Journal: Frontiers in Immunology

Article Title: Identification of the novel FOXP3-dependent T reg cell transcription factor MEOX1 by high-dimensional analysis of human CD4 + T cells

doi: 10.3389/fimmu.2023.1107397

Figure Lengend Snippet: Assessment of T reg cell specific expression of MEOX1 (A) Expression of MEOX1 in activated T reg cells and T conv cells over a time period of 360 min (n=2; dataset: GSE11929). (B) MEOX1 gene expression in different immune cells assessed by qRT-PCR and normalized to B2M (n=3). *p < 0.05 (paired Student’s t-test), ** p < 0.01 (paired Student’s t-test). (C) MEOX1 gene expression in different immune cells according to the NextBio database. (D) Application of Markov Clustering Algorithm ‘MCL’ to the consensus network generated in Figure 2 . Visualized is a subnetwork consisting of only three genes (FOXP3, HPGD, and MEOX1). (E) Analysis of MEOX1 protein expression in either unstimulated T reg cells or in T reg cells stimulated with 100 U/ml IL-2 overnight by immunoblotting. (F) MFI (left) and exemplary histogram (right) of MEOX1 expression in human T reg cells and naïve T conv cells. PBMCs were isolated from buffy coats and stimulated overnight with 100 U/ml IL-2 (n=3 of different donors). T reg cells (red) were gated on size, singlets, live, CD4 + , CD3 + , FOXP3 + (Clone 206D), CD45RA - and T conv cells (blue) were gated on size, singlets, live, CD4 + , CD3 + , FOXP3 - (Clone 206D), CD45RA + . Secondary antibody controls are depicted in light (T reg cells) and dark grey (T conv cells). *p < 0.05 (paired Student’s t-test). (G) MEOX1 gene expression in T reg cells (red), T reg cells stimulated with IL-2 (light ref), T reg cells incubated with supernatant from stimulated T conv cells (rose) and T reg cells incubated with supernatant from stimulated T conv cells in combination with anti-CD25 and anti-IL-2 antibodies (grey) assessed by qRT-PCR and normalized to B2M. Data is from one representative experiment of three (mean and s.e.m.) with cells derived from different donors. *p < 0.05 (two-way ANOVA).

Article Snippet: CD25 MicroBeads II, human , Miltenyi Biotec , Cat# 130-092-983.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Generated, Western Blot, Isolation, Incubation, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Identification of the novel FOXP3-dependent T reg cell transcription factor MEOX1 by high-dimensional analysis of human CD4 + T cells

doi: 10.3389/fimmu.2023.1107397

Figure Lengend Snippet: FOXP3 as upstream regulator of MEOX1 expression. (A) Module genes correlated with ‘T reg cell CD3/IL-2’ which exhibit a FOXP3 binding-motif in their promoter region. Genes were colored according to their respective fold change (reference: ‘T conv cell resting’). (B) FOXP3 ChIP tiling array data from human expanded cord-blood T reg cells. Data were analyzed with MAT and overlayed to the MEOX1 locus to identify binding regions (p < 10 -5 and FDR < 0.5%). Data are representative of two independent experiments with cells derived from different donors. (C) Overlay of MeDip-seq and FOXP3 ChIP-seq data (SRA : SRP006674) for the human genomic MEOX1 locus. FOXP3 binding as well as DNA methylation is depicted for T reg (red) and T conv cells (blue). (D) mRNA expression of FOXP3 and MEOX1 in T reg cells treated with scrambled (scrmbld, left) or FOXP3 specific (right) siRNA (E) mRNA expression of FOXP3 and MEOX1 in T reg cells treated with scrambled (left) or MEOX1 specific (right) siRNA (F) mRNA expression of RPS27L in T reg cells treated with scrambled, MEOX1 or FOXP3 specific siRNA. (D–F) Data were first normalized to B2M expression and shown in relation to donor-specific scrambled mRNA expression. (D,E) *p < 0.05 (Student’s t-test). (F) *p < 0.05 (two-way ANOVA). (D–F) Data are representative of three to five independent experiments (mean ± s.e.m.), each with cells derived from a different donor. (G, H) Suppression of allogeneic CD4 + CD25 - T conv cells labelled with the cytosolic dye CFSE by human T reg cells transfected with siRNA targeting MEOX1 (MEOX1) or non-targeting siRNA (scrmbld) presented as CFSE dilution in responding T conv cells cultured with CD3/CD28/anti-MHC-I antibody-coated beads and T reg cells at a ratio of 1:1 (G) , and as relative suppression (H) . Data is from one representative experiment of three with cells derived from different donors. *p < 0.05 (paired Student’s t-test). n.s. = not significant.

Article Snippet: CD25 MicroBeads II, human , Miltenyi Biotec , Cat# 130-092-983.

Techniques: Expressing, Binding Assay, Derivative Assay, Methylated DNA Immunoprecipitation, ChIP-sequencing, DNA Methylation Assay, Transfection, Cell Culture

Journal: Frontiers in Immunology

Article Title: Identification of the novel FOXP3-dependent T reg cell transcription factor MEOX1 by high-dimensional analysis of human CD4 + T cells

doi: 10.3389/fimmu.2023.1107397

Figure Lengend Snippet:

Article Snippet: CD25 MicroBeads II, human , Miltenyi Biotec , Cat# 130-092-983.

Techniques: Recombinant, Reverse Transcription, Derivative Assay, Protease Inhibitor, One Step RT-PCR, Staining, cDNA Synthesis, SYBR Green Assay, Gel Extraction, Purification, Microarray, Methylated DNA Immunoprecipitation Sequencing, Modification, Software

Journal: Oncogene

Article Title: A tumor suppressor role for srGAP3 in mammary epithelial cells.

doi: 10.1038/onc.2012.489

Figure Lengend Snippet: Figure 2. srGAP3 protein and mRNA levels are reduced in breast cancer cell lines partly through epigenetic regulation. (a) Protein levels of srGAP3 in MDA-MB-231, Hs578t, SKBR7, BT474, T47D, HCC1954, MDA-MB-468, BT549, MCF7 and SKBR3 breast cancer cell lines and two normal mammary epithelial cell lines, MCF10A and HMEC are shown on western blots. Actin is used as a control. The following cell culture conditions were used: BT474, MCF7 and T47D in DMEM supplemented with L-glutamine and 10% serum; HCC1954, SKBR7 and BT549 in RPMI1640 supplemented with L-glutamine and 10% serum; MDA-MB-231 and MDA-MB-468 in Leibovitz L15 and 10% serum; Hs578t in DMEM supplemented with L-glutamine, non-essential amino acids and 10% serum; HMECs (Lonza) immortalized with hTERT using pBabe-hygro- hTERT (Addgene plasmid 1773,37 in MEGM; SKBR3 cells in DMEM F12 supplemented with L-glutamine, 15 mM HEPES and 10% serum; MCF10A in DMEM F12 supplemented with 20 ng/ml EGF, 0.5 mg/ml hydrocortizone, 100 ng/ml cholera toxin, 10 mg/ml insulin and 5% of horse serum. All cell lines were cultured with antibiotics at 37 1C and 5% CO2 except MDA-MB-231 and MDA-MB-468, which were incubated without CO2. (b) srGAP3 mRNA expression was analyzed by Q-PCR. Total cellular RNA was isolated from cells lines with the RNAeasy Mini kit (74104 Qiagen, Valencia, CA, USA). Reverse transcription-PCR (RT–PCR) was performed using SuperScript One-Step RT–PCR kit (Invitrogen) using 200 ng of total RNA for each reaction. Quantitative-PCR was performed using taqman reagents on the 7500 real-time PCR system (Applied Biosystem, Carlsbad, CA, USA). mRNA expression levels of srGAP3 (Hs00322672_m1, Applied Biosystem) were normalized to GAPDH mRNA levels (Hs99999905_m1, Applied Biosystem). Data are represented as fold inductions relative to HMECs. **Po0.01; ***Po0.001. (c) Oncomine microarray data analysis of srGAP3 expression in human breast cancer vs normal breast tissue is shown. The Student’s t-test was conducted using the Oncomine software (http://www.oncomine.org). The boxes represent the 25th through 75th percentiles; the horizontal lines represent the medians; the points represent the end of the ranges. In the left panel, 18 samples were analyzed and compared with 6 normal samples.20 In the right histogram a total of 32 invasive ductal breast carcinomas samples were compared with 3 normal samples.21 (d) srGAP3 expression levels were measured after inhibition of DNA methylation using 5-azacytidine, (5-AzaC, Sigma A2385) or histone deacetylation using trichostatin A, (TSA, Sigma T8552) in MCF7, BT549 and BT474 breast cancer cell lines. 1 105 cells were plated the day before treatment in a six-well plate and 5 mM or 10 mM of 5-AzaC was added for 96 h and 0.5 mM of TSA was added for 20–24 h. Protein lysates and RNA levels were analyzed after treatment. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: Total cellular RNA was isolated from cells lines with the RNAeasy Mini kit (74104 Qiagen, Valencia, CA, USA).

Techniques: Western Blot, Control, Cell Culture, Plasmid Preparation, Incubation, Expressing, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, One Step RT-PCR, Real-time Polymerase Chain Reaction, Microarray, Software, Inhibition, DNA Methylation Assay

Journal: Science advances

Article Title: MAGE-A4 induces non-small cell lung cancer and tumor-promoting plasma cell accumulation.

doi: 10.1126/sciadv.ads4227

Figure Lengend Snippet: Fig. 1. MAGE-A4 is expressed in human NSCLC and is associated with loss of PTEN. (A) MAGE-A4 expression in NSCLC by mRNA from TCGA dataset, n = 989. Fragments per kilobase million ≥1 was considered positive. (B) Representative H&E of human NSCLC and serial sections show MAGE-A4pos staining (IHC) in lung adenocarcinoma (LUAD) and in lung squamous cell carcinoma (LUSC); 100× (top); scale bar, 150 μm (insets). (C) Incidence of MAGE-A4 in NSCLC array of 23 NSCLC tumors and cellular local- ization of MAGE-A4 as determined by IHC detection. LUSC/mixed includes LUSC, adenosquamous (mixed features), and large-cell carcinoma subtypes. Total MAGE-A4pos in tumor cases (left graph) and all MAGE-A4pos by subcellular localization (right side of graph). (D) The Cancer Genome Atlas (TCGA) data of 1023 cases shows MAGE-A4 expres- sion in human NSCLC is correlated with decreased PTEN, as indicated by homozygous (hom) loss of both copies, or heterozygous (het) loss of PTEN, compared with intact, nonmutated PTEN, having ≥2 copies (wt/gain). Association of MAGEA4 with PTEN loss comparing both loss groups with wt/gain by t test (P = 1 × 10−8).

Article Snippet: To generate an embryonic stem cell targeting construct, the human MAGE- A4 cDNA (OriGene, RC223991) was inserted into the targeting construct downstream of a Lox- Stop- Lox (LSL) cassette (fig. S2A).

Techniques: Expressing, Staining

Journal: Science advances

Article Title: MAGE-A4 induces non-small cell lung cancer and tumor-promoting plasma cell accumulation.

doi: 10.1126/sciadv.ads4227

Figure Lengend Snippet: Fig. 2. De novo hMAGE-A4 expression in the lung causes invasive LUAD in the absence of Pten. (A) Breeding scheme to generate mouse models and controls: CCSPWT hMAGE-A4LSL/Ptenf/f (Cre-MAG4/Pt), CCSPiCre Ptenf/f (Pt), CCSPiCre hMAGE-A4LSL (MAG4), and CCSPiCre hMAGE-A4LSL/Ptenf/f (MAG4/Pt). (B) Representative H&E-stained images of Cre-MAG4/Pt, Pt, MAG4, and MAG4/Pt lung from 6- to 7-month-old mice demonstrate tumors in MAG4/Pt but not in MAG4 or Pt alone. Arrows indicate areas with loss of basement membrane and invasion. The scale bar is 300 μm; the inset scale bar is 150 μm. (C) Tumor incidence among groups of mice at indicated ages as deter- mined by histology. n = 8 to 27 per group. (D) Representative IHC of MAGE-A4 expression in all four genotypes at 6 to 7 months of age. Black arrows in MAG4/Pt indicate areas of invasion with loss of basement membrane. The scale bar is 300 μm (left panel) and 75 μm (right insets). (E) H&E (top) and respective IHC of MAGE-A4 (bottom) of Cre-MAG4/Pt and MAG4/Pt spleen at 12 months of age; scale bar, 75 μm. (F) Summary of MAG4/Pt tumors metastasize to spleen, intestine, and cervical lymph nodes by age as determined from d-luciferin screening. (G) Detection of cgb1a1 in 12 to 13 months in Cre-MAG4/Pt versus MAG4/Pt spleen by quantitative polymerase chain reaction (qPCR), n = 4 per group, one experiment. Student’s t test, *P < 0.05

Article Snippet: To generate an embryonic stem cell targeting construct, the human MAGE- A4 cDNA (OriGene, RC223991) was inserted into the targeting construct downstream of a Lox- Stop- Lox (LSL) cassette (fig. S2A).

Techniques: Expressing, Staining, Membrane, Real-time Polymerase Chain Reaction

Journal: Science advances

Article Title: MAGE-A4 induces non-small cell lung cancer and tumor-promoting plasma cell accumulation.

doi: 10.1126/sciadv.ads4227

Figure Lengend Snippet: Fig. 5. MAGE-A4 indirectly induces CXCL12 in endothelial cells to retain PCs by CXCL12-CXCR4 axis. (A) CXCL12 concentration from whole-lung homogenate from 7- to 8-month-old mice by ELISA. Data from three independent experiments combined, n = 7 to 21 per group. (B) Featureplot of whole lung shows colocalization of Cxcl12 with Pecam1. (C) Dotplot of Cxcl12 in endothelial (Pecam1+) cells. (D) Schematic of assay design, using human umbilical vein endothelial cells (HUVEC) cocultured with human NSCLC MAGE-A4k/o or MAGE-A4pos H1299 cells for 24 hours. (E) CXCL12 gene expression quantified using qPCR. Two independent experiments combined, n = 3 to 4 per group. Significance determined by one-way ANOVA with Dunnett’s correction or Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001; ns = not significant.

Article Snippet: To generate an embryonic stem cell targeting construct, the human MAGE- A4 cDNA (OriGene, RC223991) was inserted into the targeting construct downstream of a Lox- Stop- Lox (LSL) cassette (fig. S2A).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Science advances

Article Title: MAGE-A4 induces non-small cell lung cancer and tumor-promoting plasma cell accumulation.

doi: 10.1126/sciadv.ads4227

Figure Lengend Snippet: Fig. 6. MAGE-A4pos human NSCLC exhibits increased PCs and excluded T cells. IMC was used to evaluate a microarray of 24 individual NSCLC samples. (A and B) Over- lay of gated CD19+ CD138+ CD38+ PCs onto images showing PCs (red) in (A) MAGE-A4neg and (B) colocalization of MAGE-A4 and E-Cadherin (pink) with PCs (red) in MAGE- A4pos. (C) CD19+ CD138+ PCs quantified as relative abundance of total immune cells. Total immune cells are the sum of non–B cell CD45+, B cells, and PCs. (D) Representative IMC staining of CD4 and CD8 T cells in MAGE-A4neg and MAGE-A4pos tumors showing T cell exclusion in MAGE-A4pos NSCLC. (E to H) Mean cell density of T cells in a 200 μm diameter around each tumor cell. Cell density calculation schematic (inset). Mean cell density quantification of (E) CD8 T cells, (F) CD4 T cells, (G) PD-1+ CD8, and (H) PD-1+ CD4 T cells. (I and J) TCGA data evaluated for PCs (PRDM1/IRF4/CD138) and MAGEA4. (I) PCs are significantly enriched in human MAGE-A4pos NSCLC. (J) Human NSCLC with high enrichment of PCs are positively correlated with MAGE-A4pos. Significance determined by Welch’s or Student’s t test for normally distributed data and Wilcoxon rank-sum test for non-normal data. *P < 0.05 and **P < 0.01.

Article Snippet: To generate an embryonic stem cell targeting construct, the human MAGE- A4 cDNA (OriGene, RC223991) was inserted into the targeting construct downstream of a Lox- Stop- Lox (LSL) cassette (fig. S2A).

Techniques: Microarray, Staining

Journal: Science advances

Article Title: MAGE-A4 induces non-small cell lung cancer and tumor-promoting plasma cell accumulation.

doi: 10.1126/sciadv.ads4227

Figure Lengend Snippet: Fig. 8. MAGE-A4 role in NSCLC development by immune modulation. MAGE-A4 promotes tumorigenesis and metastasis. MAGE-A4 stimulates endothelial cells to in- crease production of CXCL12, which recruits and retains cognate CXCR4+ PCs, expressing TGF-β and IL-10 and producing IgA. These immunosuppressive PCs inhibit antigen-presenting cells, including macrophages and cross-presenting CD103+ dendritic cells. Immunosuppressive PCs also inhibit the infiltration and activation of all T cell subsets. While CD163+ TAMs are increased in the MAG4/Pt tumor microenvironment, these are driven by B cells, and the absence of both immune cell types does not affect tumor burden. In contrast, PCs promote tumor progression.

Article Snippet: To generate an embryonic stem cell targeting construct, the human MAGE- A4 cDNA (OriGene, RC223991) was inserted into the targeting construct downstream of a Lox- Stop- Lox (LSL) cassette (fig. S2A).

Techniques: Expressing, Activation Assay

Journal: Frontiers in Immunology

Article Title: Identification of the novel FOXP3-dependent T reg cell transcription factor MEOX1 by high-dimensional analysis of human CD4 + T cells

doi: 10.3389/fimmu.2023.1107397

Figure Lengend Snippet: Assessment of T reg cell specific expression of MEOX1 (A) Expression of MEOX1 in activated T reg cells and T conv cells over a time period of 360 min (n=2; dataset: GSE11929). (B) MEOX1 gene expression in different immune cells assessed by qRT-PCR and normalized to B2M (n=3). *p < 0.05 (paired Student’s t-test), ** p < 0.01 (paired Student’s t-test). (C) MEOX1 gene expression in different immune cells according to the NextBio database. (D) Application of Markov Clustering Algorithm ‘MCL’ to the consensus network generated in Figure 2 . Visualized is a subnetwork consisting of only three genes (FOXP3, HPGD, and MEOX1). (E) Analysis of MEOX1 protein expression in either unstimulated T reg cells or in T reg cells stimulated with 100 U/ml IL-2 overnight by immunoblotting. (F) MFI (left) and exemplary histogram (right) of MEOX1 expression in human T reg cells and naïve T conv cells. PBMCs were isolated from buffy coats and stimulated overnight with 100 U/ml IL-2 (n=3 of different donors). T reg cells (red) were gated on size, singlets, live, CD4 + , CD3 + , FOXP3 + (Clone 206D), CD45RA - and T conv cells (blue) were gated on size, singlets, live, CD4 + , CD3 + , FOXP3 - (Clone 206D), CD45RA + . Secondary antibody controls are depicted in light (T reg cells) and dark grey (T conv cells). *p < 0.05 (paired Student’s t-test). (G) MEOX1 gene expression in T reg cells (red), T reg cells stimulated with IL-2 (light ref), T reg cells incubated with supernatant from stimulated T conv cells (rose) and T reg cells incubated with supernatant from stimulated T conv cells in combination with anti-CD25 and anti-IL-2 antibodies (grey) assessed by qRT-PCR and normalized to B2M. Data is from one representative experiment of three (mean and s.e.m.) with cells derived from different donors. *p < 0.05 (two-way ANOVA).

Article Snippet: CD45RA MicroBeads, human , Miltenyi Biotec , Cat# 130-045-901.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Generated, Western Blot, Isolation, Incubation, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Identification of the novel FOXP3-dependent T reg cell transcription factor MEOX1 by high-dimensional analysis of human CD4 + T cells

doi: 10.3389/fimmu.2023.1107397

Figure Lengend Snippet:

Article Snippet: CD45RA MicroBeads, human , Miltenyi Biotec , Cat# 130-045-901.

Techniques: Recombinant, Reverse Transcription, Derivative Assay, Protease Inhibitor, One Step RT-PCR, Staining, cDNA Synthesis, SYBR Green Assay, Gel Extraction, Purification, Microarray, Methylated DNA Immunoprecipitation Sequencing, Modification, Software