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cd3 17a2 pe cy7  (Miltenyi Biotec)


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    Miltenyi Biotec cd3 17a2 pe cy7
    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 <t>CD3</t> + 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.
    Cd3 17a2 Pe Cy7, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Exercise facilitates post-stroke recovery through mitigation of neuronal hyperexcitability via interleukin-10 signaling"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-025-62631-y

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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



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    Thermo Fisher pe-cy7-conjugated anti-cd3 (17a2)
    Fas-CD40 activates NF-κB and induces strong proliferation upon FasL binding (A) Left: upon binding FasL, Fas trimerization recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) overnight and NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) Human T cells (5 × 10 4 ) expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) for 5 days, at which point cell counts were analyzed by flow cytometry. Due to the large list of Fas-TNFR chimeras, they were tested over two separate experiments (screens 1 and 2) with the data being compiled onto one graph. The conditions were identical between screens having the same 19-ζ and FasΔDD controls. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilized recombinant FasL (20 μg/mL) for 3 days, at which point RNA was extracted and analyzed using the nCounter NanoString platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were normalized to 19-ζ alone, and the number of significantly (p < 0.05) upregulated differentially expressed genes (DEGs) were categorized by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from the experiment described in (F). (H) Volcano plot from the experiment described in (F) of Fas-CD40-19-ζ cells compared with 19-ζ alone after incubation with immobilized FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from the experiment described in (F). (J and K) 19-ζ cells were cultured in the presence or absence of immobilized FasL (20 μg/mL) for 5 days and then stained for CCR8, ICOSL, and ICOS expression by flow cytometry (I), or the cell culture supernatant analyzed for CCL1, CXCL10, and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA. (L) Top: TCF7 expression from the experiment described in (F). Bottom left: 19-ζ cells were stained for TCF-1, representative flow cytometry plots from one donor. Bottom right: TCF-1 expression from three independent donors, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA.
    Pe Cy7 Conjugated Anti Cd3 (17a2), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd3 (17a2; alexafluor488, dil. 1:400; pe-cy7, dil. 1:400  (Thermo Fisher)
    90
    Thermo Fisher cd3 (17a2; alexafluor488, dil. 1:400; pe-cy7, dil. 1:400
    Hh affects colonic T cell-fate decisions. Different cell subsets in the colonic LP were analyzed in 14-week-old Hh -free Ctr and DC-LMP1/CD40 animals. Shown are representative FACS-plots as well as pooled statistics from two experiments (mean ± SEM, n = 7), illustrating frequencies of the indicated cell subsets. (a) DCs were gated on single, live, CD45 + , MHCII + CD11c + CD64 − cells. (b) Tregs were gated on single, live, CD45 + , <t>CD3</t> + CD4 + FoxP3 + CD25 + , Helios − (iTregs) or Helios + (nTregs). Single-cell suspensions of colonic LP from 14-and 25-week-old Hh -free or Hh -colonized Ctr or DC-LMP1/CD40 (14-weeks-old, 40 days post Hh -colonization) mice were stimulated with PMA/Ionomycin and subsequently stained intracellularly for IL-17 and IFN-γ production at the indicated time points. Bar graphs represent pooled statistics from two experiments (mean ± SEM, n = 7–9) animals. (c) T cells were pre-gated on single, live, CD45 + , CD3 + CD4 + cells. Shown are representative FACS-plots as well as bar graphs, illustrating the frequencies of the indicated cell subsets within the CD4 T cell population. (d) Shown are representative histograms as well as bar graphs, illustrating the MFI of IFN-γ expression within IFN-γ + CD4 + T cells from (c) as median ± SEM. p.i.: post-inoculation
    Cd3 (17a2; Alexafluor488, Dil. 1:400; Pe Cy7, Dil. 1:400, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 1 article reviews
    cd3 (17a2; alexafluor488, dil. 1:400; pe-cy7, dil. 1:400 - by Bioz Stars, 2026-05
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    pe-cy7-conjugated anti-cd3 (17a2  (Thermo Fisher)
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    Thermo Fisher pe-cy7-conjugated anti-cd3 (17a2
    Hh affects colonic T cell-fate decisions. Different cell subsets in the colonic LP were analyzed in 14-week-old Hh -free Ctr and DC-LMP1/CD40 animals. Shown are representative FACS-plots as well as pooled statistics from two experiments (mean ± SEM, n = 7), illustrating frequencies of the indicated cell subsets. (a) DCs were gated on single, live, CD45 + , MHCII + CD11c + CD64 − cells. (b) Tregs were gated on single, live, CD45 + , <t>CD3</t> + CD4 + FoxP3 + CD25 + , Helios − (iTregs) or Helios + (nTregs). Single-cell suspensions of colonic LP from 14-and 25-week-old Hh -free or Hh -colonized Ctr or DC-LMP1/CD40 (14-weeks-old, 40 days post Hh -colonization) mice were stimulated with PMA/Ionomycin and subsequently stained intracellularly for IL-17 and IFN-γ production at the indicated time points. Bar graphs represent pooled statistics from two experiments (mean ± SEM, n = 7–9) animals. (c) T cells were pre-gated on single, live, CD45 + , CD3 + CD4 + cells. Shown are representative FACS-plots as well as bar graphs, illustrating the frequencies of the indicated cell subsets within the CD4 T cell population. (d) Shown are representative histograms as well as bar graphs, illustrating the MFI of IFN-γ expression within IFN-γ + CD4 + T cells from (c) as median ± SEM. p.i.: post-inoculation
    Pe Cy7 Conjugated Anti Cd3 (17a2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe-cy7-conjugated anti-cd3 (17a2/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    pe-cy7-conjugated anti-cd3 (17a2 - by Bioz Stars, 2026-05
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    Image Search Results


    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.

    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

    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.

    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

    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.

    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

    c-Myc reprograms fibroblasts into macrophages. A . Schematic diagram of the protocol of c-Myc reprogramming fibroblasts into macrophages. Representative bright-field images of murine MEF/c-Myc, P0 MCC intermediates, P1 MCC intermediates, and iMac. Scale bars, 100 μm. B . Flow cytometry of the percentage of CD45 + cells in MEF and P0 MCC intermediates. C . Effects of different iPSC factors on the yield of CD45 + cells derived from MEF. MEFs with overexpression of c-Myc, Klf4, Sox2, or Oct4 were cultured in an iPSC medium. D . Effects of different culture mediums on the yield of CD45 + cells derived from MEF/c-Myc. MEF-M: MEF medium. EC-M: Erythroid culture medium. GC-M: granulocyte culture medium. iPSC-M: iPS cell medium. E . Flow cytometry analysis of CD11b and F4/80 expression in MCC intermediates and iMac cells. F . Quantification of CD11b + F4/80 + macrophages in MCC intermediates and iMac cells. G . Flow cytometry of the percentage of LSK (Lin - Sca-1 + c-Kit + ) and MP (Lin - Sca-1 - c-Kit + ) cells in Lin - cells of MCC intermediates. H . The percentage of LSK, CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in MCC intermediates. I . Representative images of Wright-Giemsa staining of B220 + , CD3 + , and CD11b + cells in MCC intermediates. Scale bar, 20 μm. J . Comparison of hematopoietic colony formation between MCC intermediates and murine bone marrow cells. Hematopoietic colony formation in methylcellulose was performed using MCC intermediates and murine bone marrow-derived progenitors. CFU, colony-forming units. CFU-G, granulocyte. CFU-GM, granulocyte-macrophage. CFU-M, macrophage. CFU-GEMM, granulocyte, erythroid, macrophage, megakaryocyte. CFU-E, erythroid. K . Representative images of hematopoietic colonies ex-vivo. Scale bar, 100 μm. L . Schematic diagram for the evaluation of the engraftment ability of MCC intermediates. C57BL/6J mice were pre-treated with 8 Gy irradiation and then GFP-tagged MCC intermediates were transplanted via tail veins. Three months later, a second transplantation was conducted to further investigate the potential and safety of MCC intermediates. n = 5. M . Quantification of PB reconstitution in recipient animals at different time points after the first transplantation of GFP-tagged MCC intermediates. N . Quantification of reconstitution in different tissues of recipient mice at 3 months after the first transplantation. O . PB reconstitution of fetal liver and MCC first and second transplants at 1 month after transplantation. P . Comparison of the lineage reconstitution of MCC (second transplantation) and fetal liver transplants in the recipient mice BM. Data are presented as mean ± SEM. * P < 0.05, *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Journal: Journal of Hematology & Oncology

    Article Title: c-Myc alone is enough to reprogram fibroblasts into functional macrophages

    doi: 10.1186/s13045-024-01605-x

    Figure Lengend Snippet: c-Myc reprograms fibroblasts into macrophages. A . Schematic diagram of the protocol of c-Myc reprogramming fibroblasts into macrophages. Representative bright-field images of murine MEF/c-Myc, P0 MCC intermediates, P1 MCC intermediates, and iMac. Scale bars, 100 μm. B . Flow cytometry of the percentage of CD45 + cells in MEF and P0 MCC intermediates. C . Effects of different iPSC factors on the yield of CD45 + cells derived from MEF. MEFs with overexpression of c-Myc, Klf4, Sox2, or Oct4 were cultured in an iPSC medium. D . Effects of different culture mediums on the yield of CD45 + cells derived from MEF/c-Myc. MEF-M: MEF medium. EC-M: Erythroid culture medium. GC-M: granulocyte culture medium. iPSC-M: iPS cell medium. E . Flow cytometry analysis of CD11b and F4/80 expression in MCC intermediates and iMac cells. F . Quantification of CD11b + F4/80 + macrophages in MCC intermediates and iMac cells. G . Flow cytometry of the percentage of LSK (Lin - Sca-1 + c-Kit + ) and MP (Lin - Sca-1 - c-Kit + ) cells in Lin - cells of MCC intermediates. H . The percentage of LSK, CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in MCC intermediates. I . Representative images of Wright-Giemsa staining of B220 + , CD3 + , and CD11b + cells in MCC intermediates. Scale bar, 20 μm. J . Comparison of hematopoietic colony formation between MCC intermediates and murine bone marrow cells. Hematopoietic colony formation in methylcellulose was performed using MCC intermediates and murine bone marrow-derived progenitors. CFU, colony-forming units. CFU-G, granulocyte. CFU-GM, granulocyte-macrophage. CFU-M, macrophage. CFU-GEMM, granulocyte, erythroid, macrophage, megakaryocyte. CFU-E, erythroid. K . Representative images of hematopoietic colonies ex-vivo. Scale bar, 100 μm. L . Schematic diagram for the evaluation of the engraftment ability of MCC intermediates. C57BL/6J mice were pre-treated with 8 Gy irradiation and then GFP-tagged MCC intermediates were transplanted via tail veins. Three months later, a second transplantation was conducted to further investigate the potential and safety of MCC intermediates. n = 5. M . Quantification of PB reconstitution in recipient animals at different time points after the first transplantation of GFP-tagged MCC intermediates. N . Quantification of reconstitution in different tissues of recipient mice at 3 months after the first transplantation. O . PB reconstitution of fetal liver and MCC first and second transplants at 1 month after transplantation. P . Comparison of the lineage reconstitution of MCC (second transplantation) and fetal liver transplants in the recipient mice BM. Data are presented as mean ± SEM. * P < 0.05, *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Article Snippet: The following antibodies were used: anti-mouse CD16/32 (clone S17011E, Biolegend), APC-Cy7 anti-mouse CD45 (clone 30-F11, Biolegend), PE anti-mouse Sca-1 (clone E13-161.7, Biolegend), APC-eFluorTM 780 anti-mouse c-Kit (clone 2B8, eBioscience), PE-Cy7 anti-mouse/human B220 (clone RA3-6B2, Biolegend), PE anti-mouse Lineage Cocktail (Biolegend), PE-Cy7 anti-mouse CD3 (clone 17A2, eBioscience), APC anti-mouse CD11b (clone M1/70, eBioscience), Super BrightTM 436 anti-mouse F4/80 (clone BM8, Invitrogen), FITC anti-mouse CD11c (clone N418, eBioscience), Pacific Blue anti-mouse CD69 (clone H1.2F3, eBioscience), PE anti-mouse CD49b (clone DX5, eBioscience), PE anti-mouse CD206 (clone MR6F3, Invitrogen), Alexa FluorTM 488 anti-mouse iNOS (clone CXNFT, eBioscience), PE-Cy7 anti-human/mouse Arginase1 (clone A1exF5, eBioscience), FITC anti-mouse CD107a (clone 1D4B, Biolegend), PE anti-human CD7 (clone CD7-6B7, Biolegend), APC-Cy7 anti-human CD45 (clone 2D1, Biolegend).

    Techniques: Flow Cytometry, Derivative Assay, Over Expression, Cell Culture, Expressing, Staining, Comparison, Ex Vivo, Irradiation, Transplantation Assay, Two Tailed Test

    c-Myc upregulates MafB and promotes fibroblasts conversion to macrophages. A . A t-SNE plot of total cells from MCC intermediates, with each cell color-coded for cell cluster and cell type. B . Heatmap shows expression of marker genes in each subcluster. C . The cell proportion and inflammatory cytokines pathway activation rate of the six clusters in A. D . Pseudotime analysis of six MCC intermediates clusters indicates two developmental directions. Arrow, the process of fibroblast reprogramming. E . TF regulatory networks (regulon, x-axis) derived by SCENIC, plotted against their normalized specificity score (y-axis). F . The t-SNE plots of expression levels of MafB and c-Maf . G . ChIP-seq peaks of c-Myc on MafB gene in MEFs (GSE90893) and macrophages (GSE84520) using IGV (Integrative Genomics Viewer). H . ChIP-qPCR of c-Myc binding to MafB promoter in MEFs and MCC intermediates. I . c-Myc overexpression activated the expression of MafB. HEK293T was transfected with c-Myc plus MafB promoter-reporter wild type (WT), Site 1 mutation (Mut1), Site 2 mutation (Mut2), or an empty vector (EV). J . A schematic diagram of WT and two mutation motifs of the c-Myc binding site in MafB promoter. K . Western blotting of MafB expression during MEF/c-Myc conversion to MCC intermediates. MEF/c-Myc/tetO-shMafB was treated with or without Dox. L . Flow cytometry of CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in MCC intermediates treated with or without Dox at day 12. M . Quantification of CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in L. Data are presented as mean ± SEM. *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Journal: Journal of Hematology & Oncology

    Article Title: c-Myc alone is enough to reprogram fibroblasts into functional macrophages

    doi: 10.1186/s13045-024-01605-x

    Figure Lengend Snippet: c-Myc upregulates MafB and promotes fibroblasts conversion to macrophages. A . A t-SNE plot of total cells from MCC intermediates, with each cell color-coded for cell cluster and cell type. B . Heatmap shows expression of marker genes in each subcluster. C . The cell proportion and inflammatory cytokines pathway activation rate of the six clusters in A. D . Pseudotime analysis of six MCC intermediates clusters indicates two developmental directions. Arrow, the process of fibroblast reprogramming. E . TF regulatory networks (regulon, x-axis) derived by SCENIC, plotted against their normalized specificity score (y-axis). F . The t-SNE plots of expression levels of MafB and c-Maf . G . ChIP-seq peaks of c-Myc on MafB gene in MEFs (GSE90893) and macrophages (GSE84520) using IGV (Integrative Genomics Viewer). H . ChIP-qPCR of c-Myc binding to MafB promoter in MEFs and MCC intermediates. I . c-Myc overexpression activated the expression of MafB. HEK293T was transfected with c-Myc plus MafB promoter-reporter wild type (WT), Site 1 mutation (Mut1), Site 2 mutation (Mut2), or an empty vector (EV). J . A schematic diagram of WT and two mutation motifs of the c-Myc binding site in MafB promoter. K . Western blotting of MafB expression during MEF/c-Myc conversion to MCC intermediates. MEF/c-Myc/tetO-shMafB was treated with or without Dox. L . Flow cytometry of CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in MCC intermediates treated with or without Dox at day 12. M . Quantification of CD45 + B220 + , CD45 + CD3 + , and CD45 + CD11b + cells in L. Data are presented as mean ± SEM. *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Article Snippet: The following antibodies were used: anti-mouse CD16/32 (clone S17011E, Biolegend), APC-Cy7 anti-mouse CD45 (clone 30-F11, Biolegend), PE anti-mouse Sca-1 (clone E13-161.7, Biolegend), APC-eFluorTM 780 anti-mouse c-Kit (clone 2B8, eBioscience), PE-Cy7 anti-mouse/human B220 (clone RA3-6B2, Biolegend), PE anti-mouse Lineage Cocktail (Biolegend), PE-Cy7 anti-mouse CD3 (clone 17A2, eBioscience), APC anti-mouse CD11b (clone M1/70, eBioscience), Super BrightTM 436 anti-mouse F4/80 (clone BM8, Invitrogen), FITC anti-mouse CD11c (clone N418, eBioscience), Pacific Blue anti-mouse CD69 (clone H1.2F3, eBioscience), PE anti-mouse CD49b (clone DX5, eBioscience), PE anti-mouse CD206 (clone MR6F3, Invitrogen), Alexa FluorTM 488 anti-mouse iNOS (clone CXNFT, eBioscience), PE-Cy7 anti-human/mouse Arginase1 (clone A1exF5, eBioscience), FITC anti-mouse CD107a (clone 1D4B, Biolegend), PE anti-human CD7 (clone CD7-6B7, Biolegend), APC-Cy7 anti-human CD45 (clone 2D1, Biolegend).

    Techniques: Expressing, Marker, Activation Assay, Derivative Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay, Over Expression, Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Flow Cytometry, Two Tailed Test

    c-Myc-induced macrophages reduce leukemia and breast cancer progression. A . Treatment scheme for the evaluation of in vivo efficacy of iMac in Reh leukemia xenografts. SCID mice were pre-treated with 2 Gy irradiation and then injected with 2 × 10 5 Reh-GFP cells with vehicle (PBS) or indicated iMac by tail vein. B and C . The percentage of leukemia cells in bone marrow (B) and spleen (C) of mice at day 28 after iMac treatment. n = 5. D . Treatment scheme for the evaluation of in vivo efficacy of iMac in ALL PDX model. NCG mice were injected with 1 × 10 6 PDX cells on day 1 by tail vein. Vehicle (PBS) or iMac (2 × 10 6 ) was injected on day 14 and 21, respectively. E and F . The percentage of PDX cells in bone marrow (E) and spleen (F) of mice on day 35. n = 5. G . Treatment scheme for the evaluation of in vivo efficacy of iMac in 4T1 breast cancer xenografts. BALB/c mice were injected with 2 × 10 5 luciferase-labeled 4T1 cells by tail vein on day 1 and treated with 1 × 10 6 iMac or vehicle (PBS) on day 5. n = 5. H . Representative images of BLI at day 16. I . Quantification of the BLI activity in H. J . Kaplan-Meier survival curves of animals bearing 4T1 tumors. Data were analyzed using the log-rank Mantel-Cox test. Median survival: Vehicle (17.0 days) and iMac (20.0 days). K . Representative images of bright field (upper panel) and H&E-stained sections (lower panel) of the lung tissue with or without iMac treatment. Scale bar, 100 μm. L-M . Quantification of the number of lung metastases (L) and the weight of lungs (M) in K. N . Representative IHC staining of Ki-67 and CD68 in lung sections. Scale bar, 50 μm. O and P . Quantification of Ki-67 (O) and CD68 (P) positive cells in N. Q-U . Quantification of CD11c + CD206 − M1-like macrophages (% of CD11c + CD206 − in CD11b + F4/80 + cells) (Q), CD11c − CD206 + M2-like macrophages (% of CD11c − CD206 + in CD11b + F4/80 + cells) (R), CD69 + T cells (% of CD69 + in CD3 + cells) (S), CD107a + NK cells (% of CD49b + CD107a + in CD3 − cells) (T), and DC (% of CD11c + in CD11b + F4/80 − cells) (U) in lungs by flow cytometry. V . A model of c-Myc reprogramming of fibroblasts into macrophages. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Journal: Journal of Hematology & Oncology

    Article Title: c-Myc alone is enough to reprogram fibroblasts into functional macrophages

    doi: 10.1186/s13045-024-01605-x

    Figure Lengend Snippet: c-Myc-induced macrophages reduce leukemia and breast cancer progression. A . Treatment scheme for the evaluation of in vivo efficacy of iMac in Reh leukemia xenografts. SCID mice were pre-treated with 2 Gy irradiation and then injected with 2 × 10 5 Reh-GFP cells with vehicle (PBS) or indicated iMac by tail vein. B and C . The percentage of leukemia cells in bone marrow (B) and spleen (C) of mice at day 28 after iMac treatment. n = 5. D . Treatment scheme for the evaluation of in vivo efficacy of iMac in ALL PDX model. NCG mice were injected with 1 × 10 6 PDX cells on day 1 by tail vein. Vehicle (PBS) or iMac (2 × 10 6 ) was injected on day 14 and 21, respectively. E and F . The percentage of PDX cells in bone marrow (E) and spleen (F) of mice on day 35. n = 5. G . Treatment scheme for the evaluation of in vivo efficacy of iMac in 4T1 breast cancer xenografts. BALB/c mice were injected with 2 × 10 5 luciferase-labeled 4T1 cells by tail vein on day 1 and treated with 1 × 10 6 iMac or vehicle (PBS) on day 5. n = 5. H . Representative images of BLI at day 16. I . Quantification of the BLI activity in H. J . Kaplan-Meier survival curves of animals bearing 4T1 tumors. Data were analyzed using the log-rank Mantel-Cox test. Median survival: Vehicle (17.0 days) and iMac (20.0 days). K . Representative images of bright field (upper panel) and H&E-stained sections (lower panel) of the lung tissue with or without iMac treatment. Scale bar, 100 μm. L-M . Quantification of the number of lung metastases (L) and the weight of lungs (M) in K. N . Representative IHC staining of Ki-67 and CD68 in lung sections. Scale bar, 50 μm. O and P . Quantification of Ki-67 (O) and CD68 (P) positive cells in N. Q-U . Quantification of CD11c + CD206 − M1-like macrophages (% of CD11c + CD206 − in CD11b + F4/80 + cells) (Q), CD11c − CD206 + M2-like macrophages (% of CD11c − CD206 + in CD11b + F4/80 + cells) (R), CD69 + T cells (% of CD69 + in CD3 + cells) (S), CD107a + NK cells (% of CD49b + CD107a + in CD3 − cells) (T), and DC (% of CD11c + in CD11b + F4/80 − cells) (U) in lungs by flow cytometry. V . A model of c-Myc reprogramming of fibroblasts into macrophages. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, by two-tailed t -test or one-way ANOVA.

    Article Snippet: The following antibodies were used: anti-mouse CD16/32 (clone S17011E, Biolegend), APC-Cy7 anti-mouse CD45 (clone 30-F11, Biolegend), PE anti-mouse Sca-1 (clone E13-161.7, Biolegend), APC-eFluorTM 780 anti-mouse c-Kit (clone 2B8, eBioscience), PE-Cy7 anti-mouse/human B220 (clone RA3-6B2, Biolegend), PE anti-mouse Lineage Cocktail (Biolegend), PE-Cy7 anti-mouse CD3 (clone 17A2, eBioscience), APC anti-mouse CD11b (clone M1/70, eBioscience), Super BrightTM 436 anti-mouse F4/80 (clone BM8, Invitrogen), FITC anti-mouse CD11c (clone N418, eBioscience), Pacific Blue anti-mouse CD69 (clone H1.2F3, eBioscience), PE anti-mouse CD49b (clone DX5, eBioscience), PE anti-mouse CD206 (clone MR6F3, Invitrogen), Alexa FluorTM 488 anti-mouse iNOS (clone CXNFT, eBioscience), PE-Cy7 anti-human/mouse Arginase1 (clone A1exF5, eBioscience), FITC anti-mouse CD107a (clone 1D4B, Biolegend), PE anti-human CD7 (clone CD7-6B7, Biolegend), APC-Cy7 anti-human CD45 (clone 2D1, Biolegend).

    Techniques: In Vivo, Irradiation, Injection, Luciferase, Labeling, Activity Assay, Staining, Immunohistochemistry, Flow Cytometry, Two Tailed Test

    Fas-CD40 activates NF-κB and induces strong proliferation upon FasL binding (A) Left: upon binding FasL, Fas trimerization recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) overnight and NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) Human T cells (5 × 10 4 ) expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) for 5 days, at which point cell counts were analyzed by flow cytometry. Due to the large list of Fas-TNFR chimeras, they were tested over two separate experiments (screens 1 and 2) with the data being compiled onto one graph. The conditions were identical between screens having the same 19-ζ and FasΔDD controls. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilized recombinant FasL (20 μg/mL) for 3 days, at which point RNA was extracted and analyzed using the nCounter NanoString platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were normalized to 19-ζ alone, and the number of significantly (p < 0.05) upregulated differentially expressed genes (DEGs) were categorized by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from the experiment described in (F). (H) Volcano plot from the experiment described in (F) of Fas-CD40-19-ζ cells compared with 19-ζ alone after incubation with immobilized FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from the experiment described in (F). (J and K) 19-ζ cells were cultured in the presence or absence of immobilized FasL (20 μg/mL) for 5 days and then stained for CCR8, ICOSL, and ICOS expression by flow cytometry (I), or the cell culture supernatant analyzed for CCL1, CXCL10, and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA. (L) Top: TCF7 expression from the experiment described in (F). Bottom left: 19-ζ cells were stained for TCF-1, representative flow cytometry plots from one donor. Bottom right: TCF-1 expression from three independent donors, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

    doi: 10.1016/j.omtn.2023.04.017

    Figure Lengend Snippet: Fas-CD40 activates NF-κB and induces strong proliferation upon FasL binding (A) Left: upon binding FasL, Fas trimerization recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) overnight and NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) Human T cells (5 × 10 4 ) expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilized recombinant FasL (20 μg/mL) for 5 days, at which point cell counts were analyzed by flow cytometry. Due to the large list of Fas-TNFR chimeras, they were tested over two separate experiments (screens 1 and 2) with the data being compiled onto one graph. The conditions were identical between screens having the same 19-ζ and FasΔDD controls. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilized recombinant FasL (20 μg/mL) for 3 days, at which point RNA was extracted and analyzed using the nCounter NanoString platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were normalized to 19-ζ alone, and the number of significantly (p < 0.05) upregulated differentially expressed genes (DEGs) were categorized by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from the experiment described in (F). (H) Volcano plot from the experiment described in (F) of Fas-CD40-19-ζ cells compared with 19-ζ alone after incubation with immobilized FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from the experiment described in (F). (J and K) 19-ζ cells were cultured in the presence or absence of immobilized FasL (20 μg/mL) for 5 days and then stained for CCR8, ICOSL, and ICOS expression by flow cytometry (I), or the cell culture supernatant analyzed for CCL1, CXCL10, and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-way ANOVA. (L) Top: TCF7 expression from the experiment described in (F). Bottom left: 19-ζ cells were stained for TCF-1, representative flow cytometry plots from one donor. Bottom right: TCF-1 expression from three independent donors, error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, two-way ANOVA.

    Article Snippet: Antibodies used were: CD2-PE (300208), CD3-PE Cy7 (344816), CD34-APC (R&D Systems, FAB7227A), TCF-1-PE (655208), GD2-APC (357306), FasL-BV421 (306412), Fas-PE (305608), Fas-APC Cy7 (305636), CCR8-PE (360604), ICOSL-PE (309404), ICOS-PE (313508), CD45RA-PE Texas Red (Invitrogen, MHCD45RA17), CD62L-Pacific blue (304826), LAG3-FITC (369308), PD-1-PE (329906), TIM3-BV421 (345008), CD8-APC Cy7 (301016), and PE Mouse IgG1 Isotype Control (981804).

    Techniques: Binding Assay, Cell Culture, Recombinant, Activity Assay, Expressing, Flow Cytometry, Incubation, Staining

    Fas-TNFRs rescue FasL-mediated kill (A) Schematic of polycistronic transgene transduced into human T cells. 19-BBζ:Fmc63 binder fused to the endodomains of 4-1BB and CD3ζ via a CD8 stalk/transmembrane domain. (B) Representative flow cytometry plots from one human T cell donor transduced to express either 19-BBζ alone or co-express FasΔDD or the stated Fas-TNFRs. Statistical analyses from multiple donors are shown in (C). (C) Left: transduction percentages of T cells from eight independent donors; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to 19-BBζ). Right: median fluorescence intensity (MFI) of the Fas-TNFRs relative to FasΔDD MFI, measured from the top right quadrant in (B). Eight independent donors tested, mean being shown, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to FasΔDD). (D) 19-BBζ cells were cultured with SupT1 FasKO or SupT1 FasKO -FasL cells either for 72 h (left) or 5 h (right) at a 1:1 effector to target (E:T) ratio, at which point 19-BBζ cell survival or percentage of apoptotic cells (Annexin V + 7AAD − ) were calculated, respectively. Seven and four independent donors were tested for the cell survival and apoptotic analysis, respectively. Error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-way ANOVA. (E) 19-BBζ cells from four independent donors were stimulated with 5 × 10 4 SupT1 FasKO or SupT1 FasKO -FasL cells at an initial 1:1 E:T up to four times with cell counts being analyzed after each stimulation. (F) Cell numbers from (E) after the second round of SupT1 FasKO -FasL stimulation. ∗p < 0.05; ns, non-significant, two-way ANOVA. (G) Mean average of relative Fas expression (from C) versus mean average of relative 19-BBζ survival (from second stimulation readout in (E) and <xref ref-type=Figure S4 B), r, Pearson correlation coefficient. (H) From experiment described in (E), the percentage of surviving targets analyzed after each target stimulation. Error bars are SEM, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, non-significant, two-way ANOVA." width="100%" height="100%">

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

    doi: 10.1016/j.omtn.2023.04.017

    Figure Lengend Snippet: Fas-TNFRs rescue FasL-mediated kill (A) Schematic of polycistronic transgene transduced into human T cells. 19-BBζ:Fmc63 binder fused to the endodomains of 4-1BB and CD3ζ via a CD8 stalk/transmembrane domain. (B) Representative flow cytometry plots from one human T cell donor transduced to express either 19-BBζ alone or co-express FasΔDD or the stated Fas-TNFRs. Statistical analyses from multiple donors are shown in (C). (C) Left: transduction percentages of T cells from eight independent donors; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to 19-BBζ). Right: median fluorescence intensity (MFI) of the Fas-TNFRs relative to FasΔDD MFI, measured from the top right quadrant in (B). Eight independent donors tested, mean being shown, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to FasΔDD). (D) 19-BBζ cells were cultured with SupT1 FasKO or SupT1 FasKO -FasL cells either for 72 h (left) or 5 h (right) at a 1:1 effector to target (E:T) ratio, at which point 19-BBζ cell survival or percentage of apoptotic cells (Annexin V + 7AAD − ) were calculated, respectively. Seven and four independent donors were tested for the cell survival and apoptotic analysis, respectively. Error bars are SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-way ANOVA. (E) 19-BBζ cells from four independent donors were stimulated with 5 × 10 4 SupT1 FasKO or SupT1 FasKO -FasL cells at an initial 1:1 E:T up to four times with cell counts being analyzed after each stimulation. (F) Cell numbers from (E) after the second round of SupT1 FasKO -FasL stimulation. ∗p < 0.05; ns, non-significant, two-way ANOVA. (G) Mean average of relative Fas expression (from C) versus mean average of relative 19-BBζ survival (from second stimulation readout in (E) and Figure S4 B), r, Pearson correlation coefficient. (H) From experiment described in (E), the percentage of surviving targets analyzed after each target stimulation. Error bars are SEM, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, non-significant, two-way ANOVA.

    Article Snippet: Antibodies used were: CD2-PE (300208), CD3-PE Cy7 (344816), CD34-APC (R&D Systems, FAB7227A), TCF-1-PE (655208), GD2-APC (357306), FasL-BV421 (306412), Fas-PE (305608), Fas-APC Cy7 (305636), CCR8-PE (360604), ICOSL-PE (309404), ICOS-PE (313508), CD45RA-PE Texas Red (Invitrogen, MHCD45RA17), CD62L-Pacific blue (304826), LAG3-FITC (369308), PD-1-PE (329906), TIM3-BV421 (345008), CD8-APC Cy7 (301016), and PE Mouse IgG1 Isotype Control (981804).

    Techniques: Flow Cytometry, Transduction, Fluorescence, Cell Culture, Expressing

    Fas-CD40 optimally enhances 19-28ζ CAR efficacy (A) Schematic of polycistronic transgene transduced into human T cells. 19-28ζ:Fmc63 binder fused to the endodomains of CD28 and CD3ζ via a CD8 stalk/transmembrane domain. (B) Left: transduction percentages of T cells from 10 independent donors; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to 19-28ζ). Right: MFI of the Fas-TNFRs relative to FasΔDD MFI, measured from top right quadrant in <xref ref-type=Figure S6 A. Ten independent donors tested, bars indicate means, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA (Dunnett’s multiple comparisons test relative to FasΔDD). (C) 19-28ζ cells from four independent donors were stimulated up to 10 times with either Nalm6 FasKO or Nalm6 FasKO -FasL cells at a starting 1:8 E:T ratio, measuring for target survival and 19-28ζ cell counts after each stimulation. Effectors were stimulated with 50,000 targets for the first five stimulations and 100,000 targets for the final five stimulations, error bars are SEM. (D) Relative target survival of Nalm6 FasKO (left) and Nalm6 FasKO -FasL (right) cells after the ninth or fourth rounds of stimulation, respectively, as described in (C). ∗p < 0.05, ∗∗p < 0.01; ns, non-significant, two-way ANOVA, error bars are SEM. (E) Cell culture supernatants after the first round of target stimulation from the experiment described in (C) were analyzed for IFN-γ and IL-2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, non-significant, two-way ANOVA, error bars are SEM. (F) T cell memory phenotypes were analyzed for CD8 (top) and CD4 (bottom) cells after the fifth, seventh, and ninth stimulations from the restimulation experiment described in (C). Error bars are SEM, an “X” denotes where too few cells were present to accurately determine memory phenotype." width="100%" height="100%">

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

    doi: 10.1016/j.omtn.2023.04.017

    Figure Lengend Snippet: Fas-CD40 optimally enhances 19-28ζ CAR efficacy (A) Schematic of polycistronic transgene transduced into human T cells. 19-28ζ:Fmc63 binder fused to the endodomains of CD28 and CD3ζ via a CD8 stalk/transmembrane domain. (B) Left: transduction percentages of T cells from 10 independent donors; ns, non-significant, one-way ANOVA (Dunnett’s multiple comparisons test relative to 19-28ζ). Right: MFI of the Fas-TNFRs relative to FasΔDD MFI, measured from top right quadrant in Figure S6 A. Ten independent donors tested, bars indicate means, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA (Dunnett’s multiple comparisons test relative to FasΔDD). (C) 19-28ζ cells from four independent donors were stimulated up to 10 times with either Nalm6 FasKO or Nalm6 FasKO -FasL cells at a starting 1:8 E:T ratio, measuring for target survival and 19-28ζ cell counts after each stimulation. Effectors were stimulated with 50,000 targets for the first five stimulations and 100,000 targets for the final five stimulations, error bars are SEM. (D) Relative target survival of Nalm6 FasKO (left) and Nalm6 FasKO -FasL (right) cells after the ninth or fourth rounds of stimulation, respectively, as described in (C). ∗p < 0.05, ∗∗p < 0.01; ns, non-significant, two-way ANOVA, error bars are SEM. (E) Cell culture supernatants after the first round of target stimulation from the experiment described in (C) were analyzed for IFN-γ and IL-2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, non-significant, two-way ANOVA, error bars are SEM. (F) T cell memory phenotypes were analyzed for CD8 (top) and CD4 (bottom) cells after the fifth, seventh, and ninth stimulations from the restimulation experiment described in (C). Error bars are SEM, an “X” denotes where too few cells were present to accurately determine memory phenotype.

    Article Snippet: Antibodies used were: CD2-PE (300208), CD3-PE Cy7 (344816), CD34-APC (R&D Systems, FAB7227A), TCF-1-PE (655208), GD2-APC (357306), FasL-BV421 (306412), Fas-PE (305608), Fas-APC Cy7 (305636), CCR8-PE (360604), ICOSL-PE (309404), ICOS-PE (313508), CD45RA-PE Texas Red (Invitrogen, MHCD45RA17), CD62L-Pacific blue (304826), LAG3-FITC (369308), PD-1-PE (329906), TIM3-BV421 (345008), CD8-APC Cy7 (301016), and PE Mouse IgG1 Isotype Control (981804).

    Techniques: Transduction, Cell Culture

    Universal application of Fas-CD40 to enhance CAR T cell efficacy Cartoon illustrating how Fas-CD40 augments CAR T cell efficacy. The CAR binds the cognate antigen on the cancer cell forming an immunological synapse (1). The co-stimulatory domain and CD3ζ within the CAR undergo signal transduction delivering signals 1 and 2 to the cell (2), leading to CAR T cell activation (3). Intracellular stores of FasL are then trafficked to the plasma membrane (4), where Fas-CD40 binds to upregulated FasL on neighboring CAR T cells, as well as FasL-positive cancer cells (5), delivering signal 3 to the cell, augmenting CAR T cell activation (6). Upon activation, the CAR T cell induces target cell killing (7).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel Fas-TNFR chimeras that prevent Fas ligand-mediated kill and signal synergistically to enhance CAR T cell efficacy

    doi: 10.1016/j.omtn.2023.04.017

    Figure Lengend Snippet: Universal application of Fas-CD40 to enhance CAR T cell efficacy Cartoon illustrating how Fas-CD40 augments CAR T cell efficacy. The CAR binds the cognate antigen on the cancer cell forming an immunological synapse (1). The co-stimulatory domain and CD3ζ within the CAR undergo signal transduction delivering signals 1 and 2 to the cell (2), leading to CAR T cell activation (3). Intracellular stores of FasL are then trafficked to the plasma membrane (4), where Fas-CD40 binds to upregulated FasL on neighboring CAR T cells, as well as FasL-positive cancer cells (5), delivering signal 3 to the cell, augmenting CAR T cell activation (6). Upon activation, the CAR T cell induces target cell killing (7).

    Article Snippet: Antibodies used were: CD2-PE (300208), CD3-PE Cy7 (344816), CD34-APC (R&D Systems, FAB7227A), TCF-1-PE (655208), GD2-APC (357306), FasL-BV421 (306412), Fas-PE (305608), Fas-APC Cy7 (305636), CCR8-PE (360604), ICOSL-PE (309404), ICOS-PE (313508), CD45RA-PE Texas Red (Invitrogen, MHCD45RA17), CD62L-Pacific blue (304826), LAG3-FITC (369308), PD-1-PE (329906), TIM3-BV421 (345008), CD8-APC Cy7 (301016), and PE Mouse IgG1 Isotype Control (981804).

    Techniques: Transduction, Activation Assay, Clinical Proteomics, Membrane

    Hh affects colonic T cell-fate decisions. Different cell subsets in the colonic LP were analyzed in 14-week-old Hh -free Ctr and DC-LMP1/CD40 animals. Shown are representative FACS-plots as well as pooled statistics from two experiments (mean ± SEM, n = 7), illustrating frequencies of the indicated cell subsets. (a) DCs were gated on single, live, CD45 + , MHCII + CD11c + CD64 − cells. (b) Tregs were gated on single, live, CD45 + , CD3 + CD4 + FoxP3 + CD25 + , Helios − (iTregs) or Helios + (nTregs). Single-cell suspensions of colonic LP from 14-and 25-week-old Hh -free or Hh -colonized Ctr or DC-LMP1/CD40 (14-weeks-old, 40 days post Hh -colonization) mice were stimulated with PMA/Ionomycin and subsequently stained intracellularly for IL-17 and IFN-γ production at the indicated time points. Bar graphs represent pooled statistics from two experiments (mean ± SEM, n = 7–9) animals. (c) T cells were pre-gated on single, live, CD45 + , CD3 + CD4 + cells. Shown are representative FACS-plots as well as bar graphs, illustrating the frequencies of the indicated cell subsets within the CD4 T cell population. (d) Shown are representative histograms as well as bar graphs, illustrating the MFI of IFN-γ expression within IFN-γ + CD4 + T cells from (c) as median ± SEM. p.i.: post-inoculation

    Journal: Gut Microbes

    Article Title: Helicobacter hepaticus is required for immune targeting of bacterial heat shock protein 60 and fatal colitis in mice

    doi: 10.1080/19490976.2021.1882928

    Figure Lengend Snippet: Hh affects colonic T cell-fate decisions. Different cell subsets in the colonic LP were analyzed in 14-week-old Hh -free Ctr and DC-LMP1/CD40 animals. Shown are representative FACS-plots as well as pooled statistics from two experiments (mean ± SEM, n = 7), illustrating frequencies of the indicated cell subsets. (a) DCs were gated on single, live, CD45 + , MHCII + CD11c + CD64 − cells. (b) Tregs were gated on single, live, CD45 + , CD3 + CD4 + FoxP3 + CD25 + , Helios − (iTregs) or Helios + (nTregs). Single-cell suspensions of colonic LP from 14-and 25-week-old Hh -free or Hh -colonized Ctr or DC-LMP1/CD40 (14-weeks-old, 40 days post Hh -colonization) mice were stimulated with PMA/Ionomycin and subsequently stained intracellularly for IL-17 and IFN-γ production at the indicated time points. Bar graphs represent pooled statistics from two experiments (mean ± SEM, n = 7–9) animals. (c) T cells were pre-gated on single, live, CD45 + , CD3 + CD4 + cells. Shown are representative FACS-plots as well as bar graphs, illustrating the frequencies of the indicated cell subsets within the CD4 T cell population. (d) Shown are representative histograms as well as bar graphs, illustrating the MFI of IFN-γ expression within IFN-γ + CD4 + T cells from (c) as median ± SEM. p.i.: post-inoculation

    Article Snippet: The following antibodies were used: FoxP3 (FJK-16s; eFlour660, dil. 1:50), Helios (22F6; FITC, dil. 1:400) (eBioscience); CD25 (PC61; PerCP, dil. 1:400), CD103 (M290; PE, dil. 1:150) (BD Pharmingen); CD11b (M1/70; APC-eFluor780, dil. 1:400) (Invitrogen); CD3 (17A2; AlexaFluor488, dil. 1:400; Pe-Cy7, dil. 1:400), CD4 (RM4-5; PerCP, 1:800; GK1.5; APC-Cy7, dil. 1:400), CD11c (N418; Pe-Cy7, dil. 1:400), CD45 (30-F11; BV421, dil. 1:400), CD64 (X54-5/7.1; APC, dil. 1:200), IL-17A (TC11-18H10.1; PE, dil. 1:200), IFN-γ (XMG1.2; APC, dil. 1:400), MHC class II (I-A/I-E) (M5/114.15.2; FITC, PerCP, dil. 1:800) (BioLegend).

    Techniques: Staining, Expressing