Blood Natural Killer Cells Research Articles

ПОДАВЛЕНИЕ ПРОДУКЦИИ ПРОВОСПАЛИТЕЛЬНЫХ ЦИТОКИНОВ В ЕСТЕСТВЕННЫХ КИЛЛЕРАХ КРОВИ ПАЦИЕНТОВ С ХРОНИЧЕСКОЙ ОБСТРУКТИВНОЙ БОЛЕЗНЬЮ ЛЕГКИХ ПОД ВЛИЯНИЕМ НОРТРИПТИЛИНА

Aim. To determine the ability of nortriptyline, alone and in combination with budesonide, to inhibit the production of pro-inflammatory cytokines in the peripheral blood natural killer (NK) cells of patients with chronic obstructive pulmonary disease (COPD) and to establish the molecular mechanisms of the combined action of these drugs. Material and methods. Blood cells from COPD patients (n=21) were incubated with budesonide, nortriptyline, or their combination. Cytokine production, expression of glucocorticoid receptor β (GRβ), histone deacetylase 2 (HDAC2), and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in NK cells were analyzed by flow cytometry. Results. The combination of nortriptyline (10 μM) and budesonide (10 nM) reduced the synthesis of interleukin 4 (IL-4), IL-8, interferon γ, tumor necrosis factor α as well as the expression of GRβ and p-p38 MAPK in NK cells; it also increased the expression of HDAC2 more significantly than budesonide alone. Conclusions. Nortriptyline enhances the anti-inflammatory effect of budesonide by altering the expression of HDAC2, GRβ, p-p38 MAPK in peripheral blood NK cells of patients with COPD. The data obtained indicate the advisability of combining nortriptyline with budesonide for the treatment of COPD.

Phenotypic diversity of human adipose tissue-resident NK cells in obesity.

Natural killer (NK) cells have emerged as key mediators of obesity-related adipose tissue inflammation. However, the phenotype of NK cell subsets residing in human adipose tissue are poorly defined, preventing a detailed understanding of their role in metabolic disorders. In this study, we applied multicolor flow cytometry to characterize CD56bright and CD56dim NK cells in blood and adipose tissue depots in individuals with obesity and identified surface proteins enriched on adipose tissue-resident CD56bright NK cells. Particularly, we found that adipose tissue harbored clusters of tissue-resident CD56bright NK cells signatured by the expression of CD26, CCR5 and CD63, possibly reflecting an adaptation to the microenvironment. Together, our findings provide broad insights into the identity of NK cells in blood and adipose tissue in relation to obesity.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

CD56bright natural killer cells preferentially kill proliferating CD4+ T cells.

Human CD56br natural killer (NK) cells represent a small subset of CD56+ NK cells in circulation and are largely tissue-resident. The frequency and number of CD56br NK cells in blood has been shown to increase following administration of low-dose IL-2 (LD-IL2), a therapy aimed to specifically expand CD4+ regulatory T cells (Tregs). Given the potential clinical application of LD-IL-2 immunotherapy across several immune diseases, including the autoimmune disease type 1 diabetes, a better understanding of the functional consequences of this expansion is urgently needed. In this study, we developed an in vitro co-culture assay with activated CD4+ T cells to measure NK cell killing efficiency. We show that CD56br and CD56dim NK cells show similar efficiency at killing activated CD4+ conventional T (Tconv) and Treg cell subsets. However, in contrast to CD56dim cells, CD56br NK cells preferentially target highly proliferative cells. We hypothesize that CD56br NK cells have an immunoregulatory role through the elimination of proliferating autoreactive CD4+ Tconv cells that have escaped Treg suppression. These results have implications for the interpretation of current and future trials of LD-IL-2 by providing evidence for a new, possibly beneficial immunomodulatory mechanism of LD-IL-2-expanded CD56br NK cells.

Circulating CXCR5+ natural killer cells are expanded in patients with myasthenia gravis.

Myasthenia gravis (MG) is a classic autoantibody-mediated disease in which pathogenic antibodies target postsynaptic membrane components, causing fluctuating skeletal muscle weakness and fatigue. Natural killer (NK) cells are heterogeneous lymphocytes that have gained increasing attention owing to their potential roles in autoimmune disorders. This study will investigate the relationship between the distinct NK cell subsets and MG pathogenesis. A total of 33 MG patients and 19 healthy controls were enrolled in the present study. Circulating NK cells, their subtypes and follicular helper T cells were analysed by flow cytometry. Serum acetylcholine receptor (AChR) antibody levels were determined by ELISA. The role of NK cells in the regulation of B cells was verified using a co-culture assay. Myasthenia gravis patients with acute exacerbations had a reduced number of total NK cells, CD56dim NK cells and IFN-γ-secreting NK cells in the peripheral blood, while CXCR5+ NK cells were significantly elevated. CXCR5+ NK cells expressed a higher level of ICOS and PD-1 and a lower level of IFN-γ than those in CXCR5- NK cells and were positively correlated with Tfh cell and AChR antibody levels. In vitro experiments demonstrated that NK cells suppressed plasmablast differentiation while promoting CD80 and PD-L1 expression on B cells in an IFN-γ-dependent manner. Furthermore, CXCR5- NK cells inhibited plasmablast differentiation, while CXCR5+ NK cells could more efficiently promote B cell proliferation. These results reveal that CXCR5+ NK cells exhibit distinct phenotypes and functions compared with CXCR5- NK cells and might participate in the pathogenesis of MG.

Co-administration of tacrolimus and low molecular weight heparin in patients with a history of implantation failure and elevated peripheral blood natural killer cell proportion.

To investigate the therapeutic effect of co-administration of tacrolimus (TAC) and low-molecular-weight heparin (LMWH) or LMWH only on pregnancy outcomes in the female with a history of implantation failure and elevated peripheral blood natural killer (pNK) cell proportion in frozen-thawed embryo transfer cycles. To evaluate the pregnancy parameters for 249 patients with ≥2 implantation failures and pNK cell proportion ≥12% by analyzing a retrospective observational cohort study. Sixty patients had received the co-administration TAC and LMWH (TAC & LMWH group), 85 others had only taken LMWH (LWMH group), and the rest did not take any particular drugs (control group). The experimental finding indicated that the TAC & LMWH group and the LMWH group showed higher clinical pregnancy rates than the control group (p < 0.05), and TAC & LMWH group had a much higher live birth rate. According to the binary logistic regression analysis, the combination of TAC and LMWH was conducive to clinical pregnancy and live birth rate and reduced the possibility of miscarriage. It would not affect the result of spontaneous abortion and live birth, although the LMWH was only beneficial to clinical pregnancy. In addition, these findings were similar for these three groups' obstetrical and neonatal outcomes. The combination of TAC and LMWH can improve clinical pregnancy and live birth rates and reduce the risk of spontaneous miscarriage in patients with a history of implantation failure and elevated pNK ratio. LMWH is also beneficial to clinical pregnancy.

Preliminary Dose-Escalation Results from a Phase 1/2 Study of GEN3014 (HexaBody®-CD38) in Patients (pts) with Relapsed or Refractory Multiple Myeloma (RRMM)

Background: CD38-targeted antibodies (Abs) are foundational treatments (Tx) for pts with newly diagnosed or RRMM. GEN3014 (HexaBody®-CD38) is a novel human IgG1 anti-CD38 monoclonal Ab (mAb) with an E430G hexamerization-enhancing mutation that facilitates highly efficient complement-dependent cytotoxicity (CDC). GEN3014 showed preclinical tumor cell killing through highly potent CDC (more efficient than daratumumab) and Ab-dependent cellular cytotoxicity and phagocytosis. We present preliminary data from dose escalation in the first-in-human, phase 1/2 trial of GEN3014 in pts with RRMM (NCT04824794). Methods: Adults with RRMM were enrolled if they had ≥3 prior lines of Tx including a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), were double refractory to a PI and IMiD, or had ≥2 prior lines of Tx if 1 combined a PI and IMiD. GEN3014 was tested at 6 dose levels ranging from 0.2 to 24 mg/kg and administered intravenously in 28-d cycles (first dose split equally between D1 and D2 of cycle 1, then full doses QW through cycle 2; Q2W, cycles 3-6; Q4W, cycles ≥7). Primary objectives were to determine the recommended phase 2 dose (RP2D) and maximum tolerated dose. Other endpoints included pharmacokinetic (PK) and pharmacodynamic (PD) characterization. PD biomarker analysis included immunophenotyping and quantification of complement factors and cytokines in peripheral blood. Results: As of July 10, 2022, 24 pts with RRMM were treated (median age, 65 y; range, 45-84). Pts were heavily pretreated with a median of 7 prior lines of Tx (range, 3-13). Most pts (67%) had prior exposure to a daratumumab- or isatuximab-containing regimen, with 8 pts naive to anti-CD38 mAb. GEN3014 was well tolerated; the most common Tx-emergent AEs were infusion-related reactions (IRRs; 75.0%), neutropenia (62.5%), anemia (33.3%), diarrhea (33.3%), pyrexia (25.0%), and thrombocytopenia (25.0%). IRRs were mostly low grade (G; 62.5% G1-2, 12.5% G3, no G4), occurred mainly during the first infusion, and were manageable. There were no Tx-related deaths or dose-limiting toxicities observed at doses up to 16 mg/kg, the RP2D. Disease progression led to discontinuation in 80% of pts. Among 19 response-evaluable pts (5 naive to anti-CD38 mAb, 14 refractory to anti-CD38 mAb), preliminary antitumor activity was seen. In pts naive to anti-CD38 mAb, 2 pts achieved very good partial response (1 each at 4 and 24 mg/kg); 1 achieved minimal response (MR; 16 mg/kg). In pts refractory to anti-CD38 mAb, 2 pts achieved MR (1 each at 8 and 16 mg/kg). Five pts remained on Tx (4 naive and 1 refractory to anti-CD38 mAb). Updated data will be presented. Peak GEN3014 concentrations at the end of infusion increased roughly proportionally with dose. The interval area under the concentration-time curve showed a more than proportional increase with doses ≤8 mg/kg but a proportional or less than proportional increase at doses >8 mg/kg. These results suggest target-mediated drug disposition at lower doses and a high degree of target saturation at >8 mg/kg. Trough concentrations increased with weekly administration, most notably in pts responding to Tx, indicating target cell depletion over time. GEN3014 Tx was associated with a rapid, sustained decrease in peripheral blood natural killer cells at all doses in all pts (mean 92.4% ± 9.2% peak reduction from baseline [BL], n=21). T cells transiently decreased after administration of first doses ≥4 mg/kg, followed by expansion (≥100% increase from BL), particularly in pts naive to anti-CD38 mAb. GEN3014 induced transient reduction in complement component C2 (mean 58% ± 18% peak reduction from BL, n=18) and total complement lytic activity (CH50; mean 48% ± 24% peak reduction from BL for ≥8 mg/kg, n=11) at all evaluable doses, suggesting CDC. Complement parameters rapidly returned to BL, indicating that Tx does not exhaust complement. Plasma cytokine (IL-2, IL-6, IL-8, IL-10, IFNγ, and TNFα) levels generally remained low after Tx. Conclusions: Dose-escalation results show GEN3014 had a tolerable safety profile and clinical activity in pts with RRMM, including pts with prior anti-CD38 mAb. The PK profile is characterized by target-mediated drug disposition at lower doses and signs of saturation at doses >8 mg/kg. Biomarker analyses confirm biological activity in pts with RRMM at all evaluated doses. The expansion part of the trial evaluating the 16 mg/kg RP2D is ongoing.

B7-H3 Trike Enhances Killing of Myeloid Derived Suppressor Cells in Multiple Myeloma

Multiple myeloma (MM) is sensitive to Natural Killer (NK) cell-mediated killing but develops methods to attenuate NK cell function. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through the CD16 receptor. We developed a tri-specific killer engager (TriKE®) with a camelid single-domain antibody (sdAb) fragment against CD16 (on NK cells) as well as a linker region containing IL-15, to enhance NK cell survival. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. The second sdAb targets B7-H3 (CD276), a tumor antigen that is expressed on myeloma and is associated with decreased progression free survival. B7-H3 is differentially expressed on myeloma tumor lines MM1S, RPMI-8226, U266, and H929 but all four MM lines are significantly killed by peripheral blood NK cells in the presence of 3nM B7-H3 TriKE compared to NK cells alone. Bone marrow aspirates from 5 MM patients (3 newly diagnosed and 2 relapsed) were analyzed by flow cytometry and showed 58-96% expression of B7-H3 on CD138+ plasma cells. The bone marrow biopsy aspirates also showed monocytic myeloid derived suppressor cells (Mo-MDSC, CD14+, CD11b+) that expressed B7-H3 (38-87%) as well as the damage associated molecular pattern (DAMP) S100A8/9. In comparison, CD14+ cells from the peripheral blood of healthy donors expressed very low levels of B7-H3 or S100A8/9 (<5%). Mo-MDSC are important to the pathogenesis of MM because they support MM growth and contribute to suppression of NK in the tumor microenvironment. B7-H3 is highly expressed on mature osteoclasts and MDSC can develop into osteoclasts in mouse models of MM and breast cancer. We saw that Mo-MDSC expressing B7-H3 also co-expressed RANK, a receptor necessary for signaling osteoclastic development. We developed Mo-MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or co-culture with MM cell lines at 1:1000 ratio. Both types of MDSC suppressed NK proliferation. Peripheral blood derived MDSC expressed >80% B7-H3 and RANK. We separated B7-H3 positive and negative populations by flow cytometry and attempted to develop osteoclasts with M-CSF and RANK-L over 14 days. Tartrate resistant acid phosphatase (TRAP) staining was performed but both groups were negative and exhibited no phenotypic changes by light microscopy. We then isolated CD14+ cells from the bone marrow of an MM patient who was in complete remission (CR) after autologous stem cell transplant but had a distinct population of B7-H3+, RANK+, S100A8/9+ Mo-MDSC in her surveillance bone marrow aspirate. After fourteen days of culture with M-CSF and RANK-L, TRAP staining was positive, and cells had grown larger, consistent with osteoclastic differentiation (Figure 1A). We tested the ability of B7-H3 TriKE to eliminate MDSC by NK cells, therefore diminishing there immune suppression. Cytotoxicity assays were conducted over 48 hours with NK cells with or without B7-H3 TriKE. Killing was significantly enhanced by B7-H3 TriKE (Figure 1B). Since MDSC are known to suppress NK cell function, we assessed metabolic fitness in NK cells with MDSC at 1:1 ratio for 48 hours with or without 3nM B7-H3 TriKE and found that B7-H3 TriKE improved both the oxygen consumption and extracellular acidification rates. These data show that B7-H3 TriKE enhances NK cell killing of MM through direct targeting and eliminates suppressive Mo-MDSC. B7-H3+ Mo-MDSC can differentiate into osteoclasts and persist even in patients who are in CR, possibly contributing to relapse. B7-H3 TriKE may be particularly useful for patients with bone lesions or as consolidative therapy. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun, and a phase I trial is expected in late 2023. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pre-Clinical Efficacy of AB-101, an Allogeneic Cord-Blood Derived Natural Killer (NK) Cell Therapeutic Candidate, in Combination with Anti-CD38 Antibodies in Models of Multiple Myeloma

Introduction: AB-101 is a non-engineered, allogeneic, off-the-shelf, cryopreserved cord blood-derived natural killer (NK)-cell therapy in development as a cancer therapeutic. A highly scaled manufacturing process enables production of 1000s of doses of AB-101 from a single donor cord blood unit (CBU). AB-101 is derived from CBUs selected for KIR-B haplotype and natural high-affinity variant of CD16 (158V/V), which are associated with improved anti-tumor activity and ADCC enhancement, resulting in a highly active NK cell product without the requirement for additional engineering. We are developing AB-101 for combination with monoclonal antibodies (mAbs) to enhance antibody dependent cellular cytotoxicity (ADCC) in patients, initially in an ongoing Phase I clinical trial for NHL. To further develop AB-101 as an ADCC enhancer, we assessed anti-tumor activity in combination with CD38 mAb, against multiple myeloma (MM) cell lines. CD38 is a transmembrane glycoprotein that is uniformly and highly upregulated on MM cells. Anti-CD38 mAbs exert anti-tumor activity against MM through ADCC and other mechanisms. NK cells play a key role in ADCC responses through CD16 receptor signaling. However, the majority of NK cells express CD38 and are depleted by therapeutic CD38 mAbs which could limit their therapeutic efficacy. Combining NK cell therapies with anti-CD38 mAbs, thus necessitates CD38 knockout to prevent fratricide. AB-101 has a naturally low percent and intensity of CD38 expression. Here we present data demonstrating that AB-101 are resistant to anti-CD38 mAb-induced fratricide and can elicit significant ADCC against MM cell lines both in vitro and in vivo. Since anti-CD38 mAbs are combined with glucocorticoids in the clinic, we assessed the impact of dexamethasone and methylprednisolone on ADCC by AB-101. Here we report robust ADCC following treatment of AB-101 with glucocorticoids. Methods: AB-101 was screened for CD38 expression and incubated with anti-CD38 mAb - daratumumab or hIgG1 control (300 - 0.41 µg/ml) for 4 hours to assess fratricide. In vitro ADCC assays were performed at various effector to target ratios against control (Daudi and K562) and MM cell lines (NCI-H929, RPMI 8226, MM.1S and MM.1R) using 2 µg/ml of daratumumab or hIgG1. To study the effect of glucocorticoids, AB-101 was cultured in various concentrations of dexamethasone and methylprednisolone for 48 hours in the presence of 50 or 500 IU/ml of IL-2, prior to ADCC assay. In vivo efficacy of AB-101 in combination with daratumumab was assessed in the intraperitoneal (IP) MM.1S-Luc xenograft model in NSG mice (Figure 1A). Daratumumab and AB-101 were administered by IP injection. Tumor burden was assessed by bioluminescent imaging (BLI). Results: AB-101 was over 98% CD56+/CD3- with 87% CD56+/CD16+ population. Only 22% of GMP drug product were CD38+ with low intensity of expression compared to peripheral blood NK cells (~88%). In the presence of daratumumab AB-101 viability was unaltered compared to hIgG1 control demonstrating resistance to fratricide. Daratumumab enhanced ADCC potential of AB-101 against MM cell lines and Daudi in in vitro. ADCC against MM lines and Daudi did not decrease on exposure to glucocorticoids compared to no treatment group. In vivo anti-tumor efficacy, as evidenced by reduction in BLI signal, was observed in the MM.1S model in AB-101 + daratumumab groups compared to daratumumab and AB-101 alone (Figure 1B). AB-101 + daratumumab (1 and 3mpk) led to 98 and 99% tumor growth inhibition (TGI), respectively, relative to no treatment. Daratumumab monotherapy at 1 and 3mpk showed 21% and 87% TGI respectively, and AB-101 showed 16% TGI relative to no treatment. Conclusions: AB-101 has high and consistent expression of CD16 and low CD38 expression and is resistant to daratumumab induced fratricide and hence can be combined with daratumumab without additional engineering such as CD38 knockout. Daratumumab is combined in the clinic with glucocorticoids, immunomodulatory drugs (IMiDs) and proteosome inhibitors (PI). While IMiDs and PIs are reported to enhance NK cell function, glucocorticoids have been shown to diminish NK cell cytotoxicity. Data presented here suggests that ADCC potential of AB-101 is not negatively impacted by glucocorticoids. Taken together, the in vitro and in vivo data supports further clinical development of AB-101 in combination with daratumumab to treat MM patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

In Vivo Generation of Memory-like NK Cells for the Treatment of AML and Myelodysplastic Syndrome; Early Clinical Applications of INKmuneTM

Natural killer (NK) cells can have potent anti-tumour responses and recently highlighted potential for memory-like functions, but the best approach to generate memory-like NK (mlNK) cells remains unclear. Priming of NK cells with the pharmaceutical-grade, replication incompetent tumour cell product, INKmuneTM generates tumour-induced mlNK (TIML-NK) cells with enhanced cytokine production and cytotoxicity against multiple NK-resistant tumour target cell lines in vitro, analogous to previously reported cytokine-primed memory-like NK cells. In addition, proteomic profiling of TIML-NK cells revealed differential abundance of proteins involved in promoting mitochondrial survival and function as well as critical nutrient receptors, which may provide a unique benefit of NK cell activation whilst sparing mitochondrial damage typically associated with cytokine-mediated activation. INKmune priming increased NK glycolysis and oxidative phosphorylation whilst enhancing mitochondrial respiratory capacity and maintaining glycolytic reserve. Four patients have been treated with a cycle of three, weekly infusions of 1x108 INKmuneTM cells. All patients received all three doses without incident and without side effects. Patient 1 was a 78yr old man with A 3yr history of refractory MLD MDS who was transfusion and platelet dependent and required G-CSF. Within 7 days of first infusion, over 50% of his peripheral blood NK cells activated and this increased with each dose. By day +29, 72% of his NK cells were activated and this remained above 68% at day +119 when monitoring ended. His ECOG status fell from 2 pre-treatment to 0 at D+119 and his RAD-1 ctDNA from 45 to 38. Analysis of systemic cytokines showed increases in MIP-1a/b, TNF-a and sIL2R which paralleled the changes in percentages of CD69+ NK cells. IL6 levels peaked after the second infusion but levels remained low and there was no evidence of CRS. At one year follow-up he remained well, platelet and G-CSF independent, with reduced transfusion requirements and had resumed playing sport with friends. Subjects 2, 3 and 4, were all multiply relapsed and refractory AML patients; two of whom had relapsed after HLA-mismatched HSCT. All showed rapid activation of peripheral NK cells post infusion with sustained activity throughout follow-up. In all cases these activated NK cells killed NK-resistant tumour cells in vitro without additional activation. Subject 2 was a neutropenic 21yr old female with 70% a mixed chimera and refractory AML (M2) post VUD HSCT at time of treatment. Within 1 month of initiation of INKmune treatment she was discharged with PMN >500 and her mixed chimera resolved to full donor chimera. At Day 140 post INKmune her bone marrow NK cells remained highly activated (>60% CD69+, K562 and Raji lysis of >70%). Her AML recurred 4 months post INKmune infusion and she died from relapsed disease 8 months after INKmune treatment. Subject 3 was a 21yr old male with refractory AML (M6) after two failed HSCT (haplo m/m and VUD). He showed the same rapid generation of activated NK cells in vivo but to a lesser degree (maximum 35% CD69+, sustained increased lysis of K562 but no lysis of Raji cells) and no evidence of clinical improvement. Subject 4, a multiply relapsed AML patient has just been treated and has shown peripheral NK proliferation and maturation at D+8. The subject experienced no adverse effects post 1st and 2nd doses and further follow-up will be presented. INKmuneTM infusions proved safe and led to rapid and sustained activation of peripheral NK cells with concomitant increases in relevant systemic cytokines. The TIML-NK have a distinct phenotypic, proteomic and metabolomic signature which mirrors the activity in vitro and in vivo. This is the first time memory-like NK cells have been generated in vivo without the need for cytokine induction or support and our findings inform the development of more optimal NK cell-based immunotherapeutic strategies for cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alzheimer's disease alters the transcriptomic profile of natural killer cells at single-cell resolution.

Alzheimer's disease (AD) is the most common dementia without an effective cure at least partially due to incomplete understanding of the disease. Inflammation has emerged as a central player in the onset and progression of AD. As innate lymphoid cells, natural killer (NK) cells orchestrate the initiation and evolution of inflammatory responses. Yet, the transcriptomic features of NK cells in AD remain poorly understood. We assessed the diversity of NK cells using web-based single-cell RNA sequencing data of blood NK cells from patients with AD and control subjects and flow cytometry. We identified a contraction of NK cell compartment in AD, accompanied by a reduction of cytotoxicity. Unbiased clustering revealed four subsets of NK cells in AD, i.e., CD56bright NK cells, CD56dim effector NK cells, adaptive NK cells, and a unique NK cell subset that is expanded and characterized by upregulation of CX3CR1, TBX21, MYOM2, DUSP1, and ZFP36L2, and negatively correlated with cognitive function in AD patients. Pseudo-temporal analysis revealed that this unique NK cell subset was at a late stage of NK cell development and enriched with transcription factors TBX21, NFATC2, and SMAD3. Together, our study identified a distinct NK cell subset and its potential involvement in AD.

Anti-Ro/SSA and/or anti-La/SSB antibodies are associated with adverse endometrial status.

Anti-Ro/SSA and/or anti-La/SSB (anti-SSA/SSB) antibodies impair pregnancy outcomes, including embryo implantation and pregnancy maintenance. Optimal endometrial immune status is essential for successful pregnancy. However, whether these antibodies affect endometrial immune status is still unclear. Menstrual blood can be collected non-invasively, differs from peripheral blood, and can reflect the endometrial immune status. We herein focused on changes in subsets of natural killer (NK) cells and T cells in menstrual blood. Menstrual blood samples from anti-SSA/SSB antibody-positive (n=18) and anti-SSA/SSB antibody-negative control (n=8) women were collected, and the profile of lymphocyte subsets was analyzed. The phenotypes of menstrual blood CD49a- and CD49a+ NK cells were compared, and the abundance of NK and CD49a+ NK cells in menstrual blood of the two groups was assessed. Additionally, CD4+T and CD8+T cells were investigated for their ability to secret functional cytokines. Menstrual blood contains a large number of (mostly CD49a+) NK cells, which exhibited a more exhausted phenotype with greater expression of the immune checkpoint molecules programmed cell death protein 1 and Tim-3 compared to CD49a- conventional NK cells. CD8+T cells in menstrual blood from anti-SSA/SSB antibody-positive women produced a stronger response after stimulation, accompanied by increased interferon-γ, tumor necrosis factor-α, and granzyme B secretion (P < 0.05, separately). Menstrual blood cell composition differs between anti-SSA/SSB antibody-positive women and normal controls, especially in terms of CD49a+ NK cells and CD8+T cells, unbalancing the immune cell composition and inflammatory uterine microenvironment and possibly contributing to adverse pregnancy outcomes.

Natural killer cells in peripheral blood at diagnosis predict response to immunosuppressive therapy in severe aplastic anemia.

Immunosuppressive therapy (IST) consisting of antihuman thymocyte globulin and cyclosporine A is the first-line therapy for patients with severe aplastic anemia (AA) who are ineligible for undergoing bone marrow transplantation. The aim of the study was to evaluate the correlation between natural killer (NK) cells and response to IST in SAA patients. We retrospectively included 93 AA patients and detected NK cells in peripheral blood by flow cytometry. Both the proportion and absolute number of NK cells in newly diagnosed SAA patients were significantly lower than in controls, while the proportion and absolute number of NK cells in complete remission patients treated with IST were remarkably increased compared with treatment-naïve SAA patients. Additionally, the absolute number of NK cells at diagnosis was positively correlated with initial blood counts. For SAA patients receiving IST, the proportion of NK cells at baseline and 6months was significantly higher in responders than in non-responders. Unexpectedly, we found that the increase in the proportion of NK cells at 6months after IST was closely related to the recovery of hematopoiesis. ROC curve identified 7.3% of NK cells proportion at diagnosis as the cutoff value to predict response to IST. The response rate was higher in NK proportion high group than in NK proportion low group. Multivariate logistic regression analysis further confirmed the independent predictive value of NK cells proportion in assessing IST response. The proportion of NK cells at diagnosis may serve as a promising predictor of response to IST in patients with SAA.

INFLAMMATORY CYTOKINES PERTURB NK CELL FUNCTION AND GENE SIGNATURE IN CHRONIC MYELOID LEUKEMIA

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent anti-cancer immunotherapeutic. NK cells'mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in CML patients. The molecular drivers of this impairment during leukemic progression remain unclear. Given the established role of inflammatory cytokines (namely IL1β, IL-6, GM-CSF, TNFα) in driving myeloid malignancies, we aimed to define their effect on NK cell phenotype and function using a unique pre-clinical chimeric mouse model of CML. We hypothesize that leukemic cytokines impede NK cell maturation and cytotoxicity and shift NK cells toward a pro-tumor cytokine-producing phenotype. We establish a chimeric mouse model of CML by transplanting bone marrow BCR-ABL+ CD45.2 cells into sub-lethally irradiated CD45.1 C57BL/6 wild-type mice. For controls, mice are transplanted with non-mutant cells. Such chimeras represent a robust model to study the exposure of non-transformed host cells to leukemia. At the endpoint, splenic, blood, and bone marrow NK cells are harvested and profiled by flow cytometry, RT-qPCR, scRNA-seq, or used for degranulation assay. Blood samples are collected to obtain serum. Consistent with clinical observations, NK cells are severely reduced in numbers and frequencies and display an immature phenotype and diminished expression of activating receptors (Nkp46, Ly49D) while upregulating inhibitory molecules (Lag-3, TIGIT, NKG2A) during leukemia. Moreover, CML-exposed NK cells have a higher proliferation rate and a greater cell death indicating perturbed homeostasis. We revealed a preferential loss of terminally mature CD27−CD11b+ NK subset in CML mice, suggesting a functional shift from cytotoxic phenotype toward the immature cytokine-producing. In agreement, CML-exposed NK cells show impaired cytotoxicity as measured by target-specific degranulation ex vivo . Next, we mapped the transcriptional landscape of CML-exposed NK cells using single-cell RNA-sequencing. We confirmed decreased gene expression of NK maturation and cytotoxicity markers ( Itgam, Cx3cr1, Prf1, Gzma ) and upregulation of inhibitory molecules ( Tigit, Lag-3, Pdcd1lg1/2) and proteins involved in the cell cycle and senescence ( Mki67, Ccna, Ccnb ). Importantly, CML-exposed NK cells had significantly increased mRNA levels of inflammatory cytokines including IL1β, TNF, GM-CSF; cytokine receptors Il6st, Il-2Ra; and negative regulators of STAT signaling SOCS1/2/3 and CIS. Furthermore, IL-6-STAT3 and IL1β/TNF-signaling gene signatures were enriched in CML-exposed NK cells – an effect that is likely to be triggered by inflammatory cytokines. Thus, we next found that peripheral blood serum from CML mice dampens healthy NK cell degranulation ex vivo , compared to serum from control mice. Further ELISA analysis revealed elevated levels of IL-6, IL-1, TNF, and GM-CSF in CML serum samples. Some of these cytokines might be secreted by functionally skewed NK cells, as suggested by gene expression analysis. Altogether, our data suggest that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing optimal targets for NK-boosting CML immunotherapies.

PREDICTION AND DIAGNOSIS OF REPRODUCTIVE FAILURES BASED ON NKp46 EXPRESSION IN ENDOMETRIAL OR DECIDUAL NK CELLS

Natural Killer (NK) cells are the most abundant immune cells at the time of implantation and early stage of pregnancy in uterine endometrium and decidua. NK cells have important roles in the achievement and maintenance of pregnancy. NKp46, an NK cell receptor, is involved in cytotoxicity and cytokine production. We have previously reported the lower expression of NKp46 on uterine and peripheral blood NK cells in women with reproductive failures, such as repeated implantation failures or recurrent pregnancy loss (RPL).

756P Glypican-3 (GPC3) and NKp46 directed FLEX-NK engager antibody (CYT-303) recruits natural killer (NK) cells to tumors in a preclinical hepatocellular carcinoma (HCC) mouse model

GPC3 is an oncofetal antigen that is highly expressed in HCC while minimally expressed in adult normal tissues except placenta. CYT-303 is a multifunctional bispecific NK engager targeting NK cell activating receptor NKp46 and GPC3 expressed on tumor cells constructed with Cytovia’s Flex-NK scaffold with a fully functional Fc. Previously, we reported CYT-303 in vitro treatment of Hep3B tumor cells demonstrated cytolysis and cytokine production as well as in vivo tumor growth inhibition when combined with both PBNK and iNK cells. Here we further characterized the CYT-303 dose-response for tumor growth inhibition in vivo and its influence on NK cell distribution. CYT -303 pharmacokinetics was evaluated in tumor-bearing mice. Anti-tumor efficacy and dose response of CYT-303 in the presence of human PBNKs or iNKs was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors. Biodistribution of the NK cells were analyzed by flow cytometry. CYT-303 pharmacokinetics in tumor bearing mice was similar to normal mice suggesting no target mediated clearance. In the Hep3B tumor model in PBNK or iNK injected NSG-hIL-15 mice, CYT-303 showed dose dependent tumor growth inhibition compared to control hIgG1 treated mice. Consistent with CYT-303 tumor growth inhibition, increased NK cells were detected in the tumor compared to control animals. Blood NK cells in CYT-303 treated animals were significantly lower compared to IgG1 isotype control treated mice suggesting CYT-303 could facilitate NK cell trafficking from blood into the tumor. Blood AFP (alpha fetoprotein), a biomarker in HCC, decreased with CYT-303 tumor growth inhibition. Pharmacologically active CYT-303 doses were identified. CYT-303 can help recruit NK cells to the tumor site to achieve tumor growth inhibition.

How Can We Improve Vaccination Response in Old People? Part I: Targeting Immunosenescence of Innate Immunity Cells.

Vaccination, being able to prevent millions of cases of infectious diseases around the world every year, is the most effective medical intervention ever introduced. However, immunosenescence makes vaccines less effective in providing protection to older people. Although most studies explain that this is mainly due to the immunosenescence of T and B cells, the immunosenescence of innate immunity can also be a significant contributing factor. Alterations in function, number, subset, and distribution of blood neutrophils, monocytes, and natural killer and dendritic cells are detected in aging, thus potentially reducing the efficacy of vaccines in older individuals. In this paper, we focus on the immunosenescence of the innate blood immune cells. We discuss possible strategies to counteract the immunosenescence of innate immunity in order to improve the response to vaccination. In particular, we focus on advances in understanding the role and the development of new adjuvants, such as TLR agonists, considered a promising strategy to increase vaccination efficiency in older individuals.

The MICA-NKG2D axis in clear cell renal cell carcinoma bolsters MICA as target in immuno-oncology

ABSTRACT NKG2D is a major natural killer (NK) cell-activating receptor that recognizes eight ligands (NKG2DLs), including MICA, and whose engagement triggers NK cell effector functions. As NKG2DLs are upregulated on tumor cells but tumors can subvert the NKG2D-NKG2DL axis, NKG2DLs constitute attractive targets for antibody (Ab)-based immuno-oncology therapies. However, such approaches require a deep characterization of NKG2DLs and NKG2D cell surface expression on primary tumor and immune cells. Here, using a bioinformatic analysis, we observed that MICA is overexpressed in renal cell carcinoma (RCC), and we also detected an association between the NKG2D-MICA axis and a diminished overall survival of RCC patients. Also, by flow cytometry (FC), we observed that MICA was the only NKG2DL over-expressed on clear cell renal cell carcinoma (ccRCC) tumor cells, including cancer stem cells (CSC) that also coexpressed NKG2D. Moreover, tumor-infiltrating leukocytes (TIL), but not peripheral blood lymphoid cells (PBL) from ccRCC patients, over-expressed MICA, ULBP3 and ULBP4. In addition, NKG2D was downregulated on peripheral blood NK cells (PBNK) from ccRCC patients but upregulated on tumor-infiltrating NK cells (TINK). These TINK exhibited impaired degranulation that negatively correlated with NKG2D expression, diminished IFN-γ production, upregulation of TIM-3, and an impaired glucose intake upon stimulation with cytokines, indicating that they are dysfunctional, display features of exhaustion and an altered metabolic fitness. We conclude that ccRCC patients exhibit a distorted MICA-NKG2D axis, and MICA emerges as the forefront NKG2DL for the development of targeted therapies in ccRCC.

Circulating natural killer cells in patients with symptomatic atherosclerosis acquire the CD9+CD49A pro-inflammatory phenotype and support M1-like macrophage polarization

Background and Aims : Inflammation represents a hallmark of atherosclerosis (ATS), with monocytes/macrophages and T cells as the most investigated immune cells. Natural killer (NK) cells have been proposed as immunoregulatory cells in ATS, but the mechanisms still require investigations. We phenotypically and functionally characterized peripheral blood NK cells in patients with symptomatic atherosclerosis (sATS) and investigated their functional interactions with endothelial cells and monocyte/macrophages.

Improving NK cell function in multiple myeloma with NKTR-255, a novel polymer-conjugated human IL-15

AbstractMultiple 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 natural killer (NK) cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal to increase tumor cell recognition, avoid tumor escape and potentially enhancing the effect of other drugs. In this study, we evaluated the ability of the investigational medicine NKTR-255, an IL-15 receptor agonist, to engage the IL-15 pathway and stimulate NK cells against MM cells. We observed that incubation with NKTR-255 was able to tilt the balance toward an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of patients with MM, with increased expression of activating receptors 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. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared with 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 observed that combination of NKTR-255 with the anti-CD38 antibody, daratumumab, was effective against MM cells in vitro and in vivo. Taken together, our data suggest a significant impact of NKTR-255 in inducing NK cell function against MM cells with important translational implications.