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Journal Articles

Correction: Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology

Ian M. Mbano, Nuo Liu, Marc H. Wadsworth, II, Mark J. Chambers, Thabo Mpotje, Osaretin E. Asowata, Sarah K. Nyquist, Kievershen Nargan, Duran Ramsuran, Farina Karim, Travis K. Hughes, Joshua D. Bromley, Robert Krause, Threnesan Naidoo, Liku B. Tezera, Michaela T. Reichmann, Sharie Keanne Ganchua, Henrik N. Kløverpris, Kaylesh J. Dullabh, Rajhmun Madansein, Sergio Triana, Adrie J.C. Steyn, Bonnie Berger, Mohlopheni J. Marakalala, Gabriele Pollara, Sarah M. Fortune, JoAnne L. Flynn, Paul T. Elkington, Alex K. Shalek, Alasdair Leslie
Journal: Journal of Experimental Medicine
J Exp Med (2026) 223 (3): e2025106701072026c.
https://doi.org/10.1084/jem.2025106701072026c
Published: 14 January 2026
View article titled, Correction: Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology
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Journal Articles

Correction: Dominant negative ADA2 mutations cause ADA2 deficiency in heterozygous carriers

Marjon Wouters, Lisa Ehlers, Wout Van Eynde, Meltem Ece Kars, Selket Delafontaine, Verena Kienapfel, Mariia Dzhus, Rik Schrijvers, Petra De Haes, Sofie Struyf, Giorgia Bucciol, Yuval Itan, Alexandre Bolze, Arnout Voet, Anneleen Hombrouck, Leen Moens, Benson Ogunjimi, Isabelle Meyts
Journal: Journal of Experimental Medicine
J Exp Med (2026) 223 (2): e2025049912162025c.
https://doi.org/10.1084/jem.2025049912162025c
Published: 09 January 2026
View article titled, Correction: Dominant negative ADA2 mutations cause ADA2 deficiency in heterozygous carriers
Open the PDF for Correction: Dominant negative ADA2 mutations cause ADA2 deficiency in heterozygous carriers in another window
Images
Shows original image, before it was corrected on December 29, 2025.

Shows original image, before it was corrected on December 29, 2025.

in Correction: Dominant negative ADA2 mutations cause ADA2 deficiency in heterozygous carriers > Journal of Experimental Medicine
Published: 09 January 2026
Shows original image, before it was corrected on December 29, 2025. More about this image found in Shows original image, before it was corrected on December 29, 2025.
Images
Clinical impact of R169Q in the UK Biobank and FinnGen. Forest plots depict ORs with 95% confidence intervals for different DADA2 phenotypes in the UK Biobank (UK BB) and FinnGen. ORs, odds ratios.

Clinical impact of R169Q in the UK Biobank and FinnGen. Forest plots depic...

in Correction: Dominant negative ADA2 mutations cause ADA2 deficiency in heterozygous carriers > Journal of Experimental Medicine
Published: 09 January 2026
Figure 8. Clinical impact of R169Q in the UK Biobank and FinnGen. Forest plots depict ORs with 95% confidence intervals for different DADA2 phenotypes in the UK Biobank (UK BB) and FinnGen. ORs, odds ratios. More about this image found in Clinical impact of R169Q in the UK Biobank and FinnGen. Forest plots depic...
Journal Articles

Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology

Ian M. Mbano, Nuo Liu, Marc H. Wadsworth, II, Mark J. Chambers, Thabo Mpotje, Osaretin E. Asowata, Sarah K. Nyquist, Kievershen Nargan, Duran Ramsuran, Farina Karim, Travis K. Hughes, Joshua D. Bromley, Robert Krause, Threnesan Naidoo, Liku B. Tezera, Michaela T. Reichmann, Sharie Keanne Ganchua, Henrik N. Kløverpris, Kaylesh J. Dullabh, Rajhmun Madansein, Sergio Triana, Adrie J.C. Steyn, Bonnie Berger, Mohlopheni J. Marakalala, Gabriele Pollara, Sarah M. Fortune, JoAnne L. Flynn, Paul T. Elkington, Alex K. Shalek, Alasdair Leslie
Journal: Journal of Experimental Medicine
J Exp Med (2026) 223 (3): e20251067.
https://doi.org/10.1084/jem.20251067
Published: 05 January 2026
View article titled, Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology
Open the PDF for Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology in another window
Includes: Supplementary data
Images
Overview of the single-cell and spatial data generated from TB-diseased and control lungs. (A) Schematic showing the experimental flow for the isolation of cells from human lung tissues, generation of single-cell libraries using Seq-Well S3. Four TB-negative and nine TB-positive lung samples were processed through scRNA-seq. Shown adjacent to the process flow is a low-dimensional embedding (UMAP) of the 19,632 cells passing quality control annotated with high-level cell types (middle) or detailed cell subtype (right). (B) 10x Visium platform workflow for spatial transcriptomics profiling on FFPE samples from TB-diseased lung resections. 21 of these samples come from current TB patients with detectable M.tb; 9 came from post-TB patient, where bacteria are no longer detected in BAL TB culture after infection. Samples contain either granulomas, iBALTs, or lung LNs, representing different pathological states.

Overview of the single-cell and spatial data generated from TB-diseased and...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 1. Overview of the single-cell and spatial data generated from TB-diseased and control lungs. (A) Schematic showing the experimental flow for the isolation of cells from human lung tissues, generation of single-cell libraries using More about this image found in Overview of the single-cell and spatial data generated from TB-diseased and...
Images
Overview of tissue heterogeneity and cell type abundance in the single-cell dataset. (A) Cell type proportions by disease status (left) and patient (right, n = 7 HIV+TB+; n = 2 TB+; n = 1 HIV+, n = 3 cancer control). (B) Low-dimensional embedding (UMAP) of all scRNA-seq data colored by patient HIV status (left) and TB status (right). (C) Dot plot showing expression levels of top 2 DE genes in each of the broad-level cell types. (D) Two-sided Fisher’s exact test for abundance of major cell types between samples from patients with previous TB diagnosis and samples from control patients. Holm’s method was applied to adjust P values for multiple-testing correction. Statistical annotations: P value < 0.05 (*) and P value < 0.001 (***).

Overview of tissue heterogeneity and cell type abundance in the single-cell...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 2. Overview of tissue heterogeneity and cell type abundance in the single-cell dataset. (A) Cell type proportions by disease status (left) and patient (right, n = 7 HIV+TB+; n = 2 TB+; n = 1 HIV+, n = 3 cancer control). (B) More about this image found in Overview of tissue heterogeneity and cell type abundance in the single-cell...
Images
Single-cell transcriptomic reveals heterogeneity within monocyte and macrophage populations with disease-specific difference. (A) Monocyte/macrophage (n = 8,318) subclustering reveals 10 subclusters (left), also colored by patient ID (middle) and disease condition (right). (B) Heatmap of subtype top 10 DE genes in each of the monocyte/macrophage subcluster. (C) Expression of marker genes in monocyte/macrophage subclusters by disease conditions. (D) Two-sided Fisher’s exact test on abundance of detailed macrophage (left) and monocyte (right) subclusters between TB conditions. Holm’s method was applied to adjust P values for multiple-testing correction. Statistical annotations: P value < 0.05 (*), P value < 0.01 (**), P value < 0.001 (***), fold-change >1 (Δ), fold-change >2 (ΔΔ), and fold-change <1 (∇). (E) Cell2loc imputed macrophage (left) and monocyte (right) abundance distribution on the Visium dataset grouped by TB and granuloma status (Materials and methods). The 5% quantile of the estimated posterior distribution of cell abundance at each Visium spot is displayed, representing the value of cell abundance that the model has high confidence in. Two-sided Mann–Whitney U test without correction were used for statistical testing. Statistical annotations: P value < 0.0001 (****). (F) Similar to E, but grouped by TB status and HIV status.

Single-cell transcriptomic reveals heterogeneity within monocyte and macrop...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 3. Single-cell transcriptomic reveals heterogeneity within monocyte and macrophage populations with disease-specific difference. (A) Monocyte/macrophage (n = 8,318) subclustering reveals 10 subclusters (left), also colored by patient ID More about this image found in Single-cell transcriptomic reveals heterogeneity within monocyte and macrop...
Images
Fibroblast exhibit TB-specific phenotypes. (A) Fibroblast (n = 1,627) subclustering reveals five subclusters (left), also colored by patient ID (middle) and disease condition (right). (B) Heatmap of subtype top 10 DE genes in each of the fibroblast subcluster. Columns (cells) are annotated by fibroblast subclusters and sample source disease status. (C) Comparing annotation against literature stromal annotation from Travaglini et al. (2020). Left: Original fibroblast UMAP as seen in A colored by mapped cell types in Travaglini et al. (2020). Right: Barplot showing distributions of mapped cell type in each original subcluster. ASM, airway smooth muscle; VSM, vascular smooth muscle; MyoF, myofibroblast; FibM, fibromyocyte; AdvF, adventitial fibroblast; AlvF, alveolar fibroblast; LipF, lipofibroblast; Peri, pericyte; Meso, mesothelial. (D) Reference mapping to the HLCA. Query (all cells in this study, n = 19,632) vs. reference cells (n = 584,944) on integrated UMAP with transferred label from HLCA to query cells. (E) Query (all fibroblasts in this study that was mapped to fibroblast/myofibroblast in label transfer, n = 1,601) and reference lung fibroblast cells (n = 17,500) from HLCA colored by annotation (either “Fibroblast” or “Myofibroblast”) and TB conditions. (F) GSEA on DE genes between TB fibroblasts and TB-negative fibroblasts on HLCA-integrated data.

Fibroblast exhibit TB-specific phenotypes. (A) Fibroblast (n...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 4. Fibroblast exhibit TB-specific phenotypes. (A) Fibroblast (n = 1,627) subclustering reveals five subclusters (left), also colored by patient ID (middle) and disease condition (right). (B) Heatmap of subtype top 10 DE genes in each of More about this image found in Fibroblast exhibit TB-specific phenotypes. (A) Fibroblast (n...
Images
Fibroblast WGCNA (hdWGCNA). (A) High-dimensional WGCNA (hdWGCNA) for gene module detection in fibroblasts of this study. UMAPs are colored by eigengene of each of the seven modules. (B) Evaluation of M1 module expression in fibroblast subclusters. Bonferroni-adjusted P computed from two-sided Wilcoxon test is shown. (C) ORA by enricher on all assigned M1 module genes using MSigDB Gene Ontology Biological Processes (GOBP) gene set database. (D) Top: Bacterial burden of NHP lung granulomas by Gideon et al. (2022) grouped by the time point. Bottom: Evaluation of human TB-myofibroblast module expression in NHP TB fibroblasts on 4-wk and 10-wk samples. Two-sided Mann–Whitney U test without correction was used. Statistical annotations: P value < 0.05 (*), P value < 0.01 (**), and P value < 0.001 (***). (E) Evaluation of human TB-myofibroblast module expression in fibroblasts from granuloma vs uninvolved lungs in an independent NHP study with 4-wk post-infection (p.i) macaques (Bromley et al., 2024) (Materials and methods). Two-sided Mann–Whitney U test without correct was used. Statistical annotations: P value < 0.01 (**) and P value < 0.0001 (****).

Fibroblast WGCNA (hdWGCNA). (A) High-dimensional WGCNA (hdWGCNA) for g...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 5. Fibroblast WGCNA (hdWGCNA). (A) High-dimensional WGCNA (hdWGCNA) for gene module detection in fibroblasts of this study. UMAPs are colored by eigengene of each of the seven modules. (B) Evaluation of M1 module expression in More about this image found in Fibroblast WGCNA (hdWGCNA). (A) High-dimensional WGCNA (hdWGCNA) for g...
Images
Evidence of MMP1+CXCL5+fibroblast populations in TB-diseased human lungs. (A) Representative flow cytometry plot showing the isolation strategy of the PDPN+FAP+ fibroblast population from the CD45-EPCAM cell fraction. (B) Cumulative data on frequency of PDPN+FAP+CD90+ fibroblasts as a fraction of live lung cells from five separate lung resections. Three separate sections were taken from each TB-diseased lung, corresponding to the most diseased and least diseased tissues areas and an intermediate lung piece, according to the expert opinion of the operating surgeon. The Friedman test was used to ascertain statistical significance between proportion of PDPN+FAP+ fibroblast between severity groups. (C) Expression of human TB-myofibroblast signature and SPP1+CHI3L1+ marker genes in the TST challenge site in vivo model. Active TB TST (n = 48): biopsies from participants with microbiologically confirmed pulmonary TB disease within the first month of treatment who underwent TST; latent TB TST (n = 191): biopsies from participants lacking clinical and radiological evidence of active TB disease but with a positive peripheral blood IFN-γ release assay; saline (n = 34): biopsies from participants that received saline under the skin instead of tuberculin. Each dot corresponds to a sample; horizontal lines represent median values. Two-sided Mann–Whitney U test without correct was used. Statistical annotations: P value <0.001 (***), P value < 0.0001 (****).

Evidence of MMP1 + C...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 6. Evidence of MMP1 + CXCL5 + fibroblast populations in TB-diseased human lungs. (A) Representative flow cytometry plot showing the isolation strategy of the PDPN+FAP+ fibroblast population from the CD45-EPCAM cell fraction. (B) More about this image found in Evidence of MMP1 + C...
Images
Cell–cell interaction analysis reveals key discrepancies between TB-diseased and control lung niches. (A) Top 20 ligand–receptor (L–R) pairs from MultiNichenet analysis highlighting putative interaction pairs with upregulated interactions in TB-negative lung compared with TB-diseased lung. (B) Top 20 ligand–receptor (L–R) pairs from MultiNichenet analysis highlighting putative interaction pairs differentially communicating in TB-diseased lungs. (C) Summary of top 200 interactions in TB-diseased and TB-negative/control lungs by the number of interactions between each cell pair. Cartoons on the right of each heatmap show the suggested major modes of interactions in each condition. (D) Circos plots of significant interaction pairs in TB-diseased lungs from LIANA, where sender and receiver cell types in each condition are clustered to reflect similar patterns.

Cell–cell interaction analysis reveals key discrepancies between TB-disease...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 7. Cell–cell interaction analysis reveals key discrepancies between TB-diseased and control lung niches. (A) Top 20 ligand–receptor (L–R) pairs from MultiNichenet analysis highlighting putative interaction pairs with upregulated More about this image found in Cell–cell interaction analysis reveals key discrepancies between TB-disease...
Images
Global interaction analysis identifies key players in cellular communication within TB-diseased lung tissues. (A) Heatmap visualization of interaction flux analysis. Rows represent sender cell types; columns represent receiver cell types. Each entry represents the potential flux of interaction from sender cell to receiver cell, whereas the total flux of each sender cell type is summarized on the left. Sender cell types are sorted based on descending order of total flux (Materials and methods). (B) Top: Bar plot showing top 30 and bottom 30 ligands by log fold-change of interaction strength between TB and control lungs across all sender cell types. Bottom: Log fold-change of interaction strength between TB and control lungs in each sender cell type (Materials and methods). (C) Dot plot of top five ligand by ligand activity in TB-diseased lungs secreted by MMP1+CXCL5+ fibroblasts and their receivers (Materials and methods). (D) L–R interactions with MMP1+CXCL5+ fibroblasts in the TB-diseased lungs; rows (L–R pair) and columns (target cell types) are hierarchically clustered by correlation distance (Materials and methods). L–R, ligand–receptor.

Global interaction analysis identifies key players in cellular communicatio...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 8. Global interaction analysis identifies key players in cellular communication within TB-diseased lung tissues. (A) Heatmap visualization of interaction flux analysis. Rows represent sender cell types; columns represent receiver cell More about this image found in Global interaction analysis identifies key players in cellular communicatio...
Images
Spatial transcriptomics analysis on post- and current TB lung resections. (A) Heatmap showing the expression of human TB-myofibroblast gene signature and SPP1+CHI3L1+ macrophage markers on selective tissue slides from patients who are post-TB (top) or current TB (bottom), alongside paired H&E staining (these H&E stains are also shown in Fig. S1 A together with those other samples used for spatial transcriptomics not shown here). (B) Distribution of human TB-myofibroblast signature expression on the spatial cohort. HIV statuses are shown in different shades of blue for positive or negative. Two-sided Mann–Whitney U test without correction was used for statistical testing. Statistical annotation: P value < 0.0001 (****). (C) Distribution of SPP1+CHI3L1+ macrophage markers and human TB-myofibroblast signature on the spatial data across all Visium spots. Left two panels: Manual segmentation of the granuloma structure was done to allow separation of the Visium slide into three different regions: in granuloma, on granuloma border (cuff), and outside of granuloma (Materials and methods). Right two panels: The same as left panels with the exception that “on border” = True means on granuloma cuff and False means the rest. Two-sided Mann–Whitney U test without correction was used for statistical testing. Statistical annotation: P value < 0.0001 (****). (D) Correlation between human TB-myofibroblast signature and all macrophage subpopulations’ markers. Each circle represents a Visium sample. Boxplot of the Pearson’s r distribution is shown for each macrophage subtype. Mann–Whitney U test without correction were used for statistical testing. Statistical annotation: P value < 0.0001 (****). (E) Spatially informed ligand–receptor (L–R) analysis using LIANA+ on Visium samples. Examples are shown where SPP1(L)–CD44(R) interactions are being nominated as top L–R pairs. H&E overlaid with pathology annotation for granuloma structures are shown next to heatmap of L–R interaction scores, which are calculated at each Visium spot using spatially weighted Cosine similarity (Materials and methods).

Spatial transcriptomics analysis on post- and current TB lung resections. (...

in Single-cell and spatial profiling highlights TB-induced myofibroblasts as drivers of lung pathology > Journal of Experimental Medicine
Published: 05 January 2026
Figure 9. Spatial transcriptomics analysis on post- and current TB lung resections. (A) Heatmap showing the expression of human TB-myofibroblast gene signature and SPP1+CHI3L1+ macrophage markers on selective tissue slides from patients who are More about this image found in Spatial transcriptomics analysis on post- and current TB lung resections. (...
Journal Articles

Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance

Jiří Březina, Tomáš Brabec, David Machač, Matouš Vobořil, Ondřej Ballek, Jan Pačes, Vojtěch Sýkora, Kristína Jančovičová, Evgeny Valter, Katarína Kováčová, Jasper Manning, Valerie Tahtahová, Adéla Čepková, Martina Dobešová, Jan Dobeš, Jan Kubovčiak, Michal Kolář, Petr Kašpárek, Radislav Sedlacek, Ondřej Štepánek, Jan Černý, Sachiko Tsukita, Bernard Malissen, Graham Anderson, Dominik Filipp
Journal: Journal of Experimental Medicine
J Exp Med (2026) 223 (3): e20250970.
https://doi.org/10.1084/jem.20250970
Published: 02 January 2026
View article titled, Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance
Open the PDF for Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance in another window
Includes: Supplementary data
Images
Claudin 1 is upregulated in CAT-experienced DC1s. (A) FACS gating strategy used to perform scRNAseq of thymic myeloid cells. Thymic cells were isolated from Foxn1CreR26TdTOMATO mice, MACS-enriched for CD11c+ and CD11b+ cells, and sorted as either TdTOMATO+ or TdTOMATO−CD45+ CD11c+/CD11b+ cells. (B) UMAP of annotated thymic myeloid cells from scRNAseq excluding subsets shown in red in the legend of Fig. S1 B. Individual cell lineages are demarcated by dotted lines. DC = conventional DC; aDC = activated DC; Mac = macrophages; Mono = monocytes; prolif = proliferating. (C) UMAP of scRNAseq corresponding to Fig. 1 B projecting CAT-experienced (orange) and CAT-inexperienced (gray) cells. A dotted line shows the DC1 subset. (D) Heat map showing top 10 down- and upregulated genes in CAT-experienced over CAT-inexperienced DC1s. The heat map color scale depicts average log2 fold change. (E) Violin plots show the expression of Cldn1 and Cd36 by CAT-experienced (orange) and CAT-inexperienced (gray) DC1. All mice were bred on the B6 background. Littermates were used.

Claudin 1 is upregulated in CAT-experienced DC1s. (A) FACS gating strategy...

in Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance > Journal of Experimental Medicine
Published: 02 January 2026
Figure 1. Claudin 1 is upregulated in CAT-experienced DC1s. (A) FACS gating strategy used to perform scRNAseq of thymic myeloid cells. Thymic cells were isolated from Foxn1CreR26TdTOMATO mice, MACS-enriched for CD11c+ and CD11b+ cells, and More about this image found in Claudin 1 is upregulated in CAT-experienced DC1s. (A) FACS gating strategy...
Images
Lineage tracing and CLAUDIN 1 protein expression in thymic DC1s. (A) Schematic of the mouse model used for lineage tracing. (B) Representative flow cytometry plots show TdTOMATO expression within thymic DC subsets from XCR1iCreR26TdTOMATO mouse model. (C) Frequency of TdTOMATO+ cells within DC subsets from Fig. 2 B (mean ± SEM, n = 14 mice from three independent experiments). (D) Design of BrdU DC1 lineage tracing experiment. (E) Representative flow cytometry plots show the frequency of BrdU+ cells within DC1 subsets on indicated days after the BrdU administration related to Fig. 2 D. (F) Percentage of BrdU+ cells within DC1 lineage subsets on indicated days after BrdU administration related to Fig. 2 E (mean ± SEM, n = 3–5 mice from two independent experiments). (G) Representative flow cytometry plots show CLAUDIN 1 positivity within thymic DC1 subsets. FMO controls are shown. (H) Frequency of CLAUDIN 1+ cells and MFI of CLAUDIN 1 expression within thymic DC1 subsets related to Fig. 2 G (mean ± SEM, n = 20–35 mice from a minimum of three independent experiments). Statistical analysis in F was performed using RM one-way ANOVA with Tukey’s multiple comparisons test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls. FMO, Fluorescence minus one.

Lineage tracing and CLAUDIN 1 protein expression in thymic DC1s. (A) Schem...

in Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance > Journal of Experimental Medicine
Published: 02 January 2026
Figure 2. Lineage tracing and CLAUDIN 1 protein expression in thymic DC1s. (A) Schematic of the mouse model used for lineage tracing. (B) Representative flow cytometry plots show TdTOMATO expression within thymic DC subsets from XCR1iCreR26 More about this image found in Lineage tracing and CLAUDIN 1 protein expression in thymic DC1s. (A) Schem...
Images
Claudin 1 is involved in CAT and homeostatic DC1 maturation. (A) Representative flow cytometry plots show the acquisition of TdTOMATO by thymic DC subsets from Defa6iCreR26TdTOMATO mice. FMO controls are shown (R26TdTOMATO). (B) Frequency of TdTOMATO+ cells and MFI of TdTOMATO expression within thymic DC subsets from Fig. 3 A (mean ± SEM, n = 7 mice from two independent experiments). (C) Frequency of CLAUDIN 1+ cells and MFI of CLAUDIN 1 expression within TdTOMATO+ and TdTOMATO− DC1 subsets from thymi of Defa6iCreR26TdTOMATO mice (mean ± SEM, n = 9–14 mice from a minimum of three independent experiments). (D) Schematic of competitive BM chimera experiment assessing the role of Claudin 1 in CAT. (E) Representative flow cytometry plots show the frequency of TdTOMATO+ cells within Claudin 1–sufficient (Ly5.1 BM) and Claudin 1–deficient (XCR1iCreCldn1fl/fl BM) DC1 subsets from competitive BM chimeras in Fig. 3 D. FMO controls are shown (R26TdTOMATO mouse). (F) Frequency of TdTOMATO+ cells within Claudin 1–sufficient and Claudin 1–deficient DC1 subsets from Fig. 3 E (mean ± SEM, n = 10 mice from three independent experiments). (G) Violin plots from scRNAseq analysis (Fig. 1) show the expression of cholesterol efflux–associated genes within CAT-experienced (orange) and CAT-inexperienced (gray) DC1 (left panel) and DC1 lineage subsets (right panel). (H) Frequency of individual DC1 subsets within thymic DCs from Claudin 1–sufficient (solid circle) and Claudin 1–deficient (empty circle) BM from Fig. 3 D (mean ± SEM, n = 10 mice from three independent experiments). Statistical analysis in C, F, and H was performed using paired, two-tailed Student’s t test, *P ≤ 0.05, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls.

Claudin 1 is involved in CAT and homeostatic DC1 maturation. (A) Represent...

in Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance > Journal of Experimental Medicine
Published: 02 January 2026
Figure 3. Claudin 1 is involved in CAT and homeostatic DC1 maturation. (A) Representative flow cytometry plots show the acquisition of TdTOMATO by thymic DC subsets from Defa6iCreR26TdTOMATO mice. FMO controls are shown (R26TdTOMATO). (B) More about this image found in Claudin 1 is involved in CAT and homeostatic DC1 maturation. (A) Represent...
Images
Claudin 1 facilitates positioning and contact between DC1s and mTECs. (A) Flow cytometry gating strategy of mTEC subsets. TECs were gated as EPCAM+CD45− and further distinguished to LY51+UEA− cortical thymic epithelial cells (cTECs) and LY51−UEA+ mTECs. The latter were further separated into AIRE+ITGB4− mTECHI, AIRE–ITGB4+ mTECLO, and double-negative cells, which were comprised of keratinocyte mimetics (LY6D+) and other mimetic cells (LY6D−). Defa6+ mTECs are color-coded in orange denoting their TdTOMATO positivity. (B) Frequency of CLAUDIN 3+ cells within cell populations from Fig. S3 I (mean ± SEM, n = 9 mice from three independent experiments). (C) Frequency of CLAUDIN 3+ cells and MFI of CLAUDIN 3 expression within mTEC subsets from Fig. S3 J (mean ± SEM, n = 5–9 mice from three independent experiments). (D) Schematic of competitive BM chimera used to assess the role of Claudin 1 in positioning of DC1 lineage cells in the proximity of mTECs. Mouse models used as donors of BM are marked by the letters a and b. Note that AdigGFP recipients obtained BM either from mice a (BM mix a) or b (BM mix b). (E) Flow cytometry gating strategy used to analyze the reconstitution of competitive BM chimeras from Fig. 4 D. Thymic CD11c+XCR1+ cells (DC1s) were gated as in Fig. S4 F and either as TdTOMATO+ or TdTOMATO−. Note that TdTOMATO+ DC1s are Cldn1+/+ and TdTOMATO− DC1s are Cldn1−/− in the case of mice receiving BM mix a, and TdTOMATO+ DC1s are Cldn1−/− and TdTOMATO− DC1s are Cldn1+/+ in the case of mice receiving BM mix b. (F) Light sheet fluorescence microscopy images of analogous regions within the thymic medulla of competitive BM chimeras from Fig. 4, D and E. The top images capture the entire medullary compartment imaged. The bottom images visualize segmented objects (red, DC1s; and green, mTEC clusters) within selected regions of the whole 3D images shown above. Separate legends are shown for BM mix a and b. (G) Schematic of the analysis of the regions imaged in Fig. 4 F. The imaged area of each mTEC cluster captured was expanded by 50, 25, or 5 μm, and the percentage of DC1s from the total within these expanded clusters was counted. Note that DC1s localized up to 5 μm from mTEC clusters are considered to be in direct contact with mTECs (left panel). The percentage of DC1s that are within a defined distance of mTEC clusters related to Fig. 4 F. The number above the Cldn1−/− DC1 columns indicates the fold change reduction in the percentage of Cldn1−/− DC1 with respect to the percentage of Cldn1+/+ DC1 (right panel). (H) Violin plots showing minimum distance (μm) between DC1 and the nearest mTEC cluster related to Fig. 4 F. Medians and quartiles are shown (n = 2,259 Cldn1+/+ DC1s and 531 Cldn1−/− DC1s per representative experiment from a total of two experiments). Statistical analysis in B, C, and H was performed using unpaired, two-tailed Student’s t test, ***P ≤ 0.001, ****P < 0.0001, ns = not significant. All mice were bred on the B6 background. Littermates were used as controls.

Claudin 1 facilitates positioning and contact between DC1s and mTECs. (A) ...

in Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance > Journal of Experimental Medicine
Published: 02 January 2026
Figure 4. Claudin 1 facilitates positioning and contact between DC1s and mTECs. (A) Flow cytometry gating strategy of mTEC subsets. TECs were gated as EPCAM+CD45− and further distinguished to LY51+UEA− cortical thymic epithelial cells (cTECs) More about this image found in Claudin 1 facilitates positioning and contact between DC1s and mTECs. (A) ...
Images
Claudin 1 is critical for the expression of antigen presentation–associated genes. (A) Representative FACS gating strategy used to perform bulkSeq of Cldn1+/+ and Cldn1−/− thymic DC1 lineage cells isolated from competitive BM chimera described in Fig. 3 D (n = 3 mice). Cells were gated as CD45.1+ (that included Cldn1+/+ DC1 lin.) or CD45.2+ (that included Cldn1−/−DC1 lin.). Both CD45.1+ and CD45.2+ cells were further gated as DC1, aDC1a, and aDC1b according to Fig. S2 A. DC1 lineage cells sharing the common origin (CD45.1+ or CD45.2+) were sorted into a common collection tube and sequenced. Sorting gates are highlighted by thick lines. (B) Volcano plot of bulkSeq analysis showing up- and downregulated genes in Cldn1−/− over Cldn1+/+ DC1 lineage cells. The threshold of P value adjusted after FDR correction is 0.05 and for log2 fold change is 0.5. The baseMean cutoff was set to 10. (C) Heat map showing relative expression of genes involved in MHCII presentation across individual sequenced samples. The heat map color scale corresponds to z scores of regularized log data values. The numbers below each column in the heat map correspond to three independent biological replicates, each derived from an individual mouse. (D) GSEA of “Antigen processing and presentation of peptide or polysaccharide antigen via MHC class II” pathway between Cldn1+/+ and Cldn1−/− DC1 lineage cells. (E) Heat map showing the relative expression of Cd207 and Cd36 genes in Cldn1+/+ and Cldn1−/− DC1 lineage cells. The heat map color scale corresponds to z scores of regularized log data values. The numbers below each column in the heat map correspond to three independent biological replicates, each derived from an individual mouse. (F) Frequency of DC1 lineage subsets within B220–CD11c+ cells from I-abfl/fl (control; solid circle) and XCR1iCreI-abfl/fl (empty circle) mice (mean ± SEM, n = 5–9 mice from two independent experiments). Statistical analysis in F was performed using unpaired, two-tailed Student’s t test, *P ≤ 0.05, **P ≤ 0.01, ****P < 0.0001. All mice were bred on the B6 background. Littermates were used as controls.

Claudin 1 is critical for the expression of antigen presentation–associated...

in Claudin 1–mediated positioning of DC1 to mTECs is essential for maintenance of central tolerance > Journal of Experimental Medicine
Published: 02 January 2026
Figure 5. Claudin 1 is critical for the expression of antigen presentation–associated genes. (A) Representative FACS gating strategy used to perform bulkSeq of Cldn1+/+ and Cldn1−/− thymic DC1 lineage cells isolated from competitive BM chimera More about this image found in Claudin 1 is critical for the expression of antigen presentation–associated...