Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: S1pr1 fl/fl mice (no. 019141) were obtained from the Jackson Laboratory.

Techniques: Gene Expression, Staining, Mass Spectrometry

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Protocol for assessing the retention of CD4 + T RM cells in tissues. CD45(iv) − CD69 + KJ1 + CD4 + T RM cells or CD45(iv) + KJ1 + CD4 + T CIRC cells (8.0 × 10 4 cells) were transferred into the recipient mice. PBS or OVA was intranasally administered to the recipient mice for 6 consecutive days starting from day 36, and mice were analyzed 30 days after the last OVA challenge. ( B ) Representative plots of tissue Ex-T CIRC cells, circulating Ex-T CIRC cells, tissue-Ex-T RM cells, and circulating Ex-T RM cells in the lung of the recipient mice treated with PBS or OVA. ( C ) Comparison of global ATAC peaks in the four groups of CD4 + memory T cells. The heatmap shows the signal intensity of each ATAC peak with reads per million (RPM) normalized by z -scores. Rep: replication. ( D ) Pathway analysis (Kyoto Encyclopedia of Genes and Genomes) in gene proximal to region 1 and region 2. ( E ) Cumulative normalized tag counts of ATAC peaks in the indicated regions (left: circulating Ex-T CIRC cells; right: circulating Ex-T RM cells), plotted with ranked open chromatins. ( F and G ) Comparison of ATAC-seq signals across CD4 + T RM cell–related genes, including the (F) S1pr1 locus and (G) Il4 , Il5 , and Il13 loci. ( H ) Concentrations of IL-5 and IL-13 in supernatants of stimulated circulating Ex-T CIRC cells and circulating Ex-T RM cells. Representative data of two independent experiments. ( I ) The percentage increase in ear thickness was measured in recipient mice (4 × 10 3 circulating Ex-T RM or Ex-T CIRC cells per mouse) that received either a control antibody (Ab) or a combination of anti–IL-5 (100 μg, BE0198) and anti–IL-13 (100 μg, mil13-mab9-1) antibodies at each time point. s.c.: subcutaneously. Data from two independent experiments were pooled and are presented ( n = 2 mice per group). Data represent the means ± SEM. Significance was determined by an unpaired t test in (H). *** P < 0.001 and **** P < 0.0001.

Article Snippet: S1pr1 fl/fl mice (no. 019141) were obtained from the Jackson Laboratory.

Techniques: Comparison, Control

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Volcano plot of the differential gene expression between CD4 + T RM cells and CD4 + T CIRC cells identified in fig. S5 (A to E). ( B ) Plots of CD69 and S1PR1 in Ly5.1 + CD45(iv) − CD4 + T cells in the lung of the OVA-treated mice at day 49 shown in fig. S6A. ( C ) Absolute numbers of CD45(iv) − S1PR1 + CD4 + T cells in the mice transferred with creER T2 Cd69 +/+ T H 2 cells ( n = 9) or creER T2 Cd69 fl/fl T H 2 cells ( n = 10). Pooled data from two independent experiments. ( D ) Absolute numbers of CD45(iv) − CD4 + T cells in the three groups ( n = 4 or 5). Data of two independent experiments. ( E ) Confocal micrograph of the lungs at day 49. B: bronchiole. ( F ) Absolute numbers of Ly5.1 + memory CD4 + T cells in iBALT. Data of two independent experiments. ( G ) H&E-stained lung section (top). Lungs were collected from the mice treated as shown in fig. S2A. The vessel wall, iBALT, and a parenchyma area located 200 μm from blood vessels were chosen to measure S1P levels by mass spectroscopy. The S1P intensity in these regions is shown (bottom). ( H ) Average peak intensities of S1P in each region. ( I ) Confocal micrograph of the lungs at day 35. Lungs were collected from the mice treated as shown in fig. S6H. ( J ) Absolute numbers of CD69 + Thy1.1 + cells (left) and CD69 − Thy1.1 + cells (right) in concentric distance zones from Lyve-1 + lymphatic vessels. Data represent the means ± SEM. Significance was determined by a one-way ANOVA in (C), (D), and (F), by paired one-way ANOVA in (H), and by an unpaired U test in (J). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Briefly, cells were stained for 90 min with an anti-mouse S1PR1 antibody (7.2 μg/ml; MAB7089, R&D Systems), stained for 45 min with an anti-rat immunoglobulin G-biotin F(ab′)2 (9.5 μg/ml; 2340649, Jackson Immunoresearch), and lastly stained with streptavidin coupled with anaphase-promoting complex and the other surface membrane antibodies.

Techniques: Gene Expression, Staining, Mass Spectrometry

Journal: Science Advances

Article Title: CD69 regulates the tissue dynamics of epigenetically imprinted memory CD4 + T cells

doi: 10.1126/sciadv.adw1038

Figure Lengend Snippet: ( A ) Protocol for assessing the retention of CD4 + T RM cells in tissues. CD45(iv) − CD69 + KJ1 + CD4 + T RM cells or CD45(iv) + KJ1 + CD4 + T CIRC cells (8.0 × 10 4 cells) were transferred into the recipient mice. PBS or OVA was intranasally administered to the recipient mice for 6 consecutive days starting from day 36, and mice were analyzed 30 days after the last OVA challenge. ( B ) Representative plots of tissue Ex-T CIRC cells, circulating Ex-T CIRC cells, tissue-Ex-T RM cells, and circulating Ex-T RM cells in the lung of the recipient mice treated with PBS or OVA. ( C ) Comparison of global ATAC peaks in the four groups of CD4 + memory T cells. The heatmap shows the signal intensity of each ATAC peak with reads per million (RPM) normalized by z -scores. Rep: replication. ( D ) Pathway analysis (Kyoto Encyclopedia of Genes and Genomes) in gene proximal to region 1 and region 2. ( E ) Cumulative normalized tag counts of ATAC peaks in the indicated regions (left: circulating Ex-T CIRC cells; right: circulating Ex-T RM cells), plotted with ranked open chromatins. ( F and G ) Comparison of ATAC-seq signals across CD4 + T RM cell–related genes, including the (F) S1pr1 locus and (G) Il4 , Il5 , and Il13 loci. ( H ) Concentrations of IL-5 and IL-13 in supernatants of stimulated circulating Ex-T CIRC cells and circulating Ex-T RM cells. Representative data of two independent experiments. ( I ) The percentage increase in ear thickness was measured in recipient mice (4 × 10 3 circulating Ex-T RM or Ex-T CIRC cells per mouse) that received either a control antibody (Ab) or a combination of anti–IL-5 (100 μg, BE0198) and anti–IL-13 (100 μg, mil13-mab9-1) antibodies at each time point. s.c.: subcutaneously. Data from two independent experiments were pooled and are presented ( n = 2 mice per group). Data represent the means ± SEM. Significance was determined by an unpaired t test in (H). *** P < 0.001 and **** P < 0.0001.

Article Snippet: Briefly, cells were stained for 90 min with an anti-mouse S1PR1 antibody (7.2 μg/ml; MAB7089, R&D Systems), stained for 45 min with an anti-rat immunoglobulin G-biotin F(ab′)2 (9.5 μg/ml; 2340649, Jackson Immunoresearch), and lastly stained with streptavidin coupled with anaphase-promoting complex and the other surface membrane antibodies.

Techniques: Comparison, Control

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: Representative confocal images (20X) of S1PR1-GFP astrocytes (green) cultured alone ( A only ) or with WT neurons ( A+N ) stained with Map2 (red) for 8 (A-D) or 24 hours ( E-H ). Based on the astrocytes interaction with or without Map2+ neuronal processes in A+N cocultures, astrocytes were separated into either non-interacting astrocytes (NIA) (Blue arrow in E) or interacting astrocytes (IA) (white arrow in E). Quantification of perimeter ( B and F ) and S1PR1 expression as green signal intensity ( C and G ) per astrocyte, respectively, in A or A+N cultures. ( D and H ) Quantification of astrocyte morphological complexity as indicated by number of intersections plotted against distance from center of the soma by Sholl analyses from A or A+N cultures. ( I-L ) S1PR1 inhibition blocks neuronal contact dependent astrocyte morphological complexity. ( I ) Representative confocal image of S1PR1-GFP astrocytes treated with vehicle (DMSO) or the S1PR1 antagonist, W146, for 24 hours cultured with or without neurons (Map2, red). Perimeter ( J ) and fluorescent intensity ( K ) analyses of GFP immunofluorescence from S1PR1-GFP astrocytes. ( L ) Quantification of morphological complexity of astrocytes as indicated by number of intersections plotted against distance from center of the soma by Sholl analyses. Data represents the mean ± SEM from two- (A-H) or three (I-L) -biological replicates. * = p <0.05, *** = p < 0.001, **** = p <0.0001; one way ANOVA.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Cell Culture, Staining, Expressing, Inhibition, Immunofluorescence

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: (A) Representative confocal images (20X) of S1PR1-GFP astrocytes (green) cultured alone ( A only ) or with WT neurons ( A+N ) stained with Map2 (red) treated with vehicle (DMSO), the JAK2 inhibitor Ruxolitinib (Rux), or the Smoothened inhibitor Sonidegib (Sonid), for 24 hours. Quantification of perimeter ( B ) and level of S1PR1 expression as fluorescent signal intensity ( C ) per astrocyte respectively in A or A+N cultures. ( D, E ) Quantification of astrocyte morphological complexity as indicated by the number of intersections plotted against distance from center of the soma by Sholl analyses from A or A+N cultures treated with vehicle (DMSO) control, Rux, or Sonid. Data represents the mean ± SEM from three biological replicates. ** = p <0.01, *** = p < 0.001, **** = p <0.0001, one way ANOVA.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Cell Culture, Staining, Expressing, Control

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: ( A ) Astrocyte-specific, S1PR1 knock-out mouse model (S1PR1 ΔAST ) model. ( B ) A substantial reduction in S1PR1 mRNA levels measured by qPCR. ((n=8 and 7 mice per group). (C) Representative confocal images of S1PR1 (green) with the endothelial marker CD31 (red) from S1PR1 ΔAST or littermate control cortices at P30. Arrows indicate intact S1PR1 expression on CD31+ vasculature. ( D ) Quantification of CD31 intensity represented as area per image is not altered in S1PR1 ΔAST cortices at P30. ( F ) Blood-brain barrier integrity assay scheme (left) and quantification of tracer dyes 1 kDa Cadaverine (center) and 3 kDa Dextran (right) signal in S1PR1 ΔAST cortices at ∼P35 indicates intact BBB integrity in S1PR1 ΔAST mice. ( G ) Representative image (top) and quantifications of GFAP signal (green) in S1PR1 ΔAST cortices at P30. ( H ) Representative image of perisynaptic astrocyte marker GLT-1 (red) with S1PR1 (green) in S1PR1 ΔAST cortices at P30 (top). Quantification of GLT1 puncta measured as GLT1-spot count and -spot volume by IMARIS 3D rendering indicate no changes in GLT1 expression in S1PR1 ΔAST cortices at P30. Data represents the mean ± SEM from n=3 and 4 mice per group (D-H). *** = p < 0.001, ns=not significant; Unpaired Two-tailed student’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Knock-Out, Marker, Control, Expressing, Integrity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: ( A ) S1PR1 ΔAST or littermate controls were intracranially injected with the adenoassociated virus (AAV), pZac2.1-GfaABC1D-YFP, to sparsely label astrocytes. Sparsely labeled astrocytes from L2-3 somatosensory cortices were imaged and analyzed. ( B ) Representative confocal images and subsequent IMARIS 3D surface/filament renderings of YFP astrocytes from S1PR1 ΔAST or littermate controls at P30. (C) Quantification of astrocyte volume (left), and soma volume (right) from surface renderings generated using IMARIS. ( D ) Quantification of astrocyte territory volume (left), astrocyte process/filament length (middle) and area (right) from filament traces generated using IMARIS. ( E ) Plot depicting the proportion of astrocyte filament thicknesses grouped by mean diameter and represented as % of total filaments. ( F ) Sholl analyses of IMARIS rendered filament traces of astrocytes from S1PR1 ΔAST and littermate controls. Data represents the mean ± SEM from total 26 and 25 astrocytes from n=5 mice per group. * = p <0.05, *** = p <0.001, ns=non-significant, Unpaired Welch’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Injection, Virus, Labeling, Generated

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: ( A ) Sparsely YFP-labeled L4-5 somatosensory cortical astrocytes were imaged and analyzed. (B) Representative confocal images and subsequent IMARIS 3D surface/filament renderings of YFP astrocytes from S1PR1 ΔAST or littermate controls at P30. (C) Quantification of astrocyte volume (left) and soma volume (right) from surface renderings generated using IMARIS. ( D ) Quantification of astrocyte territory volume (left), astrocyte process/filament length (middle) and area (right) from filament traces generated using IMARIS. ( E ) Plot depicting the proportion of astrocyte filaments grouped at various mean diameter and represented as % of total filaments revealed no differences in mean dendrites in S1PR1 ΔAST astrocytes. ( F ) Sholl analyses of IMARIS rendered filament traces of astrocytes from S1PR1 ΔAST and littermate controls. Data represents the mean ± SEM of 24 and 22 astrocytes from n=5 mice per group. * = p <0.05, ** = p <0.01, ns=non-significant, Unpaired Welch’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Labeling, Generated

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: (A) Schematics showing S1PR1 deletion in sparse astrocytes by delivering AAV-GfaABC1D-YFP-P2A-Cre or AAV-GfaABC1D-YFP control in S1PR1 fl/fl mouse pups. (B) Sparsely labeled astrocytes were imaged from L2-3 somatosensory cortex from P30 pups. ( C ) Representative confocal images and subsequent IMARIS 3D surface/filament renderings of YFP astrocytes from S1PR1 ΔAST or littermate controls at P30. (D) Quantification of astrocyte volume (left) and soma volume (right) from Surface renderings generated by IMARIS. ( E ) Quantification of astrocyte territory volume (left) filament length (middle) and filament area (right) from filament traces generated by IMARIS. (F) Plot depicting the proportion of astrocyte filaments grouped by various mean diameter and represented as % of total filaments revealed no differences in mean dendrites in S1PR1 ΔAST astrocytes. ( G ) Sholl analyses of IMARIS rendered filament traces of YFP labeled sparse astrocytes from S1PR1 ΔAST and littermate controls. Data represents the mean ± SEM of 23 and 24 astrocytes from n=4 mice per group. * = p <0.05, ** = p <0.01, ns=non-significant, Unpaired Welch’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Control, Labeling, Generated

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: (A) Schematics showing S1PR1 deletion in sparse astrocytes by delivering AAV-GfaABC1D-YFP-P2A-Cre or AAV-GfaABC1D-YFP control in S1PR1 fl/fl mouse pups. Sparsely labeled astrocytes were imaged from L4-5 somatosensory cortex from P30 pups. ( B ) Representative confocal images and subsequent IMARIS 3D surface/filament renderings of YFP astrocytes from S1PR1 ΔAST or littermate controls at P30. (C) Quantification of astrocyte volume (left) and soma volume (right) from Surface renderings generated by IMARIS. ( E ) Quantification of astrocyte territory volume (left) process/filament length (middle) and area (left) from filament traces generated by IMARIS. (F) Plot depicting the proportion of astrocyte filaments grouped by mean diameter and represented as % of total filaments revealed no differences in mean dendrites in S1PR1 ΔAST astrocytes. ( G ) Sholl analyses of IMARIS rendered filament traces of GFP labeled sparse astrocytes from S1PR1 ΔAST and littermate controls. Data represents the mean ± SEM of 22 and 17 astrocytes from n=4 mice per group. * = p <0.05, ** = p <0.01, *** = p <0.001 ns=non-significant, Unpaired Welch’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: Control, Labeling, Generated

Journal: bioRxiv

Article Title: Sphingosine-1-Phosphate Receptor 1 regulates competition dependent astrocyte morphogenesis and tiling in murine cortex

doi: 10.64898/2026.03.28.714989

Figure Lengend Snippet: B) Schematics showing S1PR1 deletion in neighboring astrocytes in two-color schemes by delivering AAV-GfaABC1D-YFP-P2A-Cre plus AAV-GfaABC1D-tdTomato-P2A-Cre or AAV-GfaABC1D-YFP plus AAV-GfaABC1D-tdTomato controls in S1PR1 fl/fl mouse pups. ( C ) Example 20x confocal image to show the labelling by two color viruses. (D) Representative confocal images and subsequent IMARIS 3D surface/filament renderings to create territories of neighboring astrocytes from KO:KO or littermate WT:WT controls at P30. (E) Quantification of astrocyte territory overlap volume represented as % of total volume from two neighboring astrocytes. Data represents the mean ± SEM of 23 and 18 pairs of neighboring astrocytes from n=3 and 4 mice per group. **** = p <0.0001, Unpaired Welch’s T-test.

Article Snippet: To further investigate S1PR1’s expression and localization dynamics, we prepared primary astrocyte cultures from S1PR1 reporter mice (S1PR1 GFP/GFP ) (JAX Strain # 028623) in serum-free medium.

Techniques: