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: 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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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: Briefly, human PBMCs from healthy donors were isolated using a CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130091301), followed by stimulation with varying concentrations of human recombinant IFN-α (0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL) (Abcam, Cat. No. ab48750).

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