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