Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Upper panel: Chemical Structure of 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled PAPTP (PAPTP-NBD). Lower panel: Synthesis of PAPTP-NBD. Reagents and Conditions (i) TsCl, DMAP, pyridine, DCM, r.t., 18 h; (ii) NaN 3 , DMF, 90 °C, 3 h; (iii) Pd/C, H2, MeOH, r.t., 16 h; (iv) NBD-Cl, DIPEA, MeOH, r.t., 16 h; (v) TsCl, DMAP, pyridine, DCM, r.t., 5 h; (vi) NaI, acetone, 40 °C, 16 h; (vii) PAPTP-OH, K 2 CO 3 , DMF, r.t., 16 h. Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (1). To a solution of triethylene glycol (PEG3-OH, 77.28 g, 514.6 mmol, 8.0 equiv) in dichloromethane (DCM, 255 mL) at 0 °C were added 4-dimethylaminopyridine (DMAP, 15.7 g, 128.6 mmol, 2.0 equiv) and pyridine (10.18 g, 128.6 mmol, 2.0 equiv). After 10 min of stirring, p-toluenesulfonyl chloride (TsCl, 12.3 g, 64.3 mmol, 1.0 equiv), previously dissolved in DCM (165 mL), was added dropwise. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched by dilution with 300 mL of 1.0 M HCl, the organic layer separated and the aqueous phase was extracted with DCM (2 × 250 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using a DCM/acetone mixture (8:2) to afford 1 as a pale yellow oil (13.2 g, 43.4 mmol, yield: 67%). 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.18–4.15 (m, 2H), 3.72–3.69 (m, 4H), 3.61 (s, 4H), 3.58–3.56 (m, 2H), 2.44 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 145.01, 133.07, 129.98, 128.11, 72.59, 70.92, 70.43, 69.29, 68.85, 61.89, 21.78. ESI-MS (ion trap): m /z 305 [M + H]+. Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (2). To a solution of compound 1 (13.2 g, 43.4 mmol, 1.0 equiv) in anhydrous N,N-dimethylformamide (DMF, 100 mL), sodium azide (NaN₃, 8.5 g, 130.2 mmol, 3.0 equiv) was added. The reaction mixture was stirred at 90 °C for 3 h until thin-layer chromatography (TLC) analysis (EtOAc/PE, 6:4) indicated complete consumption of the starting material. The reaction mixture was then diluted with ethyl acetate (EtOAc, 300 mL) and washed with brine/water 1:1 (5 × 100 mL). The organic layer was dried, and the solvent was removed under reduced pressure. The flask was left under high vacuum overnight to remove residual DMF, yielding compound 2 as a pale yellow oil (6.8 g, 38.7 mmol, yield: 89%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.71 (m, 2H), 3.68–3.64 (m, 6H), 3.61–3.59 (m, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.45 (s, 1H).13 C NMR (101 MHz, CDCl3) δ 72.59, 70.74, 70.48, 70.13, 61.84, 50.74. ESI-MS (ion trap): m / z 176 [M + H]+. Synthesis of 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol (3). Palladium on carbon (Pd/C, 10% w/w, 0.70 g) was suspended in methanol (30 mL) in a round-bottom flask under nitrogen atmosphere. Compound 2 (6.8 g, 38.7 mmol, 1.0 equiv), previously dissolved in methanol (10 mL), was added to the suspension. The reaction atmosphere was then replaced with hydrogen, and the mixture was stirred at room temperature for 16 h, until thin-layer chromatography (TLC) analysis (DCM/acetone, 8:2) confirmed complete consumption of the starting material. Hydrogen was removed by nitrogen stream and the reaction mixture was filtered through a celite pad to remove the catalyst. The solvent was evaporated under reduced pressure and the crude product was purified by flash column chromatography on silica gel using a DCM/MeOH/NH3(aq) (87:12:1) as the eluent, affording compound 3 as a colorless oil (2.6 g, 17.4 mmol, yield: 45%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.70 (m, 2H), 3.68–3.62 (m, 4H), 3.61–3.58 (m, 2H), 3.56–3.53 (m, 2H), 2.88 (t, J = 5.1 Hz, 2H), 2.55 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 72.81, 72.79, 70.47, 70.24, 61.63, 41.53. ESI-MS (ion trap): m / z 150 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethan-1-ol (4). To a solution of NBD-Cl (1.0 g, 5.0 mmol, 1.0 equiv) in methanol (25 mL) at 0 °C were added N,N-diisopropylethylamine (DIPEA, 2.6 g, 20 mmol, 4.0 equiv) and compound 3 (0.82 g, 5.5 mmol, 1.1 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was then diluted with ethyl acetate (150 mL) and washed with saturated NH4Cl solution (3 × 50 mL). The organic layers were combined, dried, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using an EtOAc/MeOH mixture (99:1) as the eluent, affording compound 4 as a brown powder (1.0 g, 3.2 mmol, yield: 64%). 1H NMR (400 MHz, MeOD) δ 8.51 (d, J = 8.9 Hz, 1H), 6.43 (d, J = 8.9 Hz, 1H), 3.84–3.80 (m, 2H), 3.76 (s, 2H, broad signal), 3.71–3.66 (m, 2H), 3.66–3.62 (m, 4H), 3.56–3.53 (m, 2H).13 C NMR (101 MHz, DMSO) δ 154.82, 153.90, 153.60, 147.35, 130.29, 109.01, 81.83, 79.36, 79.22, 77.44, 69.66, 52.88. ESI-MS (ion trap): m / z 313 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (5). To a solution of compound 4 (100 mg, 0.32 mmol, 1.0 equiv) in dichloromethane (DCM, 1.5 mL) at 0 °C were added pyridine (76 mg, 0.96 mmol, 3.0 equiv), 4-dimethylaminopyridine (DMAP, 78 mg, 0.64 mmol, 2.0 equiv), and p-toluenesulfonyl chloride (TsCl, 122 mg, 0.64 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 5 h. The mixture was then diluted with brine (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using DCM/MeOH (99:1) as the eluent, affording compound 5 as a brown solid (94 mg, 0.20 mmol, yield: 63%). 1H NMR (400 MHz, (CD3)2CO) δ 8.50 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 4.19–4.11 (m, 2H), 3.84–3.80 (m, 4H), 3.71–3.66 (m, 2H), 3.64 – 3.60 (m, 2H), 3.60–3.53 (m, 2H), 2.86 (s, 1H), 2.42 (s, 3H).13 C NMR (101 MHz, (CD3)2CO) δ 145.78, 145.45, 145.08, 137.77, 134.22, 130.77, 128.63, 123.50, 99.90, 71.24, 71.07, 70.61, 69.34, 44.68, 21.49. ESI-MS (ion trap): m/z 467 [M + H]+. Synthesis of N-(2-(2-(2-iodoethoxy)ethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (6). To a solution of compound 5 (30 mg, 0.064 mmol, 1.0 equiv) in acetone (0.8 mL) was added sodium iodide (NaI, 39 mg, 0.257 mmol, 4.0 equiv). The reaction mixture was stirred at 40 °C for 16 h in a sealed vial. After completion, the mixture was diluted with ethyl acetate (EtOAc, 40 mL) and washed with brine (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1:1) as the eluent, affording compound 6 as a brown solid (14.5 mg, 0.034 mmol, yield: 54%). 1H NMR (400 MHz, (CD3)2CO) δ 8.54 (d, J = 8.8 Hz, 1H), 8.13 (s, 1H, broad signal), 6.55 (d, J = 8.8 Hz, 1H), 3.93–3.79 (m, 4H), 3.73–3.58 (m, 6H), 3.29 (t, J = 6.5 Hz, 2H).13 C NMR (101 MHz, (CD3)2CO) δ 145.81, 145.41, 145.02, 137.67, 123.43, 99.98, 72.35, 71.08, 70.67, 69.32, 44.59, 4.26. ESI-MS (ion trap): m/z 423 [M + H]+ Synthesis of (4-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)phenyl) (3-(4-(4-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)butoxy)phenyl)propyl)diphenylphosphonium (PAPTP-NBD) A solution of compound 6 (14.5 mg, 0.034 mmol, 1.1 equiv) in DMF (0.5 mL) was cooled to 0 °C, and PAPTP-OH (25 mg, 0.031 mmol, 1.0 equiv, synthesized as previously reported, 10.3390/ph14020129) and potassium carbonate (K₂CO₃, 4 mg, 0.031 mmol, 1.0 equiv) were added. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction was diluted with EtOAc (30 mL) and washed with 0.5 M HCl (2 × 10 mL) followed by brine (1 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by preparative HPLC and lyophilized to afford PAPTP-NBD as an orange solid (9.6 mg, 0.009 mmol, yield: 29%, purity UPLC > 95%). 1H NMR (400 MHz, (CD3)2CO) δ 8.42 (s, 1H, broad signal), 8.21 (dd, J = 9.8, 0.7 Hz, 1H), 7.93–7.68 (m, 13H), 7.26 (dd, J = 2.4, 1.0 Hz, 1H), 7.22 (dd, J = 9.0, 2.6 Hz, 2H), 7.16–7.08 (m, 3H), 6.82 (d, J = 8.6 Hz, 2H), 6.55 (s, 1H, broad signal), 6.19 (d, J = 9.8 Hz, 1H), 4.67 (t, J = 5.8 Hz, 2H), 4.24–4.21 (m, 2H), 4.09 (t, J = 5.8 Hz, 2H), 3.90–3.82 (m, 6H), 3.68 (s, 4H), 3.60–3.48 (m, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.12–1.98 (m, 6H). 13 C NMR (101 MHz, (CD3)2CO) δ 165.18, 159.24, 158.66, 153.86, 150.18, 146.37, 140.10, 136.81 (d, J = 11.5 Hz), 135.87 (d, J = 3.0 Hz), 134.61 (d, J = 10.0 Hz), 133.12, 131.25 (d, J = 12.6 Hz), 130.52, 120.83, 119.97, 117.48, 117.35, 115.48, 113.33, 109.39, 108.46, 107.37, 106.47, 94.02, 73.61, 71.46, 71.36, 70.02, 69.73, 69.23, 68.23, 49.07, 36.03 (d, J = 16.9 Hz), 27.59, 26.72, 25.49 (d, J = 3.7 Hz), 22.25 (d, J = 52.6 Hz). ESI-MS (ion trap): m / z 363 [M]+. ( B ) Confocal microscopy images showing the fluorescent signal in untreated cells and in those treated with fluorescent PAPTP (PAPTP-fluor) in mitochondria of CD4 + CD25 - Tconv cells isolated from healthy mice before and after treatment with 100 nM PAPTP-Fluor for 30 min. The scale bar is 10 μm. Control experiment for Fig. . The same cells shown in this representative image are also shown in Fig. at higher magnification. ( C ) Left panels: Upper panel: Downregulation of Kv1.3 in cells treated with CRIPSR/Cas9 and Kv1.3 staining in sorted cells. Yellow: CRISPR/Cas transfected, unstained. Blue: CRISPR/Cas transfected, cells positively sorted for Kv1.3. This is the fraction, which was positively sorted and then stained with FITC-anti-Kv1.3 antibodies.For sorting, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (BioLegend, #101302; 1:50 dilution) for 15 min at 4 °C, washed, and labeled with biotin-conjugated anti-Kv1.3 antibody (Alomone Labs, #APC-101B) for 30 min at 4 °C. Following a second wash, cells were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec, #130-048-101) for 30 min at 4 °C. Kv1.3-positive and -negative populations were isolated using LS columns (Miltenyi Biotec, 130-042-401). For the flow cytometry, Fc-receptors were blocked with True stain (1:50 dilution, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (Clone S17011E, BioLegend, #156604) and then an aliquot of the samples was stained with a FITC-coupled anti-rabbit IgG (1:500, Jackson Immunoresearch 711-096-152) to detect the anti-Kv1.3, which was already bound to the cells. Red: CRISPR/Cas transfected, cells negatively sorted for Kv1.3. This is the fraction, which was negatively sorted and then stained exactly as the blue fraction. The staining was done on aliquots just before retransfection of the mito-Kv1.3 construct to confirm downregulation. Lower panel: Same as above, but with aliquots that were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again and then stained. This confirms downregulation of extra- and intracellular Kv1.3. Right upper panel: Flow cytometry of sorted Kv1.3-negative cells that were re-transfected. Blue: Control-transfected (empty vector). Aliquots were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again, Fc-receptors were blocked with True stain (1:50, Biolegend, #156604) and then stained with FITC-coupled anti-Kv1.3 (Alamone, #APC-101-F). Red: EYFP-Mito-Kv1.3 transfected cells, as above. Right lower panel: Representative dot plot showing the gating strategy for the identification of EYFP-mitoKv1.3 + Annexin + and EYFP-mitoKv1.3 - Annexin + cells after PAPTP treatment. Kv1.3 - cells were transfected with EYFP-mitoKv1.3 construct and subsequently treated with PAPTP for 48 h. Cells were then analyzed for apoptosis using flow cytometry. ( D ) Representative images of longitudinal spinal-cord sections stained with luxol fast blue from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 100 μm. Demyelinated are indicated with white arrows. Right: Average ± SEM of myelin area per field ( n = 3 for mice receiving untreated lymphocytes, n = 5 for mice receiving 1 μM PAPTP-treated lymphocytes). p -value of Student’s t test. ( E ) Representative images of longitudinal spinal-cord sections stained with Bielschowsky staining from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 200 μm. Right upper panel: enlarged image from the lower EAE AT untreated sample shown on the left. Right lower panel: enlarged image from the upper EAE AT + PAPTP sample shown on the left. Please note damaged axon fibers in the EAE. ( F ) Quantification of axon damage from longitudinal sections of Blieschowsky-stained spinal cords from EAE AT and EAE AT + PAPTP animals. Quantification was performed following the method used for Fig. (left panel) and according to (Theotokis et al, ). ( G ) Representative images of a brain section of a wild-type mouse injected with MOG 35–55 activated untreated lymphocytes, stained with the indicated antibodies. The sections are from the same experiment shown in Fig. . ( H ) Average ± SEM of the number of GFAP + cells per field in brain slices of healthy animals (Ctrl) and mice receiving untreated or 1 μM PAPTP-treated lymphocytes ( n = 3 for each group). On the right, representative immunohistochemical images of GFAP + in brain slices from mice of the indicated groups. The images were taken from the same region for each animal. The scale bar corresponds to 100 μm. p -value from one-way ANOVA. ( I ) Additional examples of Klüver-Barrera dual staining performed as in Fig. . Red arrows indicate infiltrated/demyelinated zones. Please note also vacuolation, as e.g., in (Morales et al, ) in the enlarged image.

Article Snippet: Single cells suspensions were then stained using the Mouse Naive/Effector/Memory T Cell Markers Flow Cytometry Panel (Cell Signaling, #78148), with PE-anti-CD3 (clone 17A2, Cell Signaling #28306S), Cy5-anti-CD44 (clone IM7, Cell Signaling #94170S) and FITC-anti-CD62L (clone MEL14, Cell Signaling #76378S), respectively, used at the following dilutions: 1:40, 1:160, and 1:200.

Techniques: Labeling, Purification, Column Chromatography, Thin Layer Chromatography, Solvent, Suspension, Synthesized, Confocal Microscopy, Isolation, Control, Staining, CRISPR, Transfection, Incubation, Blocking Assay, Flow Cytometry, Construct, Plasmid Preparation, Injection, Immunohistochemical staining

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Quantitative results of the MitoSox Mean Fluorescence Intensity of CD4 + CD25 + CCR7 + (Naive + T CM ) and CD4 + CD25 + CCR7 - (T EM + T EMRA ) untreated hPBMCs or treated with 1 μM PAPTP for 30 min (cultured for 7 days in both cases). Data were normalized on the mean Fluorescence Intensity of untreated CD4 + CD25 + CCR7 + (Naive + TCM) cells ( n = 6 patients). P -values obtained from two-way ANOVA test are shown. ( B ) Mean Fluorescence Intensity of ShK-F6CA (fluorescent Kv1.3 inhibitor ShK) in CD4 + CD25 - and CD4 + CD25 + T cells from proliferative hPBMCs after 7 days of autoproliferation in vitro ( n = 8 patients). For each patient, the mean Fluorescence intensity of CD4 + CD25 + T cells was normalized on the mean fluorescence intensity of CD4 + CD25 - T cells. Shown are p -values of Wilcoxon test. ( C ) Strategy for analyzing apoptosis in autoproliferative lymphocytes: hPBMCs were labeled with CFSE and incubated for 7 days at 37 °C. Following the incubation period, proliferation was assessed as mean of CFSE dilution. Subsequently, cells were subjected to treatment with PAPTP, or left untreated for different timepoints based on the experiment (18 h for Annexin, 30 min for MitoSox, and 1 min for TMRM). Apoptosis, mitochondrial ROS production and mitochondrial membrane hyperpolarization were evaluated via flow cytometry analysis. ( D ) Quantitative results of the TMRM Mean Fluorescence Intensity of CD4 + CCR7 - (T EM + T EMRA ) untreated hPBMCs or treated with 1 μM PAPTP or 100 nM ShK for 60 s (cultured for 7 days in both cases). Data were normalized on the Mean Fluorescence Intensity of untreated CD4 + CCR7 - (T EM + T EMRA ) cells ( n = 9 patients). ( E ) Quantitative results of the MitoSox Mean Fluorescence Intensity of CD4 + CD25 + CCR7 - (T EM + T EMRA ) untreated hPBMCs or treated with 1 μM PAPTP or 100 nM ShK for 30 min (cultured for 7 days in both cases). Data were normalized on the Mean Fluorescence Intensity of untreated CD4 + CCR7 - (T EM + T EMRA ) cells ( n = 8 patients). Right: Representative quantitative results of the Mean Fluorescence Intensity of MitoSox in CD4 + CD25 + CCR7 - (T EM + T EMRA ) lymphocytes either treated with 1 μM PAPTP or 100 nM ShK. ( F ) Normalized apoptotic levels of proliferative CD4 + CD25 + CCR7 - T EM + T EMRA cells at 1 μM PAPTP or 100 nM ShK. For each patient, data were normalized based on the untreated sample ( n = 10 for each group). ( A , B , D – F ) Data represent average ± SEM with superimposed individual data points for each patient. Each data point represents hPBMCs derived from a distinct patient. ( D – F ) p -values of Friedman test are shown. .

Article Snippet: Single cells suspensions were then stained using the Mouse Naive/Effector/Memory T Cell Markers Flow Cytometry Panel (Cell Signaling, #78148), with PE-anti-CD3 (clone 17A2, Cell Signaling #28306S), Cy5-anti-CD44 (clone IM7, Cell Signaling #94170S) and FITC-anti-CD62L (clone MEL14, Cell Signaling #76378S), respectively, used at the following dilutions: 1:40, 1:160, and 1:200.

Techniques: Fluorescence, Cell Culture, In Vitro, Labeling, Incubation, Membrane, Flow Cytometry, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Confocal microscopy image showing the accumulation of fluorescent PAPTP (PAPTP-fluor) in Mitotracker Red stained mitochondria of CD4 + CD25 - Tconv cells isolated from healthy mice. T conv were treated with 100 nM PAPTP-Fluor for 30 min. The scale bar is 10 μm. The same cells shown in this representative image are also shown in Fig. at lower magnification. ( B ) Scheme showing the strategy for the generation of Kv1.3 knockout cells expressing the mitochondrial Kv1.3 form. Splenocytes were isolated from MOG 35–55 immunized mice and Kv1.3 was deleted using CRISPR/Cas9 technology. Cells were then sorted for Kv1.3 expression, removing splenocytes still expressing Kv1.3, and transfected with EYFP-mitoKv1.3 construct. Finally, cells were treated with 1 μM PAPTP for 48 h. Apoptosis was detected in Mito-Kv1.3 + and MitoKv1.3 - cells by flow cytometry. ( C ) Percentage of Annexin V + Mito-Kv1.3 + and Annexin V + Mito-Kv1.3 - cells after 1 μM PAPTP treatment for 48 h. Populations were gated on total cells (Kv1.3 - cells) ( n = 6 for each group). Data represent average ± SEM with superimposed individual data points for each animal. Indicated p -values refer to Mann-Whitney test. See also Fig. . ( D ) Scheme of the EAE Adoptive Transfer Model: Mice underwent immunization via subcutaneous injection of MOG 35–55 in complete Freund’s adjuvant. Ten days post-immunization, spleens were harvested, and single-cell suspensions were prepared. These splenocytes were treated for 3 days with IL-12, IL-23, and anti-IFN-γ. Subsequently, the splenocytes were subjected to a 3-day treatment with 1 μM PAPTP, or left untreated. Antigen-specificity of the cells was confirmed by additional proliferation assays of isolated donor lymphocytes (see Methods). Lymphocytes were isolated, and the induction of apoptosis was assessed using flow cytometry. Additionally, these lymphocytes were transferred into wild-type recipient animals, and the animals’ clinical scores were monitored daily for 10 days. Following the observation period, the animals were euthanized for further analysis. ( E ) Percentage of Naive, T CM and T EM cells of all sorted CD3 + T lymphocytes ( n = 6 for each group). ( F ) Percentage of apoptotic Annexin V + cells of all sorted CD3 + T cells. The Trypan Blue staining gave the same result ( n = 6 for each group). ( G ) Percentage of cell death in CD4 + CD44 - CDL62 + Naive, CD44 + CDL62 + T CM and CD44 + CDL62 - T EM subsets ( n = 6 for each group). ( H ) Data represent average ± SEM of disease scores by daily scoring of mice receiving the indicated group of lymphocytes. Days 1–6: All mice had a score of 0 ( n = 6 for each group). p -values from two-way ANOVA test are shown. ( I ) Data represent average ± SEM of the percentage of spinal cord infiltrated Naive, T CM , and T EM lymphocytes (determined as in ( G )) of all cells ( n = 4 for healthy controls; n = 5 for mice receiving untreated and n = 5 for mice receiving 1 μM PAPTP-treated lymphocytes). p -values from two-way ANOVA test are shown. ( J ) Representative image (of 5 sections) of brains (upper part of the pons region) stained with PE-anti-CD45, from wild-type mice intraperitoneally injected with either untreated or PAPTP 1 μM treated, MOG 35–55 activated lymphocytes. ( E–G ) Data represent average ± SEM with superimposed individual data points for each animal. Indicated p-values from Unpaired Student’s T test. .

Article Snippet: Single cells suspensions were then stained using the Mouse Naive/Effector/Memory T Cell Markers Flow Cytometry Panel (Cell Signaling, #78148), with PE-anti-CD3 (clone 17A2, Cell Signaling #28306S), Cy5-anti-CD44 (clone IM7, Cell Signaling #94170S) and FITC-anti-CD62L (clone MEL14, Cell Signaling #76378S), respectively, used at the following dilutions: 1:40, 1:160, and 1:200.

Techniques: Confocal Microscopy, Staining, Isolation, Knock-Out, Expressing, CRISPR, Transfection, Construct, Flow Cytometry, MANN-WHITNEY, Adoptive Transfer Assay, Injection, Adjuvant

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Mean Fluorescence intensity of MitoSox in CD4 + CD62L + (Naive+T CM ) and CD4 + CD62L - (T EM ) lymphocytes in peripheral blood from EAE mice before (Untreated) and after 30 min treatment with 1 μM PAPTP. For each animal, data were normalized on the Mitosox Mean Fluorescence Intensity of CD4 + CD62L + untreated cells. Data represent average ± SEM ( n = 4). p -values of two-way ANOVA are indicated. ( B ) Percentages of lymphocytes, monocytes, and granulocytes in peripheral blood of mice of the indicated group at the endpoint of the experiment, evaluated using flow cytometry. Populations were gated on total single cells. Data represent average ± SEM ( n = 8 for controls; n = 4 for EAE group and n = 5 for EAE + PAPTP group). ( C ) Percentages of CD11b + CD80 + and CD11b + CD80 - monocytes in peripheral blood of mice of the indicated group at the experimental endpoint. Cells were gated on C11b + monocytes. Data represent average ± SEM ( n = 9 for controls; n = 4 for EAE group and n = 5 for EAE + PAPTP group). ( D ) Gating Strategy for identification of monocytes, lymphocytes and granulocytes (top). Representative dot plot showing CD11b + CD80 + and CD11b + CD80 - monocytes in peripheral blood of mice of the indicated group at the experimental endpoint (bottom). ( E ) Gating Strategy for identification of CD3 + and CD19 + lymphocytes in peripheral blood of mice. ( F ) Representative H&E images of livers and spleens of mice of the indicated groups. Scale bar indicated in the figure. Representative Transmission Electron Microscopy images showing neuronal ( G ) and spinal cords ( H ) myelination of mice from the indicated groups at the experimental endpoint. Scale bar indicated in the figure. ( I ) Representative immunohistochemical images of Iba-1 + cells in brain slices from mice of the indicated group, sacrificed at 30 dpi. The scale bar corresponds to 100 μm. See enlarged images on Fig. . ( J ) Average ± SEM of the number of GFAP + cells per field in brain slices of mice of the indicated groups ( n = 6 for Ctrl and EAE, n = 5 for EAE + PAPTP groups). One outlier, defined with GraphPad was removed from the EAE + PAPTP group. At least 5 sections per animal were analyzed. On the right, representative immunohistochemical images of GFAP + in brain slices from mice of the indicated groups. The images were taken from the same region for each animal. The scale bar corresponds to 100 μm. ( G–J ) p -values of one-way ANOVA test are shown.

Article Snippet: Single cells suspensions were then stained using the Mouse Naive/Effector/Memory T Cell Markers Flow Cytometry Panel (Cell Signaling, #78148), with PE-anti-CD3 (clone 17A2, Cell Signaling #28306S), Cy5-anti-CD44 (clone IM7, Cell Signaling #94170S) and FITC-anti-CD62L (clone MEL14, Cell Signaling #76378S), respectively, used at the following dilutions: 1:40, 1:160, and 1:200.

Techniques: Fluorescence, Flow Cytometry, Transmission Assay, Electron Microscopy, Immunohistochemical staining

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Comparison of Th17 cells, Treg cells, and Th17/Treg ratios between stroke patients and HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques: Comparison

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 was positively associated with a Th17/Treg imbalance. Association of serum RBP4 with Th17 cells (A) , Treg cells (B) , and the Th17/Treg ratio (C) in stroke patients. Association of serum RBP4 with Th17 cells (D) , Treg cells (E) , and the Th17/Treg ratio (F) in HCs.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were negatively associated with the MMSE score at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with MMSE score at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques:

Journal: Frontiers in Neurology

Article Title: Serum retinol-binding protein 4 in stroke patients: correlation with T helper 17/regulatory T cell imbalance and 3-year cognitive function decline

doi: 10.3389/fneur.2023.1217979

Figure Lengend Snippet: Serum RBP4 and Th17/Treg imbalances were associated with the occurrence of cognitive impairment at enrollment. Association of serum RBP4 (A) , Th17 cells (B) , Treg cells (C) , and the Th17/Treg ratio (D) with cognitive impairment at enrollment in stroke patients.

Article Snippet: Then, the proportions of Th17 and Treg cells in CD4 + T cells were determined by flow cytometry (FCM) using the FlowX Human Th17 Cell Multi-Color Flow Cytometry Kit (No. Cat. FMC007B, R&D Systems, Inc., Minneapolis, Minnesota, United States) and the Regulatory T Cell (Treg) Flow Cytometry Panel (No. Cat. FMC-P-004, R&D Systems, Inc., Minneapolis, Minnesota, USA).

Techniques: