Journal: Nature

Article Title: Structures of human γδ T cell receptor–CD3 complex

doi: 10.1038/s41586-024-07439-4

Figure Lengend Snippet: a , Two molecules (cholesterol-like and unassigned densities) were embedded into the TMD of the Vγ9Vδ2 TCR–CD3 complex. The electrostatic surface potential map of the Vγ9Vδ2 TCR–CD3 complex (left) and a magnified view of the interactions between the cholesterol-like molecules and the complex (right) are shown. The cryo-EM densities are contoured at 9 σ . b , Flow cytometry analysis of CD69 expression on Jurkat-76 cells transduced with WT ( n = 3 per group) and mutant variants of Vγ9Vδ2 TCR ( n = 6 per group) after co-culture for 15 h with K562 cells expressing CD1d or ZIM3–dCas9 (ref. ) (parental). c , Quantitative analysis of cholesterol content in purified WT or mutant Vγ9Vδ2 TCR–CD3 complex using liquid chromatography coupled with tandem MS (LC–MS/MS; n = 6 per group). d , Magnified views of the TMD of the TMα and AAA Vγ9Vδ2 TCR–CD3 complex. The cryo-EM maps are shown as a black mesh and contoured at 8 σ . The position of the cholesterol binding site in the Vγ9Vδ2 TCR–CD3 complex is indicated by a dashed circle. e , Structural comparison of the TMDs of the WT, AAA and TMα Vγ9Vδ2 TCR–CD3 complex (left). Right, structural comparison of the TMDs of Vγ9Vδ2, WT αβ (PDB: 7FJD ) and gain-of-function (GOF) αβ TCR–CD3 complexes (PDB: 7FJE ) . f , Flow cytometry analysis of CD69 expression on Jurkat-76 cells that were transduced with Vγ9Vδ2 TCR and Vγ5Vδ1 TCR, with or without treatment with 0.5 μM ALOD4 and 0.5 μM ALOD4 non-binding mutant (ALOD4-mut) for 12 h. n = 4 per group. Results are representative of three ( b and f ) and two ( c ) independent experiments. Each symbol corresponds to a biologically independent experiment. Data are mean ± s.d. P values were calculated using one-way ANOVA with Dunnett’s multiple-comparison test. For c , mutant complexes were compared with the WT complex.

Article Snippet: The cells were then incubated with anti-human CD69-APC (Sino Biological, 11150-MM06-A) and CD3-PE-Cyanine7 dye antibodies (BD Pharmingen, 552127) at a dilution of 1:500 for 30 min on ice.

Techniques: Cryo-EM Sample Prep, Flow Cytometry, Expressing, Transduction, Mutagenesis, Co-Culture Assay, Purification, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Comparison

Journal: Nature

Article Title: Structures of human γδ T cell receptor–CD3 complex

doi: 10.1038/s41586-024-07439-4

Figure Lengend Snippet: a , SEC analysis of recombinant WT, R120Q and EH Vγ5Vδ1 TCR–CD3 complexes. The SEC chromatograms for WT (orange), R120Q (green), and EH (blue) Vγ5Vδ1 TCR–CD3 complexes are shown. AU: arbitrary units; WT: wild-type; R120Q: R120Q mutation in TCRγ5 chain; EH: Y106E/R120H mutations in TCRγ5 chain. b , Flow cytometry analysis of CD69 expression on WT (left) or EH (middle) or R120Q (right) γδ TCR-transduced Jurkat-76 cells cocultured with K562 cells expressing CD1d or ZIM3–dCas9 (ref. ) (parental) or without K562 cells. Numbers in plots indicate percent of gated events. APC: antigen-presenting cells. c , Flow cytometry analysis of CD3 (left) or γδ TCR (right) expression level on Jurkat-76 cells expressing WT (orange), R120Q (green), or EH (blue) Vγ5Vδ1 TCRs (n = 3 per group). The surface expression level of γδ TCRs was detected by anti-Flag antibodies. d , Flow cytometry analysis of CD69 upregulation on WT (orange), R120Q (green), or EH (blue) Vγ5Vδ1 TCR-transduced Jurkat-76 cells cocultured with anti-CD3/CD28 antibodies in 24 h (n = 6 per group). Results are presented as the proportion of CD69 + cells (%CD69 + cells) in each experimental co-culture relative to that in the control co-culture. e , Flow cytometry analysis of Jurkat-76 cells expressing the γδ TCRs of interest and stained by human CD1d–α-GalCer tetramers. Numbers in plots indicate percent of gated events. Results are representative of three independent experiments in b – d , and two independent experiments in e . In panels c and d , each symbol represents a biologically independent experiment and data are represented as mean ± SD.

Article Snippet: The cells were then incubated with anti-human CD69-APC (Sino Biological, 11150-MM06-A) and CD3-PE-Cyanine7 dye antibodies (BD Pharmingen, 552127) at a dilution of 1:500 for 30 min on ice.

Techniques: Recombinant, Mutagenesis, Flow Cytometry, Expressing, Co-Culture Assay, Staining

Journal: Frontiers in Immunology

Article Title: Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

doi: 10.3389/fimmu.2020.559810

Figure Lengend Snippet: T cell immune response after TBI progress differently in K14-VEGFR3-Ig and WT littermate mice. Panels (A, B) represent the number and frequency of TCRβ+ T cells (A) and the CD4/CD8 ratio (B) in the brain of WT and TG mice, as analyzed in the perilesional and contralateral cortices 3 days post injury (WT ipsi, n = 4; WT contra, n = 4; TG ipsi, n = 3; TG contra, n = 3). No differences between the genotypes have been observed. (C–F) Analysis of T cells infiltration in the brain of K14-VEGFR3-Ig and WT littermate mice 60 days post-injury (WT ipsi, n = 5; WT contra, n = 5; TG ipsi, n = 4; TG contra, n = 4). Box plot represents the number of infiltrating T cells, defined by expression of TCRβ (C) and stacked bargram represents the percentage of CD4+ and CD8+ T cells (D) in the perilesional areas (ipsi) and correspondent contralateral areas (contra) of WT and TG mice. Bargrams in (C, D) show respectively the frequencies of CD8+ and CD4+ T cell subpopulations, as analyzed in the perilesional cortices of WT and TG mice. In CD8+ subpopulation we observed a significant reduction in the frequency of the CD44 hi CD69+ subpopulation in K14-VEGFR3-Ig compared to WT mice, which corresponded to the increase in the frequency of CD44 neg CD69+ phenotype. In CD4+ subpopulation, instead, we did not observed differences in distribution between the two genotypes. Data are presented as median ± SD. A binomial negative regression or a linear mixed model was applied to assess statistical differences in the counts of TCRβ + T cells. The Kruskal Wallis test was used for the analysis of frequency distribution. **p < 0.01 vs. WT ipsi. #p < 0.05 vs. respective contra. In all tests, Bonferroni correction was used to adjust p-values in multiple comparisons.

Article Snippet: Antibodies used: TCRβ PE-Cy7 (1:100 or 1:200 clone H57-597), CD44 PE (1:300 clone IM7) (both BioLegend); CD8a APC-R700 (1:150 or 1:200, clone 53-6.7), CD69 BV421 (1:100, clone H1.2F3), CD25 BB515 (1:150, clone PC61) (BD Biosciences); CD4 FITC (1:500, clone RM4-5), CD4 eFluor506 (1:500, clone RM4-5), CD8 PerCP eFluor710 (1:300, clone 53-6.7), CD44 APC (1:300 or 1:400, clone IM7), FoxP3 (1:40, clone FJK-16s) (eBioscience Thermo Fisher Scientific, Waltham, MA, USA); CD69 APC (1:20, clone H1.2F3, Miltenyi Biotech).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

doi: 10.3389/fimmu.2020.559810

Figure Lengend Snippet: Analysis of CD69 and CD44 T cell activation and memory markers in CD4+ and CD8+ subpopulations. Pseudocolor dot plots (A, B) represent gated subpopulations CD69 vs. CD44 of CD8+ and CD4+, respectively. Bargrams in (C, D) show respectively the counts and frequencies of CD8+ T cell subpopulations, as analyzed in the perilesional cortices of WT and TG mice. No significant differences in CD8+ subpopulations were found between genotypes. In CD4+ subpopulation, instead, we observed a significant reduction in the counts of CD44 hi CD69+ and CD44 hi CD69- subpopulations (E) , in K14-VEGFR3-Ig compared to WT mice. However, no differences were observed in the different subpopulation frequencies (F) . Data are presented as median ± SD. A binomial negative regression was applied to assess statistical differences in the counts of total T cells between WT ipsi and TG ipsi. The Kruskal Wallis test was used for the analysis of frequency distribution. # p < 0.05; *p < 0.05 vs. WT ipsi.

Article Snippet: Antibodies used: TCRβ PE-Cy7 (1:100 or 1:200 clone H57-597), CD44 PE (1:300 clone IM7) (both BioLegend); CD8a APC-R700 (1:150 or 1:200, clone 53-6.7), CD69 BV421 (1:100, clone H1.2F3), CD25 BB515 (1:150, clone PC61) (BD Biosciences); CD4 FITC (1:500, clone RM4-5), CD4 eFluor506 (1:500, clone RM4-5), CD8 PerCP eFluor710 (1:300, clone 53-6.7), CD44 APC (1:300 or 1:400, clone IM7), FoxP3 (1:40, clone FJK-16s) (eBioscience Thermo Fisher Scientific, Waltham, MA, USA); CD69 APC (1:20, clone H1.2F3, Miltenyi Biotech).

Techniques: Activation Assay

Journal: Vaccines

Article Title: Influenza Vaccination Mediates SARS-CoV-2 Spike Protein Peptide-Induced Inflammatory Response via Modification of Histone Acetylation

doi: 10.3390/vaccines12070731

Figure Lengend Snippet: The activation of immune cell subtypes in PBMCs following IV and ex vivo stimulation. ( A ) Representative plots of the gating strategy of CD3 + T cells, CD3 - immune cells, and CD45 + immune cells from PBMCs. ( B ) Representative plots of the T-cell subset gated on total CD3 + T cells expressing CD4 and CD8 surface markers. ( C , D ) Dot plots of the frequencies of cells gated out of CD4 + and CD8 + T cells expressing the AIM marker (CD69 and CD137) and the percentage of the designated population (AIM + CD4 + and AIM + CD8 + ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( E , F ) Dot plots of the frequencies of cells gated out of CD3 - immune cells expressing the NK1.1 marker and the percentage of the designated population (NK1.1 + CD3 − ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( G , H ) Dot plots of the frequencies of cells gated out of CD3 − immune cells expressing the MHC-II marker and the percentage of the designated population (MHC-II + CD3 − ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. ( I , J ) Dot plots of the frequencies of monocytes gated out of CD45 + immune cells expressing CD11b and CD115 markers and the percentage of the designated population (CD11b + CD115 + CD45 + ) in the IV-immunized or control PBMCs stimulated with SARS-CoV-2 spike protein peptide pools or DMSO. n = 4/group. Data are presented as mean ± SEM and * p < 0.05, ** p < 0.01, and *** p < 0.001 (one-way ANOVA with Tukey’s test).

Article Snippet: Afterward, the cells were incubated with 200 μL of FACS buffer of antibody cocktail for surface marker staining, including CD3 (ThermoFisher Scientific, Waltham, MA, USA, cat# 12-0038; Biolegend, San Diego, CA, USA, cat# 100233), CD45 (ThermoFisher Scientific, cat# 11-0459), CD4 (ThermoFisher Scientific, cat# 11-0041), CD8 (ThermoFisher Scientific, cat# 25-0088), CD69 (Tonbo, Tucson, AZ, USA, cat# 20-0691), CD137 (ThermoFisher Scientific, cat# 12-1371), CD11b (Tonbo, cat# 20-0112), CD115 (Biolegend, cat# 135527), NK1.1 (ThermoFisher Scientific, cat# 45-5941), and MHC-II (ThermoFisher Scientific, cat# 17-5320).

Techniques: Activation Assay, Ex Vivo, Expressing, Marker, Control

Journal: Science Advances

Article Title: Nutrient availability regulates the secretion and function of immune cell–derived extracellular vesicles through metabolic rewiring

doi: 10.1126/sciadv.adj1290

Figure Lengend Snippet: ( A and B ) M2-Mφs were induced by IL-4/TGF-β (20 ng/ml each), and qPCR analysis was conducted to measure the expression of the M2 gene ( Arg1 , Mrc1 , and TGF- β) expression in M2-Mφs treated with EV GLN− (20 μg/ml) or EV GLN+ (20 μg/ml) for 48 hours ( n = 3; ***P < 0.001, **P < 0.01, ## P < 0.01, ### P < 0.01 versus the M2 group). ( C and D ) qPCR analysis of chemokine gene expression ( Ccl2 and Cxcl2 ) in THP-1 monocytes treated with EV GLN− preparations or EV GLN+ preparations (20 μg/ml) for 24 hours ( n = 3; ***P < 0.001, **P < 0.01 versus the “CON” group). ( E and F ) Chemotaxis evaluation of (E) conditioned culture medium from EV pretreated THP-1 cells and (F) EVs from splenocytes using a Transwell system, and the migrated cells in the lower chamber were counted using FCA ( n = 3; **P < 0.01, *P < 0.05 versus the CON group). ( G and H ) Mouse splenocytes were treated with ConA or ConA plus EV GLN− or EV GLN+ (20 μg/ml) for 72 hours, and the populations of activated CD4 + T cells and (CD3 + CD4 + CD69 + ) activated CD8 + T cells (CD3 + CD8 + CD69 + ) were determined by FCA ( n = 3). ( I and J ) Evaluation of immune responses in mice ( n = 5) intravenously injected with EV GLN− or EV GLN+ (30 μg/mouse) for 4 hours and immune cell populations (F4/80 + Mφs, Ly6C + monocytes, and Ly6G + neutrophils) in the spleen were analyzed using FCA ( **P < 0.01, *P < 0.05 versus the CON group).

Article Snippet: After treatment, cells from each group were collected and stained with fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD3e (553061, BD, Brea, CA, USA, USA), peridinin chlorophyll protein (PerCP)/Cyanine5.5-conjugated anti-mouse CD4 (100434, BioLegend), phycoerythrin (PE)–Cy7–conjugated anti-mouse CD8a (552887, BD), and allophycocyanin (APC)–conjugated anti-mouse CD69 (APC-65105, Proteintech, Wuhan, China) for 30 min. After washing with PBS, the stained cells were analyzed using a flow cytometer (LSRFortessa, BD Biosciences).

Techniques: Expressing, Gene Expression, Chemotaxis Assay, Injection