Journal: Med (New York, N.y.)

Article Title: Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset

doi: 10.1016/j.medj.2021.04.003

Figure Lengend Snippet:

Article Snippet: No-GFP-CD16.NK-92 cell line (High affinity 176V) , ATCC , PTA-6967.

Techniques: Virus, Recombinant, Staining, Luminex, Neutralization, Generated, Software

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 1. ELK3 depletion increases NK cell cytotoxicity regardless of E-cadherin expression. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA- MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is presented as 1:1, 1:5, and 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5 after 4 h of coculture with CFSE-labeled NK92MI cells. The E:T ratio is 1:10. Error bars represent standard deviation; **p < 0.01 (one-way ANOVA). (c) Immunofluorescence images of perforin and granzymeB in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is 1:10. Scale bar, 50 μm. (d) The quantified graft of granzymeB and perforin protein. Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (e) The percentage of CD107a-positive and IFNγ-positive NK92MI cells in MDA-MB231 cells expressing shCon and shELK3#1 after 4 h of coculture with NK92MI cells. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). (f-g) Quantitative analysis of indicated ligands in MDA-MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; *p < 0.05, NS is non-significant (unpaired two-tailed Student’s t-test). (h-i) Quantitative analysis of indicated receptors in MDA-MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; *p < 0.05, NS is non-significant (unpaired two-tailed Student’s t-test).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunofluorescence, Activity Assay, Labeling, Standard Deviation, Two Tailed Test

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 2. ELK3 depletion increases CXCL16 expression in TNBC cells. (a) Experimental scheme for assessing NK cell cytotoxicity using conditioned medium (CM) from MDA-MB231 organoids expressing shCon and shELK3#1. CM from MDA-MB231 organoids was applied to MDA-MB231 organoids expressing shELK3#1 for NK92MI cell coculture; conversely, CM from MDA-MB231 organoids expressing shELK3#1 was applied to MDA-MB231 organoids expressing shCon for NK92MI cell coculture. N. M. represents normal media. Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon and shELK3#1 after 4 h of coculture with CFSE-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). NS is non-significant. (c) Quantification of secreted CXCL16 protein in MDA-MB231 cells transfected with siCon and siELK3. Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (d) Quantitative analysis of CXCL16 mRNA levels in MDA- MB231 cells expressing shCon and shELK3#1. Error bars represent standard deviation; **p < 0.01 (unpaired two-tailed Student’s t-test). (e) Immunoblot analysis of the CXCL16 protein in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. GAPDH was used as the loading control. (f) Heatmap analysis of immune- related genes in MDA-MB231 cells expressing shCon and shELK3#1. (g) Correlation analysis of ELK3 and CXCL16 in various breast cancer cells. Orange line indicates TNBC

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunofluorescence, Activity Assay, Labeling, Standard Deviation, Two Tailed Test, Transfection, Western Blot, Control

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 3. The chemotactic activity of CXCL16 recruits NK cells to target TNBC cells. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 organoids expressing shCon and shELK3#1 after 4 h of coculture with FarRed-labeled NK92MI cells under the indicated conditions. The E:T ratio is 1:10. Scale bar, 100 μm. (b) Rate of target cell death in MDA-MB231 cells expressing shCon after 4 h of coculture with CFSE-labeled NK92MI cells under the indicated conditions. The final concentration of CXCL16 protein was 200 ng/ml. The E:T ratio is 1:10. Error bars represent standard deviation; *p < 0.05, ***p < 0.001 (one-way ANOVA). (c) Immunofluorescence images of migrated NK92MI cells in response to recombinant CXCL16 protein. Scale bar, 200 μm. (d) The graph indicates the number of migrated NK92MI cells in CXCL16-mediated chemotaxis. Error bars represent standard deviation; **p < 0.01 (one-way ANOVA). (e) Migrated NK92MI cells in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. Scale bar, 100 μm, 200 μm. (f) The graph indicates the number of migrated NK cells in MDA-MB231 cells expressing shCon, shELK3#1, shELK3#3, and shELK3#5. Error bars represent standard deviation; **p < 0.01, ***p < 0.001 (one-way ANOVA). (g-i) Migrated NK92MI cells in Hs578T cells expressing shCon and shELK3#1. The graph indicates the number of migrated NK92MI cells in Hs578T cells expressing shCon and shELK3#1. Scale bar, 100 μm, 200 μm. Error bars represent standard deviation; ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student’s t-test).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Immunofluorescence, Expressing, Labeling, Concentration Assay, Standard Deviation, Recombinant, Chemotaxis Assay, Two Tailed Test

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 4. The ELK3-CXCL16 axis determines the response of NK cells in TNBC cells. (a) Immunofluorescence images of caspase 3/7 activity (green) in MDA-MB231 shELK3#1 organoids transfected with siCon, siCXCL16#1, and siCXCL16#2 after 4 h of coculture with FarRed-labeled NK92MI cells. The E:T ratio is 1:10. Scale bar, 50 μm. (b) Rate of target cell death in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h of coculture with CFSE-labeled NK92MI cells. Error bars represent standard deviation; *p < 0.05, **p < 0.01 (one-way ANOVA). (c) Migrated NK92MI cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Scale bar, 200 μm. (d) The graph indicates the number of migrated NK92MI cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; *p < 0.05 (one-way ANOVA). (e) Migrated primary NK cells in MDA-MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; **** p < 0.0001 (one-way ANOVA). (f) Rate of target cell death in MDA- MB231 shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h of coculture with CFSE-labeled primary cells. Error bars represent standard deviation; *p < 0.05, **p < 0.01 (one-way ANOVA). (g) Migrated NK92MI cells in Hs578T shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; ****p < 0.0001 (one-way ANOVA). (h) Migrated primary NK cells in Hs578T shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2. Error bars represent standard deviation; ****p < 0.0001 (one-way ANOVA). (i) Rate of target cell death in Hs578t and Hs578t shELK3#1 cells transfected with siCon siCXCL16#1, and siCXCL16#2 after 4 h co-cultured with CFSE-labeled NK92MI cells. Error bars represent the standard deviation; *p < 0.05 (one-way ANOVA).

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Immunofluorescence, Activity Assay, Transfection, Labeling, Standard Deviation, Cell Culture

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 5. CXCL16 promotes NK cell chemotaxis and cytotoxicity in vivo. (a) Quantitative analysis of CXCL16 mRNA levels in MDA-MB231-GFP-luciferase cells expressing control and CXCL16 (231-control and 231-CXCL16). Error bars represent standard deviation; ***p < 0.001 (unpaired two-tailed Student’s t-test). (b) Immunoblot analysis of CXCL16 in 231-control and 231-CXCL16 cells. GAPDH was used as the loading control. (c) Migrated NK92MI cells in 231-control and 231-CXCL16 cells. Scale bar, 100 μm. The graph indicates the number of migrated NK92MI cells in 231-control and 231-CXCL16 cells. Error bars represent standard deviation; *p < 0.05 (unpaired two- tailed Student’s t-test). (d) Experimental scheme for NK cell recruitment in the experimental metastasis model. NK92MI cells were intravenously injected three times after 231-control and 231-CXCL16 cell injection. (e) The percentage of GFP-positive cells in the lungs from mice harboring 231-control and 231-CXCL16 cells after administration of NK92MI cells. GFP indicates tumor cells in the lung. FarRed indicates NK92MI cells in the lung. Error bars represent standard deviation; *p < 0.05 (unpaired two-tailed Student’s t-test). NS is non-significant. (f) Immunofluorescence images of NK92MI cells in the lungs from mice harboring 231-control and 231- CXCL16 cells after NK92MI cell administration. Scale bar, 100 μm, 200 μm. (g) Experimental scheme for NK cell recruitment and NK cell cytotoxicity in the orthotopic mouse model. NK92MI cells were intravenously injected six times after visualization of primary tumors. (h) Primary tumor growth in mice harboring 231-control and 231-

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Chemotaxis Assay, In Vivo, Luciferase, Expressing, Control, Standard Deviation, Two Tailed Test, Western Blot, Injection, Immunofluorescence

Journal: OncoImmunology

Article Title: ELK3-CXCL16 axis determines natural killer cell cytotoxicity via the chemotactic activity of CXCL16 in triple negative breast cancer

doi: 10.1080/2162402x.2023.2190671

Figure Lengend Snippet: Figure 6. Negative correlation between ELK3 and CXCL16 in human breast cancer. (a) Expression analysis of ELK3 and CXCL16 in 421 human breast cancer patient samples (TCGA database). (b-c) Kaplan–Meier survival plots showing the overall survival of breast cancer patients. (d) Heatmap analysis of ELK3, CXCL16, and NK cell- relatedgenes37 in 421 human breast cancer patient samples. The white box indicates the patient samples with high expression of the ELK3 gene and low expression of the CXCL16 gene. The yellow box indicates patient samples with low expression of the ELK3 gene and high expression of the CXCL16 gene. (e) Correlation analysis of CXCL16 and NK cell-related genes in human breast cancer patient samples. (f) The biological role of ELK3-CXCL16 axis on NK cell cytotoxicity. ELK3 depletion increased secreted CXCL16 protein to recruit NK cells and resulted in improved NK cell cytotoxicity in TNBC cells.

Article Snippet: The human NK cell line NK92MI was purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: CD99 ligation upregulates HSP70 on acute lymphoblastic leukemia cells and concomitantly increases NK cytotoxicity

doi: 10.1038/cddis.2012.164

Figure Lengend Snippet: CD99 ligation enhances NK92-mediated cytotoxicity against ALL but not ET cells. Cytotoxicity assays with ALL and ET cells (4–5 for each cell line) were performed with NK92 cells as effectors. Annexin V and 7AAD were used to detect lysed targeted cells. CD99 induced HSP70 (insets in the graphics) and in parallel increased NK92 cytotoxicity against Jurkat ( a ) and Reh ( b ) (3 h of co-incubation) cells. ( c ) Cytotoxicity of NK92 cells against primary BCP-ALL (three independent ETV6/RUNX1-positive cases are shown)

Article Snippet: NK92 cells (from DSMZ) were maintained in X-VIVO 10 medium (Biowhittaker, MD, USA) containing 20% fetal bovine serum ‘Gold' (PAA Laboratories, Pasching, Austria), 200 U/ml interleukin-2 (PeproTech, Vienna, Austria) and supplements.

Techniques: Ligation, Incubation

Journal: Transplantation and cellular therapy

Article Title: NK Cell Adoptive Immunotherapy of Cancer: Evaluating Recognition Strategies and Overcoming Limitations

doi: 10.1016/j.bbmt.2020.09.030

Figure Lengend Snippet: Sample List of Active Trials Involving Allogeneic NK Cells

Article Snippet: Anderson Cancer Center; National Cancer Institute CB-derived NK cells Rituximab Stem cell transplant Recurrent or refractory B cell non-Hodgkin lymphoma NCT03027128 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Metastatic of locally advanced solid tumors NCT03068819 Washington University School of Medicine Cytokine-induced memory-like NK cells CD3 + T cells donor lymphocyte infusion Relapsed AML NCT03136406 NantKwest, Inc. NK92 cell line ALT-803 Other immune modulators Pancreatic cancer NCT03167164 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Merkel cell carcinoma NCT03167177 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Melanoma NCT03169738 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Non-small-cell lung cancer NCT03169764 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Head and neck squamous cell carcinoma NCT03169777 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Colorectal cancer NCT03169790 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Non-Hodgkin lymphoma NCT03175666 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Triple-negative breast cancer NCT03197571 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Urothelial carcinoma NCT03197584 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Ovarian cancer NCT03300492 University Hospital, Basel, Switzerland Haploidentical NK cells Acute myeloid leukemia; myelodysplastic syndromes NCT03329248 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Pancreatic cancer NCT03383978 Johann Wolfgang Goethe University Hospital NK92 cell line gene modified to express anti-Her2 CAR-expressing CD28 and ζ Glioblastoma NCT03387085 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Triple-negative breast cancer NCT03387098 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Pancreatic cancer NCT03387111 NantKwest, Inc. NK92 cell line modified to express IL-2 and CD16 (haNK) Other immune modulators Squamous cell carcinoma NCT03415100 The Third Affiliated Hospital of Guangzhou Medical University Autologous or allogeneic NK cells transfected with mRNA expressing NKG2D-directed CAR Metastatic solid tumors NCT03420963 M.D.

Techniques: Transplantation Assay, Modification, Transfection, Expressing, Derivative Assay

Journal: JCI Insight

Article Title: Trastuzumab/pertuzumab combination therapy stimulates antitumor responses through complement-dependent cytotoxicity and phagocytosis

doi: 10.1172/jci.insight.155636

Figure Lengend Snippet: ( A and B ) Mouse FCGR signaling activation assay. KPL-4 cells were plated and treated with indicated HER2 mAbs concentrations for 1 hour. Jurkat cells containing NFAT-luciferase reporter and expressing mouse FCGR1 ( A ) and FCGR4 ( B ) were added to the KPL-4 cells containing antibodies and cocultured for 4 hours. FCGR signaling activation were assessed by luciferase activity quantification. ( C and D ) Human FCGR signaling activation was similarly quantified with Jurkat-NFAT-Luciferase reporter cells expressing human FCGR1 ( C ) and FCGR3A ( D ). ( E – G ) Mouse FCGR signaling activation assays were repeated using murinized HER2 mAbs with the IgG2A isotype. In addition to mFCGR1 ( E ) and mFCGR4 ( F ), mouse FCGR3 ( G ) was tested here since mFCGR3 can be activated by murine antibodies but not human antibodies. All data represents mean ± SEM; n = 4. ( H – J ) Antigen-binding-fragment F(ab’) 2 of the HER2 mAbs were generated and their therapeutic efficacy against HER2 + BC in vivo were compared with the parental antibody, respectively. As described before, KPL-4 cells (5 × 10 5 cells) were implanted in mammary fat pads of SCID-beige mice and treated with the indicated HER2 mAbs or F(ab’) 2 (100 μg per week). ( H ) Comparison between Trastuzumab versus Trastuzumab-F(ab’) 2 . ( I ) Comparison between Pertuzumab versus Pertuzumab-F(ab’) 2 . ( J ) Comparison between T+P versus Trastuzumab + Pertuzumab-F(ab’) 2 versus Trastuzumab-F(ab’) 2 + Pertuzumab. Numbers of mice showing total tumor regression are displayed on the right of the graph. ( H – J ) n = 8–10 for all groups. Two-way ANOVA with Tukey’s multiple-comparison post hoc test. All data represent mean ± SEM; * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: CEM.NKR cells stably expressing HER2 and luciferase (CEM.NKR-HER2), and NK.92 cells stably expression human FCGR3 of the V158 variant (NK.92-FCGR3-V158) were generated with lentivirus transduction and selected with puromycin.

Techniques: Activation Assay, Luciferase, Expressing, Activity Assay, Binding Assay, Generated, In Vivo