Journal: Aging Cell

Article Title: Role of Succinate Dehydrogenase in Age‐Related Th17 Inflammation

doi: 10.1111/acel.70451

Figure Lengend Snippet: Succinate dehydrogenase protein expression increases with age in CD4 + T cells. SDH expression in young (Y) and older (O) adults assessed via immunoblotting (a) and confocal microscopy (b). SDHA protein expression in CD4 + T cells treated with SDH inhibitor 3‐nitropropionic acid (3NP) and cell‐permeable diethyl succinate (DES); immunoblotting (c) and microscopy (d). SDHB protein expression after the different treatments (e). N = 3–4 (a–e). N = 3–4 indicates cells were obtained from either three or four individuals for each condition. Microscopy data are represented as cells in the field of view. At least 5–7 fields per slide were imaged at 63× magnification with oil immersion, on a Zeiss LSM 800 confocal microscope. In fields where numerous cells were observed, the mean fluorescence intensity of 3–4 cell groups was plotted as a single dot. Images were processed as described in the methods, and brightness was adjusted to improve clarity. Mann–Whitney Test or Kruskal–Wallis test with Dunn's post hoc test, * p < 0.05 vs. Y. # p < 0.05 vs. O or O + 3NP.

Article Snippet: Human CD4 + isolation kit , Miltenyi , Cat no. 130‐096‐533.

Techniques: Expressing, Western Blot, Confocal Microscopy, Microscopy, Fluorescence, MANN-WHITNEY

Journal: Aging Cell

Article Title: Role of Succinate Dehydrogenase in Age‐Related Th17 Inflammation

doi: 10.1111/acel.70451

Figure Lengend Snippet: Age‐induced dysregulation of TCA cycle metabolites fuel Th17 cytokine production. Waterfall plots showing ScRNA seq analysis of TCA cycle enzymes in CD4 + T cells from young (Y) and older (O) adults (a) ScRNA seq analysis of TCA cycle enzymes in Th17 subset of T cells (b). Cellular amounts of succinate (c) fumarate:succinate ratio (d) HIF1α protein in T cells from older adults (e) and HIF1α protein in T cells from younger adults after FH inhibition (f) N = 3, (a, b) N = 3–4, (c, d) N = 5–7, (e) N = 3, (f) adults in each group. One‐way ANOVA with Bonferroni test or Wilcoxon matched‐pair signed rank test. * p < 0.05 vs. O or Y.

Article Snippet: Human CD4 + isolation kit , Miltenyi , Cat no. 130‐096‐533.

Techniques: Inhibition

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: Gut microbes

Article Title: Fusobacterium nucleatum -driven CX3CR1 + PD-L1 + phagocytes route to tumor tissues and reshape tumor microenvironment.

doi: 10.1080/19490976.2024.2442037

Figure Lengend Snippet: Figure 5: Fn induces PD-L1 expression in phagocytes, and PD-L1+ neutrophils exhibit immunosuppressive functions. (a-c) flow cytometry analysis (a, b) and western blot analysis (c) of PD-L1 expression in PMNs. (d and e) flow cytometry analysis (d) and western blot analysis (e) of PD-L1 protein expression in PMNs. (f) if staining of Fn (red) and PD-L1 (green) in Fn (MOI 10:1, 12 h)-infected PMNs. Right panel: quantification of PD-L1 expression. Scale bars: 25 μm. (g) Western blot analysis of protein expression in PMNs infected with Fn (MOI 10:1) for 15, 30, 60, and 120 min. (h and i) western blot analysis of protein expression in PMNs. Cells were pretreated with 30 nM TPCA-1 (h) or 100 μM NSC74859 (i) and infected with Fn for 1 h. (j) Schematic diagram showing that CD3+ T-cells were cocultured with human peripheral blood PMNs (1:1) or with an anti-PD-L1 antibody (20 μg/ml) for 48 h. (k and l) Representative flow cytometry and statistical analysis of T-cell- proliferation (k) and iFn-γ production (l) are shown (n = 3). (m) Schematic drawing of T-cell/ crc cell coculture system. (n-p) flow cytometry assay of apoptosis rates (n) and the statistical analysis (o) or CCK-8 assay of the cell viability rate of HCT116 and RKO cells (p). (q) Numbers of viable Fn were enumerated in PMNs by the gradient dilution coating method. PBS treatment was set as control (con). Data are presented as the mean ± SEM, p values were determined by one-way ANOVA (a, b, d, k, l, and o-q), and two-sided unpaired t-test (f). ns: no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 for groups connected by horizontal lines or versus con.

Article Snippet: Human peripheral blood neutrophils (PMNs) and human peripheral blood mononuclear cells (PBMCs) were isolated by a human peripheral blood neutrophil isolation kit (Solarbio, Beijing, China), and then monocytes (MCs) were isolated from PBMCs by discarding the supernatant after adhering to the walls for 1 h. The procedure was performed according to the protocol provided by the manufacturer.

Techniques: Expressing, Flow Cytometry, Western Blot, Staining, Infection, CCK-8 Assay, Control