MOESM1 of Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization
Creators: name: Powell, Allison
name: Sridevi Yadavilli
name: Saunders, Devin
name: Pelt, Stacey
name: Chorvinsky, Elizabeth
name: Burga, Rachel
name: Shuroug Albihani
name: Hanley, Patrick
name: Zhenhua Xu
name: Yanxin Pei
name: Yvon, Eric
name: Hwang, Eugene
name: Bollard, Catherine
name: Nazarian, Javad
name: Cruz, Conrad
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Ecology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 110309 Infectious Diseases

description: Additional file 1: Figure S1. Modifying CB-derived NK cells to express TGF-β dominant negative receptor does not affect cell expansion, cytolytic activity, and cytokine secretion. A. Transduction efficiency as measured by expression of TGF-β DNR. Long bar is the mean. Each sample is represented as a circle. B. Expression of wildtype TGF-βRII in untransduced and transduced cells show increased expression in transduced NK cells, representing expression of DNR. Each sample is represented as a circle. C. Untransduced NK cells (black outlined bar) expand as well as transduced NK cells (gray outlined bar) after 12 days in culture. Each sample is represented as a circle. C. Representative dot plots of NK cell populations in non-transduced and transduced cells. D. No difference in NK cell populations are seen between transduced (gray squares) and nontransduced cells (black squares). Long bar represents the mean. E. Cytotoxicity of untransduced (black lines) and transduced (gray lines) against Daoy (solid lines) and primary medulloblastoma cells (dotted lines). F. Cytokines measured in supernatant released by untransduced (black outlined bars) and transduced (gray outlined bars) NK cells following 12 days of expansion. Error bars are standard error of the mean. Each sample is represented as a circle. Figure S2. UCB-derived NK genetically modified to express TGF-β dominant negative receptor (TGF-β DNRII) can protect against exogenous TGF-β-mediated immune suppression. A. Cytotoxicity of untransduced (gray lines) and transduced (black lines) against Daoy cells (transduced cells show 24.97 ± 4.52% killing at E:T 5:1 in the absence vs. 13.11 ± 0.79% in the presence of TGF-β, n = 6, p = 0.03) while transduced cells remained protected and did not show significantly decreased killing (19.29 ± 1.12% killing at E:T 5:1 in the absence vs. 17.09 ± 1.67% in the presence of TGF-β, n = 6, p = 0.3). Dotted lines represent cells grown in the presence of 5 ng/mL of exogenous TGF-β. B. Mean fluorescence intensity of TGF-β RII in untransduced and transduced cells, in the presence and absence of 5 ng/mL of exogenous TGF-β. No decrease in the expression of TGF-β receptor was seen in transduced cells 109,864 ± 81,857 TGF-βRII MFI from 113,693 ± 69,957, n = 7, p = 0.3), while it decreased the expression of TGF-β receptor expressing nontransduced cells (2493 ± 881 TGF-βRII MFI from 8491 ± 824, n = 7, p = 0.02). Each sample is represented as a circle. Figure S3. Cytokine secretion by transduced and non-transduced NK cells in the presence and absence of exogenous TGF-β and medulloblastoma-conditioned media. Cytokines measured in supernatant released by untransduced (solid circles) and transduced (outlined circles) NK cells following 12 days of expansion. Error bars are standard error of the mean. Each sample is represented as a circle. Black denotes cells alone, dark gray denotes cells and exogenous TGF-β, and light gray denotes cells grown in medulloblastoma-conditioned media. Figure S4. Properties of target cells. Bars show mean expression of HLA-A,B,C; PVR; and MIC A/B in Daoy cells (multiple repeats, n = 5). Error bars are standard error of the mean. Each sample is represented as a circle. Figure S5. Other Effects of Transduction and TGF-β. A. Bars show mean expression of NKG2D in different cell conditions shown on the x axis (multiple donor lines, n = 8). Error bars are standard error of the mean. Each sample is represented as a circle. B. Bars show mean expression of NKp30 in different cell conditions shown on the x axis (multiple donor lines, n = 7). Error bars are standard error of the mean. Each sample is represented as a circle. C. Bars show mean expression of NKp46 in different cell conditions shown on the x axis (multiple donor lines, n = 6). Error bars are standard error of the mean. Each sample is represented as a circle. D. Bars show mean expression of DNAM-1 in different cell conditions shown on the x axis (multiple donor lines, n = 2). Error bars are standard error of the mean. Each sample is represented as a circle. E. Bars show mean expression of IFNγ in different cell conditions shown on the x axis (multiple donor lines, n = 4). Error bars are standard error of the mean. Each sample is represented as a circle. F. Bars show mean expression of CX3CR1 in different cell conditions shown on the x axis (multiple donor lines, n = 2). Error bars are standard error of the mean. Each sample is represented as a circle. No significant differences were noted in the expression of these markers. Figure S6. No Functional Effect of CCR2 Upregulation in Transduced Cells. Migration experiments in three evaluable lines, comparing different conditions (each condition in duplicate). Migration to CCL2/CXCL12 (positive control) is shown for comparison. Bars depict mean absolute number of NK cells calculated using flow cytometry counting beads at the bottom of the transwell. Error bars are standard error of the mean.