Journal: bioRxiv

Article Title: Immune and Mutational Profile of Gene-Edited Low-Immunogenic Human Primary Cholangiocyte Organoids

doi: 10.1101/2025.01.20.628680

Figure Lengend Snippet: (a) Graphs showing percentage of positive mouse and human CD45 cells, percentage of human CD3, CD8, CD19 and human CD45 cell counts in weekly in peripheral blood (tail vein bleeds) for the two PBMC donor groups (PBMC Donor A, top; PBMC Donor B, bottom). Error bars represent mean±SEM of 4-8 mice per group.

Article Snippet: Humanization was evaluated weekly by flow cytometry of tail-vein bleeds collected in heparin-coated tubes (Sarstedt Ltd, 20.1309), followed by red cell lysis removal (StemCell Technologies, 07850) according to manufacturer’s instructions and using the following conjugated antibodies: mouse anti-human CD45-FITC (1:50, Thermo Fisher Scientific, 11-0459-42, clone [HI30]), mouse anti-mouse CD45.1-PE-Cy7 (1:100, Thermo Fisher Scientific, 25-0453-82, clone [A20]), mouse anti-human CD3-APC (1:50, Biolegend, clone [UCHT1]), mouse anti-human CD19-PE (1:50, Biolegend, 363004, clone [SJ25C1]), and mouse anti-human CD8-APC/Cy7 (1:50, Thermo Fisher Scientific, A15441, clone [RFT8]).

Techniques:

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: Lethally-irradiated B6 mice were grafted with a 1:1 mixture of B6 (H-2 b ) and B6D2F1 (H-2 bd ) bone marrow and co-injected with WT or HP1α-deficient naive CD4 + T cells alone or in the presence of ex vivo expanded WT Treg. To control engraftment, a group of mice was also injected solely with the bone marrow cell mixture. a Experimental model. b Representative dot-plots showing the frequency of syngeneic (H-2K b+ ) and semi-allogeneic (H-2K bd+ ) cells in the blood 21 days after engraftment. c Percentage of semi-allogeneic (H-2K d+ ) cells among peripheral blood mononuclear cells (PBMC) 21 days after engraftment. Data show mean ± SEM of 3 to 6 independent experiments. Each symbol represents the mean of an experiment. P values were calculated using multiple unpaired t-tests with Holm-Šidák correction. Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Irradiation, Injection, Ex Vivo, Control

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: Lethally-irradiated B6 mice were grafted with a 1:1 mixture of B6 and B6D2F1 bone marrow and co-injected with WT or HP1α-deficient naive CD4 + T cells alone or in the presence of ex vivo expanded WT Treg. a Representative dot-plots showing 21 days after engraftment the percentage of WT and HP1α KO TCR Vβ6 + CD4 + T cells producing IFN-γ and IL-17A. b – d Percentage of WT or HP1α KO TCR Vβ6 + CD4 + T cells producing IFN-γ (b), IL-17A ( c ) or both ( d ) 21 days after engraftment. e Volcano plot showing results of differential gene expression analyses between Treg that had been co-injected with WT or HP1α KO naive CD4 + T cells. Red and black dots represent genes with higher expression in Treg co-injected with HP1α KO or WT cells, respectively. Gray dots represent genes that failed to reach the FDR threshold of 0.05 and the absolute log2 fold change threshold of 1. f Absolute number of spleen cells 21 days after engraftment. Data are mean ± SD of three (Tconv) or seven (Tconv + Treg) biological replicates from two independent experiments. g Percentage of Tconv, defined as CD4 + CD45.1 - CD45.2 + H-2K d- Thy1.1 - , in the spleen 21 days after engraftment. Data are mean ± SEM of three independent experiments. h Percentage of allospecific Tconv, defined as CD4 + CD45.1 - CD45.2 + H-2K d- Thy1.1 - TCRVβ6 + , in the spleen 21 days after engraftment. Data are mean ± SEM of three independent experiments. i, j Percentage of CD73 + FR4 + ( i ) or Foxp3 + ( j ) cells among WT or HP1α KO TCR Vβ6 + CD4 + T cells, as determined in the spleen 21 days after engraftment. b – d and g – j Data show mean ± SEM of 3 independent experiments. Each symbol represents the mean of an experiment. P values were calculated using multiple unpaired t test with Holm-Šidák correction. Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Irradiation, Injection, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: a Differentially-expressed genes (DEG) between WT and HP1α-deficient T cells. b GSEA of KEGG pathways performed using transcriptomes of HP1α-deficient and WT Tconv exposed to Treg. c GO enrichment analyses of genes more highly expressed in WT Tconv than in HP1α KO Tconv exposed to Treg. d GSEA of exhaustion signature genes performed using transcriptomes of HP1α-deficient and WT Tconv exposed to Treg. e Number of differentially-accessible peaks (DAP) between WT and HP1α-deficient T cells. f Relationship between the genes more highly expressed and the genes associated with peaks more open in HP1α KO Tconv than in WT Tconv exposed to Treg. g Expression levels of the 54 genes selected from the intersection in f . Horizontal bars represent the median, and the top and bottom of the boxes the upper and lower quartiles, respectively. The whiskers go from the minimum to the lower quartile and from the upper quartile to the maximum. Statistical significance was calculated using the Pairwise Wilcoxon Rank Sum Test (two-tailed). h GO enrichment analyses of the 54 genes selected from the intersection in f . i Th1 signature enrichment analyses of the 54 genes selected from the intersection in f . j ATAC–seq and RNA-seq tracks at the Il2 locus. k UMAP plot of tumor-infiltrating CD4 + T lymphocytes from melanoma patients . Cells are colored based on 6 clusters defined using the SNN algorithm. l Relative proportions of CD4 + T cell subsets in patients who responded (R) or not (NR) to immunotherapy. m Teff:Tex ratio in R and NR patients. Statistical significance was calculated using Wilcoxon rank sum Test (two-tailed). Horizontal bars represent the median, and the top and bottom of the boxes the upper and lower quartiles, respectively. The whiskers go from the minimum to the lower quartile and from the upper quartile to the maximum. n GSEA of genes downregulated in HP1α-deficient vs control Tconv performed using transcriptomes of human Teff and Tex. Significance was estimated using rank-based gene permutations. Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Expressing, Two Tailed Test, RNA Sequencing Assay, Control

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: Lethally-irradiated B6 mice were grafted with a 1:1 mixture of B6 and B6D2F1 bone marrow and co-injected with WT or HP1α KO ( a, b ) or SUV39H1 KO ( c, d ) naive CD4 + T cells alone or in the presence of ex vivo expanded WT Treg. To control engraftment, a group of mice was also injected solely with the bone marrow cell mixture. a, c Representative dot-plots showing the frequency of syngeneic (H-2K b+ ) and semi-allogeneic (H-2K bd+ ) cells in the blood 21 days after engraftment. b, d Percentage of semi-allogeneic (H-2K d+ ) cells among PBMC 21 days after engraftment. Data show mean ± SEM of 3 to 8 independent experiments. Each symbol represents the mean of an experiment. P values were calculated using multiple unpaired t test with Holm-Šidák correction. Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Irradiation, Injection, Ex Vivo, Control

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: Lethally-irradiated B6 mice were grafted with a 1:1 mixture of B6 and B6D2F1 bone marrow and co-injected with WT (a-d and I, j) or HP1γ-deficient ( a–j ) naive CD4 + T cells alone or in the presence of ex vivo expanded WT Treg. a Representative dot-plots showing 21 days after engraftment the percentage of WT and HP1γ KO TCR Vβ6 + Tconv producing IFN-γ and IL-17A. b, c Percentage of WT or HP1γ KO TCR Vβ6 + Tconv producing IFN-γ ( b ) or IL-17A ( c ) 21 days after engraftment. d Percentage of CD73 + FR4 + cells among WT or HP1γ KO TCR Vβ6 + Tconv, as determined in the spleen 21 days after engraftment. e Percentage of cytokine-producing cells among HP1γ KO TCR Vβ6 + Tconv 21 days post-engraftment. The analysis was performed on total Tconv from mice injected with Tconv only, or on anergic (A) or non-anergic (N-A) Tconv from mice injected with Tconv and Treg. ( f ) Representative dot-plots showing 21 days after engraftment the percentage of HP1γ KO TCR Vβ6 + Tconv expressing PD-1 and TIGIT. g, h Percentage of PD-1 + , TIGIT + , LAG-3 + ( g ) or PD-1 + TIGIT + LAG-3 + ( h ) cells among HP1γ KO TCR Vβ6 + Tconv 21 days after engraftment. i Percentage of Foxp3 + cells among WT or HP1γ KO TCR Vβ6 + Tconv, as determined in the spleen 21 days after engraftment. j Percentage of spleen Foxp3 + cells among anergic or non-anergic WT and HP1γ KO TCR Vβ6 + Tconv 21 days after engraftment. b – d and I, j Data are represented as mean ± SEM of three independent experiments. Each symbol represents the mean of an experiment. P values were calculated using multiple unpaired t test with Holm-Šidák correction. e, g, h Floating bar charts represent the mean of the biological replicates as well as the minimum and maximum values. Each symbol represents individual biological replicates. P values were calculated using unpaired t test (two-tailed). Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Irradiation, Injection, Ex Vivo, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: a Pattern of expression of genes overexpressed in Treg-exposed HP1γ KO Tconv compared with WT Tconv. ( b ) Cluster 2 genes expression in indicated Tconv populations. Horizontal bars represent the median, and the top and bottom of the box the upper and lower quartile, respectively. The whiskers go from the minimum to the lower quartile and from the upper quartile to the maximum. Statistical significance was calculated using the Pairwise Wilcoxon Rank Sum Test (two-tailed). c Exhaustion signature enrichment analyses of cluster 2 genes. d GO enrichment analyses of cluster 2 genes. e Euler diagram showing the relationship between cluster 2 genes and those associated with peaks more open in Treg-exposed HP1γ KO Tconv than WT Tconv. Exhaustion signature enrichment analysis was run on genes common to both groups. f ATAC–seq and RNA-seq tracks at the Lag3 locus. g ATAC-seq signal at peaks associated with cluster 2 genes. Peaks were divided into two sets according to whether or not they were differentially open in WT and KO T cells exposed to Treg. h–k PD-1 and LAG3 expression on naive CD4 + T cells before or after two days of culture with anti-CD3 antibody. h, j Representative histograms showing the expression of PD-1 ( h ) or LAG3 ( j ) in naive and activated T cells. i, k Percentage of activated T cells expressing PD-1 ( i ) or LAG3 ( k ) (left) and average immune checkpoint expression per cell (right). l Representative dot-plots showing PD-1 versus LAG3 expression by ex vivo stimulated CD4 + T cells with or without (control) DMSO or chaetocin. m, n Percentage of CD4 + T cells, either ex vivo activated and treated or not (control) with DMSO or chaetocin, expressing PD-1 ( m ) or LAG-3 ( n ). I, k, m, n Data show mean ± SD of biological replicates from 4 independent experiments. Each symbol represents an individual biological replicate. P values were calculated using unpaired t test (two-tailed). Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Expressing, Two Tailed Test, RNA Sequencing Assay, Ex Vivo, Control

Journal: Nature Communications

Article Title: Heterochromatic gene silencing controls CD4 + T cell susceptibility to regulatory T cell-mediated suppression in a murine allograft model

doi: 10.1038/s41467-025-55848-4

Figure Lengend Snippet: HLA-B7 - WT or HP1γ-deficient human naive CD4 + T cells were injected i.v . into sublethally-irradiated NSG mice with or without ex vivo expanded HLA-B7 + human Treg. Three weeks after injection, splenocytes were isolated and the xenogeneic T cell response was analyzed by flow cytometry. a Experimental model. b , c Following genome editing by CRISPR-Cas9, the expression level of HP1γ was determined in WT and HP1γ KO human CD4 + T cells. A representative western-blot ( b ) and normalized expression levels for each experiment ( c ) are shown. d Representative dot-plots showing the percentage of HLA-B7 - WT and HP1γ KO human Tconv producing IFN-γ and GM-CSF. e – g Percentage of HLA-B7 - WT and HP1γ KO human Tconv producing IFN-γ ( e ), GM-CSF ( f ) or Granzyme B ( g ). h Expression level of T-bet in HLA-B7 - WT and HP1γ KO human CD4 + T cells. i , j Percentage of HLA-B7 - WT and HP1γ KO human CD4 + T cells producing IL-10 (i) or expressing Foxp3 ( j ). Data show values for biological replicates from two ( e – j ) or three ( c ) independent experiments. Horizontal bars represent mean ± SD ( e – j ) or mean ± SEM ( c ). P values were calculated using two-tailed unpaired t tests. Source data are provided in the Source data file.

Article Snippet: Extracellular staining was performed with BV421-coupled anti-TCRβ (H57-597, BD Biosciences), BUV395-labeled anti-CD4, APC-Cy7-conjugated anti-CD8 (eBioH35-17.2, Thermo Fisher Scientific), PE-Cy7-conjugated anti-CD44 (IM7, BD Biosciences), APC-coupled anti-CD62L (MEL-14, Thermo Fisher Scientific) and PE-conjugated anti-CD25 antibodies.

Techniques: Injection, Irradiation, Ex Vivo, Isolation, Flow Cytometry, CRISPR, Expressing, Western Blot, Two Tailed Test