Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A Correlation analyses between NK cell proportion and age ( n = 26). B Correlation analyses between NK cell numbers and age ( n = 26). C Correlation analyses between purity of the NK cell product reinfused into each individual and age ( n = 26). The spearman correlation analysis was used in all cases. The correlation was significant at 0.05 (two-sided). D CD3 + T cells were separated and SA-β-gal staining was performed pre- and post-infusion of NK cells ( n = 26). E Expression of senescence (P16 Ink4a and P21) (n = 25) and SASP markers (IL-6, monocyte chemoattractant protein-1 [MCP-1], plasminogen activator 1 [Pai1]) ( n = 26) in CD3 + T cells were analysed by qRT-PCR. Differences were assessed by the two-tailed paired Student’s t -test. ns P > 0.05, * P < 0.05, ** P < 0.01. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Staining, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: Flow cytometry to detect expression of ( A ) perforin and ( B ) CD69 was performed after 48-h co-incubation with NK cells and human adipose tissue, treated with conditional medium from control (CON) or senescent (SEN) preadipocytes for 24 h. Cells were gated in CD3 - CD56 + location. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05, ** P < 0.01. C Senescent markers for P16 and P21 were detected by Western blotting. n = 3. D Secreted proinflammatory cytokines in conditional medium from adipose tissue was collected 48-h after removing the NK cells. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Flow Cytometry, Expressing, Incubation, Control, Two Tailed Test, Western Blot

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: Flow cytometry to detect the expression of ( A ) CD69 and ( B ) IFN-γ was performed after a 24 h co-incubation with mouse NK cells and preadipocytes or irradiation-induced preadipocytes. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. ** P < 0.01. C SNC apoptosis was detected after a 24-h co-incubation with DiR-labeled NK cells by flow cytometry. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01. D Activating ligands and inhibitory ligands of NK cells on preadipocytes, doxorubicin-induced preadipocytes or irradiation-induced preadipocytes were measured by qPCR. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. * P < 0.05 (Irradiation vs CON). # P < 0.05 (Doxorubicin vs CON). E 1 × 10 6 DiR-labeled control preadipocytes or irradiation-induced senescent preadipocytes were implanted into the abdominal cavity. Representative images showing fluorescence activity in mice seven days after 5 × 10 6 NK cell treatment. Quantification of fluorescence activity. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, **** P < 0.0001. F Quantification of cytotoxicity of non-senescent and senescent counterparts induced by irradiation or adriamycin incubated with increasing NK cells for 24 h. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Flow Cytometry, Expressing, Incubation, Irradiation, Two Tailed Test, Labeling, Control, Fluorescence, Activity Assay

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A , B Cytotoxicity of mouse NK cells against SNCs supplemented with different concentrations of DA after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. C Data showing cAMP content in NK cells after 24-h incubation with SNCs supplemented with different concentrations of DA. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. * P < 0.05, *** P < 0.001, **** P < 0.0001. D Western blot analysis of phosphorylated CREB level in NK cells after 24-h incubation with SNCs supplemented with different concentrations of DA. E DR expression in NK cells was detected after co-incubation with SNCs or control cells by qPCR. n = 3. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05, ** P < 0.01. F , G Cytotoxicity of mouse NK cells against SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the two-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. H cAMP content in NK cells after incubation with SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. n = 3. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, **** P < 0.0001. I Western blot analysis of phosphorylated CREB level in NK cells after incubation with SNCs supplemented with 10 −9 M DA and SCH23300 or haloperidol after a 24-h co-incubation. Two parallel samples were set in each experiment and three independent experiments were performed for each result.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Incubation, Western Blot, Expressing, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Combining adoptive NK cell infusion with a dopamine-releasing peptide reduces senescent cells in aged mice

doi: 10.1038/s41419-022-04562-w

Figure Lengend Snippet: A Peripheral dopamine levels in young (10-week-old) and aged (20-month-old) mouse. n = 6. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. * P < 0.05. B Peripheral dopamine levels induced by 10 mg/kg Acein 1 h after Acein administration in old mice. n = 5. Data are presented as means ± SD. Differences were assessed by the two-tailed unpaired nonparametric t -test. ** P < 0.01. C , D NK cells in peripheral blood were detected by flow cytometry after a four-month treatment. n = 4. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, * P < 0.05, ** P < 0.01. E CD69 expression level in NK cells. n = 4. Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P>0.05, * P < 0.05, *** P < 0.001. F SA- β -gal staining of adipose tissue in the groin. G Expression levels of senescent markers P16 and P21 in adipose tissue in the groin. H BrdU was injected intraperitoneally at 24 and 72 h before euthanasia. BrdU + Ki67 + adipocytes in the groin were detected by flow cytometry. n = 4 . Data are presented as means ± SD. Differences were assessed by the one-way ANOVA test. ns P > 0.05, * P < 0.05. Two parallel samples were set in each experiment.

Article Snippet: Briefly, 50 mL peripheral blood was extracted from each individual, after which peripheral blood mononuclear cells (PBMCs) were isolated and transferred to a T75 flask with ExCellerateTM Human NK Cell Expansion Media (R&D Systems, USA) containing 1×Cloudz CD2/NKp46, recombinant Human IL-2 (27 ng/mL), recombinant Human IL-12 (10 ng/mL), recombinant Human IL-18 (10 ng/mL), and recombinant Human IL-21 (10 ng/mL) (R&D Systems, USA) for 48 h, and then replaced with activated medium (270 ng/mL recombinant Human IL-2, 20 ng/mL recombinant Human IL-12, 20 ng/mL recombinant Human IL-18, and 20 ng/mL recombinant Human IL-21) for further culture.

Techniques: Two Tailed Test, Flow Cytometry, Expressing, Staining, Injection

Journal: Cytotherapy

Article Title: Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

doi: 10.1016/j.jcyt.2024.07.013

Figure Lengend Snippet: Fig. 3. Twenty-day fold-expansion of peripheral and cord blood isolated NKreg-like cells. Fold-expansion of CD56brightCD16- NKreg-like cells isolated from either a 24hr healthy donor peripheral blood or cord blood course. Cells were cultured under identical expan- sion conditions over a 20-day period. The Immunocult NK Cell Expansion Media from Stemcell Technologies was supplemented with 1% penicillin/streptomycin, 10% L-gluta- mine, 10% human AB serum, 2ng/mL TGF-b1, 25ng/mL IL-2, and 300nM rapamycin (added at day 18) (mean +/- SD). P < 0.0005 = ***. Statistical analyses for expansion experi- ments were performed using Microsoft Excel version 2110 and a 2-tailed T test two- sample assuming unequal variance. The data is representative of 3 separate experiments utilizing cells from 3 unique healthy donors or 3 unique cord blood units.

Article Snippet: Cells were expanded with 25 ng/mL IL-2 (Biolegend, San Diego, CA, USA) + 2 ng/mL TGF-b1 (Stemcell Technologies, Vancouver, BC, Canada) in complete MACS NK Cell Expansion media (Miltenyi Biotec, Auburn, CA, USA), Immunocult NK Cell Expansion media (Stemcell Technologies, Vancouver, BC, Canada), or ExCellerate NK Cell Expansion media (R&D Systems, Inc, Minneapolis, MN, USA).

Techniques: Isolation, Cell Culture

Journal: Cytotherapy

Article Title: Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

doi: 10.1016/j.jcyt.2024.07.013

Figure Lengend Snippet: Fig. 2. Twenty-day fold-expansion of NKreg-like cells grown in differing expansion media. Fold-expansion of CD56brightCD16- NKreg-like cells over a 20-day culture period, utilizing various NK cell expansion media, including the ExCellerate NK Cell Expansion media (EC), MACS NK Cell Expansion media (MACS), or Immunocult NK Cell Expansion media (Immu- nocult). Each medium condition was supplemented with 1% penicillin/streptomycin, 10% L-glutamine, 10% human AB serum, 2 ng/mL TGF-b1, 25ng/mL IL-2, and 300nM rapamy- cin (added at day 18) (mean +/- SD). NS = not significant, *P < 0.05, **P < 0.005. Statistical analyses for expansion experiments were performed using Microsoft Excel version 2110 and a 2-tailed T test 2-sample assuming unequal variance. The data is representative of 3 separate experiments utilizing cells from 3 unique healthy donors.

Article Snippet: Cells were expanded with 25 ng/mL IL-2 (Biolegend, San Diego, CA, USA) + 2 ng/mL TGF-b1 (Stemcell Technologies, Vancouver, BC, Canada) in complete MACS NK Cell Expansion media (Miltenyi Biotec, Auburn, CA, USA), Immunocult NK Cell Expansion media (Stemcell Technologies, Vancouver, BC, Canada), or ExCellerate NK Cell Expansion media (R&D Systems, Inc, Minneapolis, MN, USA).

Techniques:

Journal: Cytotherapy

Article Title: Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

doi: 10.1016/j.jcyt.2024.07.013

Figure Lengend Snippet: Fig. 4. Bulk RNA sequencing of untreated CD56brightCD16- NK cells compared to expanded CD56brightCD16- NK cells. Healthy donor fresh peripheral CD56brightCD16- NK cells (N=3) and healthy donor CD56brightCD16- NK cells expanded for 20 days (N = 3) were utilized for bulk RNA sequencing, performed by Novogene via Novaseq 6000 (Illumina). The tran- scriptome of the untreated CD56brightCD16- NK cells was compared to the expanded CD56brightCD16- NK cells. (A) Volcano plot identifying the Log2 fold change and p-value of highly expressed genes by untreated CD56brightCD16- NK cells compared to expanded CD56brightCD16- NK cells. (B) Identification of individual sample differences in the gene expression of untreated CD56brightCD16- NK cells compared to expanded CD56brightCD16- NK cells. (C) A Gene set enrichment analysis was performed to identify genes signficantly expressed by untreated CD56brightCD16- NK cells compared to expanded CD56brightCD16- NK cells, and group the overrepresented genes according to Broad Institute hallmark signaling path- ways.

Article Snippet: Cells were expanded with 25 ng/mL IL-2 (Biolegend, San Diego, CA, USA) + 2 ng/mL TGF-b1 (Stemcell Technologies, Vancouver, BC, Canada) in complete MACS NK Cell Expansion media (Miltenyi Biotec, Auburn, CA, USA), Immunocult NK Cell Expansion media (Stemcell Technologies, Vancouver, BC, Canada), or ExCellerate NK Cell Expansion media (R&D Systems, Inc, Minneapolis, MN, USA).

Techniques: RNA Sequencing, Gene Expression

Journal: Cytotherapy

Article Title: Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

doi: 10.1016/j.jcyt.2024.07.013

Figure Lengend Snippet: Fig. 5. Metabolic characteristics of expanded NK cells. NK cell culture medium comparison between three commercially available NK cell expansion medias. CD56brightCD16- NKreg- like cells and CD56dimCD16+ NK cells expanded for 20-days in 3 differing media conditions (MACS NK Cell Expansion media, ExCellerate NK Cell Expansion media, or Immunocult NK Cell Expansion media), supplemented via 1% penicillin/streptomycin, 10% L-glutamine, 10% human AB serum, and appropriate cytokines (2 ng/mL TGF-b1, 25 ng/mL IL-2, and 300nM rapamycin (added at day 18) for NKreg-like cells, and 50 ng/mL IL-2 and 25 ng/mL IL-15 for CD56dim NK cells). Comparisons were made on (A) concentration of glucose and glutamine in the media. NKreg-like cells grown in the 3 media were compared with a (B) mitochondrial stress test assessing mitochondrial respiration represented by oxygen con- sumption rate (OCR) and glycolytic activity represented by extracellular acidification rate (ECAR). The test was performed using a seahorse extracellular flux analyzer with the addi- tion of inhibitors oligomycin (oligo), the proton uncoupling agent FCCP, Rotenone and Antimycin (R&A), and 2-DeoxyGlucose (2DG). The effect is (C) quantified with OCR over ECAR ratios, and spare respiratory capacity (SRC) as a percentage of the basal respiration. Comparison between NKreg-like, CD56dim NK cells, and effects of rapamycin were determined metabolically with d) OCR and ECAR, and e) OCR/ECAR ratios and SRC. Data are presented as mean § SEM and P values are determined by 2-tailed Student’s t-test (N = 3).

Article Snippet: Cells were expanded with 25 ng/mL IL-2 (Biolegend, San Diego, CA, USA) + 2 ng/mL TGF-b1 (Stemcell Technologies, Vancouver, BC, Canada) in complete MACS NK Cell Expansion media (Miltenyi Biotec, Auburn, CA, USA), Immunocult NK Cell Expansion media (Stemcell Technologies, Vancouver, BC, Canada), or ExCellerate NK Cell Expansion media (R&D Systems, Inc, Minneapolis, MN, USA).

Techniques: Cell Culture, Comparison, Concentration Assay, Activity Assay, Metabolic Labelling

Journal: Cytotherapy

Article Title: Expansion and characterization of immune suppressive CD56(bright)Perforin(-) regulatory-like natural killer cells in chronic graft-versus-host disease.

doi: 10.1016/j.jcyt.2024.07.013

Figure Lengend Snippet: Fig. 6. Evaluation of phenotypic expression of CD34+ HSPC differentiated and sorted CD56brightCD16- NKreg-like cells. Differentiated CD56brightCD16- NK cells were isolated via FACS cell sorting, cultured for 72 h via Immunocult NK Expansion media supplemented with IL-2, TGF-b1, and rapamycin. Cultured cells were stained for cell surface expression of select markers (CD56 PE, CD3 APC, CD16 FITC, Perforin Pacific Blue, Granzyme K AF647) and analyzed by flow cytometry. Lymphocytes were first gated using FSC and SSC. The data is rep- resentative of 6 separate experiments utilizing cells from unique healthy donors (N = 6).

Article Snippet: Cells were expanded with 25 ng/mL IL-2 (Biolegend, San Diego, CA, USA) + 2 ng/mL TGF-b1 (Stemcell Technologies, Vancouver, BC, Canada) in complete MACS NK Cell Expansion media (Miltenyi Biotec, Auburn, CA, USA), Immunocult NK Cell Expansion media (Stemcell Technologies, Vancouver, BC, Canada), or ExCellerate NK Cell Expansion media (R&D Systems, Inc, Minneapolis, MN, USA).

Techniques: Expressing, Isolation, FACS, Cell Culture, Staining, Cytometry