anti rat cd4  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Phenotypic changes and inflammatory factor secretion of T cells stimulated by cardiac lysate antigens (CLAs). (A) Coculture protocol for dendritic cells (DCs) and T cells. The tolerogenic DCs (tDCs) present antigens to T cells; meanwhile, programmed death ligand 1 binds to programmed cell death protein 1 on T cells and inhibits T cell activation, promoting the phenotypic conversion of T cells to regulatory T cells (Tregs). Furthermore, due to the lack of activation signals such as CD80 and CD86, some T cells exhibit a weak activation state after receiving antigen presentation, and under the regulation of key factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), they undergo phenotypic transition to Tregs. Under restimulation, tDCs phagocytose antigens and present them to Tregs. Tregs are rapidly activated, express high levels of Forkhead box P3 (FOXP3), and directly inhibit T cell activation, while secreting large amounts of anti-inflammatory factors. (B) Flow cytometry (FC) results for T cell phenotype. (C) Quantification of T cell phenotypes by FC. (D) T cell phenotype immunofluorescence staining results, where blue represents cell nuclei, red represents CD127, and green represents CD4. (E) Expression of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β after coculture of DCs and T cells. (F) Western blot analysis of the activation status of the mechanistic target of rapamycin (mTOR)-p70S6K-S6 pathway after treatment with Gel@PPM, Rapa, inverse vaccine nanomimics (IVNMs), and Gel@IVNMs under CLA stimulation. (G) The percentage of phosphorylation of mTOR, p70S6K, and S6 was statistically analyzed based on the gray values of the western blot results (compared to the unphosphorylated proteins). Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were then surface-stained with fluorophore-conjugated anti-rat CD4 (E-AB-F1105C, Elabscience, China) and CD25 (PE-30314, Finetest, China) antibodies.

Techniques: Activation Assay, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Staining, Expressing, Western Blot, Analysis, Phospho-proteomics

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Changes in dendritic cell phenotype and regulatory T cell responses in cardiac-draining lymph nodes after myocardial infarction (MI). (A) Representative flow cytometry plots showing CD11c + MHC-II + dendritic cells and programmed death ligand 1 (PD-L1) expression in the Control, Gel@PPM, Gel@PP@Rapa, Gel@OVA-IVNMs, and Gel@IVNMs groups at 7 and 56 d after MI. (B) Quantification of the proportion of PD-L1 + cells within the CD11c + MHC-II + dendritic cell population at 7 and 56 d. (C) Quantification of CD4 + CD25 + FOXP3 + regulatory T cells (Tregs) in lymph node-derived cells from the 5 treatment groups after 48 h of culture under unstimulated Control, OVA, or cardiac lysate antigen (CLA) stimulation at 7 and 56 d. (D) Representative flow cytometry plots of CD25 and Forkhead box P3 (FOXP3) expression within CD4 + T cells in the Gel@OVA-IVNMs and Gel@IVNMs groups following Control, OVA, or CLA stimulation at 7 and 56 d. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were then surface-stained with fluorophore-conjugated anti-rat CD4 (E-AB-F1105C, Elabscience, China) and CD25 (PE-30314, Finetest, China) antibodies.

Techniques: Flow Cytometry, Expressing, Control, Derivative 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 ) 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: BMC Immunology

Article Title: CD30 knockout attenuates experimental colitis by reducing inflammatory cytokine production

doi: 10.1186/s12865-026-00816-w

Figure Lengend Snippet: CD30 Knockdown Suppresses Th1 Cell Differentiation In Vitro. A Representative flow cytometry plot showing purity of isolated naive CD4 + T cells (CD62L vs. CD44 gated on CD4 + cells). Average purity: 97.2 ± 1.3%. B CD30 mRNA knockdown efficiency by qRT-PCR ( n = 6 per group). C Western blot bands of protein expression in each group. D CD30 protein knockdown efficiency by Western blot with quantification ( n = 6 per group). E Representative flow cytometry plots of IFN-γ + cells among CD4 + T cells (gated on CD4 + population). F Quantification of IFN-γ + cell percentages among CD4 + T cells ( n = 6 per group). G Quantification of total CD4 + T cell percentages among all live cells ( n = 6 per group). Data are presented as mean ± SD. ** P < 0.01 vs. Control group; or ## P < 0.01 vs. si-NC group. All experiments were independently repeated three times with similar results

Article Snippet: Single-cell suspensions were prepared by mechanical dissociation through 70 μm cell strainers (BD Biosciences, San Jose, CA, USA), and naive CD4 + T cells (CD4 + CD25-CD44lowCD62Lhigh) were isolated using magnetic-activated cell sorting (MACS) with a Naive CD4 + T Cell Isolation Kit (Cat. No. 130-104-453; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Knockdown, Cell Differentiation, In Vitro, Flow Cytometry, Isolation, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: BMC Immunology

Article Title: CD30 knockout attenuates experimental colitis by reducing inflammatory cytokine production

doi: 10.1186/s12865-026-00816-w

Figure Lengend Snippet: CD30 Promotes Th1 Differentiation Under Inflammatory Conditions. A Representative flow cytometry plots of IFN-γ + cells among CD4 + T cells (gated on CD4 + population) in PBS, LPS, si-NC + LPS, si-CD30 + PBS, and si-CD30 + LPS groups. B Quantification of IFN-γ + cell percentages among CD4 + T cells ( n = 6 per group). C mRNA expression of T-bet by qRT-PCR ( n = 6 per group). D Western blot bands of protein expression in each group. E mRNA expression of IFN-γ by qRT-PCR ( n = 6 per group). F-H Western blot analysis of T-bet ( F ), total STAT4 ( G ), and p-STAT4 ( H ) with quantification ( n = 6 per group). I-J Secreted concentrations of IFN-γ ( I ) and IL-2 ( J ) by ELISA (pg/ml) ( n = 6 per group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. PBS group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. LPS group; & P < 0.05, && P < 0.01, &&& P < 0.001 vs. si-NC + LPS group. All experiments were independently repeated three times with similar results

Article Snippet: Single-cell suspensions were prepared by mechanical dissociation through 70 μm cell strainers (BD Biosciences, San Jose, CA, USA), and naive CD4 + T cells (CD4 + CD25-CD44lowCD62Lhigh) were isolated using magnetic-activated cell sorting (MACS) with a Naive CD4 + T Cell Isolation Kit (Cat. No. 130-104-453; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay