fluorescence lifetime imaging microscopy  (Purdue University Cytometry)

Journal: Science immunology

Article Title: Transmembrane domain–driven PD-1 dimers mediate T cell inhibition

doi: 10.1126/sciimmunol.ade6256

Figure Lengend Snippet: (A to C) Thy1.1 and PD-1 expression on reconstituted PD-1−/− OT1 CD8+ T cells after RV transduction with the indicated vector. (D to F) B16-OVA tumor growth in WT C57BL/6 mice after adoptive transfer with OT1 T cells retrovirally transduced as in (A) to (C). (D) Spider chart of tumor growth in individual mice with no T cell transfer (gray), PD-1–WT reconstituted OT1 cells (purple), or PD-1−/− EV transduced OT1 cells (black). Spider chart of tumor growth with PD-1–G172W reconstituted OT1 cells (blue) or PD-1–L186W reconstituted OT1 cells (orange). (F) Cumulative B16-OVA tumor growth as in (D) and (E) with SEM indicated by transparent shading. Representative experiment with five mice in the vehicle group and seven mice in the EV, mPD-1–WT, mPD-1–G172W, and mPD-1–L186W groups. Significance was tested by two-way ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Fluorescence lifetime imaging microscopy–FRET CD8a, CD80, CD86, PD-1, PD-1–V173W, and PD-1–L184W ECDs were cloned into the monomeric enhanced green fluorescent protein (mEGFP)–N1 and mCherry2-N1 plasmids (Addgene plasmids #54767 and #54517) using the Xho I and Hind III restriction sites.

Techniques: Expressing, Transduction, Plasmid Preparation, Adoptive Transfer Assay

Journal: Science immunology

Article Title: Transmembrane domain–driven PD-1 dimers mediate T cell inhibition

doi: 10.1126/sciimmunol.ade6256

Figure Lengend Snippet: (A) FRET efficiency calculated from FLIM of the indicated mEGFP, mCherry2 fusion protein pairs expressed in baby hamster kidney cells (see fig. S6). (B) FCCS measurements of the indicated mEGFP, mCherry2 fusion protein pairs expressed in HEK293T cells (see fig. S7). Significance tested by unpaired t tests: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns, not significant.

Article Snippet: Fluorescence lifetime imaging microscopy–FRET CD8a, CD80, CD86, PD-1, PD-1–V173W, and PD-1–L184W ECDs were cloned into the monomeric enhanced green fluorescent protein (mEGFP)–N1 and mCherry2-N1 plasmids (Addgene plasmids #54767 and #54517) using the Xho I and Hind III restriction sites.

Techniques:

Journal: Science immunology

Article Title: Transmembrane domain–driven PD-1 dimers mediate T cell inhibition

doi: 10.1126/sciimmunol.ade6256

Figure Lengend Snippet: (A) The effects on dimerization as determined by TOXGREEN of Trp substitutions at the indicated positions in the hPD-1 TMD. Bars were assigned a color on the basis of the log2 scaled look-up table (right). (B) Helical model of the hPD-1 TMD color-coded as in (A) with the V173hPD-1 low and L184hPD-1 high Trp substitutions labeled. (C) FRET efficiency calculated from FLIM of the indicated pairs of mEGFP- and mCherry2-tagged constructs expressed in baby hamster kidney cells. (D) FCCS measurements of mEGFP- and mCherry2-tagged constructs expressed in HEK293T cells pooled across four biological replicates. (E and F) Analysis and color-coding of the mPD-1 TMD as in (A) and (B) revealing G172mPD-1 low and L186mPD-1 high Trp substitutions. Significance in (A), (C), and (E) was determined by unpaired t tests; significance in (D) was determined by ANOVA test with Tukey’s correction for multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Fluorescence lifetime imaging microscopy–FRET CD8a, CD80, CD86, PD-1, PD-1–V173W, and PD-1–L184W ECDs were cloned into the monomeric enhanced green fluorescent protein (mEGFP)–N1 and mCherry2-N1 plasmids (Addgene plasmids #54767 and #54517) using the Xho I and Hind III restriction sites.

Techniques: Labeling, Construct