Journal: Frontiers in Immunology

Article Title: Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells

doi: 10.3389/fimmu.2021.790775

Figure Lengend Snippet: (A) IR1 (TIRC7+) and IR1 neg Tregs (TIRC7-) frequencies as counts per 10,000 freshly isolated PBMC in peripheral blood from 22 healthy subjects. (B) Differentiation of CD25+ population shows distribution of CD4+ CD25+ TIRC7+ cells, which are mainly localized between CD25med/hi. Cells were pre-gated on CD4. (C) Two overlaid histograms (blue FMO control, red TIRC7 panel) show extracellular TIRC7 expression on Tregs from healthy donors, directly after isolation (T=0h) (upper panel) and after 72h IL-2 stimulation (lower panel). The x-axis shows TIRC7 expression. (Supplemental data for gating: 1.1 for t0, 1.2 for t72h). Post expansion (lower panel) the proportion of IR1cells is higher than pre-expansion (upper panel). (D) The x-axis shows TIRC7 expression. The Y-axis shows FOXP3 expression levels. At t0 (upper panel) IR1 cells express FOXP3. After 72 hours (lower panel) of IL-2 induction the proportion of FOXP3+IR1 cells is further increased. Cells were pre-gated on CD4, CD25, CD127lo and FOXP3. ( Supplementary Figures 1.2 , 1.3 ). (E) Treg subpopulations (IR1neg and IR1) are mapped as secreting (CD45RO+, CD25dim/med), resting (CD45RO-, CD25dim/med) and activated (CD45RO+, CD25bright) subsets of Treg at t=72h IL-2 stimulation. (See Supplementary Figure 1.4 for gating). (F) As above, Tregs are mapped to memory (CD45RO+, CD25med) and effector (CD45RO+, CD25hi) subsets of Treg. Cells were pre-gated on CD4, CD25med/high, FoxP3 ( Supplementary Figure 1.1 ) The distribution from IR1 and IR1neg to the effector (CD25high, CD45RO+) or memory group (CD25med, CD45RO+) was assessed. (G) Expression of TIRC7 vs CD45RA in CD25+CD127loFoxp3+ nonactivated Treg cells. Shown is one representative example out of five obtained from PBMC of healthy donors. IR1 cells make up 11.3% (3.0/26.5) of CD45RAhi, and 9.5% (7.0/73.5) of the more numerous CD45RAlo sets. (H) Immune phenotyping of IR1 and IR1neg cells in nonactivated CD45RAhi and CD45RAlo population in HD (n=5) (Gating Supplementary Figure 1.4 ). (I) Representative example of activation markers of IR1 and IR1neg Tregs in nonactivated CD45RAhi. (J) Representative example of activation markers of IR1 and IR1neg Tregs in nonactivated CD45ROlow in CD25+CD127loFoxp3+ Treg cells. Sample shown obtained from a healthy donors PBMC (n=5). Marker expression is measured on the y-axis versus side scatter (SSC-A) on the x-axis.

Article Snippet: Expansion of Tregs was performed by using the Human Treg Expansion Kit, from Miltenyi.

Techniques: Isolation, Control, Expressing, Activation Assay, Marker

Journal: Frontiers in Immunology

Article Title: Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells

doi: 10.3389/fimmu.2021.790775

Figure Lengend Snippet: Mean IR1 cell and IR1neg Treg counts per 10,000 live PBMC after isolation (t=0h) and after activation with IL-2 (t=72h). N=8 subjects.

Article Snippet: Expansion of Tregs was performed by using the Human Treg Expansion Kit, from Miltenyi.

Techniques: Isolation, Activation Assay

Journal: Frontiers in Immunology

Article Title: Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells

doi: 10.3389/fimmu.2021.790775

Figure Lengend Snippet: (A) IR1 cells express multiple membrane and soluble immune suppressor molecules. Surface expression of HLA-DR, CD39, LAP, ICOS, IL-10, CTLA-4, CCR7, CD62L, GARP and Helios is higher for IR1 cells than for IR1neg Tregs after 72h IL-2 induction. Violin plots were generated using statistical software Prism GraphPad. (Gating Supplementary Figure 1.2 , for overlaid histograms from marker on IR1 versus IR1neg; Supplementary Figure 2A ). Medians indicated by red lines. P-values reflect **P < 0.01, *P < 0.05 whether the ratio of IR1/IR1neg is significantly different from one. (See also Table 3 ). (B) Representative example of Immunophenotyping for two subpopulations: IR1 Treg compared to IR1neg Treg, using the above ten markers plus CD45RO. Marker expression is measured on the y-axis versus side scatter (SSC-A) on the x-axis. Smoothed pseudo-color dot plots are shown. (Gating Supplementary Figure 1.2 , for overlaid histograms from marker on IR1 versus IR1neg; Supplementary Figure 2A , MFI- Values (B) . (C) Ratio (IR1 cells/IR1neg Tregs) of median percentage Tregs with the respective marker: CD62L, LAP, IL-10, CTLA-4, HLA-DR, GARP, ICOS, HELIOS, CD39, CCR7 and CD45RO.

Article Snippet: Expansion of Tregs was performed by using the Human Treg Expansion Kit, from Miltenyi.

Techniques: Membrane, Expressing, Generated, Software, Marker

Journal: Frontiers in Immunology

Article Title: Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells

doi: 10.3389/fimmu.2021.790775

Figure Lengend Snippet: (A) Coincubation of humanized TIRC7 agonist antibody significantly induce TIRC7+ IR1 cells in human PBMC cultures after 72h compared to IgG-control ex vivo (n=7). ** mAb = p.0025. (B) Exposure of purified CD25+CD4+ cells to IL-2 plus agonist anti-TIRC7 mAb leads to profound expansion of IR1 cells after 2 weeks. The absolute numbers of purified Treg cells after 2 weeks of expansion cultures are shown. The absolute numbers of IR1 cells in the presence of TIRC7 antibody plus IL-2 exceed those achieved in the IL-2 cultures alone by several fold. (C) Stability of suppressive phenotypes as reflected based on the expression of markers LAP, CD39, GARP, IL-10 and FOXP3 remain high and unchanged in expanded IR1+ cultures after 14 days. (D) In BrdU proliferation assay, expanded human IR1+ cells show more potent inhibition of lymphocyte activation in mixed lymphocyte culture (MLC) compared to IR1neg Tregs.

Article Snippet: Expansion of Tregs was performed by using the Human Treg Expansion Kit, from Miltenyi.

Techniques: Control, Ex Vivo, Purification, Expressing, Proliferation Assay, Inhibition, Activation Assay

Journal: Journal of Immunology Research

Article Title: Decreased GZMB , NRP1 , ITPR1 , and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

doi: 10.1155/2022/3426717

Figure Lengend Snippet: Gating strategy for CD4 + CD25 + FOXP3 + Treg cells. Estimation of protein levels of CD4, and CD25. (a) Lymphocytes were gated on the basis of size and morphology. (b) CD3 + CD4 + T cells were gated on the basis of CD3 and CD4 expression.(c) Treg cells were gated on the basis of CD3, CD4, CD25, and FOXP3 expression. The purity of isolated CD3 + CD4 + CD25 + FOXP3 + Treg cells was found to be 94.22%. (d) Expression of CD3 in T cells. Representative graph shows the amount of CD3 in the T cells as mean fluorescence intensity (MFI). (e) Expression of CD4 in T cells. Representative graph shows amount of CD4 in the T cells as mean fluorescence intensity (MFI). (f) Expression of CD25 in T cells. Representative graph shows amount of CD25 in the T cells as mean fluorescence intensity (MFI). (g) Expression of FOXP3 in T cells. Representative graph shows amount of intracellular FOXP3 in the T cells as mean fluorescence intensity (MFI).

Article Snippet: CD4 + CD25 + Treg cells and CD4 + T cells were isolated from three-milliliter peripheral blood of 52 GV patients and 48 controls using MACSxpress® whole blood Treg isolation kit (Miltenyi Biotec, Auburn, CA) as mentioned previously [ ].

Techniques: Expressing, Isolation, Fluorescence

Journal: Journal of Immunology Research

Article Title: Decreased GZMB , NRP1 , ITPR1 , and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

doi: 10.1155/2022/3426717

Figure Lengend Snippet: Correlation of in vitro Treg suppression assay with ITPR1 , GZMB , NRP1 , and SERPINB9 transcripts in Tregs of GV patients. The correlation of in vitro Treg suppression assay with ITPR1 , GZMB , NRP1 , and SERPINB9 was analyzed by Spearman's correlation analysis. (a) GZMB transcripts were positively correlated with in vitro Treg mediated suppression of CD4 + T cells in GV patients' Tregs ( r = 0.61; p = 0.0012). (b) NRP1 transcripts were positively correlated with in vitro Treg-mediated suppression of CD4 + T cells in GV patients' Tregs ( r = 0.55; p = 0.021). (c) SERPINB9 transcripts were positively correlated with in vitro Treg-mediated suppression of CD4 + T cells in GV patients' Tregs ( r = 0.56; p = 0.002). (d) ITPR1 transcripts were positively correlated with in vitro Treg-mediated suppression of CD4 + T cells in GV patients' Tregs ( r = 0.54; p = 0.001). (e) GZMB transcripts positively correlated with in vitro Treg-mediated suppression of CD8 + T cells in GV patients' Tregs ( r = 0.58; p = 0.004). (f) NRP1 transcripts positively correlated with in vitro Treg-mediated suppression of CD8 + T cells in GV patients' Tregs ( r = 0.52; p = 0.0022). (g) SERPINB9 transcripts positively correlated with in vitro Treg-mediated suppression of CD8 + T cells in GV patients' Tregs ( r = 0.48; p = 0.024). (h) ITPR1 transcripts positively correlated with in vitro Treg-mediated suppression of CD8 + T cells in GV patients' Tregs ( r = 0.49; p = 0.032).

Article Snippet: CD4 + CD25 + Treg cells and CD4 + T cells were isolated from three-milliliter peripheral blood of 52 GV patients and 48 controls using MACSxpress® whole blood Treg isolation kit (Miltenyi Biotec, Auburn, CA) as mentioned previously [ ].

Techniques: In Vitro, Suppression Assay

Journal: Journal of Immunology Research

Article Title: Decreased GZMB , NRP1 , ITPR1 , and SERPINB9 Transcripts Lead to Reduced Regulatory T Cells Suppressive Capacity in Generalized Vitiligo Patients

doi: 10.1155/2022/3426717

Figure Lengend Snippet: Role of GZMB , NRP1 , SERPINB9 , and ITPR1 transcripts in GV pathogenesis. (a) The decreased ITPR1 transcripts could lead to impaired calcium-NFAT signalling pathway, which might result in decreased GZMB and NRP1 transcripts. Further, the decreased SERPINB9 transcripts may result in increased granzyme B-mediated endogenous apoptosis of Tregs. Overall, the decreased GZMB , NRP1 , SERPINB9 , and ITPR1 transcripts result into decreased Treg suppressive capacity, which could lead to unchecked CD4 + and CD8 + T cells and thereby results into melanocytes' destruction contributing to GV pathogenesis, progression, and severity. (b) Upon calcium treatment, ITPR1 mRNA expression is increased which may lead to intracellular Treg calcium influx and calcium-NFAT signalling pathway, thereby results into increased GZMB and SERPINB9 transcripts, leading to increased Treg suppressive capacity. The increased Treg suppressive capacity controls the CD8 + and CD4 + T cells proliferation and IFN- γ production and thereby contributes to melanocytes survival.

Article Snippet: CD4 + CD25 + Treg cells and CD4 + T cells were isolated from three-milliliter peripheral blood of 52 GV patients and 48 controls using MACSxpress® whole blood Treg isolation kit (Miltenyi Biotec, Auburn, CA) as mentioned previously [ ].

Techniques: Expressing

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Comparison of Th17 cells, Treg cells, and Th17/Treg ratios between stroke patients and HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques: Comparison

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 was positively associated with a Th17/Treg imbalance. Association of serum RBP4 with Th17 cells (A) , Treg cells (B) , and the Th17/Treg ratio (C) in stroke patients. Association of serum RBP4 with Th17 cells (D) , Treg cells (E) , and the Th17/Treg ratio (F) in HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were negatively associated with the MMSE score at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with MMSE score at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were associated with the occurrence of cognitive impairment at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with cognitive impairment at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

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