precipitated primarily by infections, which increase both morbidity and mortality and inflates healthcare costs. Despite the significance of exacerbations, little understanding of immune function in COPD exacerbations exists. Natural killer (NK) cells are important effectors of innate and adaptive immune responses to pathogens and NK cell function is altered in smokers and COPD. Using high-dimensional flow cytometry, we phenotyped peripheral blood NK cells from never smokers, smokers, and COPD patients and employed a non-supervised clustering algorithm to define and detect changes in NK cell populations. We identified greater than 1,000 unique NK cell subpopulations across patient groups and describe 13 altered NK populations in patients who experienced prior exacerbations. Based upon cluster sizes and associated fluorescence data, we generated a logistic regression model to predict patients with a history of exacerbations with high sensitivity and specificity. Moreover, highly enriched NK cell subpopulations implicated in the regression model exhibited enhanced effector functions as defined by in vitro cytotoxicity assays. These novel data reflect the effects of smoking and disease on peripheral blood NK cell phenotypes, provide insight into the potential immune pathophysiology of COPD exacerbations, and indicate that NK cell phenotyping may be a useful and biologically relevant marker to predict COPD exacerbations.

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

KIR Genotype and KIR Ligand Phenotypes That Improve the in Vitro Cord Blood or Autologous NK Cell Mediated Cytotoxicity Against Myeloma Marrow Plasma Cells

<h3>Instruction</h3> Killer Immunoglobulin-like Receptor (KIR) inhibition is known to influence the cytotoxic effects of natural killer (NK) cells against cancer cells. KIRs can be protective against relapse in myeloma (MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011) by NK cell licencing <i>or through</i> blocking of the inhibitory KIR receptors such as NKG2A (Mahaweni et al, 2018). <h3>Objectives</h3> In this study, the role of KIR geno/phenotypes and their ligands on autologous and cord blood (CB) derived NK cell mediated cytotoxicity against MM plasma cells (PC) were investigated. <h3>Methods</h3> Peripheral blood NK cells and marrow PCs were isolated from 20 patients with relapsed MM. PC were isolated from fresh BM aspirates using EasySep Human CD138 Positive Selection Kit (StemCell Technologies) and were subjected to cytotoxicity assays with either autologous or CB derived NK cells isolated (Human NK cell Enrichment Cocktail, Stemcell Technologies) and cultured in the presence of IL-15 (10ng/ml) for 2-8 days. KIR/KIR ligand genotyping were performed by Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) in order to detect KIR genotypes; KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A/B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, and KIR ligands; HLA-ABw4+, HLA-BBw4+Thr80, HLA-BBw4+Ile80, HLA-BBw4+Asp77,Thr80, HLA-CAsn80, and HLA-CLys80. Effector cells were stained with antibodies against NKG2A (CD159a) and CD158i (KIR2DS4) and analyzed using a Beckman Coulter Flow Cytometer. <i>Cytotoxicity assays</i>: Using <i>3,3</i> (DiO) as an indicator and 1:10 target:effector cell ratio PC death was evaluated after 120 mins of incubation, using flow cytometry. <h3>Results</h3> 20 MM patients were tested in vitro. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.0% (11.4%-51.2%). While autologous NK mediated cytotoxicity was median 17.3% (11.4%-43.6%), CB-NK cell mediated cytotoxicity was 23.2% (range: 11.5%-51.2%). Highest cytotoxicity was achieved with CB-NK cells (CB7) with a KIR AB haplotype, carrying 8 inhibitory KIR genes against PC from MM12. Effector cells with the iKIR gene number above 6 were found to cause higher cytotoxicity (13/19; p=0.031). Additionally, higher (>30%) expression of NKG2A on CB-NK cells were found to be associated with lack of cytotoxicity. <h3>Conclusion</h3> CB-derived NK cells were found to be able to induce higher PC death than autologous PB-NK cells. Also higher number of iKIR genotypes and lacking KIR2DS4 receptor was found to be associated with higher cytotoxicity. In our current study we are reporting KIR NKG2A which binds with HLA-E to play an additional role. Allogeneic NK cell mediated immunotherapies are promising due their clinical advantages ie no need for HLA matching. Furthermore certain KIR haplotypes (ie KIR2DS4⁺ or NKG2A⁻) may offer better therapeutic efficacy if confirmed by others as well.

Rapid isolation and enrichment of mouse NK cells for experimental purposes.

Natural killer (NK) cells have shown to play a critical, but as yet poorly defined, role in the process by which the immune system controls tumor progression. Indeed, NK cell-based immunotherapy, particularly NK cell adoptive transfer therapy, has become a very attractive cancer weapon against multiple types of cancers such as metastatic and hematological cancers. Unfortunately, the implementation of these therapies has been challenged by the existence of immunosuppression mechanisms that have prevented NK cell functionality. Additionally, the development of protocols to obtain purified and functional NK cells has faced some difficulties due to the limitations in the numbers of cells that can be obtained and the development of an exhaustion phenotype with impaired proliferative and functional capabilities during lengthy ex vivo NK cell expansion protocols. Thus, the development of new strategies to obtain a rapid expansion of highly functional NK cells without the appearance of exhaustion is still much needed. This is particularly true in the case of mouse NK cells, a surrogate commonly used to evaluate NK cell biology and human NK cell-based immunotherapeutic alternatives. Here, we describe a feasible and rapid protocol to produce strongly activated mouse NK cells in vivo taking advantage of the hydrodynamic delivery of a plasmid that contains interleukin-15, a cytokine known to cause NK cell expansion and activation, fused with the binding domain of the IL-15Rα ("sushi" domain) and apolipoprotein A-I.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.

Sex Hormone Contributes to Sexually Dimorphic Susceptibility in CVB3-Induced Viral Myocarditis via Modulating IFN-γ+ NK Cell Production

BackgroundViral myocarditis is a widespread cardiac disease associated with inflammation and myocardial injury and is predominantly caused by coxsackievirus B3 (CVB3) infection in humans as well as in mice. CVB3-induced myocarditis shows sexually dimorphic sensitivity and is more prevalent in male mice. Our previous studies showed that natural killer (NK) cells played an indispensable role in CVB3-induced myocarditis, and female mice exhibited less pathological cardiac interferon gamma (IFN-γ)+ NK cell infiltration than did male mice. However, the precise mechanisms were not well elucidated. MethodsWe investigated the influence of estrogen on cardiac IFN-γ+ NK cell enrichment in CVB3-induced myocarditis and explored the underlying molecular mechanism. ResultsIn this study, we found that CVB3 stimulation could clearly induce IFN-γ expression by NK cells; however, this trend could be blunted by estrogen treatment. Consistently, ovariectomized female mice with decreased estrogen levels exhibited substantially increased enrichment of cardiac IFN-γ+ NK cells and displayed significantly aggravated myocarditis. Similarly, estrogen-treated male mice showed less cardiac IFN-γ+ NK cell infiltration, accompanied by significantly alleviated viral myocarditis. In sharp contrast, sexually immature female and male mice (with similar estrogen levels) showed comparable levels of cardiac IFN-γ+ NK cell infiltration and similar levels of myocarditis severity. Upon further exploration of the underlying mechanisms, we found that estrogen could downregulate expression of Th1-specific T box transcription factor (T-bet), the key transcription factor associated with IFN-γ production, in CVB3-stimulated NK cells. ConclusionsOverall, this study might help us understand the mechanism of increased cardiac infiltration by IFN-γ+ NK cells in CVB3-infected male mice compared with that in female mice and might provide new clues for the sex bias in CVB3-induced myocarditis.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Continuous IL-15 Signaling Leads to Functional Exhaustion of Human Natural Killer Cells through Metabolic Changes That Alters Their In Vivo Anti-Tumor Activity

Natural Killer (NK) cells represent an exciting immunotherapeutic approach to treat cancer. We have shown that in vivo expansion and activation of donor NK cells supported by administration of IL-2 induces remission in patients with refractory AML. Recent clinical studies by our group have shown that IL-15 is superior to IL-2 to support NK cell persistence 14 days after adoptive transfer. However, only 36% of patients treated with 12 consecutive days of IL-15 had NK cell expansion to the level of ≥100 donor derived NK cells/µL blood compared to 10% in subjects treated with IL-2 (p=0.02). This leads us to conclude that we might not know the optimal route and interval to administer in vivo IL-15. We hypothesized that daily uninterrupted IL-15 dosing could lead to exhaustion or NK cellular stress. Therefore we designed an in vitro model system in which enriched NK cells are treated with three 3-day cycles of continuous IL-15 (IL-15cont) or were rested with a "gap" (skipping the middle cycle [IL-15gap]) before returning to the last cycle of IL-15. IL-15cont treatment yielded more proliferation and higher cell numbers compared to IL-15gap (4.8±0.44 vs. 1.9±0.26 million cells/ml, p < 0.0001) when cells were analyzed at the end of the three cycles (on day 9, where all in vitro measurements were taken). However, NK cell death, measured by flow cytometry, in the IL-15cont group was higher (18.9±2.2 vs 14.9±1.7 % cell death, p = 0.035) and this group also had an enrichment in genes involved in cell cycle checkpoint/ arrest, perhaps indicating more cellular stress in the IL-15cont. In an in vitro flow cytometric functional assay, the IL-15cont group had decreased activation when compared to the IL-15 gap group against K562 targets (43.6±2.1 vs 55.6±2.7 % CD107a [degranulation], p < 0.0001; 1.9±0.41 vs 7.1±0.93 % IFNg [inflammatory cytokine production], p = 0.0055). The decrease in NK cell activation correlated with a strong decrease in tumor target killing in an in vitro chromium release assay (Figure 1A) measuring killing of acute promyelocytic leukemia (HL-60) cell targets, in which the IL-15cont NK cells were potently outperformed by the IL-15gap cells (6.4±2.6 vs 51.5±4.8 % killing at 2.5:1 effector:target ratio, p < 0.0001). We used an in vivo xenogeneic model of AML, where conditioned NSG (NOD scid gamma) mice are engrafted with HL-60luc tumor targets 3 days prior to infusion with nothing, IL-15cont or IL-15gap human NK cells prepared within our 9 day culture system. Only the IL-15gap NK group mediated statistically significant tumor control when compared to tumor alone at two weeks following NK cell infusion (Figure 1B). To probe deeper into the functional defect we evaluated signaling after these treatments and noted decreased phosphorylation of several proteins in the IL-15cont group. These data led us to explore proteins involved in metabolism and we noted that CPT1A, a critical enzyme involved in fatty acid oxidation (FAO), was strongly increased in the IL-15gap treated NK cells (protein MFI of 15,759±2,603 [IL-15gap] vs 5,273±744 [IL-15cont], p = 0.009). Metabolic analysis using a Seahorse XFe24 analyzer showed an increased mitochondrial spare respiratory capacity (SRC) in the IL-15gap group, denoting better capability of the IL-15gap NK cells to respond to energetic demands (Figure 1C). In a separate experiment the groups were treated with etomoxir to inhibit CPT1A, and the SRC phenotype was reversed, with the IL-15gap group containing lower SRC than the IL-15cont group. To test these findings in a functional assay we repeated the IL-15cont treatment in combination with rapamycin, which can induce CPT1A through inhibition of mTORC1, and saw restoration of function to levels similar to IL-15gap (40.8±2.0 vs 49.3±2.9 % CD107a in the IL-15cont vs IL-15cont + rapamycin, p = 0.005; 2.4±0.47 vs 4.8±1.0 % IFNg in the IL-15cont vs IL-15cont + rapamycin, p = 0.03). These data indicate that NK cell functional exhaustion via continuous IL-15 signaling is mediated by a decrease in FAO. Intermittent IL-15 dosing or altering metabolism through other mechanisms may overcome this competition. These findings could impact ongoing clinical trials through simple alterations in dosing strategies in order to minimize NK cell exhaustion in the immunotherapeutic setting. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy, Other: SAB; Fate Therapeutics: Consultancy, Research Funding.

“Adherent” versus Other Isolation Strategies for Expanding Purified, Potent, and Activated Human NK Cells for Cancer Immunotherapy

Natural killer (NK) cells have long been hypothesized to play a central role in the development of new immunotherapies to combat a variety of cancers due to their intrinsic ability to lyse tumor cells. For the past several decades, various isolation and expansion methods have been developed to harness the full antitumor potential of NK cells. These protocols have varied greatly between laboratories and several have been optimized for large-scale clinical use despite associated complexity and high cost. Here, we present a simple method of “adherent” enrichment and expansion of NK cells, developed using both healthy donors' and cancer patients' peripheral blood mononuclear cells (PBMCs), and compare its effectiveness with various published protocols to highlight the pros and cons of their use in adoptive cell therapy. By building upon the concepts and data presented, future research can be adapted to provide simple, cost-effective, reproducible, and translatable procedures for personalized treatment with NK cells.