Peripheral Blood Natural Killer Cells Research Articles

Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature.

Human CMV (HCMV) is a ubiquitous pathogen that indelibly shapes the NK cell repertoire. Using transcriptomic, epigenomic, and proteomic approaches to evaluate peripheral blood NK cells from healthy human volunteers, we find that prior HCMV infection promotes NK cells with a T cell-like gene profile, including the canonical markers CD3ε, CD5, and CD8β, as well as the T cell lineage-commitment transcription factor Bcl11b. Although Bcl11b expression is upregulated during NK maturation from CD56bright to CD56dim, we find a Bcl11b-mediated signature at the protein level for FcεRIγ, PLZF, IL-2Rβ, CD3γ, CD3δ, and CD3ε in later-stage, HCMV-induced NK cells. BCL11B is targeted by Notch signaling in T cell development, and culture of NK cells with Notch ligand increases cytoplasmic CD3ε expression. The Bcl11b-mediated gain of CD3ε, physically associated with CD16 signaling molecules Lck and CD247 in NK cells is correlated with increased Ab-dependent effector function, including against HCMV-infected cells, identifying a potential mechanism for their prevalence in HCMV-infected individuals and their prospective clinical use in Ab-based therapies.

Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

CD38 Down-Regulation on Ex Vivo Activated and Expanded NK Cells for Cell Therapy Persists after Infusion

Introduction: Immunotherapies are gaining more and more importance in the treatment of multiple myeloma (MM). Antibodies directed against MM antigens like CD38, SLAMF7 or BCMA are used either in their natural form, conjugated to drugs, or in the form of bispecific T-cell engagers. Cellular therapies make use of cytotoxic lymphocytes, i.e. T cells or NK cells that can also be modified to express chimeric antigen receptors to target MM cells. Combinations of antibody and cellular therapies could further improve the outcome as, for example, NK cells can mediate antibody dependent cellular cytotoxicity (ADCC). However, NK cells also express CD38 and SLAMF7 and would be targeted by the therapeutic antibodies against these antigens. We have recently reported our clinical study infusing multiple doses of ex vivo activated and expanded autologous NK cells in six patients with MM post autologous stem-cell transplantation (EudraCT 2010-022330-83). Here, we report results of a phenotypic analysis of the ex vivo expanded NK cells and peripheral blood NK cells before and after infusion with implications for possible combination therapies.Methods: Ex vivo activated and expanded NK cells and NK cells in peripheral blood of the patients were analyzed by multiparameter flow cytometry. Peripheral blood cells were taken from the non-NK cell infusion arm before and at three different timepoints after infusion. NK-cell sub-populations within these samples were analyzed using t-SNE clustering.Results: Upon ex vivo activation and expansion, we observed that the NK cells gained a unique activated phenotype including populations of CD56 brightCD16 +Ki67 +HLA-DR + NK cells. Interestingly, these NK cells showed a reduced expression of CD38 compared to peripheral blood NK cells. Clustering analyses of data from peripheral blood samples revealed the gradual appearance of a new NK cell population with a similar phenotype in a dose-dependent fashion over four hours following infusion of the NK cell product. Infused NK cells could be detected in circulation up to four weeks after the last infusion. Like the NK cell infusion product, these cells expressed little to none CD38, high levels of NKG2D, 2B4, TIM-3, and TIGIT and similar levels of SLAMF7 compared to peripheral blood NK cells.Conclusions: The persistent high expression of CD16 and the low expression of CD38 in infused NK cells offers the choice to combine ex vivo activated and expanded NK cells with anti-CD38 antibody therapy without concern for antibody-mediated NK-cell death. Based on these findings, we have started a clinical trial testing this combined therapy (NCT04558931). DisclosuresNahi: XNK Therapeutics AB: Consultancy. Chrobook: XNK Therapeutics AB: Consultancy. Meinke: XNK Therapeutics AB: Consultancy, Current holder of stock options in a privately-held company. Gilljam: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Stellan: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Walther-Jallow: XNK Therapeutics: Other: Shareholder in the company. Liwing: XNK Therapeutics AB: Current Employment. Gahrton: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company; Fujimoto Pharmaceutical Corporation Japan: Membership on an entity's Board of Directors or advisory committees. Ljungman: Takeda: Consultancy, Other: Endpoint committee, speaker; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy; Janssen: Other: Investigator. Ljunggren: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Alici: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company.

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.

Engineering CD70-Directed CAR-NK Cells for the Treatment of Hematological and Solid Malignancies

Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen. CD70 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in >70% CAR integration/expression and >80% knockout of CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing.1 Perna et al. 2017, Cancer Cell. 32:506-519.2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772. DisclosuresChoi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.

Safety of Anti-NKG2A Blocking Antibody Monalizumab As Maintenance Therapy after Allogeneic Hematopoietic Stem Cell Transplantation: A Phase I Study

BackgroundFollowing allogeneic hematopoietic stem cell transplantation (Allo-HSCT), the phenotype and function of circulating NK Cells are altered; notably NK cells display an immature (CD56-bright/KIR -/CD57 -/NKG2A +) phenotype and a low cytotoxic activity. Since NK cells have demonstrated anti-leukemic activity in the context of Allo-HCT while less prone to induce GVHD than T cells, we hypothesized that NKG2A inhibition using the checkpoint inhibitor monalizumab (humanized IgG4 blocking monoclonal antibody) could improve NK cell mediated graft-versus-tumor effect, without triggering graft-versus-host disease. We thus performed the first clinical trial evaluating the safety of monalizumab as a prophylactic treatment after Allo-HSCT (NCT02921685).MethodThe objective was to determine the maximal tolerated dose (MTD) of a single infusion of monalizumab administered on day+75 or thereafter following Allo-HSCT. Inclusion criteria were: 1) adult patients who underwent first Allo-HSCT for hematological malignancy; 2) no prior history of GVHD, 3) reduced toxicity conditioning regimen, 4) ATG or PT-Cy based GVHD prophylaxis, and 5) GVHD prophylaxis with cyclosporine A ongoing at the time of monalizumab infusion.Four dose levels were investigated in this dose escalation study (0.04, 0.1, 0.3, and 1.0 mg/kg). Dose limiting toxicities (DLT) were defined by the occurrence of any grade 3 to 4 monalizumab-related adverse event within 28 days after infusion, including grade 3 to 4 acute GVHD and moderate to severe chronic GVHD. In case of DLT, a Bayesian continuous reassessment model (CRM) was used to predict the maximal tolerated dose (MTD) after each series of 3 patients treated at a given dose level. It was planned to treat 18 patients. Beyond clinical safety, blood samples were collected for NK and T cell immunomonitoring and receptor saturation assay.ResultsFifteen patients received monalizumab at a median time of 83 days (range: 75-103) after Allo-HSCT (3 at 0.04 mg/kg, 3 at 0.1 mg/kg, 3 at 0.3 mg/kg, and 6 at 1 mg/kg). Patients were transplanted for hematological diseases (9 AML, 3 MDS, 1 lymphoma, 1 CLL and 1 myelofibrosis), from a matched sibling (n=8), matched unrelated (n=6) or haploidentical donor (n=1).No DLT was observed during the dose escalation process or among the 6 patients treated at the highest dose level, justifying study early termination after 15 patients. Two patients developed transient low grade GVHD that spontaneously resolved without treatment (1 grade I acute GVHD on day+8 and 1 mild chronic GVHD on day+118). Systemic steroid requiring GVHD occurred in 3 patients (20%): 1 severe overlap GVHD (day+42, CR after treatment), 1 severe chronic GVHD (day+80, death from GVHD) and 1 late severe chronic GVHD after cyclosporine tapering for mixed chimerism (day+149, complete remission [CR] after treatment). No disease recurrence was observed, except for one patient with myelofibrosis who relapsed 3 years after monalizumab infusion. At a median follow up of 22 months (3-48) after monalizumab infusion, 13 out of 15 patients are in CR from their underlining malignancy without GVHD and immunosuppressive treatment at last contact.At the dose of 1 mg/kg, receptor saturation assays (RSA) showed persistent binding of monalizumab on peripheral blood NK cells. Indeed, more than 90% of NKG2A-positive NK cells were positive for monalizumab detection (Anti-IgG4-PE) until at least 14 weeks post treatment.ConclusionsMonalizumab can be safely administered after Allo-HSCT, with prolonged persistence on circulating NK cells. In the absence of DLT, MTD was not reached. Future trials will aim at measuring the clinical efficacy of monalizumab in this context, alone or in combination therapy. Disclosuresboyer Chammard: Innate Pharma: Current Employment. Chabannon: Sanofi SA: Other: Travel Support, Research Funding, Speakers Bureau; Bellicum Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel Support, Speakers Bureau; Novartis: Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Terumo BCT: Speakers Bureau; Miltenyi Biotech: Research Funding; Fresenius Kabi: Research Funding; EBMT: Membership on an entity's Board of Directors or advisory committees. Vey: Amgen: Honoraria; BMS: Honoraria; BIOKINESIS: Consultancy, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; SERVIER: Consultancy; JAZZ PHARMACEUTICALS: Honoraria; JANSSEN: Consultancy. Olive: ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Blaise: Jazz Pharmaceuticals: Honoraria.

Immune Profiling of Multiple Myeloma Patients Treated with Daratumumab Monotherapy: Acquired and Primary Daratumumab-Resistance Is Associated with NK Cell Exhaustion

Immune Profiling of Multiple Myeloma Patients Treated with Daratumumab Monotherapy: Acquired and Primary Daratumumab-Resistance Is Associated with NK Cell Exhaustion

Study of Bone Marrow Mesenchymal Stem Cells Regulating NK Cells Function through Tigit/ CD226 in Patients with Multiple Myeloma

Introduction：Bone marrow microenvironment plays a significant supporting role in the proliferation, differentiation, migration, survival and drug resistance of myeloma cells. To detect the expression of the co-inhibitory receptor TIGIT and the co-stimulatory receptor CD226 on the surface of natural killer cells (NK cells) in multiple myeloma (MM) patients, and changes in the immune phenotypes and killing function of NK cells. Furthermore, to explore the underlying mechanism of bone marrow mesenchymal stem cells (BMSCs) regulating NK cells function through TIGIT/CD226 in patients with MM. The expressions of TIGIT, CD226, activated molecules NKG2D and CD107a and functional molecules IFN-γ and Perforin on the NK cells in bone marrow or peripheral blood were detedted by FCM. BMSCs and NK cells were co-cultured at a ratio of 1:4 in vitro, which was grouped according to different monoclonal antibodies (mAbs) added. After co-cultured, the changes in the expression of NKG2D, NKp30, NKp44 and CD69 of NK cells and IFN-γ concentration were examined by FCM. NK cells and U266 were co-cultured with different E:T ratio and the apoptosis of U266 was detected to evaluate the killing function of NK cells by FCM. The expressions of CD155 on BMSCs in the bone marrow were detected by FCM. The correlations between CD155 expression on BMSCs and the production of IFN-γ and Perforin in bone marrow NK cells, as well as clinical characteristics were analyzed.Results: 1. In bone marrow, expression of TIGIT was significantly higher in patients with NDMM than those in patients with CR and HC (both P values <0.01). CD226 was significantly lower in NDMM patients than those in patients with CR and HC (both P values <0.01). NK cell surface activated molecules NKG2D and CD107a, and functional molecules IFN-γ and Perforin were significantly decreased in NDMM (Fig. A). In peripheral blood, TIGIT, CD226 and above immune phenotype expression levels were basically consistent with the trend in bone marrow. 2. In co-culture experiments of BMSCs and NK cells, only added anti-TIGIT mAbs, compared to both added anti-TIGIT and CD226 mAbs, the immune phenotypes of NK cells significantly increased (Fig. B). And the concentration of IFN-γ in the co-culture supernatants also increased (Fig. C). 3. After co-cultured with BMSCs, NK cells were co-cultured again with U266 in vitro, the apoptosis level of U266 in TIGIT mAbs group increased than those in TIGIT+ CD226 mAbs group at different E: T ratio (Fig. D). 4. The expression of CD155, the ligand of TIGIT and CD226, increased notably on the surface of BMSCs in MM patients(P<0.01) (Fig. E). 5. The expression of CD155 on BMSCs was negatively correlated with the production of IFN-γ and perforin in bone marrow NK cells with NDMM patients. And it has correlation with clinical characteristics including β2-MG, LDH, ALB, Hb, ISS stage and R-ISS stage in patients with MM.Summary: BMSCs may regulate NK cells through TIGIT/CD226. High expression of TIGIT on NK cells may mediate the inhibitory effect of BMSCs in MM patients. Blocking TIGIT could restore NK cells activation and killing function. CD155, a common ligand of TIGIT and CD226, was overexpressed on BMSCs in NDMM patients, but was low expressed on bone marrow MM cells. And it has correlation with NK cells function and clinical characteristics, indicating that CD155 may be involved in the regulation of NK cells function by BMSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytokine-Induced Memory-like NK Cells Have a Distinct Single Cell Transcriptional Profile and Persist for Months in Adult and Pediatric Leukemia Patients after Adoptive Transfer

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies.To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2).We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design. [Display omitted] DisclosuresFoltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.

Central Memory T-Cell Differentiation Correlates with Depth of Response in Relapsed/Refractory Multiple Myeloma Patients Receiving Elotuzumab in Combination with Carfilzomib, Lenalidomide and Dexamethasone (Elo-KRd)

Central Memory T-Cell Differentiation Correlates with Depth of Response in Relapsed/Refractory Multiple Myeloma Patients Receiving Elotuzumab in Combination with Carfilzomib, Lenalidomide and Dexamethasone (Elo-KRd)

PiggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells

Promising progress has been made in adoptive transfer of allogeneic natural killer (NK) cells to treat relapsed or refractory acute myeloid leukemia (AML). In this regard, chimeric antigen receptor (CAR)-modification of NK cells is considered as a compelling approach to augment the specificity and cytotoxicity of NK cells against AML. Using a non-viral piggyBac transposon technology and human peripheral blood-derived primary NK cells, we generated CAR-NK cells to target NKG2D ligands and demonstrated their in vitro activity in lysing cancer cells expressing the ligands and in vivo efficacy in inhibiting tumor growth in a xenograft KG-1 AML model. We further generated CAR-NK cells co-expressing transgenes for the NKG2D CAR and interleukin-15 (IL-15). The ectopic expression of IL-15 improved the in vitro and in vivo persistence of NKG2D CAR-NK cells, leading to enhanced in vivo tumor control and significant prolongation of mouse survival in the KG-1 AML model. Collectively, our findings demonstrate the ectopic expression of IL-15 as an important means to improve the antileukemic activity of NKG2D CAR-NK cells. Our study further illustrates the feasibility of using the piggyBac non-viral platform as an efficient and cost-effective way for CAR-NK cell manufacturing.

Human intestinal and circulating invariant natural killer T cells are cytotoxic against colorectal cancer cells via the perforin-granzyme pathway.

Invariant natural killer T (iNKT) cells are lipid‐specific T lymphocytes endowed with cytotoxic activities and are thus considered important in antitumor immunity. While several studies have demonstrated iNKT cell cytotoxicity against different tumors, very little is known about their cell‐killing activities in human colorectal cancer (CRC). Our aim was to assess whether human iNKT cells are cytotoxic against colon cancer cells and the mechanisms underlying this activity. For this purpose, we generated stable iNKT cell lines from peripheral blood and colon specimens and used NK‐92 and peripheral blood natural killer cells as cell‐mediated cytotoxicity controls. In vitro cytotoxicity was assessed using a panel of well‐characterized human CRC cell lines, and the cellular requirements for iNKT cell cytotoxic functions were evaluated. We demonstrated that both intestinal and circulating iNKT cells were cytotoxic against the entire panel of CRC lines, as well as against freshly isolated patient‐derived colonic epithelial cancer cells. Perforin and/or granzyme inhibition impaired iNKT cell cytotoxicity, whereas T‐cell receptor (TCR) signaling was a less stringent requirement for efficient killing. This study is the first evidence of tissue‐derived iNKT cell cytotoxic activity in humans, as it shows that iNKT cells depend on the perforin–granzyme pathway and both adaptive and innate signal recognition for proper elimination of colon cancer cells.

P-003: Improving NK cell function in Multiple Myeloma with NKTR-255, a novel polymer-conjugated human IL-15

<h3>Background</h3> Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of NK cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. <h3>Methods</h3> Here we investigate the potential of NKTR-255, a novel polymer-conjugated human IL-15 to engage the IL-15 pathway and overcome the inhibitory status observed in NK cells from MM patients. For this purpose, we have analyzed ex vivo and in vivo effects of NKTR-255 on phenotypic features, effector functions and cytotoxicity of NK cells against MM cells. <h3>Results</h3> We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors (NKG2D, NKp46, NKp30, DNAM-1, CD69, TRAIL) on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. For a more accurate assessment of the effect of NKTR-255 on NK cell activity in an autologous setting in the presence of the bone marrow (BM) milieu, we cultured whole BM samples from non-treated newly-diagnosed MM patients with increasing doses of NKTR-255 for 5 days. NK cells experienced a dose-dependent induction of proliferation and activation (as shown by increased expression of CD69 and NKG2D), which translated in a reduced viability of CD138+ MM cells in the presence of NKTR-255. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we also tested in vitro and in vivo efficacy of a combination of NKTR-255 with daratumumab. We observed a more efficient antibody-dependent cellular cytotoxicity against MM cells in vitro and decreased tumor growth in vivo, where NKTR-255 rescued CD38+ NK cell levels from depletion by daratumumab. <h3>Conclusions</h3> Taken together, these results support the restoration and expansion of NK cell activity in MM with NKTR-255, providing rationale for its clinical use as a novel immunotherapeutic approach for MM patients alone or in combination with monoclonal antibodies or other immunomodulatory drugs.

Evaluation of Natural Killer Cells in HCV Patients undergoing Hemodialysis

ABSTRACT Background Hepatitis C virus (HCV) is a hepatotropic virus and one of the major causes of liver disease and a potential cause of substantial morbidity and mortality worldwide. Patients on hemodialysis are at high risk for HCV, with frequency of infection several times higher than that in non-uremic patients. Natural killer (NK) cells are best appreciated for innate defense against viral infections and in tumor cell surveillance. Objective To study the percentage of peripheral blood natural killer cells among chronic hepatitis C patients undergoing hemodialysis compared to chronic HCV patients with normal kidney functionsby flowcytometry immunophenotyping. Methodology Frequency of CD3- CD56+ cells was assessed in two distinct groups. Group I: 35 chronic HCV patients with end stage renal disease and undergoing hemodialysis, group II: 35 age and sex matched chronic HCV patients with no kidney disease. Results No significant difference was observed between HCV hemodialysis patients with ESRD and HCV patients with normal kidney function regarding the frequency of both NK cells (CD3CD56+) and NKT cells (CD3+CD56+). However, group I had significantly higher percentage of CD3+ cells than group II. Conclusion no difference was found in this study between HCV patients on hemodialysis and HCV with normal kidney function regarding NK cell frequency. However, assessment of NK cell function in future studies might reveal differences.

TIMP1 and TIMP2 Downregulate TGFβ Induced Decidual-like Phenotype in Natural Killer Cells.

Simple SummaryCancer patients are characterized by NK cells with altered surface markers, such as CD56 brightness, CD9, CD49a (pro-angiogenic) and PD-1, and TIM-3 (exhaustion), that favor immune escape. Transforming growth factor-beta (TGFβ) is a major tumor-derived cytokine that favors cancer growth and supports pro-angiogenic activities in NK cells by inducing pro-angiogenic molecules. TIMP-1 and TIMP-2 play a crucial role in extracellular matrix (ECM) regulation, wound healing, pregnancy and cancer, and there is increasing evidence that they are immune-modulatory. We found that recombinant TIMP-1 and -2 can partially contrast the induction of pro-tumor/pro-angiogenic decidual-like polarization of NK cells by TGFβ.Natural Killer (NK) cells have been found to be anergic, exhausted and pro-angiogenic in cancers. NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like-phenotype, together with decreased levels of NKG2D activation marker, increased levels of TIM-3 exhaustion marker, similar to cancer-associated NK cells. Tissue inhibitors of metalloproteases (TIMPs) exert dual roles in cancer. The role of TIMPs in modulating immune cells is a very novel concept, and the present is the first report studying their ability to contrast TGFβ action on NK cells. Here, we investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16−, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Treatment with recombinant TIMP-1 or TIMP-2 showed a trend, although not statistically significant, to decrease CD49a+ and TIM-3+ expression and increase NKG2D in peripheral blood NK cells exposed to conditioned media from colon cancer cell lines. Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. Given the heterogeneity of released factors within the TME, it is clear that TGFβ stimulation represents a model to prove TIMP’s new properties, but it cannot be envisaged as a soloist NK cell polarizing agent. Therefore, further studies from the scientific community will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic–therapeutic roles.

PD-1, CTLA-4, LAG-3, and TIGIT: The roles of immune checkpoint receptors on the regulation of human NK cell phenotype and functions

The roles of immune checkpoint receptors were defined in many cancers and autoimmune diseases, while there is limited information on their functional roles in the NK cells of healthy individuals. Immune checkpoint receptor expression of NK cell subsets and their association with NK cell functions (cytotoxic capacity and cytokine production) in healthy population were investigated. PD-1, CTLA-4, LAG-3 and TIGIT expression of peripheral blood NK cells, cytokine levels (TNF-α, IFN-γ, IL-10) and cytotoxic functions (granzyme A, perforin, CD107a; with/without K562 target cell stimulation) were evaluated by flow cytometry. CD56dimCD16dim NK cells had the highest expression of TIGIT, while CD56dimCD16− NK cells had highest expression of PD-1, CTLA-4 and LAG-3. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had increased amount of IL-10 however, reduced IFN-γ and TNF-α levels. Cytotoxic granule expressions (perforin and granzyme A) were reduced in PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells. However, TIGIT expression did not alter perforin and granzyme A expressions. Degranulation capacity was reduced in three groups of NK cells (PD-1+ or LAG-3+ or TIGIT+). TIGIT+ NK cells responded strongly to target cell stimulation, while NK cells in the other groups (PD-1+ or CTLA-4+ or LAG-3+) were resistant. PD-1+ NK cells, CTLA-4+ NK cells and LAG-3+ NK cells had a regulatory phenotype, impaired cytotoxic functions, and response to target cell stimulation. In contrast, TIGIT+ NK cells had strong baseline cytotoxic activity that further increased in response to target cell stimulation.

Defining the AHR-regulated transcriptome in NK cells reveals gene expression programs relevant to development and function

AbstractThe aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in natural killer (NK) cells, we performed RNA and chromatin immunoprecipitation sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during interleukin-21–driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared with control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription, and maintained the CD56dim phenotype. However, it showed altered expression of 1004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding, we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1 and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

NK Cell Patterns in Idiopathic Inflammatory Myopathies with Pulmonary Affection.

Background: Pulmonary affection (PA) is associated with a substantial increase in morbidity and mortality in patients with idiopathic inflammatory myopathies (IIM). However, the underlying immune mechanisms of PA remain enigmatic and prompt deeper immunological analyses. Importantly, the Janus-faced role of natural killer (NK) cells, capable of pro-inflammatory as well as regulatory effects, might be of interest for the pathophysiologic understanding of PA in IIM. Methods: To extend our understanding of immunological alterations in IIM patients with PA, we compared the signatures of NK cells in peripheral blood using multi-color flow cytometry in IIM patients with (n = 12, of which anti-synthetase syndrome = 8 and dermatomyositis = 4) or without PA (n = 12). Results: We did not observe any significant differences for B cells, CD4, and CD8 T cells, while total NK cell numbers in IIM patients with PA were reduced compared to non-PA patients. NK cell alterations were driven by a particular decrease of CD56dim NK cells, while CD56bright NK cells remained unchanged. Comparisons of the cell surface expression of a large panel of NK receptors revealed an increased mean fluorescence intensity of NKG2D+ on NK cells from patients with PA compared with non-PA patients, especially on the CD56dim subset. NKG2D+ and NKp46+ cell surface levels were associated with reduced vital capacity, serving as a surrogate marker for clinical severity of PA. Conclusion: Our data illustrate that PA in IIM is associated with alterations of the NK cell repertoire, suggesting a relevant contribution of NK cells in certain IIMs, which might pave the way for NK cell-targeted therapeutic approaches.

NK Cell Subpopulation Is Altered and the Expression of TLR1 and TLR9 Is Decreased in Patients with Acute Lymphoblastic Leukemia.

NK cells represent a heterogeneous subpopulation of lymphocytes of the innate immune system, which possess powerful antitumor activity. NK cells exhibit their function through a complex collection of receptors that act synergistically to recognize, regulate, or amplify the immune response. TLRs allow cells to detect PAMPs, MAMPs, or DAMPs, which are essential for the initiation of the immune response. Studies on the different subpopulations of NK cells and their expression profile of innate immune receptors in hematological cancers are limited. In this study, the specific subpopulations of NK cells in pediatric patients with acute lymphoblastic leukemia (ALL) and the repertoire and level of expression of TLRs in cytotoxic NK cells were assessed. The results suggested that pediatric patients with ALL exhibited a significant decrease in NK cells in peripheral blood and bone marrow, in addition to alterations in the distribution of the subpopulations of cells. Regulatory and cytotoxic NK cells were diminished, whereas dysfunctional phenotype was considerably increased. Cytotoxic NK cells from children with ALL expressed all 10 TLRs, and expression of TLR1 and TLR9 was decreased compared with the controls. Interestingly, cytotoxic NK cells exhibited a higher expression of TLR1 in the bone marrow than in the peripheral blood of patients with ALL. The present study is the first to show that TLR10 was expressed in the cytotoxic NK cells and the first to assess the profile and levels of the 10 known TLRs in cytotoxic NK cells from patients with ALL. The alterations in expression levels and cellular distribution may be involved in the immune response.

P-033. Changes of natural cytotoxicity receptors of CD56dim and CD56bright NK cells in peripheral blood and decidua in preeclampsia

P-033. Changes of natural cytotoxicity receptors of CD56dim and CD56bright NK cells in peripheral blood and decidua in preeclampsia