Additional file 1 of Stearoyl-CoA desaturase in CD4+ T cells suppresses tumor growth through activation of the CXCR3/CXCL11 axis in CD8+ T cells
Creators: name: Hwang, Sung-Hyun
name: Yang, Yeseul
name: Jung, Jae-Ha
name: Kim, Jin Won
name: Kim, Yongbaek
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Supplementary figure S1. Characterization of lipid metabolism in TILs incubated with CAFs-supernatant. (A) Representative dot plot image of CD3 and CD4 staining in isolated CD4+ splenocytes and TILs in tumor tissue isolated from 4T1 mouse model. (B) Representative fluorescence image of staining for CD4 and FAP in spleen from normal mouse. Red: CD4, Green: FAP, Blue: DAPI. Scale bar: 100 μm. (C) Representative histogram of FAP staining in isolated CAFs from tumor mass in 4T1 cells. The P4 population was established using negatively isolated cells. ***P&lt;0.001. Data indicate the mean ± SEM (n = 3). (D) Transcription levels of Cd36 and Slc27a1 in CD4+ splenic T cells and TILs incubated with CAF-supernatant for 1 day. *P &lt; 0.05 and ***P &lt; 0.001. Data indicate the mean ± SEM (n = 4). (E) Representative histogram of BODIPY staining in CD4+ TILs treated with CAF-supernatant and SSO. The graph indicated the MFI of BODIPY staining. ***P&lt;0.001. Data indicate the mean ± SEM (n = 3). Supplementary figure S2. SCD regulates the expressions of marker for Treg and Th1 cells and mitochondrial function. (A) Transcription levels of Tbx21, Gata1, Rorγ, and Foxp3 in CD4+ splenic T cells and TILs. **P&lt;0.01 and **P&lt;0.01. Data indicate the mean ± SEM (n = 4). (B) Analysis of FoxP3 and T-bet staining in Jurkat T cells after treatment with CAF-supernatant for 1 day. (C) Expressions of SCD and GAPDH in parental, Control-Cas9-transfected cells, SCD-KO cells, empty-pLenti-infected cells and SCD-pLenti cells. Supplementary figure S3. The effect of SCD in CD4+ splenic T cells. (A) Expression of SCD in CD4+ splenic T cells treated with control, 1 μM CAY10566 and 10 μM T0901317 for 1 day. (B) The concentration of TGF-β and IL-2 in CD4+ splenic T cells treated with control, 1 μM CAY10566 and 10 μM T0901317. **P &lt; 0.01 and ***P &lt; 0.001. Data indicate the mean ± SEM (n = 4). (C) CFSE staining in CD4+ T cells treated with control, 1 μM CAY10566 and 10 μM T0901317 for 1 day. The fluorescent of CFSE staining was measured for 3 days. The graph indicated the MFI of CFSE staining. Data indicate the mean ± SEM (n = 4). Supplementary figure S4. Increase of CXCL11 secretion by SCD augmented the activity of CD8+ T cells dependent on binding with CXCR3. (A) Representative dot plot image of CD3 and CD8 staining in isolated CD8+ T cells from splenocytes. (B) Representative dot plot of staining for annexin-V and PI in B16F10 and CT26 cells which co-cultured with preincubated CD8+ T cells with cell-free supernatant from control, 1 μM CAY10566, and 10 μM T0901317-treated CD4+ T cells. The quadrant was divided according to untreated B16F10 and CT26 cells. Apoptotic rate: upper right + lower right. (C) Representative dot plot of staining for annexin-V and PI in 4T1, B16F10 and CT26 cells which co-cultured with CD8+ T cells treated with 1 μM CAY10566 and 10 μM T0901317 for 1 day. Apoptotic rate: upper right + lower right. (D) Transcription levels of Prf1 and Gzmb in CD8+ T cells treated with 1 μM CAY10566 and 10 μM T0901317. *P &lt; 0.05, **P &lt; 0.01, and ***P &lt; 0.001. Data indicate the mean ± SEM (n = 4). (E) The membrane of cytokine array using supernatant from CD4+ T cells treated with control, 1 μM CAY10566 and 10 μM T0901317. (F) Transcription levels of Cxcl13, Cxcl15, Fgf2, Tnfrsf19, Igfbp3, Retn and Igf1 in CD4+ splenic T cells treated with control, 1 μM CAY10566 and 10 μM T0901317. *P &lt; 0.05, **P &lt; 0.01 and ***P &lt; 0.001. Data indicate the mean ± SEM (n = 4). (G) Transcription levels of Cxcl9 and 10 in CD4+ T cells treated with control, 1 μM CAY10566 and 10 μM T0901317. Data indicate the mean ± SEM (n = 4). (K) Representative dot plot of staining for annexin-V and PI in B16F10 and CT26 cells co-cultured with preincubated CD8+ T cells with rCXCL11 and AMG487 for 1 day. The quadrant was divided according to untreated B16F10 and CT26 cells Apoptotic rate: upper right + lower right. Supplementary figure S5. CXCL3/CXCL11-axis augmented activity of CD8+ T cells. (A) Representative dot plot of staining for annexin-V and PI in 4T1 cells treated with 20 μM AMG487 for 18 hours. The quadrant was divided according to untreated 4T1 cells. Apoptotic rate: upper left + upper right. (B) Representative dot plot of staining for annexin-V and PI in B16F10 and CT26 cells co-cultured with alone, parental and CXCR3-CD8+ T cells. The quadrant was divided according to untreated B16F10 and CT26 cells. Apoptotic rate: upper right + lower right. Supplementary figure S6. Single administration of CXCR3-CD8+ T cells suppressed tumor growth in mouse model. (A) Schedule of 4T1-mice model injected with PBS, CD8+ T cells, and CXCR3-CD8+ T cells for 17 days. (B) Representative dot plot of staining for annexin-V and PI in CT26 and B16F1 cells which co-cultured with parental, pLenti- and CXCR3-CD8+ T cells. Apoptotic rate: upper right + lower right. (C) Representative histogram of staining for IFN-γ in CD8+ TILs which isolated from PBS, CD8+ T cells and CXCR3-CD8+ T cells injected mouse. The graph indicated the MFI of IFN-γ. **P &lt; 0.01. Data indicate the mean ± SEM (n = 5). (D) Representative dot plot of staining for annexin-V and PI in 4T1 cells which co-cultured with 1 μg/mL anti-PD1 antibody treated CD8+ T cells and CXCR3-CD8+ T. Apoptotic rate: upper right + lower right. (E) Schedule of 4T1-mice model injected with PBS, CD8+ T cells, and CXCR3-CD8+ T cells and anti-PD1 antibody for 17 days. (F) The bioluminescence of Luc2-4T1 cells in mouse model was monitored for 17 days following injections with PBS, 2×106 CD8+ T cells and CXCR3-CD8+ T cells and 200 μg of anti-PD1 antibody for 3 times. (G) The graph indicated bioluminescence intensity in the mice model. PBS vs ***P &lt; 0.001. #P &lt; 0.05 and #P &lt; 0.001. Data indicate the mean ± SEM (n = 5). (I) Measurement of tumor volume for 17 days in 4T1 mouse model which injected with 200 μg anti-PD1 antibody and PBS, CD8+ T cells and CXCR3-CD8+ T cells. PBS vs ***P &lt; 0.001. #P &lt; 0.05 and ##P &lt; 0.01. Data indicate the mean ± SEM (n = 5). (H) The weight of tumors separated from mice injected with anti-PD1 antibody and PBS, CD8+ T cells, and CXCR3-CD8+ T cells. *P&lt;0.05, **P&lt;0.01, and ***P &lt; 0.001. Data indicate the mean ± SEM (n=5). Supplementary figure S7. Analysis of clinical prognosis dependent on SCD in TCGA data sets. Analysis of Kaplan-Meier curve for DFS in patients with breast cancer (n=1100) who had high CD4 expression (n=533, 48.45%) and low CD4 expression (n=567, 51.55%). (A) Among high CD4 expression patients, Kaplan-Meier curve for DFS in patients with high SCD expression (n=245, 45.97%) and low SCD expression (n=244, 45.78%). P value: 0.0260. (B) Among low CD4 patients, Kaplan-Meier curve for DFS in patients with high SCD expression (n=253, 44.62%) and low SCD expression (n=255, 44.97%). P value: 0.3501. Analysis of Kaplan-Meier curve for overall survival (OS) and DFS in patients with B-cell lymphoma (n=48). (C) Kaplan-Meier curve for OS in patients with high SCD expression (n=25, 52.08%) and low SCD expression (n=23, 47.92%). P value: 0.500. (D) Kaplan-Meier curve for DFS in patients with SCD high expression (n=24, 50.00%) and SCD low expression (n=20, 41.67%). P value: 0.587. In the DFS data (n = 48), SCD expression data were not available for 4 patients (8.33%).