Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Analysis of the SARS-CoV-2-specific ADCC responses. Analysis of the kinetics of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response in plasma obtained from hospitalized patients with COVID-19 ( n = 19) collected at different time points: 3 ±1 ( n = 7), 6 ±1 ( n = 14), 9 ±1 ( n = 15), 12 ±1 ( n = 18), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptoms onset or nonhospitalized, mildly ill patients with COVID-19 ( n = 9) 28 to 31 (median: 30) days after symptom onset. Each plasma sample as well as each of 6 control samples from SARS-CoV-2 seronegative persons was individually tested against 26 different CD56 + CD16 + NK cell preparations from SARS-CoV-2 seronegative healthy blood donors and the percentage of CD107a (A), perforin (B), IFNγ (C), or TNFα (D) positive cells was assessed using flow cytometry. Percentage of all CD107a (A), IFNγ (C), or TNFα (D) positive cells, as well as only highly perforin-expressing cells (B) was assessed. All samples were normalized to the same SARS-CoV-2 seronegative control. From each sample, the median of independent experiments was calculated. Data are shown as mean values (±95% CI). ADCC positive plasma samples were identified using 6 SARS-CoV-2 seronegative controls. Fold change in positive cells at each time point were compared either between hospitalized patients with COVID-19 (indicated as a red bar) or mildly diseased nonhospitalized patients with COVID-19 (indicated as a blue bar) and seronegative controls (indicated as a black bar) using analysis of variance and Dunn’s post test (hospitalized patients with COVID-19) or Mann-Whitney test (nonhospitalized patients with COVID-19). P < .05 was considered significant. IFNγ, interferon gamma; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Control, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Impact of the FcγRIIIa-158-V/F variants on the ADCC responses. Analysis of the extent of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response with plasma obtained from hospitalized patients with COVID-19 ( n = 19) 6 ±1 ( n = 10), 9 ±1 ( n = 14), 12 ±1 ( n = 14), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptom onset (A-D) or nonhospitalized patients with COVID-19 ( n = 9) 28 to 31 (median: 30 days) days after symptom onset (E-H). Each plasma sample from hospitalized and nonhospitalized patients with COVID-19 was stimulated with CD56 + CD16 + NK cells from 26 healthy blood donors (FcγRIIIa-158-V/V: n = 5, FcγRIIIa-158-V/F: n = 12, FcγRIIIa-158-F/F: n = 9) and the MFI of CD107a (A,E), perforin (B,F), IFNγ (C,G), or TNFα (D,H) positive cells were assessed using flow cytometry. All samples were normalized to the same nonhospitalized SARS-CoV-2 seropositive control. MFI of all CD107a (A), IFNγ (C) or TNFα (D) positive cells, as well as of only high perforin-expressing cells (B) was assessed. Data are shown as mean values (±95% CI). Fold change MFI at each time point was compared between assays using FcγRIIIa-158-V/V, FcγRIIIa-158-V/F, and FcγRIIIa-158-F/F variant expressing NK cells, respectively using a ANOVA (A-D) or a paired t test (E-H). P < .05 was considered significant. ANOVA, analysis of variance; d.p.s.o, days post symptom onset; IFNγ, interferon gamma; MFI, mean fluorescence intensity; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Flow Cytometry, Control, Expressing, Variant Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin

doi: 10.3389/fimmu.2023.1275368

Figure Lengend Snippet: Primary human CD8 + T cells and NK cells express the IL-2 receptor βγ. (A) Composition of the IL-2 receptor (IL-2R). The IL-2Rα subunit (CD25) alone presents the low-affinity receptor, which lacks signalling capacity du to missing intracellular signalling domains. The intermediate-affinity receptor consists of the IL-2Rβ (CD122) and -γ (CD132) chains and the high-affinity receptor is consitituted of all three subunits. Adapted from . (B-E) The mRNA of IL-2Rα, -β, and –γ was quantified via qPCR (B) in freshly isolated CD8 + T cells from buffy coats ( N = 5), (C) freshly isolated NK cells ( N = 4), (D) primary human hepatocytes ( N = 6) and (E) the HepaRG cell line ( N = 5). Data are shown as mean ± SEM. QPCR reactions were prepared in technical duplicates and no statistical analyses was performed. NK, natural killer cells; PHHs, primary human hepatocytes; nd, not detectable.

Article Snippet: NK cells were activated with the human NK Cell Activation/Expansion Kit (anti-CD2, anti-CD335(NKp46), Miltenyi Biotec, Bergisch Gladbach, Germany) and 500 IU/mL rhIL-2 or with the human NK Cell Activation/Expansion Kit and rhIL-15 (f.c.

Techniques: Isolation

Journal: Frontiers in Immunology

Article Title: Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin

doi: 10.3389/fimmu.2023.1275368

Figure Lengend Snippet: Aldesleukin increases primary human CD8 + T and NK cell cytotoxicity and adhesion marker expression. (A) Gating strategy for surface marker analysis and determination of fractions of viable, early apoptotic and late apoptotic cells. Representative plots from one CD8 + T cell monoculture experiment are shown. (B-G) Primary human CD8 + T cells were stimulated for 5 d with aldesleukin or activation controls (act I, CD3/CD28 + rhIL-2 activation; act II, PHA-L + rhIL-2 activation). (H-M) Primary human NK cells were stimulated for 2 d with aldesleukin or activation controls (act I, CD2/CD335 + rhIL-2 activation; act II, CD2/CD335 + rhIL-15/21 activation). Per stimulation point, 0.1 x 10 6 cells were stimulated. To quantify surface marker expression, cells were analysed by flow cytometry and the mean fluorescence intensity was related to the vehicle control. The fractions of viable (Zombie Violet - /Annexin V - ), early apoptotic (Zombie Violet - /Annexin V + ), and late apoptotic (Zombie Violet + /Annexin V + ) cells were determined as described in the methods section. Number of biological replicates: N = 3 (G) ; N = 4 (D, M) ; N = 5 (E, J) ; N = 6 (B, C, F) . N = 7 (H, I, K) ; N = 9 (L) . In some cases, there are fewer biological replicates for the activation controls. One technical replicate was performed except for CFSE dilution assays. Data are shown as mean ± SEM. For statistical analysis, one-way ANOVA with Dunnett´s correction (D, E, F, I, K, L) , Kruskal Wallis test with Dunn´s multiple comparison test (B, C, H, J) or two-way ANOVA with Dunnett´s correction (G, M) , was used. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicate significant differences between aldesleukin-treated or activated and vehicle-treated samples. CFSE, Carboxyfluorescein succinimidyl ester; ICAM-1, intercellular adhesion molecule 1; TRAIL, Tumor Necrosis Factor Related Apoptosis Inducing Ligand.

Article Snippet: NK cells were activated with the human NK Cell Activation/Expansion Kit (anti-CD2, anti-CD335(NKp46), Miltenyi Biotec, Bergisch Gladbach, Germany) and 500 IU/mL rhIL-2 or with the human NK Cell Activation/Expansion Kit and rhIL-15 (f.c.

Techniques: Marker, Expressing, Activation Assay, Flow Cytometry, Fluorescence, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin

doi: 10.3389/fimmu.2023.1275368

Figure Lengend Snippet: Aldesleukin increased cytotoxicity in direct immune cell/HepaRG co-cultures. (A) For evaluation of cytotoxic effects of immune cells towards HepaRGs, direct and indirect co-cultures were established. (B, C) Primary human CD8 + T cells were stimulated with aldesleukin or activation controls (act I, CD3/CD28 + rhIL-2 activation; act II, PHA-L+ rhIL-2 activation) and co-incubated with HepaRG cells for 5 d (B) in direct co–cultures or (C) in indirect co-cultures in which CD8 + T cells were separated from HepaRG cells by a transwell insert. Colorimetric assessment of lactate dehydrogenase (LDH) activity was conducted to measure cell-mediated cytotoxicity. Samples were referred to the lysis control. (D) Expanded primary human NK cells were pre-treated with aldesleukin or activation controls (act I, CD2/CD335 + rhIL-2 activation; act II, CD2/CD335 + rhIL-15/21 activation) for 42 h and co-cultivated with HepaRG single cell suspensions for 6 h. HepaRG cell viability was determined via flow cytometry and referred to the vehicle-treated samples. (E) Gating strategy for the cytolysis assay. To discriminate HepaRG cells from immune cells, HepaRG cells were stained with CellTrace™ Violet (CTV). Data are shown as mean ± SEM. N = 5 (D) , N = 6 (B) , N = 8 (C) . LDH measurements were acquired in technical duplicates, for the cytolysis assay one technical replicate was acquired. For the cytolysis assay, samples in which activation control I did not induce lysis compared to vehicle-treated samples were excluded from the analyses, as NK cells were perceived as non-activatable. For statistical analysis of (B, D) one-way ANOVA with Dunnett´s correction and for analysis of (C) Kruskal Wallis test with Dunn´s multiple comparison test was used. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicate significant differences between aldesleukin-treated or activated and vehicle-treated samples. L, Triton-X-lysed HepaRG cells; LDH, lactate dehydrogenasse; untr., untreated HepaRG monocultures.

Article Snippet: NK cells were activated with the human NK Cell Activation/Expansion Kit (anti-CD2, anti-CD335(NKp46), Miltenyi Biotec, Bergisch Gladbach, Germany) and 500 IU/mL rhIL-2 or with the human NK Cell Activation/Expansion Kit and rhIL-15 (f.c.

Techniques: Activation Assay, Incubation, Activity Assay, Lysis, Control, Flow Cytometry, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: Immuno-inflammatory in vitro hepatotoxicity models to assess side effects of biologicals exemplified by aldesleukin

doi: 10.3389/fimmu.2023.1275368

Figure Lengend Snippet: Aldesleukin influences hepatic surface marker expression and pro-inflammatory mediator release in direct co-cultures of immune cells with HepaRGs. (A) Flow cytometry gating strategy for surface marker detection on hepatocytes following co-culture with immune cells. Representative plots of one direct CD8 + T/HepaRG cell co-culture experiment are shown. Please note that in the first step, debris is excluded from analysis via a “not-gate”. (B-I) Primary human CD8 + T cells were pre-stimulated with aldesleukin or activation controls (act I, CD3/CD28 + rhIL-2 activation; act II, PHA-L+ rhIL-2 activation) for 3 days and co-cultivated with HepaRG cells for 2 days in direct co-cultures. (J-M) Primary human NK cells were pre-incubated with aldesleukin or activation controls (act I, CD2/CD335 + rhIL-2 activation; act II, CD2/CD335 + rhIL-15/21 activation) for 44.5 h and co-cultivated with HepaRG cells for 3.5 h. (B-E, J-L) Hepatic surface marker expression was determined with flow cytometry and the mean fluorescence intensity was related to the vehicle control. (F-I, M) Pro-inflammatory mediators were quantified via flow cytometry, CRP was determined via ELISA. Data are shown as mean ± SEM. N = 4 (J-M) , N = 5 (B-L) . For (E) , one outlier was removed. Samples were acquired in one technical replicate. For statistical analysis of (C-E, G, J- L) one-way ANOVA with Dunnett´s correction and for (B, F, H, I, M) Kruskal Wallis test with Dunn´s multiple comparison test was used. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate significant differences between aldesleukin-treated or activated and vehicle-treated samples. CRP, c-reactive protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; HLA, human leukocyte antigen; TRAIL-R, TNF-related apoptosis-inducing ligand receptor.

Article Snippet: NK cells were activated with the human NK Cell Activation/Expansion Kit (anti-CD2, anti-CD335(NKp46), Miltenyi Biotec, Bergisch Gladbach, Germany) and 500 IU/mL rhIL-2 or with the human NK Cell Activation/Expansion Kit and rhIL-15 (f.c.

Techniques: Marker, Expressing, Flow Cytometry, Co-Culture Assay, Activation Assay, Incubation, Fluorescence, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cells

Article Title: Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas

doi: 10.3390/cells12131716

Figure Lengend Snippet: Optimization of BiKE production. ( A ) Schematic drawing representing BiKE mediated engagement of NK cells and IL13Rα2-positive glioma cells. The construct consists of an anti-CD16 single-domain antibody (sdCD16), interleukin-15 flanked by flexible protein linkers (IL15), an anti-IL13Rα2 single-chain fragment antibody (scFv IL13Rα2). ( B ) Optimization of BiKE production regarding the production temperature was conducted at 37 °C and 32 °C. Visualization of protein yields was conducted using Western blot, and binding to IL13Rα2 was demonstrated after production at 32 °C. ( C ) BiKE optimization for serum conditions was conducted in DMEM media with 10% FBS (10% FBS), DMEM with 1% FBS (1% FBS), and OptiMEM media. BiKE demonstrated the greatest production and maximal binding capacity to its target using the 10% FBS condition. ( D ) Schematic overview of BiKE production. Production can be efficiently completed in 6-7 days. First, BiKE cDNA was inserted into a pLVX-IRES-zsGreen1 lentiviral vector. A stable 293 T/17 cell line secreting BiKE was generated through lentivirus transfection. 293 T/17 cells producing cells were incubated overnight at 37 °C and transferred to 32 °C for another 2 to 3 days. The supernatant containing BiKE was collected, incubated with cOmplete His-Tag resin overnight at 4 °C, and isolated through His-Tag column purification. Purified BiKE was then dialyzed overnight with PBS serum, concentrated, and stored at −80 °C for future use. ** = p < 0.01, and *** = p < 0.001.

Article Snippet: PBMCs were obtained as previously described from fresh human blood and expanded ex vivo using a commercially available kit (CellXVivo Human NK Cell Expansion Kit, Cat CDK015a, Lot P309494; R&D Systems, Inc., Minneapolis, MN, USA) per manufacturer’s protocol.

Techniques: Construct, Western Blot, Binding Assay, Plasmid Preparation, Generated, Transfection, Incubation, Isolation, Purification

Journal: Cells

Article Title: Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas

doi: 10.3390/cells12131716

Figure Lengend Snippet: BiKE activates PBMC-derived NK cells towards IL13Rα2-positive gliomas. GBM6 or GBM39 spheroids were co-cultured for 24 h with either NK cells alone (NK Cells) or in combination with sdCD16 (sdCD16) or BiKE (BiKE). Expression levels of IL13Rα2 in both ( A ) GBM6 and ( B ) GBM39 are provided. BiKE significantly increased NK activation markers CD25 and CD69 in both ( C ) GBM6 and ( D ) GBM39 co-culture conditions. Treatment with BiKE resulted in significantly more killing of tumor cells than sdCD16-treated NK cells, NK cells alone, or controls in both ( E ) GBM6 and ( F ) GBM39 co-culture. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Article Snippet: PBMCs were obtained as previously described from fresh human blood and expanded ex vivo using a commercially available kit (CellXVivo Human NK Cell Expansion Kit, Cat CDK015a, Lot P309494; R&D Systems, Inc., Minneapolis, MN, USA) per manufacturer’s protocol.

Techniques: Derivative Assay, Cell Culture, Expressing, Activation Assay, Co-Culture Assay

Journal: Cells

Article Title: Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas

doi: 10.3390/cells12131716

Figure Lengend Snippet: BiKE treatment in GBM6 spheroids increased tumor cell killing via immunofluorescent detection of cleaved caspase-3 staining with PBMC-derived NK cells. GBM6 spheroids were co-cultured for 24 h with either PBMC-derived NK cells alone or treated with 10 μg of BiKE. ( A ) Representative images from left to the right demonstrate markers for DAPI shown in blue, cleaved caspase-3 shown in red, and respectfully merged DAPI and cleaved caspase-3. ( B ) Quantification of cleaved caspase-3 demonstrates a significant increase in GBM6 spheroid killing with BiKE-treated NK cells compared to NK cells alone or GBM6 spheroids alone. * = p < 0.05 and ** = p < 0.01.

Article Snippet: PBMCs were obtained as previously described from fresh human blood and expanded ex vivo using a commercially available kit (CellXVivo Human NK Cell Expansion Kit, Cat CDK015a, Lot P309494; R&D Systems, Inc., Minneapolis, MN, USA) per manufacturer’s protocol.

Techniques: Staining, Derivative Assay, Cell Culture

Journal: Cells

Article Title: Bi-Specific Killer Cell Engager Enhances NK Cell Activity against Interleukin-13 Receptor Alpha-2 Positive Gliomas

doi: 10.3390/cells12131716

Figure Lengend Snippet: BiKE extends survival of mice bearing patient-derived glioma xenografts through augmenting NK cell presence intratumorally and increasing tumor cell death. ( A ) GBM-6 xenografted animals treated with BiKE survived significantly longer than those treated with expanded NK cells alone or saline (Kaplan-Meier survival curves were compared using log-rank tests). ( B ) GBM12-xenografted animals demonstrated similar results. ( C ) Immunohistochemistry images with stain for NK cells (CD45-positive lymphocytes) and dead tumor cells (cleaved caspase-3, indicated by arrows) are shown for GBM6 mice sacrificed 1–3 days following treatment. Images shown are at 10× magnification. The scale bar is equal to 250 µm. Quantification of ( D ) CD45-positive lymphocytes and ( E ) cleaved caspase-3 positive cells are provided. Three brain slices were analyzed for each animal, and positive cells were averaged. The final cell count per treatment group is represented by the average of these individual values (n = 3). * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Article Snippet: PBMCs were obtained as previously described from fresh human blood and expanded ex vivo using a commercially available kit (CellXVivo Human NK Cell Expansion Kit, Cat CDK015a, Lot P309494; R&D Systems, Inc., Minneapolis, MN, USA) per manufacturer’s protocol.

Techniques: Derivative Assay, Saline, Immunohistochemistry, Staining, Cell Counting