cd4 pe cy7  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Exercise facilitates post-stroke recovery through mitigation of neuronal hyperexcitability via interleukin-10 signaling

doi: 10.1038/s41467-025-62631-y

Figure Lengend Snippet: a RAG1 −/− -mice were subjected to photothrombotic stroke, received different subsets of T cells or vehicle (veh), and were then subjected to running wheel training. b Running wheel training did not influence the asymmetry index in the adhesive tape removal test ( p = 0.46, two-sided t -test, n = 6 and 5 animals per group) in RAG1 −/− -mice without adoptive T cell-transfer (ex = exercise, no ex = no exercise). c Adoptive transfer of CD3 + T cells, in the absence of exercise, did not enhance recovery in RAG1 − / − mice ( p = 0.28, two-sided t -test, n = 8 and 8 animals per group). d Following adoptive CD3 + -T cells-transfer, exercise led to a significantly reduced asymmetry index (*** p = 0.0002, two-sided t -test, n = 13 and 12 animals per group), indicating improved functional outcome. e There was a strong trend towards better functional outcome after CD4 + - compared to CD8 + -T cell-transfer ( p = 0.07, two-sided t -test, n = 7 and 11 animals per group). f The transfer of FoxP3 + regulatory T cells (Tregs) resulted in significantly better functional outcomes than the transfer of FoxP3 - nonregulatory T cells (*** p = 0.0009, two-sided t -test, n = 6 and 10 animals per group). g Preventing leukocytes from entering the brain by inhibition of very late antigen-4 (VLA-4) impaired stroke recovery (* p = 0.02, two-sided t -test, n = 7 animals per group; iso = isotype antibody). h MRI-DTI fiber tracking demonstrated an increased number of interhemispheric connections in mice that received Tregs before running wheel training compared to mice that received only vehicle and running wheel training (** p = 0.0013, two-sided t -test, n = 9 and 10 animals per group). Exemplary scans of animals with a high (ex) and a low (no ex) number of interhemispheric connections. For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a file.

Article Snippet: We used the following fluorochrome-labeled antibodies: CD45 (30-11F) BV510 or FITC 1:100, CD45R/B220 (RA3-6B2) PerCP-Cy5.5 1:100, CD3 (17A2) PE-Cy7 1:200, F4/80 (BM8) APC 1:200, Ly-6G/Ly-6C (RB6-8C5) BV421 1:200, CD11c (N418) AF700 1:150, CD11b (M1/70) PE 1:800 all obtained from Biolegend and NK-1.1 and (PK136) APC-Vio770 1:200 obtained from Miltenyi Biotec, and CD3 (145-2C11) 1:200, CD4-Pacific Blue (RM4-4) 1:150, CD8a-FITC (53-6.7) 1:150, CD25-APC (PC61) 1:150, Ki-67 AF647 (B56) 1:200 from BD Biosciences, and FoxP3 (FJK-16s) PE 1:150 from eBioscience.

Techniques: Adhesive, Adoptive Transfer Assay, Functional Assay, Inhibition, Whisker Assay

Journal: Nature Communications

Article Title: Exercise facilitates post-stroke recovery through mitigation of neuronal hyperexcitability via interleukin-10 signaling

doi: 10.1038/s41467-025-62631-y

Figure Lengend Snippet: a Quantification of immune cells by flow cytometry 14 days after stroke revealed that exercise significantly reduced brain-infiltrating CD45 high immune cells and CD3 + T cells (* p = 0.04 and ** p = 0.002, two-sided t -test, n = 7–9 animals per group), but had no impact on the count of CD45 high immune cells and CD3 + T cells in cervical lymph nodes (cLN) and spleen. b Exercise did not influence the proportions of CD4 + - and CD8 + -T cells in brain parenchyma, cervical lymph nodes and spleen 14 days after photothrombotic stroke (two-sided t -test, n = 9–12 animals per group). c Exercise increased the proportion of CD25 + CD4 + -and FoxP3 + CD4 + -regulatory T cells in brain parenchyma (* p = 0.04 and p = 0.1, two-sided t -test, n = 9–12 animals per group) and cervical lymph nodes (* p = 0.01, Mann-Whitney-test and ** p = 0.01, two-sided t -test, n = 9–12 animals per group). d The expression of integrin CD49d, which facilitates leukocyte-trafficking into the brain, was upregulated in brain CD4 + -T cells after exercise (* p = 0.03, Mann-Whitney-test, n = 9–12 animals per group), but not in CD4 + -T cells isolated from cervical lymph nodes or spleen. The expression of the activation markers CD69 and CD11a on CD4 + -T cells did not differ in any of the examined compartments. e Exercise led to a significant increase in the proliferation of FoxP3 + CD4+ regulatory T cells (Tregs) in the brain (* p = 0.04, two-sided t -test, n = 4–5 animals per group). Conversely, the proliferation of FoxP3-CD4 + and CD8 + effector T cells exhibited impairment both in the brain (* p = 0.03, two-sided t -test, n = 4–5 animals per group) and cervical lymph nodes (cLN) (* p = 0.03; ** p = 0.006, two-sided t -test, n = 5–6 animals per group) as determined by Ki67 expression. For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: * p < 0.05, ** p < 0.01. Source data are provided as a file.

Article Snippet: We used the following fluorochrome-labeled antibodies: CD45 (30-11F) BV510 or FITC 1:100, CD45R/B220 (RA3-6B2) PerCP-Cy5.5 1:100, CD3 (17A2) PE-Cy7 1:200, F4/80 (BM8) APC 1:200, Ly-6G/Ly-6C (RB6-8C5) BV421 1:200, CD11c (N418) AF700 1:150, CD11b (M1/70) PE 1:800 all obtained from Biolegend and NK-1.1 and (PK136) APC-Vio770 1:200 obtained from Miltenyi Biotec, and CD3 (145-2C11) 1:200, CD4-Pacific Blue (RM4-4) 1:150, CD8a-FITC (53-6.7) 1:150, CD25-APC (PC61) 1:150, Ki-67 AF647 (B56) 1:200 from BD Biosciences, and FoxP3 (FJK-16s) PE 1:150 from eBioscience.

Techniques: Flow Cytometry, MANN-WHITNEY, Expressing, Isolation, Activation Assay, Whisker Assay

Journal: Nature Communications

Article Title: Exercise facilitates post-stroke recovery through mitigation of neuronal hyperexcitability via interleukin-10 signaling

doi: 10.1038/s41467-025-62631-y

Figure Lengend Snippet: a Heatmaps based on immunohistochemical CD3-staining did not reveal apparent differences in the spatial distribution of immigrating CD3 + -T cells between trained and untrained animals. Quantification of CD3 + -T cells in different areas of interest in lesioned cortex, Thalamus and internal capsule 14 or 49 days after ischemia did not show differences in the cell count (two-sided t -test, n = 4–8 animals per group). b – d Representative images of immunohistochemical stainings from the infarct area and the internal capsule; similar results were observed in all animals analyzed ( n = 28 across all groups). e To visualize immigrating FoxP3 + -regulatory T cells in the brain parenchyma, we employed genetically modified FoxP3 + -RFP-mice, in which FoxP3 + -cells are labeled with the red fluorescent protein and can thus be detected by fluorescence microscopy. Heatmaps based on fluorescence microscopy did not reveal apparent exercise effects on the spatial distribution of immigrating FoxP3 + -regulatory T cells in the brain parenchyma ( n = 4 and 5 animals per group). f Volcano plot analysis of RT² profiler displays differential gene expression in mice with or without exercise 14 days after experimental stroke. Each data point represents a transcript, with the abscissa displaying log2 fold change and the ordinate statistical significance (negative log10 p value). Data are generated from 8 wildtype mice (no exercise n = 4; exercise n = 4, two-sided t -test). For box and whisker plots, the box extends from the 25th to the 75th percentile, the center is the median and whiskers extend from the minimum or maximum. Each dot represents an individual biological replicate. Asterisks indicate levels of statistical significance: * p < 0.05. Source data are provided as a file.

Article Snippet: We used the following fluorochrome-labeled antibodies: CD45 (30-11F) BV510 or FITC 1:100, CD45R/B220 (RA3-6B2) PerCP-Cy5.5 1:100, CD3 (17A2) PE-Cy7 1:200, F4/80 (BM8) APC 1:200, Ly-6G/Ly-6C (RB6-8C5) BV421 1:200, CD11c (N418) AF700 1:150, CD11b (M1/70) PE 1:800 all obtained from Biolegend and NK-1.1 and (PK136) APC-Vio770 1:200 obtained from Miltenyi Biotec, and CD3 (145-2C11) 1:200, CD4-Pacific Blue (RM4-4) 1:150, CD8a-FITC (53-6.7) 1:150, CD25-APC (PC61) 1:150, Ki-67 AF647 (B56) 1:200 from BD Biosciences, and FoxP3 (FJK-16s) PE 1:150 from eBioscience.

Techniques: Immunohistochemical staining, Staining, Cell Counting, Genetically Modified, Labeling, Fluorescence, Microscopy, Gene Expression, Generated, Whisker Assay

Journal: Clinical Cancer Research

Article Title: Cemiplimab in Locally Advanced or Metastatic Secondary Angiosarcomas (CEMangio): A Phase II Clinical Trial and Biomarker Analyses

doi: 10.1158/1078-0432.CCR-25-0311

Figure Lengend Snippet: Genomic and immunologic landscape. Cell densities for CD3 + T cells, CD8 + T cells, FoxP3 + T cells, CD4 + T cells, CD20 + B cells, and CD56 + NK cells are reflected based on the total number of intratumoral cells/mm 2 . (Likely) pathogenic mutations and gene amplifications are reflected in this figure.

Article Snippet: After washing with autoMACS staining buffer (130-091-221, Miltenyi Biotec), cells were stained with the following antibodies: CD8 FITC (555366, BD Biosciences, RRID:AB_395769), TIM-3 PE (119704, BioLegend, RRID:AB_345377), CD20 PERCP (130-113-376, Miltenyi Biotec, RRID:AB_2726144), CD3 PE-Cy7 (25-0038-42, Invitrogen, RRID:AB_1582253), CD56 APC (130-113-305, Miltenyi Biotec, RRID:AB_2726084), CD4 APC-R700 (564975, BD Biosciences, RRID:AB_2744418), PD-1 BV421 (329920, BioLegend, RRID:AB_10960742), LAG-3 Super Bright 600 (63-2239-42, Invitrogen, RRID:AB_2688188), and PD-L1 BV711 (329722, BioLegend, RRID:AB_2565764).

Techniques:

Journal: Clinical Cancer Research

Article Title: Cemiplimab in Locally Advanced or Metastatic Secondary Angiosarcomas (CEMangio): A Phase II Clinical Trial and Biomarker Analyses

doi: 10.1158/1078-0432.CCR-25-0311

Figure Lengend Snippet: Multiplex imaging and data processing of AS samples. An example of one individual patient is shown in this figure, with a before sample (pretreatment) and an after sample (on treatment). A, mIHC-stained slides were multispectral imaged. Red-highlighted regions are displayed in B . B, Images were unmixed using inForm to visualize the markers, and lymphocytes were subsequently detected based on immune marker expression. Colors show inferred intensity of surface marker expression for lymphocytes detected by the algorithm. C, A tissue segmentation algorithm was trained to recognize tumor (black) versus stroma areas (gray). A distinct decrease in tumor was reported in this individual tumor sample. D, Example of gating strategy: The gating strategy first separated T cells (CD3 + CD20 − ) from B cells (CD3 − CD20 + ). Next, T cells were further separated into cytotoxic T cells (CD3 + CD8 + FOXP3 − ), regulatory T cells (CD3 + CD8 − FOXP3 + ), and Th cells (CD3 + CD8 − FOXP3 − ). NK cells (CD56 + ) were gated from cells negative for CD3 and CD20. This gating strategy was applied to lymphocytes detected in the tumor and stroma regions separately.

Article Snippet: After washing with autoMACS staining buffer (130-091-221, Miltenyi Biotec), cells were stained with the following antibodies: CD8 FITC (555366, BD Biosciences, RRID:AB_395769), TIM-3 PE (119704, BioLegend, RRID:AB_345377), CD20 PERCP (130-113-376, Miltenyi Biotec, RRID:AB_2726144), CD3 PE-Cy7 (25-0038-42, Invitrogen, RRID:AB_1582253), CD56 APC (130-113-305, Miltenyi Biotec, RRID:AB_2726084), CD4 APC-R700 (564975, BD Biosciences, RRID:AB_2744418), PD-1 BV421 (329920, BioLegend, RRID:AB_10960742), LAG-3 Super Bright 600 (63-2239-42, Invitrogen, RRID:AB_2688188), and PD-L1 BV711 (329722, BioLegend, RRID:AB_2565764).

Techniques: Multiplex Assay, Imaging, Staining, Marker, Expressing