Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule CD73 Is Critical in Renal Ischemia-Reperfusion Injury Protection

doi: 10.1681/ASN.2016020229

Figure Lengend Snippet: Nonhematopoietic CD73 is necessary for protection from IRI. Bone marrow-reconstituted chimeric mice were subjected to sham surgery or 22 minutes of ischemia followed by 24 hours of reperfusion. (A) Plasma creatinine values. (B and C) The percentage of outer medulla with (B) tubular damage scored in (C) hematoxylin and eosin-stained kidney sections. Chimeric combinations and sham versus IRI were compared in two-way ANOVA. Scale bars, 100 μm in C, left panel; 50 μm in C, right panel. (A–C) Values are shown for each individual mouse along with mean and SD (n≥4). (D) Confocal microscopy of the renal outer medulla showing CD73 localization and neutrophil (PMN) and macrophage infiltration after IRI. Parenchymal cells: PDGFR-β and collectrin are interstitial fibroblast-like cell and proximal tubular epithelial cell markers, respectively. CD73+ proximal epithelial cells (CD73 and collectrin colocalization) and fibroblast-like cells (CD73 and PDGFR-β colocalization) appear magenta and yellow, respectively. Myeloid cells: Infiltrating PMNs are Ly6G+ and CD11b+ (colocalization appears as aqua; arrows), F4/80 (red), and CD11b stain macrophages (colocalization appears as magenta). Staining of kidneys with WT (a,c) and CD73−/− (b,d) cells as donors and WT (a,b) and CD73−/− (c,d) mice as recipients are shown. Scale bars, 50 μm in c, column 1; 10 μm in columns 2 and 4. (E) Infiltrating Ly6C+Ly6G+ PMNs in kidney after IRI were quantified by flow cytometry for each chimera. The number next to the gate (outlined with a box) is PMNs as a percentage (10%–26%) of total cells in the plot of one representative mouse. The chimeric mice analyzed are designated as in (D). (F) PMN numbers from mice in each group (from three to four mice per group) calculated from flow cytometry. Cort, cortex; im, inner medulla; I, subthreshold ischemia treatment; S, sham. **P<0.01; ***P<0.001; ****P<0.001.

Article Snippet: Mice were injected intraperitoneally on day 0 with 80 μ g GR1 rat anti–mouse Ly6G/C mAb (Bioxcell, West Lebanon, NH).

Techniques: Staining, Confocal Microscopy, Flow Cytometry

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule CD73 Is Critical in Renal Ischemia-Reperfusion Injury Protection

doi: 10.1681/ASN.2016020229

Figure Lengend Snippet: Proximal tubule CD73 expression protects mice from IRI. Mice with CD73 deletion in proximal tubules (PEPCKCreCD73f/f) or CD11c-expressing cells (CD11cCreCD73f/f) or with global deletion (CD73−/−) were exposed to 22-minute kidney ischemia and 24-hour reperfusion. (A) Plasma creatinine. *P<0.05; ****P<0.001. (B) The percentage of outer medulla with tubular damage scored from outer medulla of (C) hematoxylin and eosin–stained kidney sections. Results from two representative experiments were scored. ****P<0.001. (A and B) Individual values and means±SD are shown. Statistical significance was assessed by two-way ANOVA using sham versus IRI and genotypes as variables. Sham-treated mice from each strain were analyzed separately but are shown in the same column for simplicity. No differences in plasma creatinine were found in sham-treated mice of all strains. Scale bar, 50 μm. Flow cytometry analyses of Ly6G+Ly6C+ neutrophils in kidneys with the indicated treatment are shown in D, and the quantitation is shown in E. At least three mice were analyzed in each group. The number next to the gate (outlined with a box) is gated cells as a percentage (7.2%–92%) of total cells in the plot. *P<0.05; **P<0.01; ****P<0.001. (F) Confocal microscopy of CD73 expression and neutrophil and macrophage infiltration in sham- and IR-treated mice. Infiltrating neutrophils and macrophages in kidneys were identified by expression of CD11b, a marker for both cell types (which can be discriminated by high and intermediate expressions of CD11b by flow cytometry). The white line demarcates the boundary between the cortex and medulla. In (C), (D), and (F), kidney tissues were obtained from mouse strains with the genotypes labeled for each panel. g, Glomerulus; im, inner medulla. Scale bars, 100 μm.

Article Snippet: Mice were injected intraperitoneally on day 0 with 80 μ g GR1 rat anti–mouse Ly6G/C mAb (Bioxcell, West Lebanon, NH).

Techniques: Expressing, Staining, Flow Cytometry, Quantitation Assay, Confocal Microscopy, Marker, Labeling

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Proximal Tubule CD73 Is Critical in Renal Ischemia-Reperfusion Injury Protection

doi: 10.1681/ASN.2016020229

Figure Lengend Snippet: A2aRs on bone marrow neutrophils and macrophages mediate protection from IRI by adenosine provided by renal CD73. (A–C) Large numbers of PMN found in WT mice injected with (A, b and c) control LTF-2 mAb were depleted on (B, b) day 2 and (C, a) day 3 when mice were injected with limiting amounts of GR1 anti–Ly6G/C mAb (80 μg per mouse). (A, e and f; B, e and f; and C, b) However, CD11b+ monocyte percentages were comparable in mice injected with either mAb. 7-AAD, 7-amino-actinomycin D. (D–F) WT mice were injected with control LTF-2 mAb or neutrophil–depleting GR1 mAb on day 0, reconstituted with 40×106 bone marrow cells from WT or Adora2a−/− mice or no cells (vehicle) on day 3, underwent 26-minute ischemia on day 4, and were euthanized on day 5. (D) Plasma creatinine and (E) tubular injury in the medulla scored from (F) hematoxylin and eosin–stained kidneys from reconstituted mice subjected to IRI. At least three mice were used in each group. BM, bone marrow. Scale bar, 100 μm. ***P<0.001; ****P<0.001.

Article Snippet: Mice were injected intraperitoneally on day 0 with 80 μ g GR1 rat anti–mouse Ly6G/C mAb (Bioxcell, West Lebanon, NH).

Techniques: Injection, Staining

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: a Schematic illustration of various nanoparticles tested in this study. TLP is a tannic acid core nanoparticle coated with lipid membranes. TLP was modified with IFNβ, anti-Ly6G antibody, or with both IFNβ and anti-Ly6G antibody, resulting in IFNβ@TLP, aLy6G@TLP, and aLy6G-IFNβ@TLP, respectively. For comparison, lipid nanoparticles modified with IFNβ and anti-Ly6G antibody, termed aLy6G-IFNβ@LP, were constructed. b Schematic illustration of NET formation-mediated IFNβ delivery to the inflamed CNS for ameliorating the experimental autoimmune encephalomyelitis (EAE). By binding to Ly6G on NEs, aLy6G-IFNβ@TLP can cross the BBB via NE hitchhiking and reach to the inflamed CNS. Upon activation by inflammatory factors, NET formation results in the release of aLy6G-IFNβ@TLP. Consequently, aLy6G-IFNβ@TLP can deliver IFNβ into the inflamed CNS, generating remyelination and immunomodulation effects against EAE.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: Modification, Comparison, Construct, Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: a Preparation scheme of aLy6G-IFNβ@TLP. b EDS-SEM images of aLy6G-IFNβ@TLP. Scale bar: 50 nm. The experiment was repeated twice independently with similar results and the representative data is shown. c Particle size distribution of aLy6G-IFNβ@TLP measured by dynamic light scattering. d Zeta potential of aLy6G-IFNβ@TLP. e SDS-PAGE analysis of aLy6G conjugation on various formulations. The experiment was repeated twice independently with similar results and the representative data is shown. f Flow cytometry analysis of aLy6G conjugation on various formulations. g ROS scavenging effect of aLy6G-IFNβ@TLP. h Dose-dependent superoxide anion scavenging activity of aLy6G-IFNβ@TLP, assessed by nitro blue tetrazolium assay. i Dose-dependent hydroxyl radical scavenging activity of aLy6G-IFNβ@TLP, evaluated by terephthalic acid assay. j , k Dose-dependent superoxide anion scavenging activity ( j ) and hydroxyl radical scavenging activity ( k ) of aLy6G-IFNβ@LP. l Quantification of IC 50 for superoxide anion scavenging activity ( n = 3 independent samples per group). m Quantification of IC 50 for hydroxyl radical scavenging activity ( n = 3 independent samples per group). n Evaluation of intracellular ROS generation of NEs after PMA stimulation and nanoparticle treatment by chemiluminescence assay ( n = 5 biologically independent samples per group). o Area under the curve (AUC) of chemiluminescence ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-Control < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. p Mean fluorescence intensity (MFI) of intracellular ROS upon PMA stimulation evaluated by flow cytometry ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-Control < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. q Underlying mechanism of denaturation and protection of IFNβ in aLy6G-IFNβ@LP and aLy6G-IFNβ@TLP under ROS-rich environment. r Determination of IFNβ stability released from aLy6G-IFNβ@LP and aLy6G-IFNβ@TLP in the absence and presence of H 2 O 2 ( n = 5 independent samples per group). Statistical significance was analyzed using unpaired two-sided t -test. At H 2 O 2 0 μM, NS, not significant. At H 2 O 2 10 μM, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. Data are presented as mean ± s.d. Source data are provided as a Source Data file. RFU, relative fluorescence unit. a.u., arbitrary units.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: Zeta Potential Analyzer, SDS Page, Conjugation Assay, Flow Cytometry, Activity Assay, Acid Assay, Chemiluminescence Immunoassay, Control, Fluorescence

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: a Schematic illustration of the in vitro BBB model for assessing NE binding and penetration ability of aLy6G-IFNβ@TLP across the endothelial monolayer. b Evaluation of NE binding capability of different formulations using flow cytometry. c Visualization of internalization of Fe-labeled nanoparticles in NEs using TEM. Scale bar: 2 μm. Scale bar of expanded images: 0.5 μm. The experiment was repeated twice independently with similar results and the representative data is shown. d Schematic illustration of nanoparticle-bound NEs migration across the endothelial monolayer in the presence of fMLP. e Migration of NEs and nanoparticle-treated NEs across the endothelial monolayer in the presence of fMLP ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant. f Confocal images of nanoparticle-bound NEs in the filtrate. Scale bar: 20 μm. The experiment was repeated twice independently with similar results and the representative data is shown. g Confocal images of NET formation-mediated nanoparticle release after incubation with PMA for 4 h. Scale bar, 10 μm. The experiment was repeated twice independently with similar results and the representative data is shown. h Evaluation of colocalization of MPO with nanoparticles in the NET. i Pearson’s correlation coefficient of MPO with nanoparticles in the NETs ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-Control < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001. j Visualization of Fe-labeled aLy6G-IFNβ@TLP release during the process of NET formation by EDS-SEM. Scale bar: 2 μm. Scale bar of expanded images: 1 μm. The experiment was repeated twice independently with similar results and the representative data is shown. k TEM images of NET formation-induced aLy6G-IFNβ@TLP release in the NET. Scale bar: 2 μm for left panels, 0.5 μm for right panels. The experiment was repeated twice independently with similar results and the representative data is shown. l Experimental design for monitoring the process of PMA-induced NET formation and nanoparticle release. m Release of aLy6G-IFNβ@TLP and their colocalization with Hoechst were observed over time. NEs nuclei were stained with Hoechst 33342, and aLy6G-IFNβ@TLP were labeled with Cy5. Scale bar: 5 μm. The experiment was repeated twice independently with similar results and the representative data is shown. n The underlying mechanism of IFNβ release in the inflamed CNS. o Cumulative IFNβ release from aLy6G-IFNβ@TLP in PBS or FBS solution ( n = 3 independent samples per group). Statistical significance was analyzed using unpaired two-sided t -test. P FBS-PBS = 0.0001. Data are presented as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: In Vitro, Binding Assay, Flow Cytometry, Labeling, Migration, Incubation, Control, Staining

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: a Ex vivo fluorescence imaging of brain and spinal cord in EAE mice following intravenous administration of IFNβ@TLP and aLy6G-IFNβ@TLP over time. b Whole-body molecular imaging of EAE mice at 6 h post-injection of IFNβ@TLP and aLy6G-IFNβ@TLP. Quantification of nanoparticle fluorescence in the brain ( c ) and spinal cord ( d ) of EAE mice after intravenous injection of different formulations over time ( n = 5 biologically independent samples per group). Statistical significance was analyzed using unpaired two-sided t -test. For brain samples: 1 h, NS, not significant; 6 h, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001; 12 h, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0039; 24 h, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0165. For spinal cord samples: 1 h, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0112; 6 h, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001; 12 h, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001; 24 h, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001. e Gating strategy for NEs in the spinal cord, stained with PerCP/Cy5.5-conjugated CD45, FITC-conjugated Ly6G, and PE-conjugated CD11b antibodies. f Representative flow cytometry histogram (left) and quantification (right) of MFI of nanoparticles taken up by NEs in the spinal cord 6 h post-administration ( n = 5 biologically independent samples per group). Statistical significance was analyzed using unpaired two-sided t -test. P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0013. g Fluorescence images of the spinal cord after intravenous injection of nanoparticles for 6 h. Scale bar: 20 μm. The experiment was repeated twice independently with similar results and the representative data is shown. h Colocalization analysis of Ly6G-expressing cells and nanoparticles in the spinal cord. i t-SNE plot of single cells, color-coded according to the identified cell types. j t-SNE plots of nanoparticles taken up by T cells, APC, and NEs in the spinal cord, color-coded according to nanoparticle fluorescence intensity. k Experimental schedule for intravital imaging of spinal cord-infiltrated nanoparticles in EAE mice. l Intravital microscopy images of aLy6G-IFNβ@TLP-bound NEs migration into the spinal cord. Scale bar: 50 μm. The experiment was repeated twice independently with similar results and the representative data is shown. Data are presented as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: Ex Vivo, Fluorescence, Imaging, Injection, Staining, Flow Cytometry, Expressing, Intravital Microscopy, Migration

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: a Schematic illustration of the treatment schedule in the EAE mouse model. Mice were intravenously administered various nanoparticle formulations three times, with three days between each administration, starting from day 12 after EAE immunization. Therapeutic efficacy of various formulations assessed by changes in body weight ( b ) and clinical score ( c ) ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For body weight, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ = 0.0011, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0063, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0082, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.01. For clinical score, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ = 0.0009, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0009, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.002. Analysis of IL-17 ( d ), TNF-α ( e ), IFN-γ ( f ), and IL-6 ( g ) production levels of splenocytes after stimulation with MOG 35-55 for 72 h at the end of the in vivo experiments ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For IL-17, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ = 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. For TNF-α, P aLy6G-IFNβ@TLP-PBS = 0.0057, P aLy6G-IFNβ@TLP-IFNβ = 0.0046, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.009, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0049, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0153. For IFN-γ, P aLy6G-IFNβ@TLP-PBS = 0.0001, P aLy6G-IFNβ@TLP-IFNβ < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0002, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0002. For IL-6, P aLy6G-IFNβ@TLP-PBS = 0.0058, P aLy6G-IFNβ@TLP-IFNβ = 0.0042, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0049, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0018, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.006. Determination of IFN-γ ( h ) and IL-17 ( i ) levels in the plasma at the end of the in vivo experiments ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For plasma IFN-γ, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. For plasma IL-17, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0002, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0003, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0002. j Analysis of mRNA expression levels in the spinal cord associated with the progression of EAE ( n = 3 biologically independent samples per group). Data are presented as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: Drug discovery, In Vivo, Clinical Proteomics, Expressing

Journal: Nature Communications

Article Title: NET formation-mediated in situ protein delivery to the inflamed central nervous system

doi: 10.1038/s41467-024-54817-7

Figure Lengend Snippet: Various nanoparticle formulations were intravenously administered 12 days after EAE immunization, repeating every three days for a total of three administrations. Spinal cord was collected and analyzed at 28 days post-immunization. a Representative flow cytometry data of Th1 in the spinal cord. b Relative quantification of Th1 in the spinal cord ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. c Representative flow cytometry data of Th17 in the spinal cord. d Relative quantification of Th17 in the spinal cord ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. Quantification of infiltrated helper T cells (CD45 + CD3 + CD4 + ) ( e ), cytotoxic T cells (CD45 + CD3 + CD8 + ) ( f ), DCs (CD45 + CD11c + MHCII + ) ( g ), and macrophages (CD45 + CD11b + F4/80 + ) ( h ) in the spinal cord ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. For helper T cells, P aLy6G-IFNβ@TLP-PBS = 0.0016, P aLy6G-IFNβ@TLP-IFNβ = 0.0021, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0002, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0005, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0004. For cytotoxic T cells, P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ = 0.0009, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0014, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0012, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0025. For DCs, P aLy6G-IFNβ@TLP-PBS = 0.0102, P aLy6G-IFNβ@TLP-IFNβ = 0.0226, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0042, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0043, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0175. For macrophages, P aLy6G-IFNβ@TLP-PBS = 0.0008, P aLy6G-IFNβ@TLP-IFNβ = 0.0003, P aLy6G-IFNβ@TLP-IFNβ@TLP = 0.0014, P aLy6G-IFNβ@TLP-aLy6G@TLP = 0.0003, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0015. i Representative CD45 staining in the spinal cord of EAE mice. Scale bar: 250 μm. Scale bar of expanded microscopic image: 125 μm. j Quantification of CD45-positive cells in three selected fields per sample from immunofluorescence images ( n = 3 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP = 0.0002. k Representative LFB staining of spinal cord revealing areas of myelination. Scale bar: 250 μm. Scale bar of expanded microscopic image: 125 μm. l Quantification of myelinated area from LFB staining images ( n = 5 biologically independent samples per group). Statistical significance was analyzed using one-way ANOVA with Tukey’s multiple comparisons test. P aLy6G-IFNβ@TLP-PBS < 0.0001, P aLy6G-IFNβ@TLP-IFNβ@TLP < 0.0001, P aLy6G-IFNβ@TLP-aLy6G-IFNβ@LP < 0.0001. Data are presented as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: Briefly, aLy6G (cat# BE0075-1, lot# 854523S1, 807722M1, clone 1A8, Bioxcell, Lebanon, NH, USA) and IFNβ (PBL Assay science, Piscataway, NJ, USA, cat# 12410-1) were functionalized with Traut’s reagent (Thermo Fisher Scientific, Waltham, MA, USA, cat# 26101) to incorporate sulfhydryl groups, enabling subsequent conjugation to maleimide moieties.

Techniques: Flow Cytometry, Quantitative Proteomics, Staining, Immunofluorescence