treg Search Results


beads  (Miltenyi Biotec)
97
Miltenyi Biotec beads
Beads, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Expansion+Kit%2C+mouse/pmc06124443-430-26-33
Average 97 stars, based on 1 article reviews
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treg exo ikvav  (Exosome Diagnostics)
86
Exosome Diagnostics treg exo ikvav
Schematic illustration of the fabrication and <t>evaluation</t> <t>of</t> <t>Treg-Exo-IKVAV</t> for traumatic SCI.( a ) The isolation of Treg-Exo and the preparation process of Treg-Exo-IKVAV. ( b ) Mechanism underlying Treg-Exo-IKVAV’s immunoregulatory and neuro-regenerative capabilities. ( c ) Treg-Exo-IKVAV spatiotemporally sequentially reshaped the neuro-immune microenvironment, and then promoted functional recovery of SCI mice
Treg Exo Ikvav, supplied by Exosome Diagnostics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/exo+ikvav+treg/pmc12888608-68-102-43
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cellxvivo human th2 cell differentiation kit  (R&D Systems)
94
R&D Systems cellxvivo human th2 cell differentiation kit
Figure 5. The pivotal effect of PM10-induced IL-33 for <t>Th2</t> differentiation effects of conditioned media on PM10-induced human nasal polyp derived fibroblast (NPDFs-CM) on the inflammatory cytokine production in CD4+ T cells. (A) Th2 differentiation was determined by the expression of GATA3 and ST2 (IL-33 receptor). (B,C) The protein levels of IFN-γ, IL-17A, IL-4, IL-13, and IL-10 in CD4+ T cell populations were determined by flow cytometry according to the addition of ST2. The graphic data represents the means ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.0001 as compared to the PM10-treated group.
Cellxvivo Human Th2 Cell Differentiation Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/CellXVivo+Human+Treg+Cell+Differentiation+Kit/pm35162939-220-50-56
Average 94 stars, based on 1 article reviews
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macsxpress whole blood treg isolation kit  (Miltenyi Biotec)
93
Miltenyi Biotec macsxpress whole blood treg isolation kit
A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody ( n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA ( n = 8 biological replicates per group). D <t>Treg</t> migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG ( n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.
Macsxpress Whole Blood Treg Isolation Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/MACSxpress+Whole+Blood+Treg+Isolation+Kit%2C+human/pmc12678525-359-9-16
Average 93 stars, based on 1 article reviews
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human treg expansion kit  (Miltenyi Biotec)
97
Miltenyi Biotec human treg expansion kit
(A) IR1 (TIRC7+) and IR1 <t>neg</t> <t>Tregs</t> (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. ( <xref ref-type= 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. " width="250" height="auto" />
Human Treg Expansion Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Expansion+Kit%2C+human/pmc08867398-263-8-13
Average 97 stars, based on 1 article reviews
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t regulatory cell flow cytometry  (R&D Systems)
93
R&D Systems t regulatory cell flow cytometry
(A) IR1 (TIRC7+) and IR1 <t>neg</t> <t>Tregs</t> (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. ( <xref ref-type= 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. " width="250" height="auto" />
T Regulatory Cell Flow Cytometry, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Regulatory+T+Cell+(Treg)+Flow+Cytometry+Panel/10__1007_slash_s42485___024___00149___5-71-4-12
Average 93 stars, based on 1 article reviews
t regulatory cell flow cytometry - by Bioz Stars, 2026-10
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anti cd28  (Miltenyi Biotec)
97
Miltenyi Biotec anti cd28
(A) IR1 (TIRC7+) and IR1 <t>neg</t> <t>Tregs</t> (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. ( <xref ref-type= 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. " width="250" height="auto" />
Anti Cd28, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Suppression+Inspector%2C+human/pmc04607131-223-20-25
Average 97 stars, based on 1 article reviews
anti cd28 - by Bioz Stars, 2026-10
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cellxvivo human th1 cell differentiation kit  (R&D Systems)
94
R&D Systems cellxvivo human th1 cell differentiation kit
(A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of <t>Th1</t> polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.
Cellxvivo Human Th1 Cell Differentiation Kit, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/CellXVivo+Human+Treg+Cell+Differentiation+Kit/pmc05760318-51-13-19
Average 94 stars, based on 1 article reviews
cellxvivo human th1 cell differentiation kit - by Bioz Stars, 2026-10
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foxp3 vior667  (Miltenyi Biotec)
97
Miltenyi Biotec foxp3 vior667
(A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of <t>Th1</t> polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.
Foxp3 Vior667, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Phenotyping+Kit%2C+anti-human%2C+REAfinity/pmc12256293-26-0-3
Average 97 stars, based on 1 article reviews
foxp3 vior667 - by Bioz Stars, 2026-10
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tcr bcr microfluidics yes medium dekosky 2013 198 bcr microwells  (fluidigm)
90
fluidigm tcr bcr microfluidics yes medium dekosky 2013 198 bcr microwells
(A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of <t>Th1</t> polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.
Tcr Bcr Microfluidics Yes Medium Dekosky 2013 198 Bcr Microwells, supplied by fluidigm, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Maxpar+Human+Treg+Phenotyping+Panel+Kit/10__1039_slash_c9me00071b-154-60-26
Average 90 stars, based on 1 article reviews
tcr bcr microfluidics yes medium dekosky 2013 198 bcr microwells - by Bioz Stars, 2026-10
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treg detection kit  (Miltenyi Biotec)
97
Miltenyi Biotec treg detection kit
(A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of <t>Th1</t> polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.
Treg Detection Kit, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Analysis+Kit%2C+with+FoxP3-PE%2C+REAfinity/10__1158_slash_2767___9764__crc___22___0180-127-1-4
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treg detection kit - by Bioz Stars, 2026-10
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treg surface marker analysis cocktail  (Miltenyi Biotec)
97
Miltenyi Biotec treg surface marker analysis cocktail
Figure 4. Modulation of FoxP3 pre-mRNA splicing with SSO targeting only exon 7 cis-elements does not allow us to <t>obtain</t> <t>Tregs</t> 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) <t>Treg</t> 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.
Treg Surface Marker Analysis Cocktail, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/treg/Treg+Surface+Marker+Analysis+Cocktail%2C+anti-human/pm38201281-63-13-18
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Image Search Results


Schematic illustration of the fabrication and evaluation of Treg-Exo-IKVAV for traumatic SCI.( a ) The isolation of Treg-Exo and the preparation process of Treg-Exo-IKVAV. ( b ) Mechanism underlying Treg-Exo-IKVAV’s immunoregulatory and neuro-regenerative capabilities. ( c ) Treg-Exo-IKVAV spatiotemporally sequentially reshaped the neuro-immune microenvironment, and then promoted functional recovery of SCI mice

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Schematic illustration of the fabrication and evaluation of Treg-Exo-IKVAV for traumatic SCI.( a ) The isolation of Treg-Exo and the preparation process of Treg-Exo-IKVAV. ( b ) Mechanism underlying Treg-Exo-IKVAV’s immunoregulatory and neuro-regenerative capabilities. ( c ) Treg-Exo-IKVAV spatiotemporally sequentially reshaped the neuro-immune microenvironment, and then promoted functional recovery of SCI mice

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Isolation, Functional Assay

Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV. Treg-Exo was stained by DiO and IKVAV was labeled by Cy5.5. ( i-j ) The physicochemical stability of Treg-Exo and Treg-Exo-IKVAV after a 7-day incubation period in phosphate-buffered saline (PBS) using time-dependent measurements of particle size distribution and zeta potential values ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f , g ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV. Treg-Exo was stained by DiO and IKVAV was labeled by Cy5.5. ( i-j ) The physicochemical stability of Treg-Exo and Treg-Exo-IKVAV after a 7-day incubation period in phosphate-buffered saline (PBS) using time-dependent measurements of particle size distribution and zeta potential values ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f , g ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Western Blot, Flow Cytometry, Fluorescence, Staining, Labeling, Incubation, Saline, Zeta Potential Analyzer, Comparison

In vitro immunomodulatory effects of Treg-Exo-IKVAV.( a ) Uptake of green, fluorescent FITC-labeled Treg-Exo and Treg-Exo-IKVAV into Raw 264.7 macrophages. ( b ) The typical images of iNOS (green) and CD206 (red) immunostaining. ( c ) Quantitative analysis of immunostaining ( n = 5). (d) Flow cytometry analysis of CD86 and CD206 positive cells after Treg-Exo and Treg-Exo-IKVAV treatments. ( e ) Quantification of the flow cytometry results ( n = 5). ( f ) RT-PCR detection of macrophage polarization and inflammation-related gene expression ( n = 5). ( g ) KEGG enrichment analysis. ( h ) GO analysis. ( i ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on macrophage. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f ) or two-way ANOVA ( c , e ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: In vitro immunomodulatory effects of Treg-Exo-IKVAV.( a ) Uptake of green, fluorescent FITC-labeled Treg-Exo and Treg-Exo-IKVAV into Raw 264.7 macrophages. ( b ) The typical images of iNOS (green) and CD206 (red) immunostaining. ( c ) Quantitative analysis of immunostaining ( n = 5). (d) Flow cytometry analysis of CD86 and CD206 positive cells after Treg-Exo and Treg-Exo-IKVAV treatments. ( e ) Quantification of the flow cytometry results ( n = 5). ( f ) RT-PCR detection of macrophage polarization and inflammation-related gene expression ( n = 5). ( g ) KEGG enrichment analysis. ( h ) GO analysis. ( i ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on macrophage. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( f ) or two-way ANOVA ( c , e ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, Labeling, Immunostaining, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Comparison

Treg-Exo-IKVAV promoted neuronal differentiation of NSCs in vitro.( a ) Schematic diagram of the experiment evaluating the direct effect on NSCs differentiation. ( b ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( c ) Quantification of MAP2 and GFAP immunostaining of ( b ). ( d ) Schematic diagram of the experiment evaluating the immunoregulatory effect on NSCs differentiation. ( e ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( f ) Quantification of MAP2 and GFAP immunostaining of ( e ). ( g ) Neuronal differentiation related gene expression determined by RT-PCR. ( h ) KEGG analysis. ( i ) GO analysis. ( j ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on NSCs differentiation. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA with Tukey’s multiple comparison test ( c , f , g ). ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV promoted neuronal differentiation of NSCs in vitro.( a ) Schematic diagram of the experiment evaluating the direct effect on NSCs differentiation. ( b ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( c ) Quantification of MAP2 and GFAP immunostaining of ( b ). ( d ) Schematic diagram of the experiment evaluating the immunoregulatory effect on NSCs differentiation. ( e ) Representative images of MAP2 (green) and GFAP (red) immunostaining. ( f ) Quantification of MAP2 and GFAP immunostaining of ( e ). ( g ) Neuronal differentiation related gene expression determined by RT-PCR. ( h ) KEGG analysis. ( i ) GO analysis. ( j ) Schematic illustration of the immunoregulatory mechanism of Treg-Exo-IKVAV on NSCs differentiation. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA with Tukey’s multiple comparison test ( c , f , g ). ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, Immunostaining, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Comparison

Treg-Exo-IKVAV targeted inflammation and promoted motor functional behavior recovery of SCI mice. ( a ) Schematic illustration of the in vivo experiments. ( b ) Typical fluorescence images of ICG-labeled Treg-Exo and Treg-Exo-IKVAV. ( c ) Quantitative analysis of ( b ) ( n = 3). ( d ) Ex vivo ICG signals of spinal cords harvested from SCI mice at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( e ) Ex vivo ICG signals of major organs (heart, liver, spleen, lung and kidney) harvested at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( f ) Quantitative analysis of relative fluorescence intensity in spinal cords and major organs ( n = 3). ( g ) Representative images of footprint assay on day 28 after Treg-Exo and Treg-Exo-IKVAV treatments, and quantitative analysis of toe dragging and stride length ( n = 5). ( h ) Typical images of the swimming test on day 28. ( i ) Quantification of the swimming test using Louisville Swim Scale ( n = 3). ( j ) BMS scores in the different groups ( n = 5). ( k ) H&E staining of bladder tissues in different groups on day 28, and quantitative analysis of bladder volume and bladder weight ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( g , k ) or two-way ANOVA with Tukey’s multiple comparison test ( c , f , i , j ). * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV targeted inflammation and promoted motor functional behavior recovery of SCI mice. ( a ) Schematic illustration of the in vivo experiments. ( b ) Typical fluorescence images of ICG-labeled Treg-Exo and Treg-Exo-IKVAV. ( c ) Quantitative analysis of ( b ) ( n = 3). ( d ) Ex vivo ICG signals of spinal cords harvested from SCI mice at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( e ) Ex vivo ICG signals of major organs (heart, liver, spleen, lung and kidney) harvested at 7. days after injection with Treg-Exo and Treg-Exo-IKVAV. ( f ) Quantitative analysis of relative fluorescence intensity in spinal cords and major organs ( n = 3). ( g ) Representative images of footprint assay on day 28 after Treg-Exo and Treg-Exo-IKVAV treatments, and quantitative analysis of toe dragging and stride length ( n = 5). ( h ) Typical images of the swimming test on day 28. ( i ) Quantification of the swimming test using Louisville Swim Scale ( n = 3). ( j ) BMS scores in the different groups ( n = 5). ( k ) H&E staining of bladder tissues in different groups on day 28, and quantitative analysis of bladder volume and bladder weight ( n = 3). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( g , k ) or two-way ANOVA with Tukey’s multiple comparison test ( c , f , i , j ). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Functional Assay, In Vivo, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Comparison

Treg-Exo-IKVAV attenuated macrophage infiltration and inhibited macrophage-induced inflammation response secondary to SCI.( a ) Sagittal T2-MRI images on day 7. ( b ) Immunostaining of CD68 in different groups on day 7. ( c ) Quantification of CD68 immunostaining ( n = 3). ( d ) Co-immunostaining of CD68 (green) and iNOS (red), and CD68 (green) and Arg1 (red) in the different groups. ( e-f ) Quantification of CD68, iNOS, and Arg1 immunofluorescence staining ( n = 3). ( g ) RT-PCR detection of the gene expression (iNOS, TNF-α, IL-1β, IL-10, and Arg1) ( n = 3). ( h ) Levels of TNF-α, IL-6, CXCL10, IL-4 and IL-10 detected by ELISA ( n = 5). Data are presented as mean ± SD. Statistical significance was tested by one-way ANOVA ( c , e , f , g , h ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV attenuated macrophage infiltration and inhibited macrophage-induced inflammation response secondary to SCI.( a ) Sagittal T2-MRI images on day 7. ( b ) Immunostaining of CD68 in different groups on day 7. ( c ) Quantification of CD68 immunostaining ( n = 3). ( d ) Co-immunostaining of CD68 (green) and iNOS (red), and CD68 (green) and Arg1 (red) in the different groups. ( e-f ) Quantification of CD68, iNOS, and Arg1 immunofluorescence staining ( n = 3). ( g ) RT-PCR detection of the gene expression (iNOS, TNF-α, IL-1β, IL-10, and Arg1) ( n = 3). ( h ) Levels of TNF-α, IL-6, CXCL10, IL-4 and IL-10 detected by ELISA ( n = 5). Data are presented as mean ± SD. Statistical significance was tested by one-way ANOVA ( c , e , f , g , h ) with Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Immunostaining, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Enzyme-linked Immunosorbent Assay, Comparison

Treg-Exo-IKVAV promoted neuronal differentiation of NSCs, and axonal regeneration and remyelination.( a ) Immunofluorescence staining of NeuN to label the mature neurons on day 28. ( b ) Quantification of NeuN + cells in Z1-Z4 regions adjacent to the lesion core ( n = 3). ( c ) Immunostaining of TUJ1 to label the newly born neurons on day 28. ( d ) Percent of TUJ1 + /DAPI + cells ( n = 5). ( e ) Quantification analysis of ( f ) co-immunostaining of NF-200 (green) and GFAP (red), and ( g ) co-immunostaining of MBP (green) and GFAP (red) in the different groups at 28 days after SCI. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( d ) or two-way ANOVA with Tukey’s multiple comparison test (c). * p < 0.05; ** p < 0.01; *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: Treg-Exo-IKVAV promoted neuronal differentiation of NSCs, and axonal regeneration and remyelination.( a ) Immunofluorescence staining of NeuN to label the mature neurons on day 28. ( b ) Quantification of NeuN + cells in Z1-Z4 regions adjacent to the lesion core ( n = 3). ( c ) Immunostaining of TUJ1 to label the newly born neurons on day 28. ( d ) Percent of TUJ1 + /DAPI + cells ( n = 5). ( e ) Quantification analysis of ( f ) co-immunostaining of NF-200 (green) and GFAP (red), and ( g ) co-immunostaining of MBP (green) and GFAP (red) in the different groups at 28 days after SCI. Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA ( d ) or two-way ANOVA with Tukey’s multiple comparison test (c). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: Immunofluorescence, Staining, Immunostaining, Comparison

In vitro and in vivo biosafety of Treg-Exo-IKVAV nanovesicles.( a ) Typical images of Live/Dead staining. ( b ) Viability of NSCs after cultured with Treg-Exo or Treg-Exo-IKVAV for 24 h determined by CCK-8 ( n = 5). ( c ) H&E staining of heart, liver, spleen, lung, and kidney tissues in the different groups. ( d ) Biochemical markers relevant to hepatic (ALT, AST) and kidney (UA, BUN) function ( n = 5). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA (b, d) with Tukey’s multiple comparison test. *** p < 0.001

Journal: Journal of Nanobiotechnology

Article Title: Dual-engineered Treg-Exosome-IKVAV nanovesicles spatiotemporally sequentially regulate neuro-immune microenvironment to promote spinal cord injury repair

doi: 10.1186/s12951-025-03999-3

Figure Lengend Snippet: In vitro and in vivo biosafety of Treg-Exo-IKVAV nanovesicles.( a ) Typical images of Live/Dead staining. ( b ) Viability of NSCs after cultured with Treg-Exo or Treg-Exo-IKVAV for 24 h determined by CCK-8 ( n = 5). ( c ) H&E staining of heart, liver, spleen, lung, and kidney tissues in the different groups. ( d ) Biochemical markers relevant to hepatic (ALT, AST) and kidney (UA, BUN) function ( n = 5). Data are expressed as mean ± SD. Statistical significance was tested by one-way ANOVA (b, d) with Tukey’s multiple comparison test. *** p < 0.001

Article Snippet: Fig. 2 Preparation and characterization of Treg-Exo-IKVAV. ( a ) Schematic illustration of the fabrication of Treg-Exo-IKVAV. ( b ) Typical TEM images of Treg-Exo and Treg-Exo-IKVAV. ( c ) Particle size distributions of Treg-Exo and Treg-Exo-IKVAV measured by NTA. ( d ) Exosome markers (CD9, TSG101, CD63, and CD81) detected Western Blot. ( e ) Exosome markers (CD9, CD63, and CD81) detected flow cytometry. ( f ) The average diameters of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( g ) Zeta potentials of Treg-Exo and Treg-Exo-IKVAV ( n = 5). ( h ) The representative fluorescence images of Treg-Exo and Treg-Exo-IKVAV.

Techniques: In Vitro, In Vivo, Staining, Cell Culture, CCK-8 Assay, Comparison

Figure 5. The pivotal effect of PM10-induced IL-33 for Th2 differentiation effects of conditioned media on PM10-induced human nasal polyp derived fibroblast (NPDFs-CM) on the inflammatory cytokine production in CD4+ T cells. (A) Th2 differentiation was determined by the expression of GATA3 and ST2 (IL-33 receptor). (B,C) The protein levels of IFN-γ, IL-17A, IL-4, IL-13, and IL-10 in CD4+ T cell populations were determined by flow cytometry according to the addition of ST2. The graphic data represents the means ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.0001 as compared to the PM10-treated group.

Journal: International journal of molecular sciences

Article Title: Effect of Airborne Particulate Matter on the Immunologic Characteristics of Chronic Rhinosinusitis with Nasal Polyps.

doi: 10.3390/ijms23031018

Figure Lengend Snippet: Figure 5. The pivotal effect of PM10-induced IL-33 for Th2 differentiation effects of conditioned media on PM10-induced human nasal polyp derived fibroblast (NPDFs-CM) on the inflammatory cytokine production in CD4+ T cells. (A) Th2 differentiation was determined by the expression of GATA3 and ST2 (IL-33 receptor). (B,C) The protein levels of IFN-γ, IL-17A, IL-4, IL-13, and IL-10 in CD4+ T cell populations were determined by flow cytometry according to the addition of ST2. The graphic data represents the means ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.0001 as compared to the PM10-treated group.

Article Snippet: TMCs (1 × 106 cells) were cultured with 1 μg/mL of phytohemagglutinin (PHA) in NPDF-CM for 72 h. For Th2 differentiation, CD4+ T cells from TMCs were purified using a CD4+ T Cell Isolation Kit MicroBeads (Miltenyi Biotech, Bisley, Surrey, UK), and purified CD4+ T cells were differentiated using a CellXVivo Human Th2 Cell Differentiation Kit (RnDSystems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Cytometry

A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody ( n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA ( n = 8 biological replicates per group). D Treg migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG ( n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody ( n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA ( n = 8 biological replicates per group). D Treg migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG ( n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Flow Cytometry, Binding Assay, Fluorescence, Incubation, Control, Blocking Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Migration, Comparison

A Experimental protocol of the skin sclerosis mouse model. Six-week-old female C57BL/6NcrSlc mice (MGI ID: MGI:5295404) received daily subcutaneous injections of bleomycin (BLM, 200 µg) or PBS as a control for two weeks. Mice were also treated intraperitoneally with either control (Ctrl) IgG or anti-C-C motif chemokine receptor 8 (CCR8) antibody (Ab) once on Day 8. Skin biopsies were collected at Week 2 for histological analysis and next-generation sequencing (NGS). This figure was partially created with BioRender ( https://BioRender.com/slxff50 ). Representative histological images and quantitative analyses of dermal fibrosis and Treg infiltration in mouse skin. H&E-stained B and Masson’s trichrome–stained C skin sections show increased dermal thickness and collagen deposition following BLM treatment. Images were captured at ×200 magnification, and scale bars represent 50 μm. D Quantification of dermal thickness and E Foxp3⁺ Treg counts are presented in the accompanying bar graphs ( n = 6 biological replicates for PBS group; n = 5 biological replicates for BLM-treated groups). Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Experimental protocol of the skin sclerosis mouse model. Six-week-old female C57BL/6NcrSlc mice (MGI ID: MGI:5295404) received daily subcutaneous injections of bleomycin (BLM, 200 µg) or PBS as a control for two weeks. Mice were also treated intraperitoneally with either control (Ctrl) IgG or anti-C-C motif chemokine receptor 8 (CCR8) antibody (Ab) once on Day 8. Skin biopsies were collected at Week 2 for histological analysis and next-generation sequencing (NGS). This figure was partially created with BioRender ( https://BioRender.com/slxff50 ). Representative histological images and quantitative analyses of dermal fibrosis and Treg infiltration in mouse skin. H&E-stained B and Masson’s trichrome–stained C skin sections show increased dermal thickness and collagen deposition following BLM treatment. Images were captured at ×200 magnification, and scale bars represent 50 μm. D Quantification of dermal thickness and E Foxp3⁺ Treg counts are presented in the accompanying bar graphs ( n = 6 biological replicates for PBS group; n = 5 biological replicates for BLM-treated groups). Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Control, Next-Generation Sequencing, Staining, Comparison

(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. ( <xref ref-type= 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. " width="100%" height="100%">

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

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.

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

(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 <xref ref-type= 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. " width="100%" height="100%">

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

(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.

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

(A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Indicated naïve untreated CFSE labeled CD4+ T cells were co-cultured with 0.1 μg/ml CD3ε Abs and TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours. (IV) Indicated naïve CD4+ TPam3 cells were stimulated as in (II). The corresponding CFSE dilution and IFN-γ accumulation are shown as a percent value in the inset. (B) Naïve untreated OT2 were co-cultured with OVA peptide (OVAp) bearing TLR2−/− APCs in the absence (I) or presence (II) of Th1 polarization or (III) with Th1 polarization and 10 μg/ml of Pam3Cys4 for 96 hours and assessed for IFN-γ expression. (IV) Naïve OT2Pam3 cells were stimulated as in (II). (C) Naïve human CD4+ T cells with or without Pam3Cys4 cargo were stimulated under Th1 polarizing conditions with CD3ε Ab-conjugated beads for 96 hours and assessed for IFN-γ expression. (A–C) are representative results from at least three independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Labeling, Cell Culture, Expressing

(A) Naïve B6 CD4+ T, TLR2−/− CD4+ T and OT2 cells with or without Pam3Cys4 cargo were stimulated with TLR2-deficient splenocyte-derived APCs bearing CD3ε Abs (B6 or TLR2−/− CD4+ T cells) or OVAp (OT2 cells) in the presence of IL-12 and indicated amounts of TGF-β1. After 72 hours culture supernatants were assessed for IFN-γ by ELISA. Data are representative of at least four independent experiments. (B) Naïve untreated OT2 and OT2Pam3 cells were stimulated with TLR2−/− APCs bearing OVAp cultured under Th1 polarization, 0.5 ng/ml TGF-β1 or Th1 polarization + 0.5 ng/ml TGF-β1. After 5 days of culture OT2 cells were evaluated for the percent abundance of Foxp3, T-bet and IFN-γ expression. Data shown is a representative result of three independent experiments. (C) Naïve untreated OT2 and OT2Pam3 cells were stimulated as in (A) and then fractionated from 72 hrs later, mixed at indicated ratios with responder CFSE labeled responder OT1 cells and re-cultured in triplicate wells with TLR2−/− APCs bearing peptides specific for OT2 and OT1. (Upper panel) Representative CFSE dilution plots of OT2 regulators were mixed with OT1 responders at a 1:1 ratio. (Lower panel) Mean CFSE dilution ± S.E of responders over indicated regulator to responder ratios (**p< 0.01). The result shown is representative of three independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Naïve B6 CD4+ T, TLR2−/− CD4+ T and OT2 cells with or without Pam3Cys4 cargo were stimulated with TLR2-deficient splenocyte-derived APCs bearing CD3ε Abs (B6 or TLR2−/− CD4+ T cells) or OVAp (OT2 cells) in the presence of IL-12 and indicated amounts of TGF-β1. After 72 hours culture supernatants were assessed for IFN-γ by ELISA. Data are representative of at least four independent experiments. (B) Naïve untreated OT2 and OT2Pam3 cells were stimulated with TLR2−/− APCs bearing OVAp cultured under Th1 polarization, 0.5 ng/ml TGF-β1 or Th1 polarization + 0.5 ng/ml TGF-β1. After 5 days of culture OT2 cells were evaluated for the percent abundance of Foxp3, T-bet and IFN-γ expression. Data shown is a representative result of three independent experiments. (C) Naïve untreated OT2 and OT2Pam3 cells were stimulated as in (A) and then fractionated from 72 hrs later, mixed at indicated ratios with responder CFSE labeled responder OT1 cells and re-cultured in triplicate wells with TLR2−/− APCs bearing peptides specific for OT2 and OT1. (Upper panel) Representative CFSE dilution plots of OT2 regulators were mixed with OT1 responders at a 1:1 ratio. (Lower panel) Mean CFSE dilution ± S.E of responders over indicated regulator to responder ratios (**p< 0.01). The result shown is representative of three independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Labeling

(A) Indicated naïve CD4+ T cells unactivated (black line) or activated for 10 mins with plate bound 0.1μg/ml CD3ε Ab and 1 μg/ml soluble CD28 Ab with (red line) or without (blue line) Pam3Cys4 cargo were evaluated for p-AktSer473. Left histograms are a representative result from 5 experiments. In the right panel data is shown as a individual and mean relative MFI calculated as the ratio of the MFI with Pam3Csy4 cargo to the MFI of untreated CD4+ cells ± S.D. where **p < 0.01. (B) B6 CD4+ TPam3 left untreated and unstimulated (black line) or pretreated for 30 mins with either indicated concentrations of Ly2924002 (green lines) or DMSO vehicle (red line) and then TCR stimulated as in (A) and assessed for p-AktSer473. The result is representative of 2 independent experiments. (C) B6 CD4+ T and B6 CD4+ TPam3 cells were pretreated Ly2924002 as in (B) and activated as in (A) under Th1 polarization & 0.5 ng/ml TGF-β1 and assessed for indicated transcripts 36 hrs after culture by semiquantitative real time RT-PCR. Data is normalized to transcript levels in freshly isolated untreated naïve B6 CD4+ T cells and the result shown is representative of 3 independent experiments. (D) Indicated naïve CD4+ T cells were pretreated with DMSO vehicle or 0.3 μM of Ly2924002 for 30 mins and stimulated as in (C) for 72 hours and assessed for percent abundance of Foxp3 and T-bet expression. Data are representative of 3 independent experiments.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: (A) Indicated naïve CD4+ T cells unactivated (black line) or activated for 10 mins with plate bound 0.1μg/ml CD3ε Ab and 1 μg/ml soluble CD28 Ab with (red line) or without (blue line) Pam3Cys4 cargo were evaluated for p-AktSer473. Left histograms are a representative result from 5 experiments. In the right panel data is shown as a individual and mean relative MFI calculated as the ratio of the MFI with Pam3Csy4 cargo to the MFI of untreated CD4+ cells ± S.D. where **p < 0.01. (B) B6 CD4+ TPam3 left untreated and unstimulated (black line) or pretreated for 30 mins with either indicated concentrations of Ly2924002 (green lines) or DMSO vehicle (red line) and then TCR stimulated as in (A) and assessed for p-AktSer473. The result is representative of 2 independent experiments. (C) B6 CD4+ T and B6 CD4+ TPam3 cells were pretreated Ly2924002 as in (B) and activated as in (A) under Th1 polarization & 0.5 ng/ml TGF-β1 and assessed for indicated transcripts 36 hrs after culture by semiquantitative real time RT-PCR. Data is normalized to transcript levels in freshly isolated untreated naïve B6 CD4+ T cells and the result shown is representative of 3 independent experiments. (D) Indicated naïve CD4+ T cells were pretreated with DMSO vehicle or 0.3 μM of Ly2924002 for 30 mins and stimulated as in (C) for 72 hours and assessed for percent abundance of Foxp3 and T-bet expression. Data are representative of 3 independent experiments.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Quantitative RT-PCR, Isolation, Expressing

Both TCR/CD28 engagement and TLR2-MyD88 signaling stimulates PI3 kinase-mediated Akt activation as measured by phosphorylation at serine 473. Akt activation induces mTORC1 to promote T-bet activation to stimulate Th1 lineage determination. TGF-β–signaling leads to SMAD2/3 phosphorylation, which in turn drives Foxo1/Foxo3a mediated transcription of Foxp3 to specify iTreg development. SMAD3 and Foxo1/Foxo3a activity are antagonized by Akt activation. TCR/CD28 activated CD4+ TPam3 cells exposed to a TGF-β + Th1 cytokine environment (e.g., IL-12) favor Th1 over iTreg lineage determination due to additional Akt activation contributed by TLR2-MyD88 dependent PI3 kinase activation.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Naïve CD4 + T cells carrying a TLR2 agonist overcome TGF–β-mediated tumor immune evasion

doi: 10.4049/jimmunol.1700396

Figure Lengend Snippet: Both TCR/CD28 engagement and TLR2-MyD88 signaling stimulates PI3 kinase-mediated Akt activation as measured by phosphorylation at serine 473. Akt activation induces mTORC1 to promote T-bet activation to stimulate Th1 lineage determination. TGF-β–signaling leads to SMAD2/3 phosphorylation, which in turn drives Foxo1/Foxo3a mediated transcription of Foxp3 to specify iTreg development. SMAD3 and Foxo1/Foxo3a activity are antagonized by Akt activation. TCR/CD28 activated CD4+ TPam3 cells exposed to a TGF-β + Th1 cytokine environment (e.g., IL-12) favor Th1 over iTreg lineage determination due to additional Akt activation contributed by TLR2-MyD88 dependent PI3 kinase activation.

Article Snippet: T h 1 polarization for human CD4 + T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations.

Techniques: Activation Assay, Phospho-proteomics, Activity Assay

Figure 4. 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.

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: Figure 4. 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

Figure 5. 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.

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: Figure 5. 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

Figure 8. 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) CD4High, (B) CD25High, (C) CD127Low, and (D) CD152High. Cell membrane markers associated with Treg suppressive activity were (E) CD39High

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: Figure 8. 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) CD4High, (B) CD25High, (C) CD127Low, and (D) CD152High. Cell membrane markers associated with Treg suppressive activity were (E) CD39High

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

Figure 11. 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.

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: Figure 11. 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