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
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![( 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 <t>CD4</t> + , 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0887/pmc13220887/pmc13220887__sciadv.aeh1671-f1.jpg)
![( 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)].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0887/pmc13220887/pmc13220887__sciadv.aeh1671-f4.jpg)
