Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Identification of regenerating factor as a regulator of therapeutic genes for Parkinson's disease therapy. A) Conceptual diagram outlining the basis of an epigenetic regulator. B) Comparative gene expression heatmap of substantia nigra (SN) in wild type control versus 6‐OHDA‐induced Parkinson's disease (PD) mouse model. C) Heatmap showing gene expression profiles in the caudate and putamen regions of healthy individuals (HI) and a cohort of human PD patients. BG: Basal Ganglia. D) Immunofluorescence images showing TUJ1‐ and MAP2‐positive cells under each condition. Scale bar = 50 µm. E) Immunochemistry and Sholl analysis of TH‐labeled neurons. Left panel: morphology of individual neurons. Right panel: Sholl analysis showing the number of neurite intersections as a function of distance from the soma. Scale bar = 100 µm. The data are presented as mean ± SEM ( n = 5 – 6 cells per group). F) Representative traces of action potentials evoked by depolarizing current injections under each condition (sham, 6‐OHDA, 6‐OHDA+NDST3). G) Dot plot showing the top 14 GO Biological Process terms from enrichment analyses: 6‐OHDA versus Sham (left side) and 6‐OHDA+NDST3 versus 6‐OHDA (right side). H) Pearson correlation matrix of transcriptomic among samples.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Gene Expression, Control, Immunofluorescence, Labeling

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Therapeutic efficacy of NDST3 and retrograde tracing with CTB in mice. A) Schematic diagram of in vivo experimental design involving CTB injection in the PD mouse model. B) Representative immunofluorescence images of CTB, TH, and NDST3 expression in the SN of Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm and 10 µm (Magnified image). C) Quantification of CTB‐, TH‐, and NDST3‐positive cells shown in Figure . Data are presented as mean ± SEM ( n = 6 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns = not significant. D) Immunofluorescence images showing GIRK2‐ and TH‐positive cells in the Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm and 10 µm (Magnified image). E) 3D Z‐stack analysis (IMARIS) of TH‐positive neurons obtained via confocal microscopy. F) DAB‐DAT staining in the SN.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Drug discovery, Retrograde Tracing, In Vivo, Injection, Immunofluorescence, Expressing, Confocal Microscopy, Staining

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Efficacy and electrophysiological properties of NDST3 in chemical‐induced PD model. A) Representative traces of spontaneous firing currents recorded from DA neurons of the SNpc in brain slices from each group. B) Cumulative fractions curves showing shortened inter‐event intervals, indicating a higher frequency of spontaneous firing in the 6‐OHDA + NDST3 group compared to the 6‐OHDA group. The inner bar graph showed mean inter‐event intervals in the ipsilateral of SNpc of each group. Data are presented as mean ± SEM ( n = 6 – 8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. *** p < 0.001. C) Quantification of DA neuronal firing rates in the ipsilateral SNpc of each group. The data are presented as mean ± SEM ( n = 6–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, and ** p < 0.01. D) Representative in vivo recording traces from the SNpc of live animals in each condition. E) Instantaneous firing frequencies during the recorded period. ( n = 4–6 independent animals per group; repeated measures) Two‐way ANOVA with Tukey's multiple comparisons test, * p < 0.05. F) Comparison of action potential waveforms among DA neurons across conditions. G) Representative image of DAB‐TH staining in ST and SN. Scale bar = 1 mm. H) Immunofluorescence images showing GIRK2‐ and TH‐positive cells in the Sham, MPTP‐induced PD mice, NDST3‐treated PD mice, and NDST3 only‐treated mice. Scale bar = 50 µm and 10 µm (Magnified image). I) Error count during the challenging beam traversal test for each experimental condition. The data are presented as mean ± SEM. ( n = 7 – 8 independent animals per group) Two‐way ANOVA with Tukey's multiple comparisons test. **** p < 0.0001. J) Errors per step during the challenging beam traversal test across conditions. The data are presented as mean ± SEM ( n = 7 – 8 independent animal per group). One‐way ANOVA with Tukey's multiple comparisons test. **** p < 0.0001. K) Fall latency in the wire‐hanging test. The data are presented as mean ± SEM ( n = 7–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. *** p < 0.001 and **** p < 0.0001. L) Time to orient downward (T‐turn) and M) time to descend to the base (T‐total). The data are presented as mean ± SEM ( n = 7–8 independent animals per group). One‐way ANOVA with Tukey's multiple comparisons test. * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: In Vivo, Comparison, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Molecular mechanisms of NDST3 in the PD model. A) One‐way hierarchical clustering heatmap based on Z‐score of normalized expression value for 5629 genes selected with fold change ≥ 2 and raw p ‐value < 0.05. B) Principal component analysis (PCA) analysis of RNA‐seq data to visualize sample‐to‐sample variation. C) Volcano plot showing differentially expressed genes between 6‐OHDA and Sham group; Down‐regulated genes marked in blue. D) Volcano plot showing differentially expressed genes between 6‐OHDA+NDST3 and 6‐OHDA; Up‐regulated genes marked in red. E) Dot plot of top 14 GO cellular component terms from GO enrichment analyses: 6‐OHDA+NDST3 versus 6‐OHDA. Heatmap showing gene expression patterns in F) pre‐synaptic neurons, G) post‐synaptic neurons, and H) glia compartments. I) UMAP visualizing cluster identity. J) UMAP representation comparing cellular composition in 6‐OHDA and 6‐OHDA+NDST3. K) Branched trajectory analysis illustrating cell state transitions in a 2D state‐space, where each dot represents a single cell, color‐coded by group identity.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Expressing, RNA Sequencing, Gene Expression, Single Cell

Journal: Advanced Science

Article Title: NDST3‐Induced Epigenetic Reprogramming Reverses Neurodegeneration in Parkinson's Disease

doi: 10.1002/advs.202507323

Figure Lengend Snippet: Comprehensive analysis of spatial transcriptomics and epigenetic modulation following NDST3 treatment in a PD model. A) Heatmap showing gene expression patterns in each cluster. ** p < 0.01, and **** p < 0.0001. B) Gene concept network plot displaying genes enriched in catabolic, metabolic, and wound healing GO categories. The top 30 most differentially expressed genes comparing 6‐OHDA versus Sham and 6‐OHDA+NDST3 versus 6‐OHDA. Node color intensity represents the log2 fold‐change of gene expression. C) Cell‐cell communication network plot illustrating interactions among three distinct cell clusters in 6‐OHDA‐induced PD model (left panel) and NDST3‐treated PD model (right panel), based on ligand–receptor pair probabilities using the CellChat database. Line thickness indicates proportionality to the number of interactions. D) Spatial localization of dopamine‐related markers. E) Spatial mapping of dopaminergic lineage markers identified via scRNA‐Seq. F) Heatmap visualization of CUT&RUN and ATAC‐Seq signal intensity ±2 kb around the TSS. G) Immunofluorescence images showing H3K27ac and TH‐positive cells in the Sham, 6‐OHDA‐induced PD mice, and NDST3‐treated PD mice. Scale bar = 50 µm. H) Venn diagram illustrating overlapping genes among DEGs from RNA‐Seq, scRNA‐Seq Cluster 9, CUT&RUN peak, and ATAC‐Seq peak. Average signal plot of I) CUT&RUN and J) ATAC‐seq signals at over‐enriched TSS regions of the Ncoa7 gene. K) Structure of NDST3‐NCOA7‐H3K27ac complex. Blue – NDST3, Green – NCOA7, and Red – H3K27ac. The yellow boundary represents the interaction region.

Article Snippet: Slices were incubated with primary antibodies targeting dopaminergic neuron markers TH (Merck Millipore, AB152, Lot# 4127053; Merck Millipore, MAB318, Lot#3990619), GIRK2 (Abcam, ab259909, Lot# GR3401320‐4), NDST3 (Novus Biologicals, NBP2‐19501, Lot# 40723), DAT (Merck Millipore, MAB369) and histone modification marker H3K27ac (Abcam, AB4729, Lot# 1059037‐6).

Techniques: Spatial Transcriptomics, Gene Expression, Immunofluorescence, RNA Sequencing

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) Representative images and quantification of CCR7 + DCs (panCK − HLA-DR + LAMP3 + , yellow) near BVs (CD31 + PDPN − , magenta), or LVs (CD31 + PDPN + , cyan) in human tumors (HNSCC, NSCLC, and EC). Scale bar represents 20 μm. Whole-tumor sections were analyzed for EC and NSCLC. Numbers of fields of view (FOVs) analyzed per HNSCC sample are as follows: HNSCC1–04 n = 7; HNSCC1–06 n = 16; HNSCC1–07 n = 11; HNSCC2–01 n = 126; HNSCC2–06 n = 455; HNSCC2–09 n = 180; HNSCC2–11 n = 122; HNSCC2–12 n = 79; HNSCC2–15 n = 205; HNSCC2–26 n = 293; HNSCC2–35 n = 175. One bar = one patient . (B) Representative images and quantification of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) or LVs (CD31 + LYVE-1 + ; cyan) in mouse tumors (MC38, B16F10, and D4M3.A-OVA). Scale bar represents 10 μm. Whole-tumor sections were analyzed. One bar = one mouse. (C) Frequencies of BV-, LV- and non-vessel-associated CCR7 + DCs in mouse MC38 tumors 3 days post anti-CD40 or anti-PD-1 treatment. Whole-tumor sections were analyzed. One bar = one mouse. (D) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) in MC38 tumors inoculated in Ccr7 ko/wt and Ccr7 ko/ko mice, 3 days post anti-PD-1 treatment. (Right) Distribution of the area of CCR7 + DC surfaces in clusters relative to their distance to closest BVs and plotted as percentage of total CCR7 + DC cluster area. CCR7 + DC surfaces from clusters associated with LVs and those not in clusters were excluded from the analysis. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = average value of all clusters in each genotype ( Ccr7 ko/ko n = 5 mice, 56 clusters; Ccr7 wt /ko n = 6 mice, 28 clusters; and Ccr7 wt /wt n = 3 mice, 19 clusters). Two-way ANOVA with multiple comparisons, mean with SEM; **** p < 0.0001 for comparison at 10 and 20 μm from closest BVs. (E) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) and Ccl19 ( Ccl19 -eYFP + Tomato + ; white) in Ccl19 -ieYFP reporter mice (left image) or CCL21 (white, right image) in MC38 tumors. (Right) Frequencies of perivascular CCR7 + DC clusters associated with Ccl19 -covered BVs or within CCL21 + areas of the tumors among total perivascular CCR7 + DC clusters. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one mouse. Unpaired t test, mean with SEM; *** p < 0.001. (F) (Left) Representative images of CCR7 + DCs (FSCN1 + ; yellow) located near BVs (CD31 + LYVE-1 − ; magenta) in MC38 tumors inoculated in Ccl19 wt/wt and Ccl19 ko/ko mice, 2 days post anti-PD-1treatment. (Right) Quantification of BV- or LV-associated CCR7 + DC clusters in MC38 tumors from Ccl19 wt/wt and Ccl19 ko/ko mice. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one mouse, whiskers represent min to max. Unpaired t test; * p < 0.05. (G) Heatmap depicts log 2 -transformed averaged expression of Ccl19 in indicated immune and non-immune populations in the TME of multiple mouse tumor models (breast, , lung [and GSE201247 ], and pancreatic , ). (H) (Left) Synthetic images of CCR7 + DCs (yellow), blood endothelial cells (BECs; magenta), lymphatic endothelial cells (LECs; cyan), and CCL19 + fibroblasts (green) in one representative NSCLC patient analyzed by spatial transcriptomics. (Right) Box plots depict the enrichment scores of CCL19 + fibroblasts within the neighborhood of BV-associated CCR7 + DCs, in four human NSCLC. Data are shown for both permuted (median enrichment scores from 1,000 permutations) and observed datasets. Scale bar represents 20 μm. Whole-tumor sections were analyzed. One dot = one sample. Paired t test, whiskers represent mean to max; * p < 0.05. (I) Heatmap depicts log 2 -transformed averaged expression of CCL19 in indicated immune and non-immune populations in the TME of multiple human cancer types (HNSCC, n = 40, n = 18 patients; CRC, n = 23, n = 64 patients; ESCC, n = 58 patients ; NSCLC, n = 32, n = 7 patients; BRCA, n = 29 patients ; and PRCA, n = 18 patients ). A cross indicates that the cellular population was not detected. See also – .

Article Snippet: InVivoMAb anti-mouse CD40 (clone FGK45) , BioXcell , Cat#BE0016-2.

Techniques: Comparison, Transformation Assay, Expressing, Spatial Transcriptomics

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) (Left) Scheme outlining the experimental setup for bulk RNA-seq analyses of tumor-derived CCR7 + DCs. (Right) GO pathway enrichment analyses performed on differentially expressed genes (DEGs) in CCR7 + DCs in MC38 tumors ( n = 4) from Treg-depleted ( FoxP3 -DTR) compared with Treg-sufficient (WT) mice. Bar plot indicates the −log 10 raw binomial p -values of the top 10 most enriched pathways in CCR7 + DCs. (B) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptides-loaded CCR7 + DCs isolated from WT or Treg-depleted tumors. As a control, CCR7 + DCs without OVA 257–264 peptides were used. Two-way ANOVA with multiple comparisons, whiskers represent min to max; ** p < 0.01. (C) (Left) Relative gene expression levels analyzed by bulk RNA-seq. Each dot represents one mouse ( n = 4), whiskers represent mean to max. Unpaired t test with multiple comparisons; * p < 0.05. (Right) Representative histogram of CD40 protein expression and relative mean fluorescence intensity (MFI) measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 18), whiskers represent min to max. Unpaired t test; ** p < 0.01. (D) Analyses of cDCs in tumor-draining lymph nodes. Absolute cell counts (left, n = 10) and MFI of CD40 expression (right, n = 18) measured by FACS in migratory cDCs (CCR7 + CD8α − ) from WT or Treg-depleted mice. Whiskers represent mean to max. (E) (Left) Experimental setup for ex vivo analyses of tumor CCR7 + DCs isolated from anti-PD-1-treated mice that received or not αCD25 NIB mAbs. (Right) CD40 protein expression measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 4 WT and n = 6 FoxP3-DTR), whiskers represent min to max. Unpaired t test; ** p < 0.01. (F) (Left) Overall survival analyses of MC38 tumor-bearing mice treated, or not treated, with αPD-1 and αCD25 NIB mAbs, and in which CD4 + or CD8 + cells were depleted or not ( n = 8 or 9 mice/group). Log-rank Mantel-Cox test; * p < 0.05, *** p < 0.001, and *** p < 0.0001. (Right) Percentage of tumor-free mice on day 60 in the indicated treatment groups. (G) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs as in (B). The DCs were obtained from mice receiving anti-PD-1 immunotherapy and that were treated or not with αCD25 NIB mAbs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptide-loaded CCR7 + DCs. Each dot represents one mouse ( n = 8 and n = 7), whiskers represent min to max. Two-way ANOVA with multiple comparisons; * p < 0.05. (H) (Left) Scheme outlining bone marrow chimeras with inducible Cd40 -deficiency in cDCs and the treatment schedule. (Right) Growth curves of MC38 tumors inoculated in zDC iDTR : Cd40 WT and zDC iDTR : Cd40 KO bone marrow chimeras treated with αPD-1, αCD25 NIB , or αPD-1 + αCD25NIB combination ( n = 8–10 mice/group). Mean with SEM. Two-way ANOVA with multiple comparisons; * p < 0.05 and **** p < 0.0001. See also and .

Article Snippet: InVivoMAb anti-mouse CD40 (clone FGK45) , BioXcell , Cat#BE0016-2.

Techniques: RNA Sequencing, Derivative Assay, Ex Vivo, Isolation, Control, Gene Expression, Expressing, Fluorescence

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: (A, C) Total and (B, D) virus-specific ASCs were quantified in the liver at week 4 post-infection for (A, B) RHV and (C, D) LCMV clone 13 infection with (E, F) representative ELISpot images showing (A, B) representative and (C, D) pooled data from (A-D) n = 2 independent experiments. Mice were either untreated (WT) or splenectomized and treated with FTY720 prior to infection with treatment being maintained throughout (splX + FTY720 [D -1]). Virus localization was determined by viral transcript density for individual portal and central zones at week 1 post-infection during (G) RHV and (H) LCMV clone 13 infection from n = 10 portal and n = 10 central zone selections from a Xenium 5 K spatial transcriptomics run of n = 1 mouse liver per group. Transcript density of genes associated with oxidative phosphorylation usage were quantified in (I) portal zone hepatocyte regions and (J) immature lymphocytic clusters at week 1 post-infection. Following (K) upstream staining, (L-O) individual transcript localization was determined in the liver at week 1 post-infection. (A-D, G-J) Mean + SEM. (A-D, G-J) Two-tailed, unpaired t-tests were performed. (A) p = 0.9168, (B) p = 0.1987, (C) p = 0.0008, (D) p < 0.0001, (G) p = 0.0261, (H) p = 0.0282. Statistical significance was denoted as *=(p ≤ 0.05), **=(p ≤ 0.01), ***=(p ≤ 0.001), and ****=(p ≤ 0.0001).

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Virus, Infection, Enzyme-linked Immunospot, Phospho-proteomics, Staining, Two Tailed Test

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: a – c , H&E staining of formalin-fixed, paraffin-embedded (FFPE) spleen ( a ) and mesenteric lymph node ( b ) sections at 4 weeks post-LCMV infection and a liver section at 4 weeks post-RHV infection ( c ). Regions of interest (ROIs) are enlarged on the right. One tissue sample from each condition was utilized for Visium HD spatial transcriptomics based on similar morphological H&E staining with limited interindividual variability for n = 4 ( a ), n = 4 ( b ) and n = 5 ( c ) mice. d – h , Visium HD spatial transcriptomic of slides shown in a – c . Individual transcript localization is shown as log 2 -scaled heat maps of 8-μm bins for Ms4a1 ( d ), H2afx ( e ), Cd3g ( f ), Ccl21a ( g ) and Xbp1 ( h ). i , Graph-based subclustering of clusters of interest from sections in a – c were manually annotated and cross-validated with ACT and PanglaoDB cell annotation databases. IFZ, interfollicular zone; SCS, subcapsular sinus. Colours indicate cell type and anatomical zones.

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Staining, Formalin-fixed Paraffin-Embedded, Infection

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: (A-F) Visium HD spatial transcriptomic outputs at 4 weeks post-RHV infection in the liver displayed as log2-scaled heatmaps of 8 μm bins for single-parameter panels and feature sums for multiple-parameter lists. (A) Transcripts characteristic of central zone (zone 3) are shown in orange ( Glul, Cyp2e1 ) while those characteristic of portal zone (zone 1) are shown in green ( Hal, Arg1 ). (B) Co-expression of central (orange) and portal (green) zone-related transcripts shown in (A) as combined feature sums. (C) Merged expression of central (red, Glul and Cyp2e1 ) and portal (green, Hal and Arg1 ) combined transcripts. (D) Expression of various plasma cell-related transcripts as shown in blue ( Xbp1 , Derlr3 , Jchain , Irf4 ). (E) Merged expression of transcripts indicative of the central zone (red, Glul and Cyp2e1 ) and plasma cells ( Xbp1 , Derl3 , Jchain , Irf4 ). (F) Merged expression of transcripts indicative of the portal zone (green, Hal and Arg1 ) and plasma cells ( Xbp1 , Derl3 , Jchain , Irf4 ). (F) Co-expression of plasma cell-related transcripts ( Xbp1 , Derl3 , Jchain , Irf4 ) as feature sum list. (A, F) From morphologically similar H&E staining with limited interindividual variability conducted on n = 5 mice, data is shown from n = 1 mouse liver tissue with which Visium HD spatial transcriptomics was conducted.

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Infection, Expressing, Clinical Proteomics, Staining

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: a – c , Visium HD spatial transcriptomics from liver tissue at four weeks post-RHV infection. a , Transcript feature sums of log 2 -scaled heat maps of 8-μm bins for osteopontin ( Spp1 ), Cxcl12 , type I collagen ( Col1a1 and Col1a2 ( Col1a1/2 )), Icam2 , fibronectin ( Fn1 ), Cd44 , Cxcr4 , CD138 ( Sdc1 ), LFA-1 ( Itga4 / Itgb1 ) and VLA-4 ( Itgal/Itgb7 ). b , Merged transcript localization of feature sum lists from a . c , H&E image with portal vein (blue) and central vein (yellow) ROIs (left) with associated transcript localization (right). d , Cartoon diagram representing plausible molecular factors responsible for intrahepatic plasma cell retention. LSEC, liver sinusoidal endothelial cell. Created in BioRender. Grakoui, A. (2025) https://BioRender.com/ppmu1j5 . e , f , Intrahepatic total ( e ) and E2-specific ( f ) ASCs at 4 weeks post-infection with or without acute blockade of anchoring molecules at days 26 and 27 post-infection. AMD, AMD3100; anti-V/L, anti-VLA-4 plus anti-LFA-1; anti-V/L/S, anti-VLA-4, anti-LFA-1 plus anti-SPP1. n = 2 independent experiments. Control versus anti-V/L + AMD: P = 0.0003 ( e ), P = 0.0182 ( f ). g – i , Xenium Prime 5K spatial transcriptomics on liver tissue at three weeks post-infection with upstream morphological staining ( g ), virus, vasculature and plasma cell transcript localization ( h ), and virus, GC-associated and plasma cell transcript localization in periportal regions ( i ). j , RHV RNA in serum plotted against intrahepatic E2-specific ASC frequencies at four weeks post-infection. μMT, B6.129S2-Ighmtm1Cgn/J mice lacking mature B cells; dpi, days post infection. k , l , Bulk IgH BCR sequencing at 4 weeks post-infection from n = 3 RHV-infected mice, n = 3 LCMV-infected mice and n = 1 naive mouse. SHM accrual is plotted as nucleotide divergence from germline sequences among distinct clonotypes ( k ) and IgH V–J gene pairing chord diagrams ( l ). m – o , Intrahepatic common Igkc transcript localization alongside unique Igkv gene family transcripts with upstream morphological staining ( m ) and segmented cell borders showing transcript localization of Igkc with Igkv4-51 ( n ) and Igkc with Igkv15-103 ( o ). e , f , j , Data are representative or pooled values from at least two independent experiments of at least three mice per group. e , f , k , Data are mean + s.e.m. One-way ANOVA with Tukey’s multiple comparisons test ( e , f , k ); two-tailed nonparametric Spearman correlations with Pearson’s r ( j ).

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Infection, Clinical Proteomics, Control, Staining, Virus, Sequencing, Two Tailed Test

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: (A) Number of total IgG + ASCs in the liver at week 4 post-RHV infection following splenectomy one week prior to infection or FTY720 administration beginning at day 11 post-infection onward with representative ELISpot image. (B) Correlation between % plasma cells (CD38 low CD138 + of total B cells) and % GC B cells (CD38 low CD95 + of total B cells) in the liver at 4 weeks post-infection with RHV. (C) Representative H&E staining of FFPE sections from spleen at week 4 post-LCMV infection and liver at week 4 post-RHV infection. (D) Immunofluorescence staining of FFPE sections at 4 weeks post-RHV infection. Localization of representative Ig kappa gene families displayed as log2-scaled heatmaps of 8 μm bins at 4 weeks post-infection in (E) spleen (LCMV), (F) mesenteric lymph nodes (LCMV) and (G) liver (RHV). (H) From liver tissue at 4 weeks post-infection with RHV following FTY720 treatment, H&E (top) is shown for corresponding ROIs where Igkv19–93 localization is shown as log2-scaled heatmaps of 8 μm bins (bottom). (I) Correlation between intrahepatic CD38 low CD95 + GC B cells and RHV serum viremia at 4 weeks post-infection. (A-B) Data shown are representative or pooled values from 2-3 independent experiments of 3–9 mice per group. Visium HD spatial transcriptomics was conducted with liver tissue from n = 1 representative mouse following similar H&E morphological staining with limited interindividual variability from (C) n = 4 (spleen) and n = 4 (liver), (E) n = 4, (F) n = 4, (G) n = 5, and (H) n = 4 mice. (D) Representative image shown from immunofluorescent staining that was performed with liver tissue from n = 4 mice. (A) Mean + SEM. Statistical tests performed were (A) one-way ANOVA with Tukey’s multiple comparisons test and (B, I) two-tailed nonparametric Spearman correlation with Pearson’s r coefficient. (A) Uninfected vs. WT: p < 0.0001, WT vs. FTY720 D + 11: p = 0.9939, FTY720 D + 11 vs. Splenectomy: p = 0.9853. Statistical significance was denoted as ****=(p ≤ 0.0001).

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Infection, Enzyme-linked Immunospot, Clinical Proteomics, Staining, Immunofluorescence, Two Tailed Test

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm 2 of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection. Data are mean + s.e.m. d – g , One-way ANOVA with Tukey’s multiple comparisons test. AIH versus HBV: P = 0.0101 ( g ). h , Quantification of cell types and their direct contact partners within leukocytic aggregates from annotated spatial transcriptomics with subcellular resolution and cell segmentation during mouse RHV (orange) and human HCV (blue) infection. HSC, hepatic stellate cell. i – n , RHV-infected mouse liver ( i , k , m ) and HCV-infected human liver ( j , l , n ) tissue. Generative GC-like structures were characterized upstream staining ( i , j ) and GC-associated transcript localization ( k , l ) and colour-coded cell-type annotation with selected overlaid transcripts ( m , n ). o – t , RHV-infected mouse liver ( o , q , s ) and HCV-infected human liver ( p , r , t ) tissue. Areas of intrahepatic plasma cell residency were characterized by upstream staining ( o , p ) and plasma cell and hepatic stellate cell and fibroblast-associated transcripts ( q , r ) with colour-coded cell-type annotation ( s , t ). Based on similar morphological H&E staining with limited interindividual variability of n = 2 HCV-infected humans and n = 4 RHV-infected mice, Xenium 5K was performed on tissue from n = 2 human and n = 1 mouse livers, from which n = 1 representative tissue of each are shown in i – t .

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Infection, Staining, Clinical Proteomics

Journal: Nature

Article Title: iHALT unlocks liver functionality as a surrogate secondary lymphoid organ

doi: 10.1038/s41586-025-09803-4

Figure Lengend Snippet: Xenium Prime 5 K spatial transcriptomics was performed with liver tissue obtained during human (A, B) AIH and (C, D) HBV infection. Depicted are regions of interest showing (A, C) upstream morphological staining and (B, D) expression of various color-coded transcripts and annotated cell types. Data shown from (A, B) n = 1 individual with AIH and (C, D) n = 1 individual chronically infected with HBV from which liver tissue was selected for spatial transcriptomics conducted on (A, B) n = 1 individual and (C, D) n = 2 individuals with limited interindividual variability.

Article Snippet: Fig. 5 Hepaciviral infection in mouse and human induce intrahepatic lymphoid structures with highly similar cellular composition, organizational microarchitecture and cell–cell contacts. a , b , Xenium Prime 5K spatial transcriptomics of liver tissue from healthy, AIH, HBV-infected and HCV-infected humans with upstream staining ( a ) and selected transcript localization ( b ). c – g , Quantitative analyses from spatial transcriptomics demonstrating number of leukocytic aggregates per mm of tissue ( c ), aggregate area of leukocytic aggregates ( d ), and lymphocytic cell-type proportions of B cells ( e ), CD4 + T cells ( f ) and CD8 + T cells ( g ) observed in leukocytic aggregates from individuals with AIH ( n = 1), chronic HBV ( n = 2) and chronic HCV ( n = 2) infection.

Techniques: Infection, Staining, Expressing