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: 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

Journal: NPJ Vaccines

Article Title: Enhanced germinal center reaction by targeting vaccine antigen to major histocompatibility complex class II molecules

doi: 10.1038/s41541-019-0101-0

Figure Lengend Snippet: Targeting antigen to MHC class II molecules increases proliferation of T and B cells in vitro. a Symbols. Naive T and B cells were enriched by negative selection from the spleens of TCR Tg and anti-Id DKI mice (Supplementary Fig. ), or BALB/c mice. b – h Either T cells or B cells were irradiated (irr.), and indicated mixtures of 5 × 10 4 T cells and 1 × 10 5 B cells were seeded with titrated amounts of indicated vaccine proteins. Proliferation was assayed by 3 HTdR incorporation. i , j Id-specific T cells and anti-Id B cells were CFSE-labeled and cultured (1:1, 5 × 10 5 ) together with 1 nM of the indicated vaccine proteins for 5 days. i Flow cytometry analysis of CFSE signal and expression of CD69 on Id-specific T cells. j Anti-Id IgM levels in supernatant. All experiments are representatives from single experiments repeated two or three times. b – h n = 3 per group. j n = 4 per group. Mean ± SEM. * p < 0.05 and ** p < 0.01, ( b–h ; αMHCII-scFv 315 vs. αNIP-scFv 315 ) unpaired two-tailed Student’s t test

Article Snippet: The following reagents were used from BD Biosciences (Franklin Lakes, NJ, USA): CD45.2 (560695) 7 μg/ml (561874) 10 μg/ml, B220 (552771) 7 μg/ml, IgD (565348) 7 μg/ml, CD138 (553714) 5 μg/ml, CD95 (740367) 7 μg/ml, CD154 (740685) 7 μg/ml, CXCR5 (560528) 7 μg/ml, MHCII (553623) 5 μg/ml, TCR Vβ 8 (553861) 5 μg/ml, CD14 (553739) 5 μg/ml, and BrdU staining kit (552598); eBioscience (San Diego, CA, USA): CXCR4 (17-9991-82) 7 μg/ml, CD69 (25-0691-82) 7 μg/ml, and CD49b (11-5971) 5 μg/ml; Tonbo biosciences (San Diego, CA, USA): CD4 (75-0041) 5 μg/ml, Ly-6G (35-1276-U100) 5 μg/ml, CD19 (35-0193) 5 μg/ml, CD3 (75-0032) 5 μg/ml, and CD11b (65-0112) 7 μg/ml; BioLegend (San Diego, CA, USA): CD64 (139306) 4 μg/ml, CD11c (117318) 4 μg/ml, F4/80 (123130) 5 μg/ml, GL7 (144603) 4 μg/ml (144614) 5 μg/ml, CD38 (102718) 4 μg/ml, PD-1 (135224) 7 μg/ml, MHCII (107622) 7 μg/ml, CD69 (104522) 7 μg/ml, CD19 (115520) 5 μg/ml, and streptavidin–phycoerythrin (405204) 4 μg/ml; Thermo Fisher Scientific: CD49b (15-5971-82) 5 μg/ml, PNA (L21409) 10 μg/ml, and Cell Trace CFSE (C34554) 5 μM; Abcam (Cambridge, UK): Anti-6x His tag antibody (ab133714) 6 μg/ml; Southern Biotech (Birmingham, AL): CD86 (1735-09) 5 μg/ml.

Techniques: In Vitro, Selection, Irradiation, Labeling, Cell Culture, Flow Cytometry, Expressing, Two Tailed Test