Higher Natural Killer Cell Research Articles

Non-invasive image-based cytometry for high throughput NK cell cytolysis analysis

Natural Killer (NK) cells are lymphocytes that are the first line of defense against malignantly transformed cells, virally infected cells and other stressed cell types. To study the cytolytic function of NK cells in vitro, a cytotoxicity assay is normally conducted against a target cancerous cell line. Current assay methods are typically performed in mixed 2D cocultures with destructive endpoints and low throughput, thereby limiting the scale, time-resolution, and relevance of the assay to in vivo conditions. Here, we evaluated a novel, non-invasive, quantitative image-based cytometry (qIBC) assay for detection of NK-mediated killing of target cells in 2D and 3D environments in vitro and compared its performance to two common flow cytometry- and fluorescence-based cytotoxicity assays. Similar to the other methods evaluated, the qIBC assay allowed for reproducible detection of target cell killing across a range of effector-to-target ratios with reduced variability. The qIBC assay also allowed for detection of NK cytolysis in 3D spheroids, which enabled scalable measurements of cell cytotoxicity in 3D models. Our findings suggest that quantitative image-based cytometry would be suitable for rapid, high-throughput screening of NK cytolysis in vitro, including in quasi-3D structures that model tissue environments in vivo.

Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+ /CD54+ NK cells.

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25+/CD54+ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25+/CD54+ NK cells for adoptive cell therapy should be considered.

Efficacy of natural killer cell activity as a biomarker for predicting immunotherapy response in non-small cell lung cancer.

BackgroundThe establishment of biomarkers that can be used to predict the response to immunotherapy for malignancy is extremely important. In particular, noninvasive analysis of immune cells from peripheral blood before treatment has gained increased attention, and natural killer (NK) cell activity has been shown to be related to treatment response. Here, we aimed to confirm the relationship between the response to immunotherapy and NK cell activity.MethodsIn this prospective observational study, patients with advanced NSCLC who were scheduled for immunotherapy from October 2018 to December 2019 were enrolled. Baseline NK cell activity was compared according to the best clinical response to immunotherapy.ResultsA total of 54 patients with advanced NSCLC were enrolled, and 34 patients were analyzed. The baseline NK cell activity was significantly higher in the non‐PD group than in the PD group (P = 0.002). At the cutoff level of ≥1200 pg/mL, baseline NK cell activity yielded a sensitivity of 80% and a specificity of 68.4% in predicting the response to immunotherapy (AUC = 0.8, P < 0.003). The median progression‐free survival (PFS) was significantly better in the high NK group (P = 0.003), and correlation between baseline NK cell activity and PFS was also confirmed (r = 0.517, P = 0.002).ConclusionsBaseline NK cell activity was related to the response to immunotherapy and the PFS. We suggest that NK cell activity from peripheral blood before immunotherapy is a noninvasive, simple, and novel way to predicting the treatment response in patients with NSCLC.Key pointsSignificant findings of the study The response to immunotherapy was significantly better in patients with high baseline NK cell activity, and there was a significant correlation between baseline NK cell activity and PFS. What this study adds This study demonstrates the efficacy of baseline NK cell activity from peripheral blood as a biomarker for predicting immunotherapy response.

Identifying prostate cancer and its clinical risk in asymptomatic men using machine learning of high dimensional peripheral blood flow cytometric natural killer cell subset phenotyping data

We demonstrate that prostate cancer can be identified by flow cytometric profiling of blood immune cell subsets. Herein, we profiled natural killer (NK) cell subsets in the blood of 72 asymptomatic men with Prostate-Specific Antigen (PSA) levels < 20 ng ml-1, of whom 31 had benign disease (no cancer) and 41 had prostate cancer. Statistical and computational methods identified a panel of eight phenotypic features ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text]) that, when incorporated into an Ensemble machine learning prediction model, distinguished between the presence of benign prostate disease and prostate cancer. The machine learning model was then adapted to predict the D'Amico Risk Classification using data from 54 patients with prostate cancer and was shown to accurately differentiate between the presence of low-/intermediate-risk disease and high-risk disease without the need for additional clinical data. This simple blood test has the potential to transform prostate cancer diagnostics.

Blood natural killer cells during treatment in recurrent ovarian cancer

Objective Recent research indicated favorable prognostic impact of intratumoral natural killer (NK) cells in ovarian carcinoma (OC). The role of NK cells during chemotherapy in OC is unknown. We investigated impact of NK cells in OC patients treated with palliative chemotherapy. Methods Participants receiving palliative chemotherapy for recurrent OC (N = 72) had prospectively blood samples at baseline and before cycle 2. NK cell counts were quantified by flow cytometry. NK cell activity was measured by the NK Vue® assay, estimating interferon-gamma production. Overall survival (OS) was the primary endpoint. Cutoffs were predefined, NK numbers (≥184 × 106 cells/L vs. <184 × 106 cells/L) and NK activity (<200 pg/mL vs. ≥200 pg/mL). Results Median OS in patients with low vs. high NK cell count at baseline was 7.1 months vs. 15.6 months (p = .028), respectively, and before cycle 2 was 5.7 vs. 17.3 months, p < .001, respectively. The difference in restricted mean survival (ΔRMST) was 5.7 months (95% CI: 3.3–8.0) at cycle 2 vs. 2.5 months (95% CI: −0.6 to 5.6) at baseline, showing a significant difference with no overlap of confidence intervals. In multivariate analyses, low NK cell count remained significant with a hazard ratio (HR)=2.83, 95% CI: 1.53–5.22, p = .001 (baseline) and HR = 3.34, 95% CI: 1.67–6.71, p = .001 (before cycle 2). Patients with both low NK count and NK activity at baseline (N = 20) had median OS 6.5 months vs. 11.5 months in patients with either high activity, high count or both (p = .007). In parallel, patients with both low NK activity and count at cycle 2 (N = 18) had a median survival of 4.0 months vs. 15.4 months (p < .001). Conclusions A low blood NK cell count in recurrent metastatic ovarian cancer during chemotherapy is associated with unfavorable prognostic impact. Early increase in survival difference based on NK cell status suggests an association between NK cell count and treatment benefit.

Improved Relapse-Free Survival in Patients With High Natural Killer Cell Doses in Grafts and During Early Immune Reconstitution After Allogeneic Stem Cell Transplantation.

Mature immunocompetent cells from the stem cell graft as well as early robust immune reconstitution are essential for the graft-vs. -tumor (GVT) effect to eliminate residual malignant cells after allogeneic hematopoietic stem cell transplantation (HSCT). In this prospective study we characterized graft composition of T- and NK cell subsets in 88 recipients of peripheral blood stem cell grafts with multicolor flowcytometry. Our primary aim was to analyze the impact of graft composition on immune reconstitution and clinical outcomes after transplantation. Patients transplanted with graft NK cell doses above the median value of 27 × 106/kg had significantly increased relapse-free-survival compared to patients transplanted with lower doses, HR 2.12 (95% CI 1.01–4.45, p = 0.04) Peripheral blood concentrations of NK cells obtained from donors before G-CSF mobilization were significantly correlated to graft NK cell doses (Spearman's ρ 0.53, p = 0.03). The dose of transplanted NK cells/kg correlated significantly with NK cell concentrations in patients early after transplantation (Spearman's ρ 0.26, p = 0.02, and ρ = 0.35, p = 0.001 for days 28 and 56, respectively). Early immune reconstitution above median values of NK cells was significantly associated with improved relapse-free survival (HR 2.84 [95% CI 1.29–6.28], p = 0.01, and HR 4.19 [95% CI 1.68–10.4], p = 0.002, for day 28 and 56, respectively). Early concentrations above the median value of the mature effector CD56dim NK cell subset were significantly associated with decreased relapse incidences at 1 year, 7% (95% CI 1.8–17) vs. 28% (95% CI 15–42), p = 0.04, and 7% (95% CI 1.8–18) vs. 26% (95% CI 14–40) %, p = 0.03, for days 28 and 56, respectively. The results suggest a protective effect of high doses of NK cells in grafts and during early immune reconstitution and support the perception of NK cells as innate effector cells with anti-tumor effects in the setting of allogeneic stem cell transplantation.

Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint destruction. Previous studies have shown that natural killer (NK) cells may play an important role in the pathogenesis of RA. Interleukin (IL)-15, a pro-inflammatory cytokine which induces proliferation and differentiation of NK cells, is overexpressed in RA. In this present study, we examine various NKRs and adhesion molecule expression on NK cells from RA patients and their response to IL-15 stimulation. We also sought to study cytokine-induced memory-like (CIML) NK cells in RA patients. We established that 1. RA patients had higher NK cell percentages in peripheral blood and their serum IL-15 levels were higher compared to healthy volunteers; 2. NK cells from RA patients showed lower NKp46 expression and an impaired CD69 response to IL-15; 3. NK cells from RA patients showed higher CD158b and CD158e expression but lower CD62L expression; 4. exogenous IL-15 up-regulated CD69, CD158b, CD158e but down-regulated NKp46 and CD62L expression in RA; 5. As to CIML NK cells, restimulation - induced NK cytotoxicity and IFN-γ production was impaired in RA patients, 6. Reduced NKp46, perforin, and granzyme B expression on NK cells was found in RA patients with bone deformity and erosion, 7. RA disease activity (DAS28) showed inverse correlation with the percentages of CD56+CD3− NK cells, and NKp46 and perforin expression on NK cells, respectively. Taken together, our study demonstrated differential expression of various NK receptors in RA patients. NKp46, CD158e, and perforin expression on NK cells may serve as markers of RA severity.

Peripheral blood natural killer cell proportion and ovarian function in women with recurrent implantation failure

Current knowledge of the association between peripheral natural killer (NK) cell proportion and ovarian function in reproductive-age women is limited. We explored the association between NK cell proportion and ovarian function in women who underwent in-vitro fertilization (IVF) treatment. This was a retrospective cohort study using the data of 20–44-year-old women with recurrent implantation failure (RIF) who were tested for NK cell proportion and anti-Müllerian hormone (AMH). Indicators of ovarian function included AMH, observed-to-(age-appropriate) reference AMH ratio, high FSH, peak E2 and total number of oocytes during the first IVF cycle following the test. We used different model specification controlling for women’s age, and body mass index. Among a total of 936 women, majority showed lower AMH compared to age-appropriate level. Average NK cell proportion was 13.5 ± 5.7%. Number of oocytes showed positive association with NK cell (ß = 0.040, p = .025). In the subgroup with NK ≥ 18%, NK cell proportion was negatively associated with AMH (−0.106, p = .012), AMH ratio (−0.049, p = .014) and number of oocytes (−0.021, p < .001) while the associations with others remain close to null. High NK cell proportion may be harmful to ovarian reserve or function.

A simple method for in vitro preparation of natural killer cells from cord blood

BackgroundCord Blood (CB) has been considered a promising source of natural killer (NK) cells for cellular immunotherapy. However, it is difficult to expand the large numbers of highly pure NK cells from CB without cell sorting and feeder cells/multiple cytokines. In this study, we try to develop a simple, safe and economical method for ex vivo expansion and purification of NK cells from CB without cell sorting and feeder cells/multiple cytokines.ResultsThe large numbers (mean: 1.59 × 1010) of highly pure (≥90%) NK cells from CB could be obtained through interleukin-2, group A streptococcus and zoledronate stimulation of mononuclear cells using the 21-day culture approach. When compared to resting NK cells, expanded NK cells were a higher expression of activating receptors CD16, NKG2D, NKp30, NKp44, NKp46 and activating markers CD62L and CD69, while the inhibitory receptors, CD158a and CD158b remained largely unchanged. In addition, these cells showed a higher concentration of IFN-γ, TNF-α and GM-CSF secretion and cytotoxicity to K562 cells and acute myeloid leukemia targets than resting NK cells.ConclusionWe develop a simple, safe and economical method to obtain high yield, purity, and functionality NK cells from CB without cell sorting and feeder cells/multiple cytokines.

High Natural Killer Cell Count at Diagnosis Predicts a Good Response to Immunosuppressive Therapy in Aplastic Anemia

Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, &lt; 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P&lt;0.001, P&lt;0.001, respectively). On the other hand, the recoveries in the number of CD3+, CD4+, CD8+, and Tregs cells were similar in both groups (Figure 2). Three relapses (among 21 responders) in higher NK cells group, and four relapses (among 28 responders) in lower NK cells group were observed. No patients developed clonal evolution in both groups. Two patients in the lower NK cell group died after IST. The etiologies of death included infection and unknown causes. Transplantation-free survival (TFS), failure-free survival (FFS), and overall survival (OS) at two years were similar between the higher and lower NK cell groups [TFS, 90% (95% CI, 64%−97%) vs. 78% (95% CI, 63%−87%); P = 0.147; FFS, 66% (95% CI, 45%−81%) vs. 50% (95% CI, 35%−62%); P = 0.147; OS, 100% (95% CI, 100%−100%) vs. 94% (95% CI, 76%−98%); P = 0.277]. Conclusions: The current study indicates that NK cells play a role in the pathogenesis of AA and may be the predictors of response to IST. Recovery of NK cell count correlated with hematopoietic recovery following IST, indicating that they may be used as markers to assess disease activity, treatment response and relapse. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

1907P - Low NK cell abundance correlates with high expression of PD-1 in CD8+ T cells

BackgroundRenal cell carcinoma (RCC) is considered an immunogenic cancer with a high frequency of indel mutations, immuno-oncological (IO) sensitivity, and infiltration of T cells. Because natural killer (NK) cells are a lesser known population compared to their T cell counterparts, we aimed to investigate the intratumoral phenotype of the cells to assess the overall immune landscape of RCC. MethodsThe tumor, matching peripheral blood (PB) and healthy kidney tissue samples were obtained from 40 RCC patients that underwent radical nephrectomy and were phenotyped using multi-parameter flow cytometry and a comprehensive immunostaining panel containing 56 fundamental markers to cancer immunology. ResultsUsing hierarchical clustering and correlation analysis, we discovered that our cohort clustered into two distinct subgroups defined by the abundance of NK cells from the intratumoral lymphocyte population, defined as NKhigh (n=17; mean 24.3%) and NKlow (n=18; mean 7.5%). Consequently, an increased abundance of NK cells correlated with a lower expression of PD-1, particularly in the CD8+ T cells (r=-0.5722, p=0.0003), but not with LAG-3. Contrastingly, clinical parameters such as tumor grade, gender and age did not differ between the two NK subgroups. However, patients with high NK cell abundance significantly had more often necrosis present in the tumors (p=0.04). We also independently phenotyped two regions of the same tumor sample (n=10) to compare the periphery and core lymphocytes. Surprisingly, both spatially different regions displayed similar quantities of immune cell populations and immunophenotypic markers. Overall, the tumor immune landscape in RCC resulted in a proportion of tumors carrying a higher abundance of NK cells than their T cell-rich corresponding PB and healthy tissue samples, which supports our findings that a proportion of tumors accumulate NK cells. ConclusionsOur study has led to the discovery of two distinct RCC tumoral NK cell subgroups, NKhigh and NKlow. Next, we aim to further explore the transcriptional differences of the two subgroups with ongoing bulk RNA sequencing (RNAseq). Furthermore, prospective single-cell RNA sequencing (sc-RNAseq) will be carried out to investigate the immunological heterogeneities in higher resolution. Legal entity responsible for the studyThe authors. FundingUniversity of Helsinki, Hematology Research Unit Helsinki. DisclosureAll authors have declared no conflicts of interest.

Availability of NK cell expansion agent combined with recombinant IL‑2 and IL‑15 stimulation on the expansion and high‑purity of NK cells in patients with immune‑related pancytopenia invitro.

Natural killer (NK) cells are a group of large granular lymphocytes that play an important regulatory role in innate immunity and adaptive immunity. Immune‑related pancytopenia (IRP) is a type of pancytopenia resulting from bone marrow hematopoietic cells that were destroyed or suppressed by auto‑antibodies. The specific mechanism of IRP is not clear. In the present study, it was identified that the percentage of NK cells in peripheral blood lymphocytes was decreased in patients with IRP. Subsequently, high purity NK cells were extracted from 6 untreated patients with IRP using the immunomagnetic beads sorting, magnetic‑activated cell‑sorting method, which were then cultured then in RPMI‑1640 medium containing 20%FBS. NK cell expansion agents, with or without recombinant interleukin (IL)‑15, were used to amplify high‑purity NK cells on the basic of recombinant IL‑2. Expression of the activated receptors NKG2‑D typeII integral membrane protein (NKG2D) and natural killer cell p46‑related protein (NKp46), and the inhibitory receptors CD158a and NKG2‑A/NKG2‑B typeII integral membrane protein (NKG2A), in CD56+ NK cells were detected by flow cytometry before and after cell culture. It was observed that treatment with an NK cell expansion agent combined with the stimulation of recombinant IL‑2 and recombinant IL‑15 could increase the number whilst maintaining the purity of NK cells. There were no significant changes in the expression of NKG2D, NKp46, NKG2A and CD158a in patients with IRP before and after NK cell culture. This new amplification method lays a foundation for clinical NK cell immunotherapy and anti‑tumor applications.

Effects of regulatory T cells, natural killer cells, and natural killer T cells on immunosuppression therapy in patients with recurrent embryo implantation failure

Objective: This retrospective study aims to investigate the effects of immunosuppressive therapy on the expression of regulatory T (Treg) cells, natural killer (NK) cells, and natural killer T (NKT) cells in patients with recurrent embryo implantation failure (RIF). Materials and Methods: In vitro fertilization and embryo transplantation (IVF-ET) at Jinhua Municipal Central Hospital from July 2013 to November 2015 of 30 RIF patients were enrolled into RIF group, and peripheral blood Treg, NK, and NKT were detected before entering the embryo transfer cycle. Meanwhile, 20 normal non-pregnant women were enrolled into control group. Twenty-four patients in RIF group were administered prednisone; Treg, NK, and NKT cell contents were detected twice before and after the embryo transfer cycle, respectively. The remaining six patients with high NK cell contents were treated with gamma globulin via intramuscular injection, and Treg, NK, and NKT cell contents were detected twice before the embryo transfer cycle and after treatment. Results: Treg cell content was significantly lower in RIF group than in control group, while NK cell proportion was significantly higher in RIF group than in control group before treatment. There was no statistical difference in NKT cells. After treatment, expression rate of Treg in RIF group was 7.1 ± 1.8%, which compared with that before treatment (2.8±1.6%) was significantly higher (p &lt; 0.01). In addition, NK cell proportion was significantly lower than pretherapy (20.9 ± 3.6% vs. 38.6 ± 8.1%, p &lt; 0.05) and NKT cell percentage did not present the obvious difference before and after treatment. Conclusion: The occurrence of RIF may be related to the decrease in Treg expression and increase of NK cells. Immunotherapy can upregulate the expression of Treg and decrease NK cell proportion, thereby regulating maternal fetal immune tolerance and reducing the rejection effect of NK cells on fetal foreign bodies, which are conducive to embryo implantation.

Enumeration of peripheral blood NKp46 positive NK lymphocytes reflects NK cytotoxic activity in vitro

Natural killer (NK) cells are the predominant innate lymphocyte subsets that mediate anti-tumor and anti-viral responses. The monitoring of NK cells function is important in various physiological and pathological conditions. Different approaches have been used to directly or indirectly evaluate NK cells activities.The purpose of this study was to investigate the correlation between the number of NK cells and cytotoxic activity of NK cells and to determine whether NKp46+NK cells reflect NK cytotoxicity status.In our study, we retrospectively analyzed laboratory data on NK cytotoxicity and NK lymphocyte levels of 4896 infertile women which underwent routine immunology investigation after IVF failures. In healthy women, NKp46 expression was assessed on NK cells (n = 214) and cytotoxicity activity was evaluated with regard to NKp46 expression. We found that despite a significant correlation coefficient (n = 4689, r = 0.447), the correlation with cytotoxicity is maintained only within the zones with a low or high NK cells frequency. NK cells frequency has no significant prognostic value for their cytotoxicity – within the medium NK frequency zone the samples may have any cytotoxicity, both reduced and elevated.However, our data demonstrate that NKp46+NK cells frequency correlates with cytotoxicity activity even more significantly than the NK cells frequency (n = 214, r = 0.67 and r = 0.62, respectively) and has significant prognostic value for the abnormal NK cytotoxicity status indications, both low and increased.Our results further support an important role of NKp46 in NK cells killing and afford grounds for using the measurement of the NKp46+NK cells frequency as an alternative method for abnormal NK cytotoxicity status indication, which is responsive, simple and reliable.

Intrinsic Resistance to Immune Checkpoint Blockade in a Mismatch Repair–Deficient Colorectal Cancer

Immunotherapy with checkpoint inhibitors, such as the programmed death-1 (PD-1) antibodies pembrolizumab and nivolumab, are effective in a variety of tumors, yet not all patients respond. Tumor microsatellite instability-high (MSI-H) has emerged as a biomarker of response to checkpoint blockade, leading to the tissue agnostic approval of pembrolizumab in MSI-H cancers. Here we describe a patient with MSI-H colorectal cancer that was treated with this immune checkpoint inhibitor and exhibited progression of disease. We examined this intrinsic resistance through genomic, transcriptional, and pathologic characterization of the patient's tumor and the associated immune microenvironment. The tumor had typical MSI-H molecular features, including a high neoantigen load. We also identified biallelic loss of the gene for β2-microglobulin (B2M), whose product is critical for antigen presentation. Immune infiltration deconvolution analysis of bulk transcriptome data from this anti-PD-1-resistant tumor and hundreds of other colorectal cancer specimens revealed a high natural killer cell and M2 macrophage infiltration in the patient's cancer. This was confirmed by single-cell transcriptome analysis and multiplex immunofluorescence. Our study provides insight into resistance in MSI-H tumors and suggests immunotherapeutic strategies in additional genomic contexts of colorectal cancer.

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1–2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log −3.6 after CD3 depletion and log −3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2–8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector–target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

Clinical factors associated with pregnancy outcome in women with recurrent pregnancy loss

The aim of this prospective cohort study was to evaluate clinical factors associated with pregnancy outcomes in women with recurrent pregnancy loss (RPL). Women with a history of two or more pregnancy losses underwent workups for clinical factors of RPL and their pregnancies were followed-up with informed consent. Two hundred eleven (81.5%) of 259 women with RPL became pregnant. The multivariable analyses demonstrated that age (p < .01, OR 0.9, 95%CI 0.97–0.83), uterine abnormality (p < .05, OR 0.3, 95%CI 0.11–0.8), and protein C (PC) deficiency (p < .01, OR 0.14, 95%CI 0.03–0.6) were independent factors for becoming pregnancy in women with RPL. The number of previous pregnancy loss (p < .01, OR 0.57, 95%CI 0.43–0.75) and natural killer (NK) cell activity ≥33% (p < .01, OR 0.31, 95%CI 0.13–0.73) were independent factors for live birth in the subsequent pregnancy. Advanced age, the presence of uterine abnormality, and PC deficiency were risk factors for reduced pregnancy rate in women with RPL. Increased number of previous pregnancy loss and high NK cell activity were risk factors for miscarriage in the subsequent pregnancy. These results involve important information and are helpful for clinical practitioners.

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies. There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.

Direct and antibody-dependent cell-mediated cytotoxicity of head and neck squamous cell carcinoma cells by high-affinity natural killer cells

High affinity natural killer cells (haNKs) are a cell therapy product capable of mediating both direct and antibody-dependent cell-mediated cytotoxicity (ADCC). These cells may be particularly useful in tumors that escape T-cell anti-tumor immunity by harboring antigen processing and presentation defects. Here, we demonstrated that haNKs directly kill both HPV-positive and negative head and neck squamous cell carcinoma cells. Variable tumor cell sensitivity to haNK direct cytotoxicity did not correlated with MHC class I chain-related protein A or B (MICA or MICB) expression. Importantly, haNK killing was significantly enhanced via ADCC mediated by cetuximab or avelumab in cells with higher baseline EGFR or PD-L1 expression, respectively. The ability of IFNγ to induce tumor cell PD-L1 expression correlated with enhanced PD-L1-specific ADCC. IFNγ induced neither tumor cell EGFR expression nor EGFR-specific ADCC. Although a single dose of 8 Gy IR did not appear to directly enhance susceptibility to haNK killing alone, enhanced PD-L1- and EGFR-mediated ADCC after IR correlated with increased PD-L1 and EGFR expression in one of four models. This pre-clinical evidence supports the investigation of haNK cellular therapy in combination with ADCC-mediating mAbs, with or without IR, in the clinical trial setting for patients with advanced HNSCCs. Given the MHC-unrestricted nature of this treatment, it may represent an opportunity to treat patients with non-T-cell inflamed tumors.

Ruxolitinib Treatment for Steroid Refractory ACUTE and Chronic GRAFT Versus Host Disease in Children: Clinical and Immunological Results

Background Ruxolitinib is a promising treatment for steroid refractory graft-versus-host disease (GvHD). However, data concerning effects on T cells are probably involved in increased risk of opportunistic infections. We analyzed clinical and immunological changes in children with GvHD taking ruxolitinib. Patients and Methods Twenty-two children that underwent transplantation and received ruxolitinib were included. Ruxolitinib indication was acute and chronic GvHD in 13 and 9 patients, respectively. Results Overall response rate in acute GvHD and chronic GvHD was high, of 77% and 89%, respectively. Ruxolitinib was associated with an increase in CD4 effector memory, and decrease of CD4 central memory percentage (Figure 1). CD4 regulatory T cells percentage decreased significantly (Figure 2). Patients who achieved complete response to ruxolitinib had higher natural killer cells before ruxolitinib that patients who did not respond. Also there was increase of CD4 lymphocytes percentage, with decrease of CD8 and natural killer cells percentage in responders against non-responders. There were 54%, 18% and 13% infections caused by virus, bacteria and fungi, respectively. Cumulative incidence of relapse and non relapse mortality was 19±9%and 28±10%, respectively. Overall survival and disease free survival rate at 2 years were 62±11% and 58±11%, respectively. Conclusions Ruxolitinib is a promising treatment for GvHD with a high overall response rate of 77% and 89%, respectively. It produces important changes in immune system, such as increase of CD4 effector memory cells and decrease in natural killer and regulatory T cells. Now we need pharmacokinetic studies to determine ruxolitinib dose in children and close surveillance and antimicrobial prophylaxis. Keywords Ruxolitinib, GvHD, children, immune reconstitution