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: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Th1 vs Th2 BILs. (A) Flow cytometry demonstrating percent expression of T-bet, IFN-γ, and IL-12R β2 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant increases in all 3 Th1 markers. (***P < 0.0005, **P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.) (B) Flow cytometry demonstrating percent expression of GATA3, the IL-4 receptor, and IL-5 on CD4+ lymphocytes 18 days after GL261 tumor implantation by treatment group. Combination immunotherapy is associated with significant decreases in all 3 Th2 markers. (**P < 0.005, *P < 0.05, absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Expressing, Tumor Implantation, Comparison

Journal: Neuro-Oncology

Article Title: Agonist OX40 immunotherapy improves survival in glioma-bearing mice and is complementary with vaccination with irradiated GM-CSF–expressing tumor cells

doi: 10.1093/neuonc/nox125

Figure Lengend Snippet: Day 18 flow cytometry of BILs shows that vaccination improves the intratumoral CD8+/FoxP3+ lymphocyte ratio, which is little affected by treatment with anti-OX40 immunotherapy. (**P < 0.005, *P < 0.05; absence of an asterisk denotes not significant comparison.)

Article Snippet: For the analysis of T helper cell subsets, we used Multi-Color Flow Cytometry Kits (R&D Systems) for mouse Th1 cells (conjugated antibodies to T-bet-PerCP, IFN-gamma-fluorescein, IL-12 R beta 2-APC, and CD4-PE) and Th2 cells (conjugated antibodies to CD4-PerCP, IL-4 R-fluorescein, STAT6-APC, and IL-5-PE).

Techniques: Flow Cytometry, Comparison

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on whole-cell Ca 2+ -activated K + current ( I K(Ca) ) in mHippoE-14 hippocampal neurons. In these experiments, cells were bathed in normal Tyrode’s solution, the composition of which was described under Section “Materials and Methods.” The recording pipette was filled with K + -containing solution. (A) Superimposed I K(Ca) traces obtained in the control (middle part) and during cell exposure to 10 μM PIO (bottom part). The upper part indicates the voltage protocol applied, and arrowheads are zero current level. (B) Averaged I–V relationships of I K(Ca) obtained in the control ( ), during the exposure ( ) to 10 μM PIO and after washout ( ) of PIO (mean ± SEM; n = 11 for each point). ∗ Significantly different from control groups taken at the same level of voltage pulse. (C) Bar graph showing summary of the effect of PIO, PIO plus TRAM-39, PIO plus apamin, and PIO plus paxilline, and PIO plus tolbutamide on I K(Ca) amplitude (mean ± SEM; n = 10–12 for each bar). Current amplitude was measured at +50 mV. (a) Control; (b) 10 μM PIO; (c) 10 μM PIO plus 3 μM TRAM-39; (d) 10 μM PIO plus 200 nM apamin; (e) 10 μM PIO plus 1 μM paxilline; (f) 10 μM PIO plus 30 μM tolbutamide. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO alone group ( P < 0.05) ( n = 9–10 for each bar).

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on BK Ca channel activity in mHippoE-14 hippocampal neurons. (A) Original current traces of BK Ca channels obtained in the absence (left) and presence (right) of 10 μM pioglitazone (PIO). The examined cells were bathed in symmetrical K + solution (145 mM). Under inside-out current recordings, the potential was held at +60 mV and bath medium contained 0.1 μM Ca 2+ . The upward deflection represents the opening event of the channel. The lower part indicates the expanded trace recorded from the uppermost part in the control and during exposure to PIO. (B) BK Ca -channel trace obtained after washout of PIO. (C) Concentration-dependent increase in channel open probability (mean ± SEM; n = 9–11 for each point). Channel activity measured at +60 mV during the exposure to 100 μM PIO was taken to be 100%. The values for EC 50 , Hill coefficient and maximal percentage increase of BK Ca channels in the presence of PIO were 7.6 μM 1.3 and 100%, respectively.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Activity Assay, Control, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on mean open- (A) and closed-time (B) histograms of BK Ca channels recorded from mHippoE-14 hippocampal neurons. The holding potential was set at +60 mV, and inside-out configuration was performed. In control (left side), the open-time histogram of the channel was fitted by a single exponential function (indicated by red smooth line) with a mean open time of 1.9 ms, while the closed-time histogram was by a sum of a two-exponential function with a mean closed time of 3.5 and 47.5 ms. After addition of 10 μM PIO (right side), the mean open time was increased to 2.7 ms, and the slow component of closed time was shortened to 28.7 ms; however, minimal change in the fast component of closed time (i.e., 3.4 ms) in the presence of this compound. Of note, the abscissa and ordinate in each histogram indicate the logarithm of open or closed time (ms) and the square root of even number, respectively. Data were taken from a measurement of 100 channel openings. The vertical black dashed lines are placed at the values of mean open or closed time for BK Ca channels.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on the I–V relation of BK Ca channels in mHippoE-14 hippocampal neurons. The experiments on BK Ca channels were conducted with symmetrical K + -rich concentration (145 mM). Under inside-out configuration, the potential was held at +60 mV and bath medium medium contained 0.1 μM Ca 2+ . (A) Original current traces obtained in the control and during exposure to 10 μM PIO. The labels in the rightmost side indicate the holding potential applied. Arrowhead in each trace corresponds to zero current level, and the upper deflection indicates the opening event of the channel. In (B) , the single-channel conductance in the absence ( ) and presence ( ) of 10 μM PIO is nearly identical. Each point represents mean ± SEM ( n = 9–10). The dashed red lines obtained with or without addition of PIO are pointed toward the values of the reversal potential (i.e., 0.0 ± 0.1 mV, n = 8). (C) The relationship between relative open probability of BK Ca channels and membrane potential obtained with or without addition of 10 μM PIO. The ramp pulses were applied from 0 to +80 mV with a duration of 1 s. Under inside-out current recordings, PIO (10 μM) was applied to the intracellular surface of the excised patch. The smooth lines represent the best fit to the Boltzmann equation as detailed in Section “Materials and Methods.”

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Concentration Assay, Control, Membrane

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of linopirdine and linopirdine plus flupirtine on the amplitude of M-type K + current [ I K ( M ) ] in mHippoE-14 hippocampal neurons. In this set of experiments, cells were bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K(M) traces obtained in the control (a) and during the exposure to 10 μM linopirdine (b), and 10 μM linopirdine plus 10 μM flupirtine (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of linopirdine and linopirdine plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9 for each bar). ∗ Significantly different from control ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Control

Journal: Frontiers in Pharmacology

Article Title: Pioglitazone, a PPAR-γ Activator, Stimulates BK Ca but Suppresses IK M in Hippocampal Neurons

doi: 10.3389/fphar.2018.00977

Figure Lengend Snippet: Effect of PIO on I K ( M ) amplitude in mHippoE-14 hippocampal neurons. These experiments were conducted in cells bathed in high K + , Ca 2+ -free solution and the recording pipette was filled with K + -containing solution. (A) Superimposed I K ( M ) traces obtained in the absence (a) and presence of 3 μM (b), and 10 μM PIO (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of PIO, linopirdine, and PIO plus flupirtine on I K ( M ) amplitude (mean ± SEM; n = 9–11 for each bar). The I K ( M ) amplitude elicited by membrane depolarization from –50 to –10 mV was measured. (a) Control; (b) 10 μM PIO; (c) 10 μM linopirdine; (d) 10 μM PIO plus 10 μM flupirtine. ∗ Significantly different from control ( P < 0.05) and ∗∗ significantly different from PIO (10 μM) alone group ( P < 0.05). LINO, linopirdine; FLUP, flupirtine.

Article Snippet: Embryonic mouse hippocampal cell line (mHippoE-14; CLU198) was obtained from Cedarlane CELLutions Biosystems, Inc. (Burlington, ON, Canada) ( ).

Techniques: Transferring, Membrane, Control

Journal: bioRxiv

Article Title: RetroCHMP3 Blocks Budding of Enveloped Viruses Without Blocking Cytokinesis

doi: 10.1101/2020.08.30.273656

Figure Lengend Snippet: (A) Schematic of putative regulatory elements upstream of retroCHMP3 ORF in Mus musculus identified by transcription factor binding site prediction tools. Numbers indicate nucleotides relative to retroCHMP3 start codon. LTR – long terminal repeat, MuRRS – murine retrovirus-related sequence, ERV – endogenous retrovirus. (B) RT-PCR detection of retroCHMP3 RNA in mouse cardiac endothelial cells (MCECs) with and without interferon stimulation. RetroCHMP3 bands were excised, cloned, and sequenced to verify a match with the retroCHMP3 sequence. Representative gel from three independent biological repeats. RT – reverse transcriptase. (C) Droplet digital PCR (ddPCR) detection of retroCHMP3 (red) and RNAse L (grey, positive control) RNA in mouse cardiac endothelial cells (MCECs) with and without interferon stimulation. The housekeeping gene succinate dehydrogenase complex, subunit A (SDHA) was used for normalization. Each line represents one independent biological repeat.

Article Snippet: MCEC cells (mouse cardiac endothelial cells, CLU510, sex: unknown) were obtained from Cedarlane.

Techniques: Binding Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Reverse Transcription, Digital PCR, Positive Control

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Clinical trial data and human organoids confirm that blocking CXCR4 enhances antitumor immunotherapy efficacy. A Schematic overview of clinical trial NCT02826486 , including treatment timeline and peripheral blood sampling schedule. B Longitudinal analysis of lymphocyte counts, CXCR4 + CD4 + T cells, CD4 + CD25 + FOXP3 + regulatory T cells, and CD69 + CD4 + T cells following CXCR4 antagonist monotherapy and combination therapy. Gray lines denote geometric means; blue and red lines indicate the lower and upper bounds of the 95% confidence intervals, respectively. M, monotherapy; Pre, pre-treatment; Post, post-treatment; D, day. Statistical comparisons were performed using two-sided t -tests based on geometric means and derived standard deviations. Significance thresholds were adjusted using Bonferroni correction. C Schematic of RNA-seq workflow from trial NCT04516616 . Cervical cancer patients receiving neoadjuvant chemotherapy plus anti-PD-1 therapy were stratified into pathological complete response (pCR) and non-pCR groups, and tumor samples were subjected to transcriptomic profiling. D Heatmap showing immune cell-type enrichment scores, as assessed by xCell algorithm analysis, comparing pCR and non-pCR groups. E Paired comparisons of gene expression and immune cell scores before and after immune checkpoint blockade (ICB) in pCR and non-pCR groups. p values calculated by paired t -test in GraphPad Prism. F Correlation analysis of gene expression profiles of pre- and post-ICB in non-pCR patients. G Schematic of experimental setup in which PBMCs from non-pCR patients were co-cultured with cervical cancer-derived organoids. H Representative images of cervical cancer organoid and PBMC co-culture system under different treatments. Tumor cell apoptosis was evaluated by caspase-3 staining. ***, p < 0.001

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Blocking Assay, Sampling, Derivative Assay, RNA Sequencing, Gene Expression, Cell Culture, Co-Culture Assay, Staining

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Single-cell transcriptomic analysis identified that CXCR4 expression was associated with T reg cell developmental trajectories. A UMAP plot of CD4 + T cells ( n = 45,363) in lung adenocarcinoma. B Correlation analysis of gene expression ( CXCR4 and CTLA4 ) and the proportion of CD4-CTLA4-T reg cells. C UMAP plot of CD4 + T cells ( n = 11,166) in non-small cell lung cancer. D Correlation analysis of gene expression ( CXCR4 and FOXP3 ) and the proportion of CD4-CTLA4-Treg cells. E UMAP plot of CD4 + T cells ( n = 63,965) in multi-cancer. F Correlation analysis of CXCR4 expression and the proportion of CD4-LEF1-T reg and CD4-CTLA4-T reg cells. G Developmental trajectories of T reg cells in breast cancer from GSE156728 via diffusion map. Expression levels of marker genes and CXCR4 were shown. H Developmental trajectories of T reg cells in pancreatic cancer from GSE156728 via diffusion map analysis. Expression levels of marker genes and CXCR4 were shown

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Expressing, Gene Expression, Diffusion-based Assay, Marker

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Blocking CXCR4 in CD4 + T cells reduces the activated T reg phenotypes in vivo. A Gross anatomy of subcutaneous tumors (U14) in mice treated with CXCR4 antagonists. B Flow cytometry and statistical analysis of tumor models (4T1 and ID8) under different treatments; p values were calculated using the unpaired, parametric t -test. C UMAP plot of CD4 + T meta-cell clusters of scRNA-seq data from pan-cancer analysis. Exhausted clusters are indicated. T n , naïve T cells; T m , memory T cells; T em , effector memory T cells; T emra , terminally differentiated effector memory or effector; Tfh, follicular helper T cell; Th1, T helper 1; ISG, interferon-stimulated genes. D Violin plots showing the expression levels of canonical marker genes across CD4 + T cell clusters. E Schematic diagram illustrating the experimental design for CXCR4 antagonist treatment in U14 tumor-bearing immunocompetent syngeneic mice. Flow cytometry analysis ( F ) and corresponding quantification ( G ) of CD4 + T cell subsets in tumors across different treatment groups. H Schematic diagram and experimental design for tumor-bearing (U14) Cxcr4 -cKO and control mice. Flow cytometry analysis ( I ) and quantification ( J ) of CD4. + T cell subsets in spleens from tumor-bearing control and cKO mice. p values were determined by unpaired, parametric t -test. ** p < 0.01; *** p < 0.001

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Blocking Assay, In Vivo, Flow Cytometry, Expressing, Marker, Control

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Blocking CXCR4 + CD4 + T cells enhances anti-PD-1 immunotherapy efficacy by reprogramming T reg -mediated immunosuppressive TME. A Mean tumor volumes and gross anatomy of U14 tumors in primary recipients with various treatments (saline and IgG, CXCR4 antagonist, anti-PD-1, combination). p values were assessed using two-way ANOVA. B Tumor volume analysis of 4T1-tumor-bearing animals treated with indicated drugs; each line represents an individual mouse. After 40 days of combination treatment, mice showing complete response to combined treatment were rechallenged with 4T1 tumors and treated with CXCR4 agonist and control vehicle. p values were assessed using two-way ANOVA. C RNA heatmaps of marker genes in U14 tumors from different treatments. D UMAP plots of 11 cell clusters and treatment samples based on scRNA-seq data. E UMAP plots of five CD4 + T cell subtypes and their treatment samples based on scRNA-seq data. F Venny plot of DGEs in CD4 + T cells compared to controls. The bottom panel shows pathway enrichment analysis of the overlapped DEGs. G Pseudotime analysis of differentially expressed heatmaps for CD4 + T cells. H Violin plot of gene expression in DC and T reg cells from different treatments based on scRNA-seq data. I Schematic illustrating the mechanism by which blocking CXCR4 + CD4 + T cells regulates T reg -mediated immunosuppression. CXCR4 inhibition reduces the recruitment and suppressive function of T reg cells, characterized by downregulation of key immunosuppressive cytokines (e.g., IL-10, TGF-β) and immune checkpoint molecules (e.g., CTLA-4, TIM-3). This leads to diminished T reg –APC interactions and decreased expression of co-inhibitory ligands (e.g., PD-L1, CD80, CD86) on APCs. *** p < 0.001

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Blocking Assay, Saline, Control, Marker, Gene Expression, Inhibition, Expressing

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Phosphoproteome and ChIP-seq analyses reveal that targeting CXCR4 + CD4 + T cells reduces T reg -associated suppressive genes via modulation of the Rho-GTPase/NF-κB signaling axis. A Experimental design for phosphoproteome analysis in human T reg cells treated with CXCR4 antagonist. Human T reg cells were purified by FACS and stimulated in vitro. B Heatmap displaying the differentially regulated phosphorylation sites in T reg cells before and after CXCR4 antagonist treatment. Each row represents a phosphorylation site, and each column represents a biological replicate. C Pathway enrichment analysis based on proteins with different phosphorylation sites in T reg cells pre- and post-CXCR4 antagonist treatment. D Overview of phosphorylation level changes associated with the Rho-GTPase/NF-κB signaling axis induction by CXCR4 antagonist treatment. Differentially regulated phosphorylation sites related Rho-GTPase/NF-κB signaling axis were indicated. Protein–protein interaction networks represent differentially regulated phosphorylation sites. E The ex vivo co-culture system using human cervical cancer organoids and PBMCs derived from two patients at different treatment timepoints. Patient-derived PBMCs were co-cultured with autologous organoids under various treatment conditions. F Violin plots showing quantification of apoptosis rates in cervical cancer organoids under different treatment conditions, as assessed by caspase-3 staining. Each dot represents an independent measurement from different patient-derived samples. p values were calculated by the Mann–Whitney test in GraphPad Prism. G ChIP-seq analysis of chromatin occupancy in T reg -associated genes based on NFKB2 and RelB ChIP-seq data. H ChIP-PCR validation of NFKB2 and RelB binding to the promoters of selected T reg signature genes in T reg cells before and after CXCR4 antagonist exposure. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; ns, no significance

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: ChIP-sequencing, Purification, In Vitro, Phospho-proteomics, Ex Vivo, Co-Culture Assay, Derivative Assay, Cell Culture, Staining, MANN-WHITNEY, Biomarker Discovery, Binding Assay

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Single-cell multi-omic analyses reveal that blocking CXCR4 + CD4 + T cells epigenetically reprogram T reg -associated suppressive genes. A tSNE plots of 14 cell clusters based on scRNA-seq and scATAC-seq data from U14 tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. B Chromatin accessibility analysis of marker genes in cell clusters. C Representative mIHC staining of spleens from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. D tSNE plots of four subtypes of CD4 + T cells based on scRNA-seq and scATAC-seq data from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. E tSNE and violin plots displaying CXCR4 expression levels in T reg cells based on scRNA-seq analysis. p values were determined by the Wilcoxon rank-sum test. F Motif scores in T reg cells from scATAC-seq analysis. G Chromatin accessibility analysis of marker genes in T reg cells. H Pathway enrichment analysis based on the top 50 different peaks in T reg cells between Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. I Chromatin accessibility analysis of marker genes in B cells, macrophages, and DCs. J Representative mIHC staining of tumors from tumor-bearing Cxcr4 flox/flox Lck Cre and Cxcr4 flox/flox mice. ***, p < 0.001

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Blocking Assay, Marker, Staining, Expressing

Journal: Genome Medicine

Article Title: Blocking CXCR4 + CD4 + T cells reprograms T reg -mediated immunosuppression via modulating the Rho-GTPase/NF-κB signaling axis

doi: 10.1186/s13073-025-01515-8

Figure Lengend Snippet: Main content analysis and findings of this study. This study integrates multi-omic profiling and functional validation experiments to investigate how CXCR4 + CD4 + T cells contribute to tumor immunosuppression and how CXCR4 blockade can reprogram this process. Approaches included single-cell RNA-seq, single-cell multi-omics, bulk RNA-seq, phosphoproteomics, mouse tumor models, patient-derived organoid PBMC co-culture systems, and clinical cohort analyses. Mechanistically, CXCR4 antagonism disrupted the Rho GTPase/NF-κB signaling pathways, leading to reduced transcription of T reg -associated suppressive molecules (e.g., CTLA-4, PD-1, TIM-3, and TNFRSF members). This downregulation impaired T reg -mediated suppression, ultimately rebalancing the tumor immune microenvironment in favor of antitumor immunity and enhancing the efficacy of anti-PD-1 immunotherapy

Article Snippet: Cells were activated using the T Cell Activation/Expansion Kit (Miltenyi Biotec, Cat. No. 130–093-627) for 48 h to stimulate proliferation.

Techniques: Functional Assay, Biomarker Discovery, RNA Sequencing, Phospho-proteomics, Derivative Assay, Co-Culture Assay, Protein-Protein interactions