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: Nature

Article Title: Antibody prophylaxis may mask subclinical SIV infections in macaques

doi: 10.1038/s41586-024-08500-y

Figure Lengend Snippet: Intracellular cytokine staining was performed on PBMCs to assess T cell responses to (A, C) SIV Gag and (B, D) Env peptide pools. (A, B) Samples assessed were collected before antibody infusion (pre-immune) and 2, 6 and 16 weeks post the dominant virus infection (see Tables S – ). Circles and squares represent individual animals. Dotted lines are equal to 0%. The overlaid boxes and middle line indicate the IQR and median, respectively, and the whiskers are the range. N = 4 (control group) or n = 6 (ITS102.03 and ITS103.1-treatment groups) animals. (C, D) For animals where a viral blip was observed (animal IDs shown below horizontal axis and day post-first virus challenge of relevant blip in parentheses where appropriate), PBMCs collected 2 weeks after the blip were assayed and are shown relative to individual’s pre-immune measurement. Solid, horizontal lines are equal to 0%. (A–D) CD4+ T cell Th1 responses (IFNγ, IL-2, or TNF), CD4+ T cell Th2 responses (IL-4 or IL-13), and CD8+ T cell responses (IFNγ, IL-2, or TNF) are shown from left to right, respectively. Responses are background-subtracted.

Article Snippet: CD4 + T cells were enriched by positive selection (CD4 MicroBeads, NHP, Miltenyi Biotec).

Techniques: Staining, Virus, Infection, Control

Journal: Nature

Article Title: Antibody prophylaxis may mask subclinical SIV infections in macaques

doi: 10.1038/s41586-024-08500-y

Figure Lengend Snippet: Flow cytometry gating strategy used to identify and quantify antigen-specific T cells in PBMCs is shown from a representative sample. Sequential gating was used to identify T cells, followed by memory CD4+ and CD8+ T cells. Cytokine-expressing cells were then identified by co-expression of each cytokine with upregulated CD69 expression.

Article Snippet: CD4 + T cells were enriched by positive selection (CD4 MicroBeads, NHP, Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing