Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Modulation of FoxP3 pre-mRNA splicing with SSO targeting only exon 2 cis -elements does not allow us to obtain Tregs expressing a single splice variant. ( A ) Two splicing regulator proteins, SRp40 (shown as green ellipses), interact with their binding sites (shown in bold green font) within exon 2 and are responsible for the inhibition of exon 2 insertion in mature FoxP3 mRNA. The splicing regulator proteins SF2/ASF (shown as a red ellipse) interact with its binding site (shown in bold red font) within intron 2 and are responsible for the inhibition of exon 2 deletion from mature mRNA. ( B ) Treg transfection with #Ins2, a 36-mer-specific antisense SSO (presented in green italics font), blocks SRp40 from binding to its sensitive cis -elements and induces the insertion of exon 2 into the mature mRNA. ( C ) Treg transfection with #Del2, a 36-mer-specific antisense SSO (presented in red italics font), blocks SF2/ASF from binding to its sensitive cis -elements and induces the deletion of exon 2 from the mature mRNA. FoxP3 splice variant mRNA levels in cells 96 h after transfection with ( D ) #Ins2 or ( E ) #Del2 SSOs. The levels of investigated mRNAs were normalized to the mean expression of three reference genes: 18S, GAPDH, and beta-actin. N = 4. The results are shown as the mean ± SD. FL, full-length splice variant; ∆2, splice variant with deleted exon 2; ∆7, splice variant with deleted exon 7; ∆2∆7, splice variant with deleted both exon 2 and exon 7. ND, not detected.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Binding Assay, Inhibition, Transfection

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Modulation of FoxP3 pre-mRNA splicing with SSO targeting only exon 7 cis -elements does not allow us to obtain Tregs expressing a single splice variant. ( A ) Two splicing regulator proteins, SC35 and SRp75 (shown as green ellipses), interact with their binding sites (shown in bold green font) within exon 7 and are responsible for the inhibition of exon 7 insertion in mature FoxP3 mRNA. The splicing regulator proteins SF2/ASF (shown as a red ellipse) interact with its binding site (shown in bold red font) within intron 7 and are responsible for the inhibition of exon 7 deletion from mature mRNA. ( B ) Treg transfection with #Ins7, a 36-mer-specific antisense SSO (presented in green italics font), blocks both SC35 and SRp75 from binding to their sensitive cis -elements and induces the insertion of exon 7 into the mature mRNA. ( C ) Treg transfection with #Del7, a 36-mer-specific antisense SSO (presented in red italics font), blocks SF2/ASF from binding to its sensitive cis -elements and induces the deletion of exon 7 from the mature mRNA. FoxP3 splice variant mRNA levels in cells 96 h after transfection with ( D ) #Ins7 or ( E ) #Del7 SSOs. The levels of investigated mRNAs were normalized to the mean expression of three reference genes: 18S, GAPDH, and beta-actin. N = 4. The results are shown as the mean ± SD. FL, full-length splice variant; ∆2, splice variant with deleted exon 2; ∆7, splice variant with deleted exon 7; ∆2∆7, splice variant with deleted both exon 2 and exon 7. ND, not detected.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Binding Assay, Inhibition, Transfection

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Modulation of FoxP3 pre-mRNA splicing with SSOs targeting both exon 2 and exon 7 cis -elements allowed us to obtain Tregs expressing a single splice variant. FoxP3 splice variant mRNA levels in Treg cells 96 h after transfection with ( A ) control nonspecific 36-mer nucleotides #Con1 & #Con2; ( B ) SSOs #Ins2 & #Ins7, which could induce the expression of the FL variant only; ( C ) SSOs #Del2 & #Ins7, which could induce the expression of the ∆2 splice variant only; ( D ) SSOs #Ins2 & #Del7, which could induce the expression of the ∆7 splice variant only; and ( E ) SSOs #Del2 & #Del7, which could induce the expression of the ∆2∆7 splice variant only. N = 4. The results are shown as the mean ± SD. * p ≤ 0.001 by Mann–Whitney U test. FL, full-length splice variant; ∆2, splice variant with deleted exon 2; ∆7, splice variant with deleted exon 7; ∆2∆7, splice variant with deleted both exon 2 and exon 7. ND, not detected. ( F ) Western blotting results demonstrate the induction of the ∆2 splice variant. Clone 150D is exon 2 specific, while clone 259D recognizes an epitope after exon 2 common for all FoxP3 splice variants.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Transfection, Control, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Immunophenotype of Tregs expressing only one FoxP3 splice variant. The expression of Treg-associated cell markers was determined by flow cytometry in Tregs four days after transfection with each of the SSOs. Cell membrane markers were ( A ) CD4 High , ( B ) CD25 High , ( C ) CD127 Low , and ( D ) CD152 High . Cell membrane markers associated with Treg suppressive activity were ( E ) CD39 High and ( F ) CD223 High . Nuclear markers associated with Treg stability: ( G ) FoxP3 High and ( H ) Helios High . n = 4. Black horizontal lines indicate the mean ± SEM. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005 vs. cells transfected with #Con1 & #Con2 oligonucleotides by the Mann–Whitney U test.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Flow Cytometry, Transfection, Membrane, Activity Assay, MANN-WHITNEY

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Levels of mRNA of molecules associated with Treg suppressive activity. Total mRNA was isolated from Tregs transfected with oligonucleotides and analyzed by real-time RT-PCR. The mRNA levels of ( A ) CTLA4, ( B ) LGALS9, and ( C ) NRP1 were normalized to the mean expression of three reference genes: 18S, GAPDH, and ACTB. n = 4. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005 vs. cells transfected with #Con1 & #Con2 oligonucleotides by the Mann–Whitney U test.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Activity Assay, Isolation, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

doi: 10.3390/cells13010077

Figure Lengend Snippet: Treg-associated cytokine concentrations in cell culture from Tregs expressing a single FoxP3 splice variant. Concentrations of ( A ) IL-10, ( B ) IL-12 (p40), ( C ) IL-12 (p70), ( D ) IL-19, ( E ) IL-20, ( F ) IL-22, ( G ) IL-26, ( H ) IL-27 (p28), ( I ) IL-28A/IFN-λ2, ( J ) IL-29/IFN-λ1, and ( K ) IL-35 were determined by Bio-Plex assay. n = 4. Black horizontal lines indicate the mean ± SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005 vs. cells transfected with #Con1 & #Con2 oligonucleotides by the Mann–Whitney U test.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Cell Culture, Expressing, Variant Assay, Plex Assay, Transfection, MANN-WHITNEY