Journal: Bioengineering

Article Title: RNU ( Foxn1 RNU -Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats

doi: 10.3390/bioengineering8010012

Figure Lengend Snippet: CD68+ staining is increased in RNU DMM-treated sites compared to Sprague Dawley. CD68 staining was quantified and showed increased levels in RNU rats ( A ). Yet, CD163 ( B ) and FoxP3 ( C ) were unaltered. CD4 staining was increased in RNU rats ( D ) while CD8 staining was lower ( E ). In addition, CD4, CD8, and FoxP3 levels were low. DRAQ5 showed no change in DNA staining ( F ). Data shown are means ± SEM of 8 animals. Letters not shared indicated a significant difference ( p < 0.05, Tukey).

Article Snippet: Primary antibodies used in this experiment were: mouse anti-Pax7 (ab55494, Abcam, Cambridge, UK); rabbit anti-nicotinic acetylcholine receptor-epsilon (AChR-ε, ab65180, Abcam); mouse anti-nicotinic acetylcholine receptor-gamma (AChR-γ, MA3-043, Thermo Fisher Scientific, Waltham, MA, USA); mouse anti-myosin heavy chain-fetal (fMyHC, SC-53097, Santa Cruz Biotechnology); CD68 (ab125212, Abcam); CD163 (ab87099, Abcam); CD8 (MAB116, R&D Systems), CD4 (MAB554, R&D Systems), FoxP3 (MAB8214, R&D Systems) and were diluted in PBS with 1% BSA and 0.3% Tween-20.

Techniques: Staining

Journal: Oncotarget

Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

doi: 10.18632/oncotarget.16221

Figure Lengend Snippet: Indicated APCs isolated from BALB/c mice were cultured with allogeneic CD4SP Foxp3 RFP− thymocytes isolated from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) in presence of IL-2 for six days. A. Expression of Foxp3 in cultured CD4 + CD90 + thymocytes was analyzed by flow cytometry. Numbers indicate frequency of Foxp3 + cells. Representative data from one out of eight independent experiments are shown. B. Graph shows frequency of Foxp3 + allo-iTregs from cultures with indicated APCs. Data are summarized from eight independent experiments (mean ± SD) and tested for significance using Mann-Whitney test; ** p < 0.01; *** p < 0.001. C. At day 6, Foxp3 RFP+ allo-iTregs were sorted from indicated cultures, and genomic DNA isolated from these cells was analyzed for the methylation status of TSDR, Eos , Ctla4 and Gitr . Representative data from one out of six (TSDR), two ( Gitr ) or three ( Eos and Ctla4 ) independent experiments are depicted. Genomic DNA from CD4SP Foxp3 − thymocytes was analyzed as input control for the methylation status of TSDR (three independent samples) and Eos , Ctla4 and Gitr (unicate). Each bar represents one CpG motif. The degree of methylation at each CpG motif is represented according to the color code.

Article Snippet: For the alloantigen-specific system, 10×10 4 CD4 + Foxp3 − cells derived from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) or Foxp3 hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days.

Techniques: Isolation, Cell Culture, Expressing, Flow Cytometry, MANN-WHITNEY, Methylation, Control

Journal: Oncotarget

Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

doi: 10.18632/oncotarget.16221

Figure Lengend Snippet: APCs were enriched from thymi and spleens of BALB/c mice by enzymatic digestion and gradient centrifugation. Expression of CD86, CD70, CD83, CD137L, OX40L and CD40 was analyzed on gated CD45 − EpCAM + Ly51 − mTECs, CD45 + CD11c hi Lin − t-DCs and CD11c hi Lin − sp-DCs (Lin defined as CD90, CD49b, F4/80 and CD19) by flow cytometry. Gated CD4SP-Foxp3 − thymocytes were taken as control. Representative histograms from one out of four (CD86 and CD70), five (CD40) or three (CD83, CD137L and OX40L) independent experiments are depicted.

Article Snippet: For the alloantigen-specific system, 10×10 4 CD4 + Foxp3 − cells derived from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) or Foxp3 hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days.

Techniques: Gradient Centrifugation, Expressing, Flow Cytometry, Control

Journal: Oncotarget

Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

doi: 10.18632/oncotarget.16221

Figure Lengend Snippet: A. Thymocytes and splenocytes from CD11c Cre xCD40 fl/fl (DC-specific CD40 knockout, filled circles) and CD11c WT xCD40 fl/fl mice (CD40 competent control, open circles) were analyzed by flow cytometry. Graphs show frequency of CD4SP thymocytes, Foxp3 + cells among CD4SP thymocytes, CD4 + CD3 + splenocytes and Foxp3 + Tregs cells among CD4 + CD3 + splenocytes. Data are summarized from two independent experiments (mean ± SD) and tested for significance using Mann-Whitney test; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. (B-D) Indicated APCs (BALB/c) were cultured with CD4SP-Foxp3 RFP− thymocytes (C57BL/6) in presence of IL-2 and either anti-CD40 or isotype control antibodies for six days. B. Expression of Foxp3 in cultured CD4 + CD90 + thymocytes was analyzed by flow cytometry. Numbers indicate frequency of Foxp3 + cells. Representative data from one out of five independent experiments are depicted. C. Graph shows frequency of Foxp3 + allo-iTregs from cultures with indicated APCs in presence of anti-CD40 (filled circles) or isotype control antibodies (open circles). Data are summarized from five independent experiments (mean ± SD) and tested for significance using Mann-Whitney test; ns, not significant. D. At day 6, Foxp3 RFP+ allo-iTregs were sorted from indicated cultures, and genomic DNA isolated from these cells was analyzed for the methylation status of TSDR. Representative data from one out of three independent experiments are depicted. Each bar represents one CpG motif. The degree of methylation at each CpG motif is represented according to the color code.

Article Snippet: For the alloantigen-specific system, 10×10 4 CD4 + Foxp3 − cells derived from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) or Foxp3 hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days.

Techniques: Knock-Out, Control, Flow Cytometry, MANN-WHITNEY, Cell Culture, Expressing, Isolation, Methylation

Journal: Oncotarget

Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

doi: 10.18632/oncotarget.16221

Figure Lengend Snippet: Indicated APCs (BALB/c) were cultured with CD4SP Foxp3 RFP− thymocytes (C57BL/6) in presence of IL-2 for six days. A. At day 6, cells were restimulated with phorbol 12-myristate 13-acetate and ionomycin for detection of IFN-γ, and expression of IL-10 GFP (top) and IFN-γ (bottom) was assessed by flow cytometry in gated Foxp3 RFP+ allo-iTregs from indicated cultures. Numbers indicate frequency of IL-10 + and IFN-γ + cells. Representative data from one out of four independent experiments are depicted. B. At day 6, expression of CCR7 (top) and CXCR3 (bottom) was directly assessed by flow cytometry on gated Foxp3 RFP+ allo-iTregs from indicated cultures. Ex vivo isolated CD4SP Foxp3 − thymocytes served as controls. Numbers indicate frequency of CCR7 + and CXCR3 + cells. Data were taken from one out of two independent experiments. C. At day 6, Foxp3 RFP+ allo-iTregs were sorted from indicated cultures by flow cytometry. Freshly isolated, CTV-labeled naïve CD4 + T cells were stimulated with anti-CD3/anti-CD28 beads in presence of indicated allo-iTregs at a ratio of 1:4 (Tregs to naïve T cells). Naïve T cells stimulated in absence of allo-iTregs (stim Tnaive) as well as naïve T cells receiving no stimulus (unstim Tnaive) served as controls. After four days, proliferation of naïve T cells was assessed by measuring CTV dilution in living naïve CD4 + CD90.2 + CD45.1 + T cells by flow cytometry. Numbers indicate frequency of cells in indicated gates. Representative data from one out of four independent experiments are depicted.

Article Snippet: For the alloantigen-specific system, 10×10 4 CD4 + Foxp3 − cells derived from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) or Foxp3 hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days.

Techniques: Cell Culture, Expressing, Flow Cytometry, Ex Vivo, Isolation, Labeling

Journal: Oncotarget

Article Title: Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells

doi: 10.18632/oncotarget.16221

Figure Lengend Snippet: Indicated APCs (BALB/c) were cultured with CD4SP Foxp3 RFP− thymocytes (C57BL/6) in presence of IL-2 for six days. One day prior to transplantation, allo-iTregs from indicated cultures were sorted by flow cytometry and injected into Rag2 −/− mice. Mice were additionally injected with CD4 + CD90.2 + CD45.1 + CD25 − CD44 − CD62L hi naïve T cells. Mice receiving no cells (w/o) or naïve T cells only served as controls. One day later, allogeneic skin transplantation was performed. The following days animals were monitored for signs of rejection until day 100. Relative graft survival for each group is depicted and data are cumulative of three independent experiments; w/o ( n = 6, black diamond), Tnaive only ( n = 13, red circle), + mTEC allo-iTregs ( n = 11, green triangle), + t-DC allo-iTregs ( n = 12, orange square) and + sp-DC allo-iTregs ( n = 8, blue diamond). Significance of survival graph was calculated between mice receiving Tnaive only versus mice co-transferred with different APC-induced allo-iTregs using log-rank (mantel cox) test. *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: For the alloantigen-specific system, 10×10 4 CD4 + Foxp3 − cells derived from Foxp3 RFP x IL-10 GFP double reporter mice (C57BL/6 background) or Foxp3 hCD2 (BALB/c background) were plated with either mTECs (APC to T cell ratio 1:50 if not indicated otherwise) or DCs (APC to T cell ratio 1:10 if not indicated otherwise) in presence of 100 ng/ml recombinant mouse IL-2 (R&D systems) for six days.

Techniques: Cell Culture, Transplantation Assay, Flow Cytometry, Injection

Journal: Oncoimmunology

Article Title: A phase I dose-escalation clinical trial of a peptide-based human papillomavirus therapeutic vaccine with Candida skin test reagent as a novel vaccine adjuvant for treating women with biopsy-proven cervical intraepithelial neoplasia 2/3

doi: 10.1080/2162402X.2015.1031439

Figure Lengend Snippet: (A) Circulating immune cells before, after two, and after four vaccinations in all vaccine recipients. (B) Circulating immune cells in responders (•) and non-responders (▪). Percentages of CD4 cells positive for CD4 and Tbet are shown for Th1 cells, positive for CD4+ and GATA3 are shown for Th2 cells, and positive for CD4+, CD25, and FoxP3 for Tregs. The bars represent standard error of means.

Article Snippet: 47,48 Slides of LEEP samples were pre-treated with a target retrieval solution (Dako Corporation, #S2369), peroxidase block (Dako Corporation, #S2003), and serum-free protein block (Dako Corporation, #X0909) prior to performing immunohistochemistry with primary goat anti-human polyclonal antibody against FoxP3 (R&D Systems, #AF3240) at 1:400 dilution.

Techniques:

Journal: Oncoimmunology

Article Title: A phase I dose-escalation clinical trial of a peptide-based human papillomavirus therapeutic vaccine with Candida skin test reagent as a novel vaccine adjuvant for treating women with biopsy-proven cervical intraepithelial neoplasia 2/3

doi: 10.1080/2162402X.2015.1031439

Figure Lengend Snippet: Regulatory T cells in lesional cervical epithelium and the underlying stroma. FoxP3 nuclear staining cells, in lesions (cervical intraepithelial neoplasia 1, 2, and/or3) remaining after vaccination or representative region if no lesions remaining, were counted. The FoxP3 nuclear staining cells were also counted in the underlying stroma. The bars represent stand error of means.

Article Snippet: 47,48 Slides of LEEP samples were pre-treated with a target retrieval solution (Dako Corporation, #S2369), peroxidase block (Dako Corporation, #S2003), and serum-free protein block (Dako Corporation, #X0909) prior to performing immunohistochemistry with primary goat anti-human polyclonal antibody against FoxP3 (R&D Systems, #AF3240) at 1:400 dilution.

Techniques: Staining

Journal: bioRxiv

Article Title: The CD4 T cell epigenetic JUNB+ state is associated with proliferation and exhaustion

doi: 10.1101/2024.01.05.573875

Figure Lengend Snippet: ( a ) Detailed expression patterns on UMAP, showing the gene specificity. ( b) Cell cycle phase per clusters, shown as fractions and (c) on UMAP, for all cells and JUNB+ reclustered cells. (d) UMAP of JUNB+ cells, dimensionally reduced separately. (e) Heatmap of key genes across JUNB+ subclusters. (f) CTCF motif activity and gene expression. (g) Representation of a transcriptionally activate domain (TAD) model with CTCF, which guides the cohesin loops and thus 3D structure. (h) Detected regulators of Foxp3, from a murine Treg CRISPR screen dataset (Cortez2020) . CTCF and JUNB act antagonistically to FOXP3, which is low in the JUNB state. (i) Telomemore analysis predicts that Tfh cells have increased chromatin condensation. (j) Detected regulators of exhaustion in CD8 T cells in a CRISPR screen vs DE genes in our JUNB cluster. Top scoring chromatin remodelling factors are shown in green, and TCR-related proteins in purple. JUNB is among the highest scoring genes for exhaustion.

Article Snippet: Following antibodies were used for the labeling of aforementioned transcription factors: Human T-bet/TBX21 Alexa Fluor 488-conjugated Antibody (1:20, IC53851G, R&D Systems), Gata-3 Monoclonal Antibody (TWAJ), PE-Cyanine7 (1:20, 25-9966-42, Thermo Fisher Scientific), Human/Mouse ROR gamma t/RORC2/NR1F3 PE-conjugated Antibody (1:10, IC6006P, R&D Systems), and Human/Mouse/Rat FoxP3 PE-conjugated Antibody (1:10, IC8970P, R&D Systems).

Techniques: Expressing, Activity Assay, Gene Expression, CRISPR