Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: C57BL/6 mice were treated with CPT-11, and immune responses were determined by flow cytometry (FCM). a Total number of immune cells in spleen and lymph nodes (LNs) of mice treated with PBS (control) or CPT-11 ( n = 4 mice per group). b , c Bar graphs show the frequency of Ki67 + CD4 + and Ki67 + CD8 + T cells in the indicated groups. d Representative fluorescence-activated cell sorting (FACS) plots of indicated groups. e – j Bar graphs showing frequencies of IFN-γ + CD4 + T (Th1) cells, T-bet + CD4 + T (Th1) cells, IFN-γ + CD8 + T cells, IL-17 + CD4 + T (Th17) cells, IL-4 + CD4 + T (Th2) cells, and FoxP3 + CD4 + Treg cells in indicated groups. Data are representative of two independent experiments. Summary data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; by unpaired two-tailed Student’s t-tests. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Flow Cytometry, Control, Fluorescence, FACS, Two Tailed Test

Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: C57BL/6 mice were challenged with CFA (subcutaneous injection) and treated with CPT-11 or PBS, and immune responses in spleen and LNs were determined using FCM. a Total number of immune cells in the spleen and LNs of mice treated with PBS (control) or CPT-11. ( n = 4 mice per group). b Representative FACS plots of indicated groups. c – g Bar graphs showing frequencies of Ki67 + CD4 + and Ki67 + CD8 + T cells, IFN-γ + CD4 + Th1 cells, IL-17 + CD4 + Th17 cells, and IFN-γ + CD8 + cells from indicated mice. Data are representative of two independent experiments. Summary data are presented as mean ± s.d. ** p < 0.01, *** p < 0.001, **** p < 0.0001; by unpaired two-tailed Student’s t-tests. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Injection, Control, Two Tailed Test

Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: CD4 + CD25 − CD62L high (naive) T cells isolated from spleen and LNs of C57BL/6 mice were cultured with anti-CD3 and anti-CD28, with or without CPT-11 for 1-3 d. Cell proliferation, cell apoptosis, and cell differentiation were determined using FCM ( n = 3). a , b Representative FACS plots ( a ) and bar graph ( b ) showing non-proliferative T cell frequencies in T cells cultured for 3 d. c , d Representative FACS plots ( c ) and bar graph ( d ) showing apoptotic T cell frequencies in T cells cultured for 24 h. e , f Representative FACS plots ( e ) and bar graph ( f ) showing the frequency of Th1 cells in T cells cultured for 3 d in the presence of IL-12. g , h Representative FACS plots ( g ) and bar graph ( h ) showing frequencies of Th17 cells among T cells cultured for 3 d in the presence of TGF-β and IL-6. Data are representative of three independent experiments ( a , c , e , g ) or are pooled from three independent experiments ( b , d , f , h ). Summary data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; by unpaired two-tailed Student’s t-tests. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Isolation, Cell Culture, Cell Differentiation, Two Tailed Test

Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: C57BL/6 mice were administered IMQ cream on a 2.5 cm × 2.5 cm patch of shaved back skin daily for 7 consecutive days, and were injected with CPT-11 or PBS intraperitoneally once per day ( n = 12 mice per group). a Statistical analysis of epidermal thickness. b Representative histological skin images. c – k Bar graphs showing frequencies of Ki67 + CD4 + T cells ( c ), Ki67 + CD8 + T cells ( d ), IL-17 + CD4 + Th17 cells ( e ), RORγt + CD4 + Th17 cells ( f ), IFN-γ + CD4 + Th1 cells ( g ), T-bet + CD4 + Th1 cells ( h ), IFN-γ + CD8 + T cells ( i ), IL-4 + CD4 + Th2 cells ( j ) and FoxP3 + CD4 + Treg cells ( k ) in the spleen (SPL) and draining lymph nodes (DLN) of indicated groups. Data are representative of three independent experiments ( a , b ) or are pooled from three independent experiments ( c – k ). Summary data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; by a one-way analysis of variance (ANOVA) with Tukey’s post hoc test. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Cream, Injection

Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: C57BL/6 mice were subcutaneously immunized with MOG peptide 35–55 emulsified in complete Freund’s adjuvant to induce EAE, and treated with CPT-11 or PBS daily from day 9. a EAE clinical scores of the indicated groups ( n = 10 mice per group). b Representative Luxol Fast Blue (LFB) staining of cervical spinal cord sections. c Representative histological images of cervical spinal cord sections. d , e Representative FACS plots ( d ) and bar graph ( e ) showing frequencies of CD3 + T cells in the brain and spinal cord. f , g Representative FACS plots ( f ) and bar graph ( g ) showing frequencies of IFN-γ + CD4 + Th1 cells in brain and spinal cord. h – k Representative FACS plots ( h , j ) and bar graphs ( l , k ) showing frequencies of Th17 cells in brain and spinal cord. l Bar graph showing frequencies of Foxp3 + Treg cells in brain and spinal cord. Data are representative of two independent experiments ( a – c ) or are pooled from two independent experiments ( d – l ). Summary data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; by unpaired two-tailed Student’s t-tests. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Adjuvant, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

doi: 10.1038/s41420-024-01983-8

Figure Lengend Snippet: C57BL/6 mice were administered IMQ cream on shaved 2.5 cm × 2.5 cm patches of back skin daily for 7 consecutive days and were injected with CPT-11 or PBS intraperitoneally once per day. Approximately 5 weeks after psoriasis induction and treatment, the mice were injected with B16 cells to establish a tumor-bearing model ( n = 7 mice per group). a Experimental scheme of the B16 tumor-bearing model after psoriasis induction and treatment. b Tumor growth curves. c – j Representative FACS plots ( c , e , g , i ) and Bar graphs ( d , f , h , j ) showing frequencies of Ki67 + CD4 + T cells ( c , d ), IFN-γ + CD4 + Th1 cells ( e , f ), IFN-γ + CD8 + cells ( g , h ), and FoxP3 + CD4 + Treg cells ( i , j ). Data are representative of two independent experiments ( b – d ) or are pooled from two independent experiments ( e – j ). Summary data are presented as mean ± s.d. * p < 0.05, ** p < 0.01, **** p < 0.0001; by unpaired two-tailed Student’s t-tests. See also Supplementary Fig. .

Article Snippet: The following chemicals were purchased from the indicated manufacturers: purified anti-mouse CD3 (145–2C11, Bio X Cell, # BE0001–1), purified anti-mouse CD28 (37.51, Bio X Cell, # BE0015–1), recombinant mouse IL-12 (R&D Systems, #419-ML-500), Freund’s adjuvant, incomplete (IFA) (BD/Difco Laboratories, # 263910), Mycobacterium tuberculosis (BD Biosciences, #231141), DNase I (Millipore Sigma, # DN25), Collagenase IV (Thermo Fisher Scientific, # # 17104–019), PMA (Millipore Sigma, #P8139), Ionomycin calcium salt (Millipore Sigma, #13909), Golgi-Plug Protein Transport Inhibitor (BD Biosciences, #555029), CD4 + CD62L + T cell Isolation Kit, mouse (Miltenyi Biotec, #130–106-643), Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, #00–5523-00), Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit (BD Biosciences, #554714), CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, # C34554), Seahorse XF Cell Mito Stress Test Kit (Agilent, # 103015–100), fluorochrome-conjugated antibodies (zombie yellow [Biolegend, #423104]), anti-mouse CD45 (30-f11, Thermo Fisher Scientific, #56-0451-82), anti-mouse TCRβ (H57-597, Thermo Fisher Scientific, # 47–5961-82), anti-mouse CD4 (RM4-5, Thermo Fisher Scientific, #45-0042-82), anti-mouse CD8α (53-6.7, Thermo Fisher Scientific, #11-0081-85), anti-mouse CD8β (H35-17.2, Thermo Fisher Scientific, 11-008S-85), anti-mouse CD62L (MEL-14, Thermo Fisher Scientific, #11-0621-85), anti-mouse CD44 (IM7, Thermo Fisher Scientific, #17-0441-83), anti-mouse CD25 (PC61.5- Thermo Fisher Scientific, #45-0251-82), anti-mouse CD69 (H1.2F3, Thermo Fisher Scientific, #12-0691-83), anti-mouse IFN-γ (XMG1.2, Thermo Fisher Scientific, #48-7S11-82), anti-mouse IL-17(eBio17B7, Thermo Fisher Scientific, #25-7177-82), anti-mouse IL-4 (11B11, Thermo Fisher Scientific, #25-7041-82), anti-mouse Ki67 (SolA15, Thermo Fisher Scientific, #25-5698-82), anti-mouse RORγt (B2D, Thermo Fisher Scientific, #17-6981-82), anti-mouse T-bet (eBio4B10, Thermo Fisher Scientific, #12-5825-82), anti-mouse FoxP3 (FJK-16s, Thermo Fisher Scientific, #48-577S-82), 7-AAD Viability Staining Solution (Thermo Fisher Scientific, #00-6993-50), and Annexin V (Thermo Fisher Scientific, #BMS306PE-100), anti-mouse CD16/32 (Biolegend, #101302).

Techniques: Cream, Injection, Two Tailed Test

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Compound Kushen Injection inhibits epithelial-mesenchymal transition of gastric carcinoma by regulating VCAM1 induced by the TNF signaling pathway.

doi: 10.1016/j.phymed.2023.154984

Figure Lengend Snippet: Fig. 3. CKI improves immunity in the tumor xenograft model in cooperation with DDP. (A) Mean thymus and spleen indices of different groups. (B) Representative flow cytometry images of CD3+ T cells, CD4+ T cells, CD8+ T cells in tumor tissues. (C) Representative flow cytometry images of CD3+ T cells, CD4+ T cells, CD8+ T cells in spleen tissues. (D) H & E staining of tumor sections. Data are presented as mean ± SD of 6 mice per group. *p < 0.05, **p < 0.01 vs. model group. #p < 0.05, ##p < 0.01 vs. DDP group. CKIMD, CKI middle dose; CKIHD, CKI high dose.

Article Snippet: BB515 Rat Anti-Mouse CD45 (BD, USA), APC/FireTM 750 antimouse CD3, APC anti-mouse CD4, and PE/Cyanine7 anti-mouse CD8a (BioLegend, USA) were used to label CD3+, CD4+, and CD8+ T cells, and measurement was performed by flow cytometry (Cytek, USA).

Techniques: Flow Cytometry, Staining

Journal: Vaccines

Article Title: Immunogenicity and Protection against Mycobacterium caprae Challenge in Goats Vaccinated with BCG and Revaccinated after One Year

doi: 10.3390/vaccines8040751

Figure Lengend Snippet: Gating strategy for the determination of frequency of the IFN-γ positive lymphocyte subsets. Peripheral blood mononuclear cells (PBMC) from all goats were cultured in the presence of M. bovis tuberculin (PPD-B). ( A , B ) Singlet lymphocytes were identified based on the degree of cellular differentiation determined by forward scatter (FSC) and side scatter (SCC). ( C ) Representative frequencies of the CD4/CD45RO cell populations. ( D ) Representative frequencies of intracellular IFN-γ staining of cells gated from prelabelled CD4 + CD45RO + .

Article Snippet: Cells were stained with mouse monoclonal antibodies (mAb) CC8 (IgG2A, conjugated to FITC), which recognizes bovine CD4, and mAb IL-A116 (IgG3, conjugated to RPE), which recognizes bovine CD45RO (both from Bio-Rad Laboratories Inc., Hercules, CA, USA).

Techniques: Cell Culture, Cell Differentiation, Staining

Journal: iScience

Article Title: Fluorescence tracking Treg movement identifies anti-CCR8 and radiation as a therapeutic combination

doi: 10.1016/j.isci.2025.114572

Figure Lengend Snippet: Tregs traffic from the tumor to the TdLN at higher proportions and are equally susceptible to radiation as non-Treg CD4 T cells (A) Kaede mice were injected with either MC38, MOC1, or MOC2 cancer cells subcutaneously. Tumors were photoconverted on day 14 via ultraviolet light. TdLNs were harvested on day 3 post-photoconversion. All cell populations were pre-gated on live singlets. (B) Photoconverted CD8 T cells were defined as CD3 + CD4 − CD8 + Red + . Photoconverted Tregs were defined as CD3 + CD8 − CD4 + CD25 + Red + . Photoconverted non-Treg CD4 T cells were defined as CD3 + CD8 − CD4 + CD25 − Red + . (C) Frequencies of photoconverted CD8 T cells, Tregs, and non-Treg CD4 T cells were quantified via flow cytometry. (D) Mice were injected with MC38 cancer cells subcutaneously. Tumors were treated with 12 Gy RT on day 14 and harvested for analysis via flow cytometry on day 1–3 post-RT. Non-Treg CD4 T cells and Tregs frequencies as a percentage of live cells were quantified. (E) MC38-bearing Kaede mouse tumors were photoconverted on day 14 followed by immediate treatment with 12 Gy RT. (F) TdLNs were harvested on day 1–3 and percentages of photoconverted non-Treg CD4 T cells and Tregs were analyzed via flow cytometry. A total of 5–6 mice were analyzed per group. Data are represented as mean ± SD. Statistics were performed using an unpaired Student’s t test between groups. ns = not significant, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05.

Article Snippet: Samples were enriched magnetically using CD4/CD8 TIL MicroBeads (Miltenyi Biotec, 130-116-480).

Techniques: Injection, Flow Cytometry