Journal: Nature Neuroscience

Article Title: Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T reg –microglia crosstalk

doi: 10.1038/s41593-025-01877-7

Figure Lengend Snippet: (a) Flow cytometry analysis and quantification of CD4 + T cells, CD4+FoxP3 + and (b) CD8 + T cells, CD4 + LAP + , Th1, and Th17 at 1,3,7,14, and 30 days post TBI and nasal anti-CD3 treatment in the cervical lymph nodes, meninges, and Ipsilateral brain hemisphere. (Sham-Iso n = 4, TBI-Iso n = 6, TBI-aCD3 n = 6). Data shown as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons for every individual timepoint (c) Flow cytometry analysis and quantification of neutrophils, monocytes, classical monocytes (7 days post-TBI), and NK cells at 1,3,7,14, and 30 days post TBI and nasal anti-CD3 treatment in Ipsilateral brain hemisphere. (Sham-Iso n = 4, TBI-Iso n = 6, TBI-aCD3 n = 6). Data shown as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparisons for every individual timepoint. All data are biological replicates and are representative from two independent experiments.

Article Snippet: The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting.

Techniques: Flow Cytometry

Journal: Nature Neuroscience

Article Title: Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T reg –microglia crosstalk

doi: 10.1038/s41593-025-01877-7

Figure Lengend Snippet: a , b , Flow cytometry analysis and quantification of CD4 + ( a ) and CD4 + FoxP3 + ( b ) T reg cells in the meninges and ipsilateral hemisphere at 1, 3, 7, 14 and 30 d (D) after TBI and treatment. c , Quantification of CD4 + subsets at the same time points. d , Analysis of CD11b + -infiltrated cells across these intervals. Groups included sham-iso ( n = 4), TBI-iso ( n = 6) and TBI-aCD3 ( n = 6). Data are analyzed by one-way ANOVA with Tukey’s multiple comparisons for every individual timepoint. Data are shown as mean ± s.e.m. representing biological replicates from two independent experiments per timepoint for a – d . e , Immunofluorescence of meninges (2 d post-TBI) and brain (7 d post-TBI) samples from FoxP3-GFP mice with DAPI (blue), CD3 (pink), FoxP3 (green). Scale bars, 100 µm. IHC, immunohistochemistry. f , PCA plot of brain and blood T reg cells 7 d post-TBI. Brain and blood samples are pools of 5 mice and sham-iso brain samples are pools of 20 mice. Due to the low number of FoxP3⁺ cells recruited to the brain and ethical considerations, we limited the study to two biological replicates, following practices from previous studies in the field . Despite this limitation, the consistent and robust results observed support the validity of our findings. g , h , Heatmaps of DEGs from blood ( g ) and brain ( h ) T reg cells at 7 d post-TBI using DESeq2 (FDR-corrected P < 0.05, n = 2 pooled samples per group). i , GSEA of GOBP 7 d post-TBI for brain T reg cells. The asterisks indicate enriched terms ( q < 0.05). NES, normalized enrichment score. j , IPA analysis of DEGs from brain T reg cells in TBI-aCD3 versus TBI-iso using DESeq2 analysis (two-sided Wald’s test, FDR-corrected P < 0.05). One-sided Fisher’s exact test was used: * P < 0.05, ** P < 0.01, *** P < 0.001. Results with FDR-corrected P < 0.05 were selected. k , Predicted upstream regulators using IPA for TBI-aCD3 versus TBI-iso. l , Quantification of FoxP3 + IL-10 + T reg cells in the ipsilateral hemisphere 7 d post-TBI. Groups included sham-iso ( n = 4), TBI-iso ( n = 6) and TBI-aCD3 ( n = 6). Data are shown as box plots (min., max., IQR, median), analyzed by one-way ANOVA with Tukey’s multiple comparisons. Data are from biological replicates and represent two independent experiments.

Article Snippet: The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting.

Techniques: Flow Cytometry, Immunofluorescence, Immunohistochemistry

Journal: Nature Neuroscience

Article Title: Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T reg –microglia crosstalk

doi: 10.1038/s41593-025-01877-7

Figure Lengend Snippet: ( a) Gating strategy used to identify and sort CD4 + FoxP3(GFP) + from the ipsilateral hemisphere of the brain and blood. (b) Overlap in differentially expressed genes in injured brain vs. sham blood Treg cells after injury with another study investigating the transcriptomic effects of stroke in brain vs. blood Treg cells . (c) Selected top predicted regulators using IPA based on DEGs in TBI-aCD3 vs. TBI-Iso blood Treg cells at 7 days post-TBI identified using DESeq2 analysis (two-sided Wald test, FDR-corrected P < 0.05). One-sided Fisher’s exact test. * P < 0.05, ** P < 0.01, *** P < 0.001. Results with FDR-corrected P < 0.05 were selected. (d) Predicted upstream regulator using IPA analysis based on DEGs of brain Tregs in TBI-aCD3 vs. TBI-Iso identified using DESeq2 analysis (two-sided Wald test, FDR-corrected P < 0.05). Due to the low number of FoxP3⁺ cells recruited to the brain, and ethical considerations, we limited the study to two biological replicates, following practices from previous studies in the field . Despite this limitation, the consistent and robust results observed support the validity of our findings.

Article Snippet: The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting.

Techniques:

Journal: Nature Neuroscience

Article Title: Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T reg –microglia crosstalk

doi: 10.1038/s41593-025-01877-7

Figure Lengend Snippet: (a) Gating strategy for IL-10 expression on CD4 + , FoxP3 + , 4D4+ microglia, NK1.1 + , Ly6C + , and Ly6G+ cell and their fluorescence minus one (FMO) control. (b) Bar plot of Quantitative PCR of microglia sorted from the ipsilateral hemisphere at 7 days post TBI for microglia specific IL-10ra knockout Tmem119 CreETR2 :IL-10ra Flx/Flx and their littermate controls Tmem119 WT :IL-10ra Flx/Flx . Mice were treated with tamoxifen for 5 straight days and were given a 2-week rest period before TBI. Expression was normalized to GAPDH. Data shown as mean ± SEM, n = 3 mice/group. Data was analyzed by two-sided unpaired Student’s t-test. The data are biological replicates and are representative from three independent experiments. (c) Gating strategy showing CD4 + FoxP3 GFP + and the population of CD4 + FoxP3 GFP - from the spleen/cLN that was selected for the adoptive transfer experiments in Fig. and Extended Data Fig. .

Article Snippet: The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting.

Techniques: Expressing, Fluorescence, Control, Real-time Polymerase Chain Reaction, Knock-Out, Adoptive Transfer Assay

Journal: Nature Neuroscience

Article Title: Nasal anti-CD3 monoclonal antibody ameliorates traumatic brain injury, enhances microglial phagocytosis and reduces neuroinflammation via IL-10-dependent T reg –microglia crosstalk

doi: 10.1038/s41593-025-01877-7

Figure Lengend Snippet: (a) Schematic representing experimental timeline of adoptive transfer experiment. Created with BioRender.com . (b) Behavioral testing of rotarod, Morris water maze, probe trial, and anxiety like behavior (measured by the open field) was assessed between WT Sham (DPBS treated, baseline), Iso-total CD4 + , aCD3-total CD4 + , and aCD3-FoxP3 - GFP groups. Morris water maze analyzed by two-factor repeated measures two-way ANOVA (group x time); others by one-way ANOVA with Tukey’s multiple comparisons. Data shown as mean ± SEM. (c) Visual representing an experiment where splenic CD4 + cells from 7 days treated TBI (FoxP3-GFP) animals were injected into untreated, but TBI-injured (CD45.1) animals and the %CD45.2 cells were analyzed by fluorescence-activated cell sorting (FACS) at 3 days after injection. Flow cytometry gating of brain, cervical lymph node, and spleen of (CD45.1) animals showing the percent of CD45.2 cell infiltration. n = 5 mice and the brain was a pool of 5 ipsilateral hemispheres. Created with BioRender.com . (d) Heatmap of DEGs from microglia 30 days following TBI and adoptive transfer identified using DESeq2 analysis (two-sided likelihood ratio test, n = 4 mice/group, FDR-corrected P < 0.05). Clusters of genes were functionally annotated using enriched GO Biological Process (BP) terms (q-value < 0.05). (e) GSEA analysis of GO Biological Process (BP) 30 days post-TBI based on the following comparisons: aCD3-Total CD4 + vs. Iso-Total CD4 + , and aCD3-FoxP3 - GFP vs. Iso-total CD4 + . Asterisk (*) indicates enriched terms (q-value < 0.05). NES, normalized enrichment score; NS, not significant. (f) Volcano plot of DEGs in aCD3-total CD4 + vs. aCD3-FoxP3 - GFP 30 days post-TBI identified using DESeq2 (two-sided Wald test, n = 4 mice/group). Labeled genes have an FDR-corrected P < 0.05. (g) GSEA analysis of GO Biological Process (BP) comparing aCD3-total CD4 + vs. aCD3-FoxP3 - GFP 30 days post-TBI. NES, normalized enrichment score . (h) Predicted top upstream regulators using IPA analysis based on DEGs in aCD3-total CD4 + vs. aCD3-FoxP3 - GFP 30 days post-TBI. (i) RT- qPCR of ipsilateral hemisphere 30 days post-TBI and expression normalized to GAPDH. Data shown as mean ± SEM, n = 5 mice/group and analyzed by one-way ANOVA with Tukey’s multiple comparisons. All data are biological replicates and are representative from two independent experiments. n.s . = non-significant.

Article Snippet: The sample was purified and enriched using CD4 cell isolation microbead kit (Miltenyi Biotech, cat. no. 130-104-454) on a magnetic MACS separator before sorting.

Techniques: Adoptive Transfer Assay, Injection, Fluorescence, FACS, Flow Cytometry, Labeling, Quantitative RT-PCR, Expressing

Journal: Molecular imaging and biology

Article Title: 2 nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature

doi: 10.1007/s11307-023-01840-7

Figure Lengend Snippet: Study design and number of animals for each experiment

Article Snippet: About ten million lung cells per animal were used for CD31 + enrichment using the CD31 Microbeads kit (cat# 130–109–680, Miltenyi) as per the manufacturer’s instructions.

Techniques: In Vivo, Imaging, Ex Vivo, Expressing, Immunostaining

Journal: Molecular imaging and biology

Article Title: 2 nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature

doi: 10.1007/s11307-023-01840-7

Figure Lengend Snippet: SS.BN3 rats have reduced Dll4 expression and reduced susceptibility to late radiation-induced morbidity. a Consomic map illustrating the SS.BN3 rat is generated by the substitution of chromosome 3 from the Brown Norway rat into the Dahl-SS genetic background. b mRNA expression of Dll4 from CD31+ endothelial cells in non-irradiated SS (Dll4-high) and consomic SS.BN3 (Dll4-low) rats (n = 4 (SS) or 5 (SS.BN3) rats per group). c Survival following 13 Gy partial body irradiation (PBI) in adult SS (n = 13) and SS.BN3 rats (n = 19). P value for log-rank analysis: P = 0.0002 for SS vs SS.BN3 following 13 Gy PBI. d Change in body weight relative to pre-irradiation body weight for the rats represented in the survival curve (**P = 0.0066, ****P < 0.0001, two-way ANOVA Sidak’s multiple testing correction). Note the increased drop in body weight at 90 and 120 days in SS as compared to SS.BN3 rats, that occurs during pneumonitis and nephropathy, respectively

Article Snippet: About ten million lung cells per animal were used for CD31 + enrichment using the CD31 Microbeads kit (cat# 130–109–680, Miltenyi) as per the manufacturer’s instructions.

Techniques: Expressing, Generated, Irradiation

Journal: Molecular imaging and biology

Article Title: 2 nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature

doi: 10.1007/s11307-023-01840-7

Figure Lengend Snippet: SS.BN3 (Dll4-low) rats have reduced radiation-induced loss of CD31+ endothelial cells. a Left, representative FACS contour plots showing the percentage of CD45−CD31+ lung ECs at day 70 following 13 Gy partial body irradiation in SS (Dll4-high) and SS.BN3 (Dll4-low) rats. Right, quantification of percent CD45−CD31+ cells (n = 9 rats per group). b Left, representative FACS contour plots showing the percentage of CD45−CD31+ kidney ECs at day 70 following 13 Gy partial body irradiation in SS (Dll4-high, n = 4) and SS.BN3 (Dll4-low, n = 5) rats. Right, quantification of percent CD45−CD31+ cells

Article Snippet: About ten million lung cells per animal were used for CD31 + enrichment using the CD31 Microbeads kit (cat# 130–109–680, Miltenyi) as per the manufacturer’s instructions.

Techniques: Irradiation

Journal: Leukemia

Article Title: Spred1 deficit promotes treatment resistance and transformation of chronic phase CML

doi: 10.1038/s41375-021-01423-x

Figure Lengend Snippet: A Expression of SPRED1 in BM CD34+ cells from patients with BC CML and CP CML by Q-RT-PCR (n=8 samples for BC CML and n=12 samples for CP CML) and western blot and in BM by immunohistochemistry staining (one of three independent experiments with similar results was shown) (left), and expression of miR-126 in CD34+ and CD34+CD38− cells from BC CML (n=6 samples) and CP CML (n=10 samples) patients by Q-RT-PCR (right). B SPRED1 mRNA expression by Q-RT-PCR and protein expression by western blot, miR-126 levels by Q-RT-PCR, cell cycling by Ki-67 and DAPi staining (top) or by cell trace violet staining (bottom) followed by flow cytometry analysis in CML CD34+ cells transduced with SPRED1 siRNA to knock-down (KD) SPRED1 or with a non-targeting control siRNA (Ctrl). UND: undivided cells, G0; DIV: division. C Representative colonies and quantification of colony forming cells (CFC) in CML CD34+ (left) and CD34+CD38− (right) cells transduced with Spred1 siRNA to KD SPRED1 or with ctrl siRNA (n=3). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human CD34 + cells were selected using the indirect CD34 microbead kit (Miltenyi Biotec, San Diego, CA) and CD34 + CD38 − cells were sorted after staining with human antibodies against CD34 and CD38 ( Supplementary Table 1 ) or selected using CD34+CD38- cell isolation kit (Miltenyi Biotec, San Diego, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Flow Cytometry, Transduction, Knockdown, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 1. The expression of CD133 in lung cancer and normal lung tissues. Original values are presented as log2 ratios. Spots showing the level of CD133 mRNA in NSCLC and normal tissues. (A) GSE10072. (B) GSE40275. (C) GSE63459; *p<0.05, **p<0.01 and ***p<0.001. (D) Correlation between CD133 and CXCR4 in GSE30219.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 2. CD133 and CXCR4 are highly expressed in NSCLC patients with metastasis. (A) Representative images of immunohistochemistry staining (mag- nification, x400) of NSCLC patients. (a) CD133 low expression; (b) CD133 high expression; (c) CXCR4 low expression; (d) CXCR4 high expression. Positive staining ratio of CD133 (B)/CXCR4 (C) in metastatic and non-metastatic NSCLC patients. Positive staining score of CD133 (D)/CXCR4 (E) in metastatic and non-metastatic NSCLC patients. (F) Correlation results of CD133 and CXCR4. (G) Correlation analysis between CD133/CXCR4 co-expression and disease- free survival (red and black curves indicate high and low CD133/CXCR4 co-expression groups of patient death, respectively; *p<0.05).

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 3. CD133 enhances A549 cells proliferation. (A) Effectiveness of magnetic cell sorting was verified by immunofluorescence assay (magnification, x400). Red and blue indicate CD133 expression and cell nucleus, respectively. (B) Formation of colonies by A549 cell lines after 2-week incubation. (C and D) Cell proliferation capacity was determined in A549 cells, CD133+ A549 cells and CD133-A549 cells by CCK8 assays.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: FACS, Immunofluorescence, Expressing, Incubation

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 4. CXCR4 is upregulated by CD133. Silencing effectiveness of CD133 siRNA was verified by qPCR (A) and western blot analysis (B). (C) Expression of CXCR4 after treatment with CD133 siRNA (50 nM, 48 h) by qPCR analysis. (D) Western blot analysis for the expression of CXCR4 after treatment with CD133 siRNA (50 nM, 72 h). β-actin as a loading control. (E) The statistics of (D). All experiments were performed in triplicate. ***p<0.001.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Western Blot, Expressing, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 5. CD133+CXCR4+ promotes EMT process in NSCLC cells. (A) Transwell assay for the invasion of CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (B) Western blot assay for the expression of E-cadherin and Vimentin in CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (C) qPCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC, A549/CD133+ shRNA-CXCR4 and A549/CD133+ with amd3100. (D and E) RT-PCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC and A549/CD133+

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Transwell Assay, Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 6. Vimentin is positively associated with CD133/CXCR4 co-expression. (A) Representative images of immunohistochemistry staining (magnification, x400) of NSCLC patients. (a) E-cadherin low expression; (b) E-cadherin high expression; (c) Vimentin low expression; (d) Vimentin high expression. Positive staining ratio of E-cadherin (B)/Vimentin (C) in metastatic and non-metastatic NSCLC patients. Correlation analysis for E-cadherin (D) or Vimentin (E) with CD133/CXCR4 co-expression in NSCLC patients.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing, Immunohistochemistry, Staining