Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Flow cytometric plot showing CD25 and Foxp3 expression of control aged mice and aged mice treated with intraperitoneal injection of IL-2/anti-IL-2 complexes. B Quantification of CD25 + Foxp3 + natural Treg proportion in a CD4 + T cell population from the spleen control aged mice and aged mice treated with intraperitoneal injection of IL-2/anti-IL-2 complexes ( n = 3, unpaired two-tailed Student’s t test after arcsin conversion, t = 5.49, ** P = 0.0054). C Quantification of CD25 + Foxp3 + natural Treg proportion in a CD4 + T cell population from the blood control aged mice and aged mice treated with intraperitoneal injection of IL-2/anti-IL-2 complexes ( n = 3, unpaired two-tailed Student’s t test after arcsin conversion, t = 3.679, * P = 0.0212). D Representative images of OLIG2 (cyan) and CC1 (magenta) immunostaining in young control and Treg expanded demyelinated lesions at 7dpl (scale bar = 50 µm, lesion area demarcated with the dotted line). E Quantification of OLIG2 + cell density in the demyelinated area of young control and Treg expanded mice ( n = 5 mice (control), n = 4 mice (Treg expanded), unpaired two-tailed Student’s t test, t = 0.2018, P = 0.8458). F Quantification of OLIG2 + CC1 + cell density in the demyelinated area of young control and Treg expanded mice ( n = 5 mice (young), n = 4 mice (Treg expanded), unpaired two-tailed Student’s t test, t = 3.59, ** P = 0.0089). G Representative images of OLIG2 (cyan) and CC1 (magenta) immunostaining in aged control and Treg expanded demyelinated lesions at 10dpl (scale bar = 50 µm, lesion area demarcated with the dotted line). H Quantification of OLIG2 + cell density in the demyelinated area of aged control and Treg expanded mice ( n = 7 mice (control and Treg expanded), unpaired two-tailed Student’s t test, t = 0.3267, P = 0.7495). I Quantification of OLIG2 + CC1 + cell density in the demyelinated area of aged control and Treg expanded mice ( n = 7 mice (control and Treg expanded), unpaired two-tailed Student’s t test, t = 1.07, P = 0.3055). J Representative images of NFH (cyan) and MBP (magenta) immunostaining in aged control and Treg expanded demyelinated lesions at 10dpl (scale bar = 50 µm, lesion area demarcated with the dotted line). K Quantification of NFH + axon density in the demyelinated area of aged control and Treg expanded mice ( n = 7 mice (young), n = 6 (aged), unpaired two-tailed Student’s t test, t = 0.8018, P = 0.4397). L Quantification of NFH + MBP + axon density in the demyelinated area of aged control and Treg expanded mice ( n = 7 mice (young), n = 6 (aged), unpaired two-tailed Student’s t test, t = 0.3268, P = 0.75). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: Expressing, Injection, Two Tailed Test, Immunostaining

Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Representative images of OPCs co-cultured with young and aged natural Treg and immunostained for OLIG2 (cyan) as a pan oligodendrocyte lineage marker and the proliferation marker Ki67 (magenta) (scale bar = 100 µm). B Quantification of OPC proliferation when co-cultured with young and aged natural Treg ( n = 6 mice, 2 independent experiments, 1-way ANOVA after arcsin conversion, F = 0.4279, P control vs young = 0.9487, P control vs aged = 0.9121, P young vs aged = 0.6418). C Representative images of OPCs co-cultured with young and aged natural Treg and immunostained for OLIG2 (cyan) and differentiation markers CNP (grey) and MBP (magenta) (scale bar = 100 µm). D Quantification of the proportion of OPCs reaching early stage-differentiation when exposed to young and aged natural Treg, as indicated by CNPase staining ( n = 9 mice, 3 independent experiments, 1-way ANOVA after arcsin conversion, F = 0.8282, P control vs young = 0.8925, P control vs aged = 0.9995, P young vs aged = 0.8439). E Quantification of the proportion of OPCs expressing late-stage differentiation marker MBP in control OPCs and OPCs treated with young and aged natural Treg ( n = 9 mice, 3 independent experiments, 1-way ANOVA, Sidak’s multiple comparisons test after arcsin conversion, F = 3.87, ** P control vs young = 0.0094, P control vs aged = 0.8951, * P young vs aged = 0.0422). F Immunohistochemistry of control and young and aged Treg treated cerebellar slices (MBP, magenta and NFH, green, scale bar = 100 µm). G Quantification of myelination index (ratio between MBP and NFH colocalization area and NFH area) in neonatal cerebellar slices ( n = 9 mice, 1 brain slice per mouse, 3 independent experiments, 1-way ANOVA after arcsin conversion, Sidak’s multiple comparison tests, F = 4.946, * P control vs young = 0.0147, P c ontrol vs aged = 0.5215, P young vs aged = 0.2203). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: Cell Culture, Marker, Staining, Expressing, Immunohistochemistry, Slice Preparation, Comparison

Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Diagram explaining the experimental design of in vivo Treg depletion, Treg adoptive transfer and spinal cord demyelination. Representative images of immunostaining identifying oligodendrocytes by the co-localisation of the pan-oligodendrocyte lineage marker OLIG2 (green) with CC1 (magenta, B ) or ASPA (magenta, C ) at 14 dpl (scale bar = 100 µm, demyelination area is indicated by the white line). D Bar graph showing the quantification of total number of oligodendrocyte lineage cells in the demyelinated lesions of PBS control mice ( n = 8 mice), natural Treg-depleted mice ( n = 8 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 2.134, P PBS vs depleted = 0.2143, P PBS vs young > 0.9999, P PBS vs aged = 0.4726, P young vs aged = 0.4562). E Bar graph showing the quantification of total number of CC1-expressing oligodendrocytes in the demyelinated lesions of PBS control mice ( n = 8 mice), natural Treg-depleted mice ( n = 8 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 6.773, ***P PBS vs depleted = 0.0006, P PBS vs young = 0.4065, P PBS vs aged = 0.5240, P young vs aged > 0.9999). F Bar graph showing the quantification of total number ASPA-expressing oligodendrocytes in the demyelinated lesions of PBS control mice ( n = 8 mice), natural Treg-depleted mice ( n = 8 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 4.901, **P PBS vs depleted = 0.0088, P PBS vs young = 0.9985, P PBS vs aged = 0.9597, P young vs aged = 0.9933). G Representative images of immunostaining for neurofilament-H (NFH, green) and MBP (magenta) to quantify myelin wrapping as an early marker of remyelination at 14 dpl (scale bar = 100 µm, demyelination area is indicated by the white line). H Quantification shows the total number of axons in the demyelinated lesions of PBS control mice ( n = 6 mice), natural Treg-depleted mice ( n = 9 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 8 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 1.689, P PBS vs depleted = 0.1740, P PBS vs young = 0.3410, P PBS vs aged = 0.8304, P young vs aged = 0.8793). I Quantification shows the density of MBP-wrapped axons in the demyelinated lesions of PBS control mice ( n = 6 mice), natural Treg-depleted mice ( n = 9 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 8 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 7.342, *** PPBS vs depleted = 0.0004, P PBS vs young = 0.0738, P PBS vs aged = 0.2766, P young vs aged = 0.9273). J Quantification shows the percentage of MBP-wrapped axons from the total number of axons in the demyelinated lesions of PBS control mice ( n = 6 mice), natural Treg-depleted mice ( n = 9 mice) and mice depleted of endogenous Treg that received young ( n = 8 mice) or aged natural Treg ( n = 8 mice) by adoptive transfer (Kruskal-Wallis test, Dunn’s multiple comparisons test, Kruskal Wallis statistic = 14.16, **P PBS vs depleted = 0.0015, P PBS vs young > 0.9999, P PBS vs aged = 0.3523, P young vs aged > 0.9999). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: In Vivo, Adoptive Transfer Assay, Immunostaining, Marker, Expressing

Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Representative images of immunostaining identifying oligodendrocytes by the co-localisation of the pan-oligodendrocyte lineage marker OLIG2 (green) with CC1 (magenta) at 14 dpl (scale bar = 100 µm, demyelination area is indicated by the white dotted line). B Bar graph shows the quantification of total number of oligodendrocyte lineage cells in the demyelinated lesions of PBS-treated control aged mice ( n = 6 mice), natural Treg-depleted aged mice ( n = 5 mice) and aged mice depleted of endogenous Treg that received young ( n = 5 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 5.859, *P PBS vs depleted = 0.0199, **P PBS vs young = 0.0057, *P PBS vs aged = 0.0343, P young vs aged = 0.8230). C Bar graph shows the quantification of total number of Ki67 + proliferating OPCs in the demyelinated lesions of PBS-treated control aged mice ( n = 6 mice), natural Treg-depleted aged mice ( n = 5 mice) and aged mice depleted of endogenous Treg that received young ( n = 4 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, 1.207, P PBS vs depleted = 0.3939, P PBS vs young = 0.6326, P PBS vs aged = 0.4502, P young vs aged > 0.9999). D Bar graph shows the quantification of CC1 + oligodendrocytes in the demyelinated lesions of PBS-treated control aged mice ( n = 6 mice), natural Treg-depleted aged mice ( n = 5 mice) and aged mice depleted of endogenous Treg that received young ( n = 5 mice) or aged natural Treg ( n = 6 mice) by adoptive transfer (1-way ANOVA, Sidak’s multiple comparisons test, F = 8.562, ***P PBS vs depleted = 0.0005, P PBS vs young = 0.1168, **P PBS vs aged = 0.0084, P young vs aged= 0.7576). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: Immunostaining, Marker, Adoptive Transfer Assay

Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Representative image of immunostaining showing cell-to-cell contact between OPCs and Treg in OPC-Treg co-cultures in vitro. OPCs are identified by the co-staining of OLIG2 (cyan) and NG2 (grey), while Treg are identified by CD3 (red) (scale bar = 50 µm). B Representative images showing Treg (CD4 + (green), Foxp3 + (magenta)) in close proximity or direct contact with OLIG2 + oligodendrocyte lineage cells in demyelinated lesions in mice with EAE, highlighted by the yellow arrows (scale bar = 25 µm). C Representative images of immunostaining of OPCs directly co-cultured with young Treg and OPCs cultured with young Treg in a transwell (OLIG2 (cyan) and MBP (magenta), scale bar = 100 µm). D Quantification of MBP-expressing oligodendrocytes in control OPCs, OPCs directly co-cultured with young Treg and OPCs cultured with young Treg in a transwell ( n = 6 mice, 2 independent experiments, Kruskal-Wallis test, Dunn’s multiple comparison’s test, KW statistic = 12.23, **P control vs Treg no transwell = 0.0022, P control vs Treg transwell > 0.9999, *P Treg no transwell vs Treg transwell = 0.0415). E Diagram summarising bioinformatic approaches to identify protein–protein interactions between OPCs and Treg. F Graph showing 21 protein candidates expressed in the Treg plasma membrane, that are downregulated in aged Treg and have potential bindings partners enriched in OPCs vs oligodendrocytes ( n = 4 mice young, n = 6 mice aged, Wald test, Bonferroni multiple test correction). Log2 Change, -Log10 (Padj) and the number of OPC binding partners are indicated (see legend). G Bar graphs showing RNAseq normalised count values for the top 6 candidates ( n = 4 mice young, n = 6 mice aged, Wald test, Bonferroni multiple test correction, *** P Ccr7 = 0.0002, *** P Itga2 < 0.00001, * P Klrb1c = 0.0215, *** P Ly6c1 < 0.00001, *** P Mcam < 0.00001, *** P Sell = 0.0004). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: Immunostaining, In Vitro, Staining, Cell Culture, Expressing, Membrane, Binding Assay

Journal: Nature Communications

Article Title: Ageing impairs the regenerative capacity of regulatory T cells in mouse central nervous system remyelination

doi: 10.1038/s41467-024-45742-w

Figure Lengend Snippet: A Representative images of immunostaining showing OPC differentiation in co-culture with young Treg in the presence or absence of neutralising antibodies against candidate cell surface mediators (scale bar = 100 µm). B Bar graph showing the quantification of OPC differentiation measured by the fold change in percentage of MBP + cells ( n = 7 mice, 2 independent experiments, 2-way ANOVA, Dunnett’s multiple comparison tests, F raw factor(experiment) =2.271, P raw (experiment) =0.0706, F column (treatment) =14.26, *** P column (treatment) <0.0001; ** P Control vs Treg isotype = 0.0052, P Treg vs Treg isotype = 0.1164, ** P Treg isotype vs Treg anti-Mcam = 0.0025, * P Treg isotype vs Treg anti-Itga2 = 0.0160). C Bar graph showing the quantification of OPC differentiation measured by the fold change in percentage MBP + area per well ( n = 7, 2-way ANOVA, Dunnett’s multiple comparison tests, F raw factor(experiment) =3.058, * P raw (experiment) =0.0229, F column (treatment) =6.108, **P column (treatment) =0.0016; ** P Control vs Treg isotype = 0.0011, P Treg vs Treg isotype = 0.6375, * P Treg isotype vs Treg anti-Mcam = 0.0102, * P Treg isotype vs Treg anti-Itga2 = 0.0128). D Representative images of immunostaining showing OPC differentiation in neonatal OPCs treated with recombinant ITGA2 (OLIG2 (cyan), CNPase (grey) and MBP (magenta), scale bar = 50 µm). E Bar graph showing quantification of the proportion of MBP-expressing oligodendrocytes in rITGA2-treated neonatal OPCs at 3 days in vitro ( n = 7 mice, 2 independent experiments, Kruskal Wallis test, Dunn’s multiple comparison test, KW statistic=4.199, P VH vs 0.01µg/mL ITGA2 = 0.8079, P VH vs 0.1 µg/mL ITGA2 > 0.9999, P VH vs 1 µg/mL ITGA2 > 0.9999). F Bar graph showing quantification of MBP + cell area per well in rITGA2-treated neonatal OPCs at 3 days in vitro n = 7 mice, 2 independent experiments, 1-way ANOVA, Dunnett’s multiple comparison test, F = 5.336, P VH vs 0.01µg/mL ITGA2 = 0.9920, P VH vs 0.1µg/mL ITGA2 = 0.8916, **P VH vs 1µg/mL ITGA2 = 0.0080. G Sholl analysis showing the number of intersections as a function of the distance from the cell body to determine changes in the morphology of MBP-expressing oligodendrocytes ( n = 7 mice, 2 independent experiments, graph shows the average of 3 wells per mice and 6–9 cells per well, 2-way ANOVA, F interaction = 7.143, *** P interaction < 0.0001; F distance = 38.42, *** P distance < 0.0001; F treatment = 8.946, * P treatment = 0.0113; Data shown as individual data points and a non-linear regression fitting curve). Data are represented as mean ± SEM. Source data are provided as a File.

Article Snippet: Then spinal cord sections were incubated with primary antibodies against Olig2 (1:500; Bio-Techne, polyclonal), anti-APC (1:400, Abcam, clone CC1), anti-ASPA (1:300, Millipore, polyclonal), anti-NFH (1:500; Abcam, polyclonal), anti-MBP (1:500; Millipore, clone 12) or anti-Ki67 (1:300; Abcam, clone SP6) overnight at 4 °C.

Techniques: Immunostaining, Co-Culture Assay, Comparison, Recombinant, Expressing, In Vitro

Journal: Nucleic acids research

Article Title: Genome-wide transformation reveals extensive exchange across closely related Bacillus species.

doi: 10.1093/nar/gkad1074

Figure Lengend Snippet: Figure 1. Replacement accumulation experiment performed with four donor species B. spizizenii , B. vallismortis , B. atrophaeus and G. thermoglucosidasius . ( A ) Genomic distance between recipient Bs166 and donor species is measured as fraction of core genome and its sequence identity. ( B ) In each cycle of the experiment, one random colony is picked from an agar plate, cells are grown to exponential growth phase, transformed for 2 h with genomic DNA from a donor species, and plated overnight. ( C ) For each donor species, the experiment is performed for eight replicates in parallel. A dditionally, w e run a control experiment where cells do not transform with donor DNA. Starting with the recipient Bs166, single transformation cycles are performed consecutively.

Article Snippet: As donor species, e use B. spizizenii 2A9, Bacillus vallismortis DSM 11031, . atrophaeus 11A3, Geobacillus thermoglucosidasius 2542 ild types obtained from the BGSC and DSMZ.

Techniques: Sequencing, Transformation Assay, Control

Journal: Nucleic Acids Research

Article Title: Genome-wide transformation reveals extensive exchange across closely related Bacillus species

doi: 10.1093/nar/gkad1074

Figure Lengend Snippet: Replacement accumulation experiment performed with four donor species B. spizizenii , B. vallismortis , B. atrophaeus and G. thermoglucosidasius . ( A ) Genomic distance between recipient Bs166 and donor species is measured as fraction of core genome and its sequence identity. ( B ) In each cycle of the experiment, one random colony is picked from an agar plate, cells are grown to exponential growth phase, transformed for 2 h with genomic DNA from a donor species, and plated overnight. ( C ) For each donor species, the experiment is performed for eight replicates in parallel. Additionally, we run a control experiment where cells do not transform with donor DNA. Starting with the recipient Bs166, single transformation cycles are performed consecutively.

Article Snippet: As donor species, we use B. spizizenii 2A9, Bacillus vallismortis DSM 11031, B. atrophaeus 11A3, Geobacillus thermoglucosidasius 2542 wild types obtained from the BGSC and DSMZ.

Techniques: Sequencing, Transformation Assay, Control