Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: Synapse engulfment was analyzed in the LGN from the same mouse EAE tissue (A-C,F, see also Figs. 2,​,3,3, and S2,3) and the same mouse DTA tissue (D,E,G, see also Figs. 2, and S5) where synapse loss was observed. (A-C) Representative immunofluorescence images and 3D-surface rendering of P2RY12+-microglia (green) containing engulfed (A) VGluT2+-retinogeniculate inputs (red, inserts), (B) VGluT1+corticothalamic inputs (red, inserts), or PSD-95+-postsynaptic compartments (red, inserts) within CD68-labeled microglial lysosomes (blue) in the EAE model. (Ai-Ci) Quantification of presynaptic inputs within microglial lysosomes in EAE vs. CFA control mice. (D,E) Representative confocal images and 3D rendering of P2RY12 (green), CD68 (blue) and (D) VGluT2 (red) or (E) VGluT1 (red) in the DTA model. (Di-Ei) Quantification of presynaptic inputs within microglial lysosomes in the DTA model. (F) Representative images and 3D-surface rendering of ALDH1L1+-astrocytes (green) and VGluT2+-retinogeniculate inputs (red, inserts) within LAMP2-labeled lysosomes (blue) in the LGN of EAE and CFA control mice. (Fi) Quantification of VGluT2 engulfment within lysosomes of reactive astrocytes in the EAE model. (G,H) Quantification of VGluT2+-retinogeniculate inputs within LAMP2+-lysosomes of ALDH1L1-labeled astrocytes in the (G) mouse DTA model and (H) in the marmoset EAE model. (A-C,F) n=4 mice per condition from one experiment, (D,E,G) n= 6 ctrls./5 DTA mice per condition from two experiments, (H) n=6 marmosets per condition from two experiments. (A-F) Scale bars, 10 μm. Data represent mean ± SEM, significant differences with ****P< 0.0001, t-test. See also Figs. S2,3, and S2,3,5.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Immunofluorescence, Labeling

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: (A) Timeline of in vivo AAV-rescue experiments. (B-N) Analyses of adjacent LGN sections of onset EAE mice shown in Fig. 6. (B-D) Quantification of (B) MOG, MAG and MBP, (C) NeuN+-neuron, and (D) neurofilament+-axon density following intraocular AAV injection in the EAE model. (E-G) Quantification of (E) microglia soma size, (F) density of P2RY12+-microglia and (G) Clec7a intensity AAV-treated mice in the EAE model. Further quantification of (H) GFAP+-astrocytes, (I) CD3+-T cells and (J) CD45+-leukocytes in AAV-treated mice in the EAE model. (K) Representative images and 3D-surface rendering of P2RY12+-microglia (green) and engulfed VGluT2+-retinogeniculate inputs (red) within CD68-labeled microglial lysosomes (blue). (Ki) Quantification of engulfed VGluT2+-presynaptic inputs (red, inserts) and (L) VGluT1+-presynaptic inputs within microglial lysosomes. (M,N) Representative confocal images of the LGN of AAV-EGFP and AAV-Crry treated EAE mice immunostained against presynaptic (M) VGluT2, or (N) VGluT1 and (Mi,Ni) respective quantification. (O) Visual acuity measured as spatial frequency threshold (cycles/degree) in the optomotor test before the induction of EAE (three left columns) and at the onset of clinical symptoms (three right columns) of the same control and AAV-EGFP or AAV-Crry transduced mice as shown in B-N. (B-J) n=4, (K-N) n=5, (O) n=5–8 mice from one experiment. Scale bars, (K) 10 μm, (M,N) 5 μm. Data represent mean ± SEM, significant differences with *P<0.05, **P<0.01, ***P <0.001, ****P < 0.0001, ANOVA with Tukey’s post hoc test. See also Figs. 6, and S7.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: In Vivo, Injection, Labeling

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Recombinant, Electron Microscopy, Clone Assay, Plasmid Preparation, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: (A,B) Coronal sections of the LGN from human controls (ctrl.) without neurological disease and from MS cases are shown. (A,Ai) Quantification of VGluT2+-retinogeniculate presynaptic input density (red) and (B,Bi) engulfment of VGluT2+-presynaptic inputs within (arrows) Iba1+-microglia/macrophages (green). (Ai) n=5 human subjects per condition. (Bi) n=3 ctrl./5 MS subjects. Scale bars, 10 μm. (C-F) Representative images of the LGN from non-EAE or EAE animals with no detectable lesions (control, no lesions) and marmosets that developed EAE with demyelinating lesions in the optic nerve and tract (EAE with ON/OT lesions). (Ci) Quantification of VGluT2+-input density (red) and (Di) engulfment of VGluT2+-presynaptic RGC inputs (Di) within Iba1+-cells (green) in the LGN from marmosets that developed ON/OT lesions following EAE compared to controls. (D) Inset shows phagocytic cups within Iba1+-cell (green), containing VGluT2+-retinogeniculate synaptic material (red). (E,F) Immunostaining and quantification of VGluT1+-corticothalamic presynaptic inputs (E) density and (F) engulfment within Iba1+-cells (arrows denote engulfed VGluT1+-corticothalamic inputs). (C-F) n=6 marmosets per condition from two experiments. Scale bars, 10 μm. Data represent mean ± SEM, significant differences with ***P< 0.001, ****P <0.0001, t-test. See also Fig. S1 & Table S1+2.

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Immunostaining

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary monoclonal (mAb) and polyclonal (pAb) antibodies have been used: mouse mAb α-ALDH1L1 (clone N103/39, Millipore, MABN495, 1:1000), mouse mAb α-APP (clone 22C11, Millipore, MAB348, 1:200), rabbit mAb α-C1q (clone 4.8, Abcam, ab182451, 1:100), rat mAb α-C3 (clone 11H-9, Abcam, ab11862, 1:500), rabbit pAb α-CASPR (provided by Matthew N. Rasband, 1:100), rabbit mAb α-CD3 (clone SP7, Abcam, ab16669, 1:100), mouse mAb α-CD8 (clone 8/114B, Thermo Fisher Scientific, MA5–13473, 1:20), rat mAb α-CD45 (clone IBL-3/16, BioRad, MCA1388, 1:100), mouse mAb α-CD68 (clone KP1, Abcam, ab955, 1:200), rat mAb α-CD68 (clone FA-11, AbD Serotec, MCA1957, 1:1000), rat mAb α-Clec7a (InvivoGen, mabg-mdect, 1:200), rabbit pAb α-Clec12a (LSBio, LS-C377776, 1:500), rabbit pAb α-cleaved caspase-3 (Cell Signaling Technologies, #9661, 1:200), mouse mAb α-Crry (clone TLD-1C11, Santa Cruz, sc-53530, 1:100), chicken pAb α-EGFP (Abcam, ab13970, 1:500), mouse mAb α-FoxP3 (clone 206D, BioLegend, 320102, 1:50), mouse mAb α-GFAP (clone G-A-5, Sigma, G3893, 1:500), rabbit pAb α-Homer1 (Synaptic Systems, #160003, 1:1000), rabbit pAb α-Iba1 (Wako Chemicals, #019–19741, 1:500), rat mAb α-LAMP2 (clone GL2A7, Abcam, ab13524, 1:200), rat mAb α-Ly6C (clone HK1.4, BioLegend, 128016, 1:300), mouse mAb α-MAG (clone 513, Millipore, MAB1567, 1:100), rat mAb α-MBP (clone 12, Millipore, MAB386, 1:500), mouse mAb α-MOG (clone 8–18C5, Millipore, MAB5680, 1:200), rabbit pAb α-Neurofilament 200 (Sigma, N4142, 1:1000), chicken pAb α-NeuN (Millipore, ABN91, 1:1000), rabbit pAb α-P2RY12 (AnaSpec, AS-55043A, 1:2000), rat mAb α-P2RY12 (clone S16007D, BioLegend, 848002, 1:100), mouse mAb α-PSD-95 (clone 6G6–1C9, Millipore, MAB1596, 1:100), guinea pig pAb α-RBPMS (PhosphoSolutions, 1832-RBPMS, 1:500), rabbit pAb α-βIV-spectrin (provided by Matthew N. Rasband, 1:100), guinea pig pAb α-VGluT1 (Millipore, ab5905, 1:2000), and guinea pig pAb α-VGluT2 (Millipore, ab2251, 1:2000).

Techniques: Recombinant, Electron Microscopy, Clone Assay, Plasmid Preparation, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: Recombinant, Electron Microscopy, Clone Assay, Plasmid Preparation, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: (A) Timeline of in vivo AAV-rescue experiments. (B-N) Analyses of adjacent LGN sections of onset EAE mice shown in Fig. 6. (B-D) Quantification of (B) MOG, MAG and MBP, (C) NeuN+-neuron, and (D) neurofilament+-axon density following intraocular AAV injection in the EAE model. (E-G) Quantification of (E) microglia soma size, (F) density of P2RY12+-microglia and (G) Clec7a intensity AAV-treated mice in the EAE model. Further quantification of (H) GFAP+-astrocytes, (I) CD3+-T cells and (J) CD45+-leukocytes in AAV-treated mice in the EAE model. (K) Representative images and 3D-surface rendering of P2RY12+-microglia (green) and engulfed VGluT2+-retinogeniculate inputs (red) within CD68-labeled microglial lysosomes (blue). (Ki) Quantification of engulfed VGluT2+-presynaptic inputs (red, inserts) and (L) VGluT1+-presynaptic inputs within microglial lysosomes. (M,N) Representative confocal images of the LGN of AAV-EGFP and AAV-Crry treated EAE mice immunostained against presynaptic (M) VGluT2, or (N) VGluT1 and (Mi,Ni) respective quantification. (O) Visual acuity measured as spatial frequency threshold (cycles/degree) in the optomotor test before the induction of EAE (three left columns) and at the onset of clinical symptoms (three right columns) of the same control and AAV-EGFP or AAV-Crry transduced mice as shown in B-N. (B-J) n=4, (K-N) n=5, (O) n=5–8 mice from one experiment. Scale bars, (K) 10 μm, (M,N) 5 μm. Data represent mean ± SEM, significant differences with *P<0.05, **P<0.01, ***P <0.001, ****P < 0.0001, ANOVA with Tukey’s post hoc test. See also Figs. 6, and S7.

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: In Vivo, Injection, Labeling, Control

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: Virus, Recombinant, Adjuvant, Electron Microscopy, Cloning, Plasmid Preparation, RNAscope, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: Synapse engulfment was analyzed in the LGN from the same mouse EAE tissue (A-C,F, see also Figs. 2,​,3,3, and S2,3) and the same mouse DTA tissue (D,E,G, see also Figs. 2, and S5) where synapse loss was observed. (A-C) Representative immunofluorescence images and 3D-surface rendering of P2RY12+-microglia (green) containing engulfed (A) VGluT2+-retinogeniculate inputs (red, inserts), (B) VGluT1+corticothalamic inputs (red, inserts), or PSD-95+-postsynaptic compartments (red, inserts) within CD68-labeled microglial lysosomes (blue) in the EAE model. (Ai-Ci) Quantification of presynaptic inputs within microglial lysosomes in EAE vs. CFA control mice. (D,E) Representative confocal images and 3D rendering of P2RY12 (green), CD68 (blue) and (D) VGluT2 (red) or (E) VGluT1 (red) in the DTA model. (Di-Ei) Quantification of presynaptic inputs within microglial lysosomes in the DTA model. (F) Representative images and 3D-surface rendering of ALDH1L1+-astrocytes (green) and VGluT2+-retinogeniculate inputs (red, inserts) within LAMP2-labeled lysosomes (blue) in the LGN of EAE and CFA control mice. (Fi) Quantification of VGluT2 engulfment within lysosomes of reactive astrocytes in the EAE model. (G,H) Quantification of VGluT2+-retinogeniculate inputs within LAMP2+-lysosomes of ALDH1L1-labeled astrocytes in the (G) mouse DTA model and (H) in the marmoset EAE model. (A-C,F) n=4 mice per condition from one experiment, (D,E,G) n= 6 ctrls./5 DTA mice per condition from two experiments, (H) n=6 marmosets per condition from two experiments. (A-F) Scale bars, 10 μm. Data represent mean ± SEM, significant differences with ****P< 0.0001, t-test. See also Figs. S2,3, and S2,3,5.

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: Immunofluorescence, Labeling, Control

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: (A) Timeline of in vivo AAV-rescue experiments. (B-N) Analyses of adjacent LGN sections of onset EAE mice shown in Fig. 6. (B-D) Quantification of (B) MOG, MAG and MBP, (C) NeuN+-neuron, and (D) neurofilament+-axon density following intraocular AAV injection in the EAE model. (E-G) Quantification of (E) microglia soma size, (F) density of P2RY12+-microglia and (G) Clec7a intensity AAV-treated mice in the EAE model. Further quantification of (H) GFAP+-astrocytes, (I) CD3+-T cells and (J) CD45+-leukocytes in AAV-treated mice in the EAE model. (K) Representative images and 3D-surface rendering of P2RY12+-microglia (green) and engulfed VGluT2+-retinogeniculate inputs (red) within CD68-labeled microglial lysosomes (blue). (Ki) Quantification of engulfed VGluT2+-presynaptic inputs (red, inserts) and (L) VGluT1+-presynaptic inputs within microglial lysosomes. (M,N) Representative confocal images of the LGN of AAV-EGFP and AAV-Crry treated EAE mice immunostained against presynaptic (M) VGluT2, or (N) VGluT1 and (Mi,Ni) respective quantification. (O) Visual acuity measured as spatial frequency threshold (cycles/degree) in the optomotor test before the induction of EAE (three left columns) and at the onset of clinical symptoms (three right columns) of the same control and AAV-EGFP or AAV-Crry transduced mice as shown in B-N. (B-J) n=4, (K-N) n=5, (O) n=5–8 mice from one experiment. Scale bars, (K) 10 μm, (M,N) 5 μm. Data represent mean ± SEM, significant differences with *P<0.05, **P<0.01, ***P <0.001, ****P < 0.0001, ANOVA with Tukey’s post hoc test. See also Figs. 6, and S7.

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: In Vivo, Injection, Labeling, Control

Journal: Immunity

Article Title: Targeted complement inhibition at synapses prevents microglial synaptic engulfment and synapse loss in demyelinating disease

doi: 10.1016/j.immuni.2019.12.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TX, USA N/A Rabbit mAb α-CD3 (clone SP7) Abcam Cat. #ab16669 Mouse mAb α-CD8 (clone 8/114B) Thermo Fisher Scientific Cat. #MA5–13473 Rat mAb α-CD45 (clone IBL-3/16) BioRad Cat. #MCA1388 Mouse mAb α-CD68 (clone KP1) Abcam Cat. #ab955 Rat mAb α-CD68 (clone FA-11) AbD Serotec Cat. #MCA1957 Rat mAb α-Clec7a InvivoGen Cat. #mabg-mdect Rabbit pAb α-Clec12a a-Clec12a LSBio Cat. #LS-C377776 Rabbit pAb α-cleaved caspase-3 Cell Signaling Technologies Cat. #9661 Mouse mAb α-Crry (clone TLD-1C11) Santa Cruz Cat. #sc-53530 Chicken pAb α-EGFP Abcam Cat. #ab13970 Rabbit pAb α-EGFP Millipore Cat. #ab3080P Mouse mAb α-FoxP3 (clone 206D) BioLegend Cat. #320102 Goat pAb α-GAPDH Abcam Cat. #ab9483 Mouse mAb α-GFAP (clone G-A-5 Sigma Cat. #G3893 Rabbit pAb α-Homer1 Synaptic Systems Cat. #160003 Rabbit pAb α-Iba1 Wako Chemicals Cat. #019–19741 Goat pAb α-Iba1 (human/marmoset tissue) Abcam Cat. #ab5076 Rat mAb α-LAMP2 (clone GL2A7) Abcam Cat. #ab13524 Rat mAb α-Ly6C (clone HK1.4) BioLegend Cat. #128016 Mouse mAb α-MAG (clone 513) Millipore Cat. #MAB1567 Chicken pAb α-MAP2 (marmoset tissue) EnCor Biotechnology Cat. #CPCA-MAP2 Rat mAb α-MBP (clone 12) Millipore Cat. #MAB386 Mouse mAb α-MOG (clone 8–18C5 Millipore Cat. #MAB5680 Rabbit pAb α-Neurofilament 200 Sigma Cat. #N4142 Chicken pAb α-NeuN Millipore Cat. #ABN91 Rabbit pAb α-P2RY12 AnaSpec Cat. #AS-55043A Rat mAb α-P2RY12 (clone S16007D) BioLegend Cat. #848002 Mouse mAb α-PSD-95 (clone 6G6–1C9) Millipore Cat. #MAB1596 Guinea pig pAb α-RBPMS PhosphoSolutions Cat. #1832-RBPMS Rabbit pAb α-βIV-spectrin Matthew N. Rasband, Baylor College of Medicine, Houston.

Techniques: Virus, Recombinant, Adjuvant, Electron Microscopy, Cloning, Plasmid Preparation, RNAscope, Multiplex Assay, In Situ, Sequencing, Generated, Software, Imaging

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Orthotopic CT26-FL3 tumor is resistant to anti-PD-L1 mAb therapy. a Establishment of orthotopic colorectal tumor models. CT26-FL3 (RFP/Luc) cells were inoculated into the mouse cecum wall, and the tumor burden was monitored by bioluminescent analysis. b Treatment scheme and tumor growth curves of orthotopic CT26-FL3 tumors in PBS and α-PD-L1 treated groups ( n = 5 mice per group). c Masson’s trichrome and immunofluorescence staining of orthotopic CT26-FL3 tumor tissues in PBS group using 4′,6-diamidino-2-phenylindole (DAPI, blue) and anti-CD3 antibody (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. Significant differences were assessed in b using two-way ANOVA. Results are presented as mean (SD). ns, not significant

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP induces immune microenvironment changes and synergizes with α-PD-L1 in CT26-FL3 tumor therapy. a CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of the PBS and OxP-treated groups on day 28, analyzed by flow cytometry ( n = 4). b Relative mRNA expressions of various cytokines in tumors of the OxP-treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). c Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP treatment using DAPI (blue), anti-CD3 (red), anti-CD11c (red), anti-CD274 (red), and anti-IL-10 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. d Treatment scheme and tumor growth curves of orthotopic CT26-FL3 tumors in PBS, α-PD-L1, OxP, and OxP + α-PD-L1 treated groups ( n = 5 mice per group). e Th17 cell ratios in the splenocytes of the mice after various treatments on day 28 ( n = 4). Significant differences were assessed in d using two-way ANOVA with multiple comparisons and in a , b , and e using t test. Results are presented as mean (SD). ns not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Design of locally and transiently expressed PD-L1 trap. a Scheme showing the tribody interaction of PD-L1 trap protein. b Preparation scheme of PD-L1 trap plasmid loaded LPD. c Images and quantitative results of the DiI-loaded LPD in major organs and the CT26-FL3 tumor at 24 h after injection ( n = 3). d PD-L1 trap protein expression in major organs and the CT26-FL3 tumor at 48 h after injection. The PD-L1 trap expression was measured using ELISA by targeting the His (6×)-tag engineered at the C-terminus of the PD-L1 trap ( n = 3). e PD-L1 trap protein expression in tumors on days 1, 2, 4 and 7 after injection. Significant differences in c , d , and e were assessed using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Plasmid Preparation, Injection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Combination of OxP and LPD-PD-L1 trap gene therapy on orthotopic CT26-FL3 tumor model. a OxP and PD-L1 trap combination treatment scheme. b Tumor growth curves of orthotopic CT26-FL3 tumors in PBS, LPD-pGFP, PD-L1 trap, OxP and OxP+PD-L1 trap gene treated groups ( n = 5 mice per group). c TSR% results on day 35. d Mice survival curves. e CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of mice after various treatments, analyzed by flow cytometry ( n = 4). f Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP+PD-L1 trap treatment using DAPI (blue) and anti-CD3 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. g Relative mRNA expressions of cytokines in tumors of the OxP+PD-L1 trap treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). h Th17 cell ratios in the splenocytes of mice after various treatments on day 28 ( n = 4). Significant differences were assessed in b using two-way ANOVA with multiple comparisons, in d using log rank test and in c , e , g , and h using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP and LPD-PD-L1 trap gene therapy on B16F10 and 4T1 tumor models. a Treatment scheme and tumor growth curves of B16F10 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). b Treatment scheme and tumor growth curves of 4T1 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). c CD4 + and CD8 + T cell ratios in the B16F10 tumors after various treatments, analyzed by flow cytometry ( n = 4). d CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the B16F10 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). e CD4 + and CD8 + T cell ratios in the 4T1 tumors after various treatments, analyzed by flow cytometry ( n = 4). f CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the 4T1 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). Significant differences were assessed in a and b using two-way ANOVA with multiple comparisons and in c , d , e , and f using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP induces immune microenvironment changes and synergizes with α-PD-L1 in CT26-FL3 tumor therapy. a CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of the PBS and OxP-treated groups on day 28, analyzed by flow cytometry ( n = 4). b Relative mRNA expressions of various cytokines in tumors of the OxP-treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). c Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP treatment using DAPI (blue), anti-CD3 (red), anti-CD11c (red), anti-CD274 (red), and anti-IL-10 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. d Treatment scheme and tumor growth curves of orthotopic CT26-FL3 tumors in PBS, α-PD-L1, OxP, and OxP + α-PD-L1 treated groups ( n = 5 mice per group). e Th17 cell ratios in the splenocytes of the mice after various treatments on day 28 ( n = 4). Significant differences were assessed in d using two-way ANOVA with multiple comparisons and in a , b , and e using t test. Results are presented as mean (SD). ns not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Combination of OxP and LPD-PD-L1 trap gene therapy on orthotopic CT26-FL3 tumor model. a OxP and PD-L1 trap combination treatment scheme. b Tumor growth curves of orthotopic CT26-FL3 tumors in PBS, LPD-pGFP, PD-L1 trap, OxP and OxP+PD-L1 trap gene treated groups ( n = 5 mice per group). c TSR% results on day 35. d Mice survival curves. e CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of mice after various treatments, analyzed by flow cytometry ( n = 4). f Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP+PD-L1 trap treatment using DAPI (blue) and anti-CD3 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. g Relative mRNA expressions of cytokines in tumors of the OxP+PD-L1 trap treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). h Th17 cell ratios in the splenocytes of mice after various treatments on day 28 ( n = 4). Significant differences were assessed in b using two-way ANOVA with multiple comparisons, in d using log rank test and in c , e , g , and h using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP and LPD-PD-L1 trap gene therapy on B16F10 and 4T1 tumor models. a Treatment scheme and tumor growth curves of B16F10 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). b Treatment scheme and tumor growth curves of 4T1 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). c CD4 + and CD8 + T cell ratios in the B16F10 tumors after various treatments, analyzed by flow cytometry ( n = 4). d CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the B16F10 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). e CD4 + and CD8 + T cell ratios in the 4T1 tumors after various treatments, analyzed by flow cytometry ( n = 4). f CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the 4T1 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). Significant differences were assessed in a and b using two-way ANOVA with multiple comparisons and in c , d , e , and f using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP induces immune microenvironment changes and synergizes with α-PD-L1 in CT26-FL3 tumor therapy. a CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of the PBS and OxP-treated groups on day 28, analyzed by flow cytometry ( n = 4). b Relative mRNA expressions of various cytokines in tumors of the OxP-treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). c Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP treatment using DAPI (blue), anti-CD3 (red), anti-CD11c (red), anti-CD274 (red), and anti-IL-10 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. d Treatment scheme and tumor growth curves of orthotopic CT26-FL3 tumors in PBS, α-PD-L1, OxP, and OxP + α-PD-L1 treated groups ( n = 5 mice per group). e Th17 cell ratios in the splenocytes of the mice after various treatments on day 28 ( n = 4). Significant differences were assessed in d using two-way ANOVA with multiple comparisons and in a , b , and e using t test. Results are presented as mean (SD). ns not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: Combination of OxP and LPD-PD-L1 trap gene therapy on orthotopic CT26-FL3 tumor model. a OxP and PD-L1 trap combination treatment scheme. b Tumor growth curves of orthotopic CT26-FL3 tumors in PBS, LPD-pGFP, PD-L1 trap, OxP and OxP+PD-L1 trap gene treated groups ( n = 5 mice per group). c TSR% results on day 35. d Mice survival curves. e CD8 + T cells, CD4 + T cells, activated DCs and PD-L1 levels in tumors of mice after various treatments, analyzed by flow cytometry ( n = 4). f Masson’s trichrome and immunofluorescence staining of the orthotopic tumors after OxP+PD-L1 trap treatment using DAPI (blue) and anti-CD3 (red). Yellow dotted line indicates the border between intestinal mucosa and the orthotopic tumor. Scale bar represents 50 μm. g Relative mRNA expressions of cytokines in tumors of the OxP+PD-L1 trap treated group compared to PBS group on day 28, detected by quantitative RT-PCR ( n = 4). h Th17 cell ratios in the splenocytes of mice after various treatments on day 28 ( n = 4). Significant differences were assessed in b using two-way ANOVA with multiple comparisons, in d using log rank test and in c , e , g , and h using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Synergistic and low adverse effect cancer immunotherapy by immunogenic chemotherapy and locally expressed PD-L1 trap

doi: 10.1038/s41467-018-04605-x

Figure Lengend Snippet: OxP and LPD-PD-L1 trap gene therapy on B16F10 and 4T1 tumor models. a Treatment scheme and tumor growth curves of B16F10 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). b Treatment scheme and tumor growth curves of 4T1 tumors in PBS, PD-L1 trap, OxP and OxP + PD-L1 trap treated groups ( n = 5 mice per group). c CD4 + and CD8 + T cell ratios in the B16F10 tumors after various treatments, analyzed by flow cytometry ( n = 4). d CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the B16F10 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). e CD4 + and CD8 + T cell ratios in the 4T1 tumors after various treatments, analyzed by flow cytometry ( n = 4). f CD4 + T cell, CD8 + T cell and Th17 cell ratios in splenocytes of the 4T1 tumor-bearing mice after various treatments, analyzed by flow cytometry ( n = 4). Significant differences were assessed in a and b using two-way ANOVA with multiple comparisons and in c , d , e , and f using t test. Results are presented as mean (SD). ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PBS, OxP (6.0 mg kg -1 , i.p.), anti-mouse PD-L1 mAb (α-PD-L1, Bioxcell, clone 10 F.9G2, 100 μg per mouse, i.p.), LPD-GFP plasmid (pGFP, 50 μg plasmid per mouse, i.v.), LPD-PD-L1 trap plasmid (PD-L1 trap, 50 μg plasmid per mouse, i.v.), OxP + α-PD-L1, OxP + PD-L1 trap, anti-mouse CD8α (α-CD8, Bioxcell, clone 53-6.72, 200 μg per mouse, i.p.) or anti-mouse CD4 (α-CD4, Bioxcell, clone GK1.5, 200 μg per mouse, i.p.) were given at respective schedules.

Techniques: Flow Cytometry