Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

doi: 10.1073/pnas.2315592121

Figure Lengend Snippet: Fig. 1. A Cy3-specific γδ TCR recognizes multiple ligands of different molecular nature. (A) The NX7 γδ TCR CDR3 sequences (with positively charged amino acid residue in bold) and the chemical structures of Cy3, 5-HT (serotonin), HIAA, and IPA. (B–D) Representative FACS plots of NX7/58α−β− cells stained with (B) Cy3-OVA (1 μg/mL), PE-OVA (3 μg/mL), serotonylated or unmodified Rac1 peptide (Biotin-Ahx-GGS GGD TAG QED GGD TAG QED GGD TAG QED, with the Rac1 sequence in bold. Ahx: 6-aminohexanoic acid.) coupled with FITC SAv-dextramer (0.45 μM, SAv concentration), HIAA-PE (1 μg/mL) or PE (1 μg/mL), IPA-PE (10 μg/mL) or PE (10 μg/mL); (C) biotinylated 3-tyrosine peptide (3Y), 4-lysine peptide (4K), HA98-106 peptide (abbreviated as HA), HA98-106 mutant peptide (abbreviated as HA mutant), and murine CCR236-42 peptide (abbreviated as CCR2) coupled with FITC SAv-dextramer (0.45 μM, SAv concentration). The peptide sequences were shown in bold; (D) surface cleaved hemagglutinin protein-coupled PE SAv tetramer (2 μM, SAv concentration) or PE-labeled SAv (2 μM, SAv concentration), Ym1 protein-coupled FITC SAv tetramer (10 μM, SAv concentration) or FITC labeled SAv (10 μM, SAv concentration). Hemagglutinin and Ym1 were randomly biotinylated. The results in B–D were representative of at least three independent experiments.

Article Snippet: Cy3- conjugated ovalbumin (OVA) (Sigma) and Cy3- conjugated chicken gamma globulin (Rockland) were prepared as previously described (29).

Techniques: Residue, Staining, Sequencing, Concentration Assay, Mutagenesis, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: γδ T cell antigen receptor polyspecificity enables T cell responses to a broad range of immune challenges.

doi: 10.1073/pnas.2315592121

Figure Lengend Snippet: Fig. 3. Commensal microbiota metabolite IPA maintains polyspecific innate-like Vγ6Vδ1 T cell homeostasis and response to infection. (A) Schematic of the experiment: C57BL/6 mice were treated with vancomycin, followed by WT or fldC mutant C. sporogenes colonization. Mice were either analyzed directly after bacteria colonization (C and D) or subjected to influenza virus infection and analyzed on day 3 after infection (I). (B) Plasma IPA level from mice of indicated groups as determined by liquid chromatography-mass spectrometry (LC–MS) measurement. Ctrl: mice without treatment, Vanco: mice treated with vancomycin, Vanco + C. sporogenes: mice treated with vancomycin followed by WT or fldC mutant C. sporogenes colonization. (C and D) A representative FACS plot of 17D1 (which identifies Vγ6Vδ 1 γδ T cells) staining of (C) lung and (D) peritoneal γδ T cells (Left), and the summary of total lung γδ T cell (Middle) and Vγ6Vδ1 (17D1+) γδ T cell (Right) numbers from vancomycin-treated mice after WT or fldC mutant C. sporogenes colonization. (E) Cell counts of γδ T cells in the lungs of C57BL/6 mice at indicated days after influenza virus infection. (F) A representative FACS plot of Ki67 staining (Left) and the summary of Ki67+ cell frequencies (Right) of lung γδ T cells from uninfected and influenza virus-infected mice at Day 3 after infection. (G) A representative FACS plot of Cy3 and HA staining of lung γδ T cells from uninfected and influenza virus-infected mice at Day 3 after infection (Left), PE-OVA and HA mutant staining of lung γδ T cells (Middle), and the frequencies of Cy3+HA+ cells among total γδ T cells in indicated groups (Right). Enriched lung γδ T cells were stained with Cy3-OVA (2 μg/mL), PE-OVA (2 μg/mL), HA coupled AF405-dextramer (1.35 μM), and HA mutant coupled AF405-dextramer (1.35 μM). (H) Cell counts of IL-17A-producing γδ T cells (Left) and the percentage of γδ T cells in total IL-17A+ cells (Right) in the lungs of C57BL/6 mice at indicated days after influenza virus infection. (I) A representative FACS plot of lung IL-17A+ γδ T cells (Left) and the summary of total γδ T and IL-17A+ γδ T cell numbers (Right) from mice treated as indicated in (A) followed by influenza virus infection for 3 d. (J) Cell counts of total lung γδ T cells (Left), and FACS analysis of IL-17A (Middle) and Ki67 (Right) staining of lung γδ T cells from isotype or anti-γδTCR (GL3) antibody treated TCRδ-GFP mice at day 3 after influenza virus infection. GFP+ cells were gated as γδ T cells. Results were shown as the mean ± SEM. Each data point represented the result from one mouse. The P values in B–D and I were determined using one-way ANOVA with Tukey’s multiple comparisons test. The P values in F, G, and J were determined using the Mann–Whitney test. The results were representative of at least 2 independent experiments.

Article Snippet: Cy3- conjugated ovalbumin (OVA) (Sigma) and Cy3- conjugated chicken gamma globulin (Rockland) were prepared as previously described (29).

Techniques: Infection, Mutagenesis, Bacteria, Virus, Clinical Proteomics, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Staining, MANN-WHITNEY