Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A ) RNA splicing of human FOXP3 and mouse Foxp3 pre-mRNA. ( B ) Alignment of human FOXP3 (hFOXP3) and mouse Foxp3 (mFoxp3) exon 2 and nearby 50-bp intron sequences. ( C ) Mutations in mouse intron according to corresponding human sequences. Minigene plasmids were transfected into 293 cells. Exon 2 splicing was analyzed by RT-PCR. ( D ) A minigene system contains human genomic sequence from exon 1 to exon 3 of FOXP3 gene (wt). The corresponding sequence of mt4 was mutated to mouse sequence (mmt). Exon 2 splicing was analyzed by RT-PCR. ( E ) The Foxp3 humanized mouse (Foxp3-Hu) was generated by replacing C57BL/6N mouse Foxp3 exon 2 and adjacent 50-bp intron sequences with human corresponding sequences. ( F ) Foxp3 exon 2 skipping in Foxp3-Hu T reg cells was confirmed by RT-PCR and DNA sequencing. ( G ) Total FOXP3 (all-Foxp3) or full-length FOXP3 (full-Foxp3) expression in T reg cells was analyzed by flow cytometry. Full-length FOXP3 protein is recognized by FJK-16s antibody. Total FOXP3 protein is recognized by NRRF-30 antibody. The histograms showed quantification of T reg cells and the mean fluorescent intensity (MFI) of total FOXP3 or full-length FOXP3 ( n = 3). ( H to J ) A total of 0.5 × 10 5 MC38 cells were injected subcutaneously into left axilla of humanized or WT mice. Tumor sizes were measured every 2 to 3 days. * P < 0.05 and ** P < 0.01. [(I) and (J)] Mice were euthanized at day 28, and tumors were isolated and weighted. ( K and L ) A total of 2 × 10 5 MC38 cells were injected subcutaneously into humanized or WT mice. Mice were euthanized at the humane endpoints. ( L ) The intratumoral populations of CD4 + , CD8 + , or T reg cells were analyzed with flow cytometry. Histogram summarized amounts of intratumoral CD8 + and T reg cells and total FOXP3 MFI in T reg cells. Statistical significance was determined by an unpaired t test or a Mann-Whitney test. Survival analysis was performed with a log-rank test. n.s., not significant.

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated, DNA Sequencing, Expressing, Flow Cytometry, Injection, Isolation, MANN-WHITNEY

Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A to H ) Single cells were isolated from fresh cancer or adjacent normal tissues. Cells were labeled with anti-CD4 and anti-TCR antibodies, followed by intracellular anti-FOXP3 and anti-SRSF3 labeling. [(A) and (E)] Gating strategy to identify TCR + CD4 + FOXP3 + T reg cells and representative fluorescence-activated cell sorting (FACS) plots showing the expression levels of FOXP3 and SRSF3 in T reg cells isolated from oral squamous cell carcinoma (A) and breast cancer (E) or their adjacent normal tissues, respectively. [(B) and (F)] Summary of SRSF3-positive population percentage of T reg cells in oral squamous cell carcinoma (A) or breast cancer (E) tissues. [(C), (D), (G), and (H)] Summary of FOXP3 and SRSF3 MFI of T reg cells in oral squamous cell carcinoma [(C) and (D)] ( n = 5) or breast cancer [(G) and (H)] ( n = 8) tissues. Data are mean ± SEM. ( I ) Human T reg cells were purified from PBMCs and then transfected with siRNA [anti-SRSF3 or nonspecific (NS)]. PBMCs from the same donor were labeled by CFSE and mixed with T reg cells as the indicated ratio. Cells were cultured for 4 days in the presence of anti-human CD3 antibody (0.5 μg/ml). Then, cells were stained with an anti-CD8 antibody. The proliferation of CD8 + cells was measured by FACS. Data are mean ± SEM, n = 3. ( J ) Down-regulation of SRSF3 released the inhibition of T reg cell on the expression of TNF-α, IFN-γ, and IL-2 by CD8 + T cells. The expression levels of TNF-α, IFN-γ, and IL-2 in CD8 + T cells after in vitro suppression assay were analyzed by intracellular cytokine staining and FACS. Data are mean ± SEM, n = 5. P values are from a two-sided unpaired t test [(I) and (J)] or a paired t test [(B), (C), (D), (F), (G), and (H)].

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Isolation, Labeling, Fluorescence, FACS, Expressing, Purification, Transfection, Cell Culture, Staining, Inhibition, In Vitro, Suppression Assay

Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A ) Srsf3-flox mice were crossed with the Foxp3 YFP-cre mice to produce T reg cell–specific Srsf3-KO mice, including both Foxp3 YFP-Cre Srsf3 flox/flox homozygous (Srsf3-cKO) and Foxp3 YFP-cre Srsf3 flox/+ heterozygous (Srsf3 +/− ) KO mice. ( B ) Genotyping of Srsf3 gene KO in Srsf3-cKO mice. ( C ) T reg cells of Srsf3-cKO mice were isolated and checked for genomic deletion of Srsf3 gene by PCR (the presence of cleaved DNA fragment). CD8 + T cells were used as the non-KO control. WT are WT mice control. CD4 gene was used as DNA template control. ( D ) Srsf3-cKO and Srsf3 +/− mice at postnatal day 28. ( E ) Srsf3-cKO mice showed significant lower body weight than Srsf3 +/− mice at day 21. ( F ) Survival analysis of Srsf3-cKO and Srsf3 +/− mice. ( G ) FACS analyses of Srsf3 expression and population of T reg cells in the thymuses of Srsf3-cKO and Srsf3 +/− mice. ( H ) Serum anti-dsDNA antibody levels in Srsf3 cKO or Srsf3 +/− mice were analyzed by enzyme-linked immunosorbent assay. ( I ) Representative images (specimens and/or hematoxylin and eosin staining) of skin, thymus, spleen, lymph node, and liver from Srsf3-cKO and Srsf3 +/− mice. Histograms show thymus weight ( n = 4), spleen weight/body weight ( n = 4), and lymph node weight ( n = 4). Scale bar, 20 μm. ( J ) T reg cells in spleens or lymph nodes from Srsf3-cKO and Srsf3 +/− mice ( n = 4 or 5). ( K ) The expression levels of CD62L and CD44 in CD8 + T cells from the spleens or lymph nodes of Srsf3-cKO and Srsf3 +/− mice ( n = 3). Statistical significance was determined by a two-sided unpaired t test. Survival analysis was performed with a log-rank test.

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Isolation, Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Inflammation and Regeneration

Article Title: Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury

doi: 10.1186/s41232-024-00319-4

Figure Lengend Snippet: Detection of migrating immune cells in transplanted area. Transplanted areas of the auto, sensory TP, and NC groups at day 7 in nude (top) and SD (bottom) rats were analyzed by flow cytometry. a - c display FACS profiles of T cell populations present at the transplant site. An electronic gate was placed on hematopoietic cells based on their FSC and SSC scatters, and then the proportions of CD3 + CD4 + (CD4 + T cells) were analyzed. d - f FACS profiles show the analysis of macrophages present in the transplant area. CD11b + CD163 + cells were defined as infiltrating M2 macrophages

Article Snippet: The cells were stained with immune cell markers as described below; PE conjugated mouse anti-rat CD3 (BD Pharmingen, Clone G4.18, Catalog No:554833), FITC conjugated mouse anti-rat CD4 (BD Pharmingen, Clone OX-35, Catalog No:554837), Catalog No:561588), PE conjugated mouse anti-rat CD11b/c (BD Pharmingen, Clone OX-42, Catalog No:554862), and Alexa Fluor® 647 conjugated mouse anti-rat CD163 (Bio-Rad Laboratories, Inc., Clone ED2, Catalog No: MCA342A647).

Techniques: Flow Cytometry

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Deficiency Accelerates the Development of Insulin-Deficient Diabetes in Non-Obese Diabetic Mice

doi: 10.1371/journal.pone.0075385

Figure Lengend Snippet: Unseparated spleen cells suspensions from female TLR4 +/+ and TLR4 −/− NOD mice were stained with FITC-conjugated anti-CD4 antibodies and PE-conjugated anti-Foxp3 antibodies. The resulting signals were quantified by FACS analysis. The diagrams show the results of a representative FACS analysis ( a ). CD4 + CD25 + Treg cells isolated from TLR4 +/+ (squares) and TLR4 −/− NOD mice (triangles) were exposed to increasing concentrations of LPS ( b ). After 6 d of culture the metabolic activity of the cells was determined by their capacity to convert the tetrazolium salt WST-1 into its water-soluble formazan product. The data show means ± SD from three experiments performed in triplicates. ** p <0.01 compared to the TLR4 −/− cells. ( c ) To assess the inhibitory capacity of Treg, CD4 + CD25 + Treg cells and CD4 + CD25 − responder cells were isolated from the spleens of TLR4 +/+ (solid bars) and TLR4 −/− NOD mice (open bars) by magnetic bead separation. Treg cells and responder cells were cocultivated at various ratios for 5 d in the presence of anti-CD3 antibodies and IL-2. The metabolic activity of the cocultures was assessed by their WST-1 conversion capacity. * p <0.05 compared to the cocultures of the TLR4 +/+ cells; n.s. not significant.

Article Snippet: To confirm the purity, cells were stained with a FITC-conjugated monoclonal rat anti-mouse CD4 antibody (BD Pharmingen, Heidelberg, Germany) and with a phycoerythrin (PE)-conjugated monoclonal rat anti-mouse CD25 antibody (BD Pharmingen).

Techniques: Staining, Isolation, Activity Assay