Journal: Brain, behavior, and immunity

Article Title: Functional circuitry of neuro-immune communication in the mesenteric lymph node and spleen.

doi: 10.1016/j.bbi.2019.08.188

Figure Lengend Snippet: Confocal microscopy was conducted on tissue sections ChAT-GFP (C57BL/6 background) mice for T-cells (anti-CD3), ChAT (anti-GFP), and sympathetic axons (anti-TH). The section was identified and overlapping tiles were acquired from 943 µm x 454 µm area of MLN (upper panel, scale bar =100 µm). Areas indicated in the bounding boxes are shown in the middle (CD3, ChAT-GFP, TH) and lower panels (ChAT-GFP, TH, scale bar = 20 μm). Region of the hilus in the MLN is within box “i”. Representative images obtained from 8 mice.

Article Snippet: Primary antibodies used in this study were rabbit Anti-TH (Millipore, AB152, Billerica, MA), goat anti-GFP (Rockland Immunochemicals, Limerick, PA), rabbit anti-TNF (Abeam, AB6671) and rat anti-CD3 (clone CD3-12, Bio-Rad, Hercules, CA).

Techniques: Confocal Microscopy

Journal: Molecular Medicine Reports

Article Title: Itgb3-integrin-deficient mice may not be a sufficient model for patients with Glanzmann thrombasthenia

doi: 10.3892/mmr.2021.12088

Figure Lengend Snippet: Spleen extramedullary hematopoiesis and iron deficiency of Itgb3 −/− mice. (A) Relative ratio of each organ to whole body weight in different genotype mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. (B) Relative ratio of spleen to whole body weight in Itgb3 −/− , Itgb3 +/− , Itgb3 +/+ and CHM mice. *P<0.01 vs. Itgb3 +/+ mice; ✩ P<0.01 vs. Itgb3 +/− mice. Immunohistochemical study of spleen biopsy from Itgb3 +/+ , Itgb3 −/− mice. Anti-CD3 antibody was used to label T lymphocytes, anti-CD19 antibody to label B lymphocytes and anti-CD71 antibody to label erythrocytes. Magnification (C) ×100 and (D) ×600. (E) Concentration of plasma ferritin in Itgb3 −/− mice. (F) OD value of fecal occult blood in Itgb3 −/− mice using ELISA. CHM, chronic hemorrhagic model; H&E, hematoxylin and eosin; Itgb3, integrin β3; OD, optical density; ns, not significant.

Article Snippet: Rabbit anti-CD3 monoclonal antibody (cat. no. MAB4841) was purchased from R&D Systems China Co., Ltd. and rat anti-transferrin R (CD71) monoclonal antibody (8D3; cat. no. NB 100-64979-0.05mg) was purchased from Novus Biologicals, LLC.

Techniques: Immunohistochemical staining, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: Gain-of-function mutations in β TMR (A) CD3 surface expression of 1G4-WT and mutants. x̄ ± SD of CD3 + cells, n = 3–8. Ala (red) and Leu (blue) substitution. (B) pErk response of 1G4-WT and β mutants stimulated with (6V-A2) 4 . x̄ ± SEM of pErk + cells; n = 3–6; unpaired t test, p = 0.0011 (βA290), p = 0.0092 (βA291). (C) pErk response of CD8 − J76 1G4-WT and 1G4-βA291 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.82 (WT), 0.89 (βA291); EC 50 = 19.5 ± 5.5 (WT), 19.0 ± 3.1 (βA291). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.0001. See also Figure S1 E. (D) (6V-A2) 4 binding to 1G4-WT or 1G4-βA291. Top, (6V-A2) 4 dose-dependent association, n = 3, non-linear regression fit, R 2 = 0.98 (WT), 0.97 (βA291), F-test (ns). Bottom, (6V-A2) 4 dissociation rate, n = 5, non-linear regression fit, R 2 = 0.84 (WT), 0.72 (βA291), F-test (ns). (E) pζ response of J76 1G4-WT or 1G4-βA291 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 MFI versus pζ MFI, n = 3, R 2 = 0.95 (WT), 0.96 (βA291). Right, x̄ ± SD of max. pζ, n = 4, unpaired t test p = 0.0078. See also Figure S1 F. (F) Basal pζ in J76 1G4-WT or 1G4-βA291. pζ MFI normalized to surface CD3 MFI, n = 8, unpaired t test p < 0.0001. See also H and S1I. (G) FRAP of 1G4-WT or 1G4-βA291 treated (right) or not (left) with A770041. x̄ ± SD of diffusion coefficient, D (μm 2 /s), n ≥ 20 cells, t test (ns). (H) Lateral distribution by dSTORM of 1G4-WT or 1G4-βA291 treated (right) or not (left) with A770041. Plots represent pair auto-correlation analysis (g), x̄ ± SD of ≥ 25 cells. Histograms show DBSCAN cluster analysis, x̄ ± SD of cluster size per cell, t test (ns).

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques: Expressing, Binding Assay, Diffusion-based Assay

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: βY291 contribution to TCR-CD3 quaternary structure cohesion (A) Anti-HA (β-HA) pull-down (PD), and IB of 1G4-WT or 1G4-βA291. Top panels: left, representative IB, arrows indicate β-isoforms; right, x̄ ± SD of ε/β T and ζ/β 2 , n = 5, unpaired t test p < 0.0001. Bottom panels: input lysates, left, representative IB; right, x̄ ± SD of β T /actin, ε/actin and ζ/actin, n = 5, unpaired t test (ns). (B) β-HA PD and IB of 1G4-WT or 1G4-βA291. NR (non-reducing) conditions. Left, representative IB. Right, x̄ ± SD of γ/β T , n = 4, unpaired t test p < 0.0001. (C) β-HA PD and IB of 1G4-WT or 1G4-βA291. NR conditions. Left, representative IB. Right, x̄ ± SD of δ/β T , n = 4, unpaired t test p < 0.0001. (D) All-atom MDS of TCR-CD3 TMRs. TCRα (ochre), TCRβ (gray), CD3δ (blue), CD3ε (yellow), CD3γ (green), ζ (red). Left, snapshot of TCR-CD3 TMRs. Right, βY291 interactions with TCR-CD3 TMRs. Liquorice sticks show significant contacts of βY291 with TCRα, CD3γ, and CD3ε. See also Figure S2 H.

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques:

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: Loosening ζ association enhances signaling (A) TCR-CD3 expression of 1G4-WT, 1G4-βA291, 1G4-βL291, and 1G4-βF291 in CD8 − J76. Left, x̄ ± SEM of CD3 MFI in HA low gate, n = 3, unpaired t test p < 0.0001. Right, x̄ ± SEM of β-HA MFI in HA low gate, n = 3, t test (ns). (B) β-HA PD and IB of 1G4-WT or 1G4-β mutants. Left, representative IB, the arrow indicates β 2 isoform. Middle, x̄ ± SD of ζ/β 2 , n = 3, unpaired t test WT versus βA291, WT versus βL291 p < 0.0001, WT versus βF291 p < 0.01. Right, x̄ ± SD of ε/β T , n = 3, unpaired t test p < 0.0001. (C) pErk response of CD8 − J76 1G4-WT or 1G4-βF291 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.915 (WT), 0.910 (βF291). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.05. See also Figure S3 A (left). (D) pErk response of CD8 − J76 1G4-WT or 1G4-βL291 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.827 (WT), 0.910 (βL291). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.0001. See also Figure S3 A (right). (E) TCR-CD3 expression in CD8 − J76 2H5-WT or 2H5-βA291. Left, x̄ ± SEM of CD3 MFI in HA low gate, n = 3, unpaired t test p < 0.0001. Right, x̄ ± SEM of β-HA MFI in HA low gate, n = 3, t test (ns). (F) β-HA PD and IB of 2H5-WT or 2H5-βA291. Left, representative IB. Middle, x̄ ± SD of ζ/β, n = 3, unpaired t test p < 0.0001. Right, x̄ ± SD of ε/β, n = 3, unpaired t test p < 0.01. (G) pErk response of CD8 − J76 2H5-WT or 2H5-βA291 stimulated with (MART-1-A2) 4 . Left, non-linear regression fit of (MART-1-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.94 (WT), 0.94 (βA291). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.001. See also Figure S3 B.

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques: Expressing

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: Loosening ζ association enhances signaling (A) Snapshot from all-atom MDS of TCR-CD3 TMRs. Contacts between TCRβ (gray), ζ 1 (light red), and ζ 2 (dark red) TMRs. See also Figure S4 A. βY291 is represented as liquorice stick for reference and does not contact ζζ. (B) Snapshot from all-atom MDS of TCR-CD3 TMRs. Left, ζ 1 (light red) and ζ 2 (dark red) residues contacting TCRα TMR (in transparency). Right, TCRα (ochre) residues contacting ζ 1 and ζ 2 TMRs (in transparency). See also Figure S4 B. (C) Snapshot from all-atom MDS of TCR-CD3 TMRs. Left, contacts between ζ 2 (red) and CD3γ (green) TMRs. Right, top view of ζ 2 I41 (red) contacts with CD3γ (green). See also C and S4F. (D) Snapshot from all-atom MDS of TCR-CD3 TMRs. Left, contacts between ζ 1 (red) and CD3ε (δε) (yellow) TMRs. Right, top view of ζ 1 I38 (red) contacts with CD3ε (δε) (yellow). See also D and S4E. (E) Snapshot from all-atom MDS of TCR-CD3 TMRs carrying ζA38 (lines) aligned to ζWT (cartoon) at the end of 1,250 ns MDS. TCRα (ochre), TCRβ (gray), CD3δ (blue), CD3ε (yellow), CD3γ (green), ζ (red). See also G–S4J. (F) Normalized spatial distributions of the Cα atoms of ζζ relative to the Cα atoms of TCRαβ in ζWT and ζA38. (G) Snapshot from all-atom MDS of TCR-CD3 TMRs carrying ζA41 (lines) aligned to ζWT (cartoon) at the end of 1,250 ns MDS. TCRα (ochre), TCRβ (gray), CD3δ (blue), CD3ε (yellow), CD3γ (green), ζ (red). See also K–S4N. (H) Normalized spatial distributions of the Cα atoms of ζζ relative to the Cα atoms of TCRαβ in ζWT and ζA41.

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques:

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: Loosening ζ association enhances signaling (A) TCR-CD3 expression in J76-1G4WT-ζKO expressing ζWT or ζA38 or ζA41. Left, x̄ ± SEM of CD3 MFI in HA low gate, n = 3, unpaired t test p < 0.0001. Middle, representative IB. Right, x̄ ± SD of ζ/actin, n = 3, unpaired t test (ns). (B) β-HA PD and IB of 1G4-WT carrying ζWT or ζA38 or ζA41. Left, representative IB, the arrow indicates β 2 isoform. Middle, x̄ ± SD of ε/β T , n = 4, unpaired t test (ns). Right, x̄ ± SD of ζ/β 2 , n = 4, unpaired t test p < 0.0001. See also Figure S5 A. (C) pErk response of J76-1G4WT-ζKO expressing ζWT or ζA38 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.98 (WT), 0.99 (ζA38). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.0001. See also Figure S5 B. (D) pErk response of J76-1G4WT-ζKO expressing ζWT or ζA41 stimulated with (6V-A2) 4 . Left, non-linear regression fit of (6V-A2) 4 nM versus pErk MFI, n = 3, R 2 = 0.96 (WT), 0.97 (ζA41). Right, x̄ ± SD of max. pErk, n = 3, F-test p < 0.0001. See also Figure S5 C.

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques: Expressing

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet: Monovalent pMHC in solution triggers TCR-CD3 untying and intracellular signaling (A) J76 wtc51 ± A770041 stimulated or not with sm-9V-A2 were lysed and subjected to PD with anti-HA Ab or Talon beads. First panel, anti-HA (β-HA) (lanes 1, 3, 5) or Talon beads (His) (lanes 2, 4, 6) PD and IB for β and ζ. The arrow indicates β 2 isoform. Second panel, x̄ ± SD of ζ/β 2 , n ≥ 3, unpaired t test p < 0.0001 and p < 0.01. Third panel, representative pErk IB. Fourth panel, x̄ ± SD of pErk, n ≥ 3, unpaired t test p < 0.0001. (B) CD8 − J76 wtc51 stimulated or not with sm-9V-A2 were processes as in (A). First panel, β-HA (lanes 1, 3) or His (lanes 2, 4) PD and IB for β and ζ. The arrow indicates β 2 isoform. Second panel, x̄ ± SD of ζ/β 2 , n = 3, unpaired t test p < 0.01. Third panel, representative pErk IB. Fourth panel, x̄ ± SD of pErk, n = 3, unpaired t test p < 0.05. (C) J76 868 stimulated or not with sm-6I-A2 were processed as in (A). First panel, β-HA (lanes 1, 3) or His (lanes 2, 4) PD and IB for β and ζ. The arrow indicates β 2 isoform. Second panel, x̄ ± SD of ζ/β 2 , n = 3, unpaired t test p < 0.01. Third panel, representative pErk IB. Fourth panel, x̄ ± SD of pErk, n = 3, unpaired t test p < 0.0001. (D) CD8 − J76 868 stimulated or not with sm-6I-A2. Left, representative pErk IB. Right, x̄ ± SD of pErk, n = 3, unpaired t test p < 0.05. (E) Primary T cells expressing wtc51 stimulated or not with sm-9V-A2 were processed as in (A). First panel, β-HA (lanes 1, 3) or His (lanes 2, 4) PD and IB for β and ζ. The arrow indicates β 2 isoform. Second panel, x̄ ± SEM of ζ/β 2 , n = 2, unpaired t test p < 0.05. Third panel, representative pErk IB. Fourth panel, x̄ ± SEM of pErk, n = 2, unpaired t test p < 0.0001. (F) J76 wtc51 were lysed, incubated or not with sm-9V-A2, and subjected to PD by anti-HA or Talon beads. Left, β-HA (lanes 1, 3) or His (lanes 2, 4) PD and IB for β and ζ. The arrow indicates β 2 isoform. Right, x̄ ± SD of ζ/β 2 , n = 5, unpaired t test p < 0.0001.

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques: Expressing, Incubation

Journal: Cell Reports

Article Title: Allosteric activation of T cell antigen receptor signaling by quaternary structure relaxation

doi: 10.1016/j.celrep.2021.109375

Figure Lengend Snippet:

Article Snippet: Rat anti-human CD3ε mAb , Cell Signaling Technology , Cat# 4443, RRID: AB_560945.

Techniques: Virus, Recombinant, Protein Extraction, Cell Isolation, Saline, Suspension, Sterility, Expressing, Software