Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a) Gene targeting strategy. The auxin-inducible degron (AID) sequence was fused to the N-terminus of Foxp3 via a seven-amino-acid flexible linker. An IRES-ZsGreen-T2A-iCre-Frt-neo-Frt cassette was inserted into the 3’ UTR. Arrows indicate the locations of PCR primers used to distinguish Foxp3 WT and Foxp3 AID alleles. IRES: internal ribosome entry sequence. iCre: codon improved Cre recombinase. PGK: PGK promoter. Neo: neomycin-resistant gene. pA: bGH polyA sequence. (b) PCR validation of the knock-in allele. Data is representative of over 200 mice from each genotype. (c) Expression pattern of the ZsGreen reporter in Foxp3 AID/WT heterozygous females. ZsGreen expression was restricted to CD25⁺ CD4 T cells, consistent with Foxp3 expression. (d) ZsGreen⁺ Foxp3 AID T reg cells suppressed naïve CD4⁺ T cell proliferation comparably to Foxp3 GFP T reg cells in vitro . Line graph represents mean ± SEM of two biological replicates. (e) Naïve CD4⁺ T cells from Foxp3 AID mice were cultured under T reg inducing conditions and transduced with either a TIR1-encoding retrovirus or the empty vector control. AID-tagged Foxp3 protein was selectively degraded in TIR1-transduced induced T reg (iT reg ) cells upon indole acetic acid (IAA) treatment. Scatter plots represents mean ± SEM. Each point represents cells from a unique mouse.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Sequencing, Biomarker Discovery, Knock-In, Expressing, In Vitro, Cell Culture, Transduction, Plasmid Preparation, Control

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a ) Gene targeting strategy. WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element; DTA: Diphtheria toxin fragment A). (b) Southern blot validation of heterozygous ROSA26 TIR1/+ mice using the hybridization probe shown in ( a ). Data is representative of two independent F1 mice. (c) PCR validation of ROSA26 TIR1/+ mice. Data are representative of over 100 mice from each genoype. (d–f) Naïve CD4⁺ T cells from ROSA26 TIR1/+ mice were co-transduced with retroviruses expressing Cre and AID-Foxp3 ( d ). TIR1 expression was induced in a Cre-dependent manner ( e ), resulting in AID-Foxp3 degradation upon IAA treatment ( f ). Data are representative of two independent experiments. (g) Guide RNA (gRNA) design for CRISPR-mediated F74-to-G mutation in TIR1. The gRNA seed sequence is shown in gray; the PAM sequence is in pink. The F74G mutation creates a KasI restriction site. (h–i) Validation of the F74G mutation by KasI digestion ( h ) and Sanger sequencing ( i ). (j–k) The TIR1 F74G mutation enables in vivo protein degradation in response to 5-ph-IAA.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Virus, Southern Blot, Biomarker Discovery, Hybridization, Transduction, Expressing, CRISPR, Mutagenesis, Sequencing, In Vivo

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , Experimental design of scRNA-seq and functional assays. Each genotype and time point consisted of four independent biological replicates. b , UMAP visualization of scRNA-seq data from Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) T reg cells before and 3 or 7 days after 5-ph-IAA-induced Foxp3 degradation. c , UMAP visualization of the same scRNA-seq data, colored by identified clusters. d , Fraction of each cluster within the total pool of Foxp3 AID R26 WT or Foxp3 AID R26 TIR1(F74G) T reg cells separated by time point. Each point represents a unique mouse. e , In vitro suppression assay of T reg cells sorted from Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) mice after 7 days of in vivo 5-ph-IAA treatment. 5-ph-IAA was included in culture to sustain Foxp3 degradation. Line graph represents mean ± s.e.m. Data are pooled from two independent experiments and analyzed using two-sided multiple t -tests. NS, not significant. f , Experimental design of the bulk RNA-seq analysis. Each genotype and time point consisted of three independent biological replicates. g , Gating strategy for sorting resting and activated T reg cells. h , Schematic comparison of the Foxp3 GFPKO reporter-null allele and the functional Foxp3 GFP allele. i , Scatter-plots and bar graphs showing the number of DEGs in resting or activated T reg cells caused by Foxp3 protein degradation or genetic Foxp3 deficiency.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Functional Assay, In Vitro, Suppression Assay, In Vivo, RNA Sequencing, Comparison

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a) UMAP visualization of scRNA-seq data from Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) T reg cells on days 0, 3 and 7 of 5-ph-IAA-induced Foxp3 degradation. (b) UMAP visualization of representative genes from the single-cell RNA-seq dataset colored by expression level. (c) MA plot showing differentially accessible ATAC-seq peaks induced by Foxp3 degradation (left) and Foxp3 gene deficiency (right). Red points represent differentially accessible regions.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: RNA Sequencing, Expressing

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , T reg cells from the scRNA-seq dataset were classified as resting or activated based on exceeding the threshold for resting or activated gene signature scores and were subsequently analyzed. b , Scatter-plot showing the correlation of gene expression changes induced by Foxp3 degradation at day 3 and day 7 in resting and activated T reg cells. FC, fold change. c , UMAP visualization of resting and activated T reg cells colored by gene signature scores for the ‘TIR1-up’ and ‘TIR1-down’ gene sets, up- and downregulated upon Foxp3 degradation, respectively. d , Dot plot summarizing statistically significant DEGs in resting or activated T reg cells following 3 or 7 days of 5-ph-IAA-induced Foxp3 degradation. The color represents the log 2 fold change of R26 TIR1(F74G) versus R26 WT and the size represents the Benjamini–Hochberg adjusted P value of the differential expression test. e , Flow cytometry analysis of Foxp3 protein and mRNA levels (reported by ZsGreen) in Foxp3 AID T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. Scatter-plots represent mean ± s.e.m. Data are pooled from two independent experiments and analyzed using a two-way ANOVA. pLN, peripheral lymph nodes. f , Flow cytometry analysis of CD25, CD122, OX40, GITR and FR4 protein levels in Foxp3 AID T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. g , Flow cytometry analysis of CD127 and TCF1 protein levels in Foxp3 AID T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. Scatter-plots represent mean ± s.e.m. ( f , g ). Each point represents a unique mouse. Data are pooled from two independent experiments and analyzed with two-sided multiple t -tests.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Gene Expression, Quantitative Proteomics, Flow Cytometry

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a) The composition of Foxp3 AID R26 WT and Foxp3 AID R26 WT(F74G) T reg cells within resting and activated subsets from day 0, day 3, and day 7 of Foxp3 degradation. (b) Number of resting and activated T reg cells utilized for the differential gene expression analysis in Fig. . (c) Flow cytometry analysis of Foxp3 protein and mRNA levels (reported by ZsGreen) in Foxp3 WT T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. Scatter plots represent mean ± SEM. Data are pooled from two independent experiments. Two-way ANOVA. (d) Bulk RNA-seq read counts of indicated genes in Foxp3 AID T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. Each point represents a unique mouse. Bar graphs represent mean ± SEM. (e) Flow cytometry analysis of CD25, CD122, OX40, GITR, and FR4 protein levels in Foxp3 WT T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. (f) Flow cytometry analysis of CD127 and TCF1 protein levels in Foxp3 AID T reg cells from heterozygous Foxp3 AID/WT R26 WT and Foxp3 AID/WT R26 TIR1(F74G) females after 7 days of Foxp3 degradation. ( e - f ) Scatter plots represent mean ± SEM. Each point represents a unique mouse. Data are pooled from two independent experiments. Multiple t -tests.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Gene Expression, Flow Cytometry, RNA Sequencing

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , Bar graphs showing the proportion of ATAC-seq peaks near Foxp3 degradation-induced DEGs bound by Foxp3 (ref. ). Genes are stratified by statistical significance ( P values) in resting and activated T reg cells. Bars represent mean ± s.e.m. Data were analyzed using a Mann–Whitney U -test. b , Bar graphs showing the proportion of ATAC-seq peaks near Foxp3-dependent DEGs bound by Foxp3. Genes are stratified by P values in resting and activated T reg cells. Bars represent mean ± s.e.m. Data were analyzed using a Mann–Whitney U -test. c , H3K27Ac and H3K27me3 ChIP-seq signals at Foxp3-bound ATAC-seq peaks near Foxp3 degradation-induced DEGs. Line graph represents mean ± s.e.m. Data were analyzed using a Mann–Whitney U -test. d , Dot plot showing transcription factor motif enrichment within Foxp3-bound regions near Foxp3 degradation-induced DEGs. e , Schematic diagram illustrating the ‘on’ and ‘off’ states of the reversible reporter-null Foxp3 LSL allele. f , Experimental design of the gain-of-function experiment to induce Foxp3 expression in T reg ‘wannabe’ cells. Each genotype and time point consisted of two independent biological replicates. g , Line graph depicting gene expression changes of TIR1-up or TIR1-down genes below a specific P value cutoff across different time points following Foxp3 induction in T reg ‘wannabe’ cells. The line and shading represent the mean ± s.e.m. Data were analyzed using a Mann–Whitney U -test.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: MANN-WHITNEY, ChIP-sequencing, Expressing, Gene Expression

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a) Experimental design of single-cell RNA-seq experiment. Each genotype consisted of four independent biological replicates. (b) Density contour plots of Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) T reg cells from SLO, liver, lung and LILP overlayed on UMAP embeddings. (c) UMAP visualization of T reg cells from depicted tissues colored by “resting” and “activated” gene signature scores based on the scoring strategy from Fig. . (d) Bar plots showing the number of DEGs between R26 WT and R26 TIR1(F74G) in resting and activated T reg cells from indicated tissues, colored by up- or downregulation. (e) Pairwise Pearson correlation coefficients for log fold changes of DEGs between activated and resting T reg cells from each tissue. Rows and columns are hierarchically clustered. (f) Heatmap of log fold changes in Foxp3 degradation DEGs separated by T reg cell activation status and tissue of origin. Genes are clustered by k-means clustering and cluster numbers are indicated by the color bars on the left. (g) Heatmap of average log fold changes for each k-means cluster in T reg cells separated by tissue and activation status. Rows and columns are hierarchically clustered. (h) Leiden clustering results visualized by UMAP for LILP T reg cells. Clusters are labeled as peripheral T reg cell (pT reg ), dividing T reg cell, lymphoid tissue thymic T reg cell (LT-tT reg ) and non-lymphoid tissue thymic T reg cell (NLT-tT reg ) based on differences in their gene expression profile. (i) Heatmap of scaled mean UMI-normalized expression values of indicated genes in each cluster. (j) Number of DEGs between R26 WT and R26 TIR1(F74G) T reg cells in pT reg , LT-tT reg and NLT-tT reg cells, colored by up- or downregulation. (k) Log fold changes of “Foxp3-repressed” and “Foxp3-activated” genes in pT reg , LT-tT reg and NLT-tT reg cells. Each point represents a gene from the Foxp3-repressed or Foxp3-activated gene set identified in Fig. . Horizontal line marks the median and box marks the interquartile range. Mann-Whitney U test. (l) Scatter-plot of log fold changes of DEGs between pT reg and NLT-tT reg cell clusters (left-top), pT reg and LT-tT reg cell clusters (left-bottom) and LT-tT reg and NLT-tT reg cell clusters (right-top). Points are colored by their direction and populations in which they are altered. Correlations were calculated with Pearson correlation over all DEGs.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: RNA Sequencing, Activation Assay, Labeling, Gene Expression, Expressing, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , Experimental design for transcriptional profiling of developing thymic T reg cells. Each genotype consisted of three independent biological replicates. b , Gating strategy used to sort CD73⁻ nascent thymic T reg cells. c , Bar graph comparing the number of Foxp3 degradation-induced DEGs in thymic, resting and activated T reg cells from Foxp3 AID/WT mice. d , Pearson correlation between Foxp3 degradation-induced and Foxp3-dependent DEGs in thymic, resting and activated T reg cells. e , Scatter-plot and cumulative distribution function (CDF) plots comparing Foxp3 degradation-induced and Foxp3-dependent DEGs across the three T reg populations. Data were analyzed using a Mann–Whitney U -test. f , Metacell analysis of thymocyte scRNA-seq data correlating UMI-normalized Foxp3 expression levels in each metacell with the expression of TIR1-up and TIR1-down gene signatures identified in a – c . UMAP plots are colored by scaled expression levels of TIR1-up, TIR1-down and UMI-normalized counts of Foxp3. Dashed red line depicts line of best fit. Correlations and corresponding P values were calculated with Pearson correlation over all genes. g , Experimental design for in vivo Foxp3 degradation in 1-day-old neonatal Foxp3 AID mice and adult Foxp3 AID mice. h , CD4 + and CD8 + T cell activation in adult and neonatal Foxp3 AID mice following Foxp3 degradation. i , Expansion of eosinophils and neutrophils in adult and neonatal Foxp3 AID mice after Foxp3 degradation. j , k , Representative H&E staining ( j ) and histology scores of liver inflammation ( k ) in neonatal Foxp3 AID mice following Foxp3 degradation. l , In vitro suppression assay of T reg cells sorted from Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) neonatal mice after 7 days of in vivo 5-ph-IAA administration. 5-ph-IAA was also included in culture to maintain Foxp3 degradation. Each point represents a unique mouse ( h – l ). Data are pooled from two independent experiments. Scatter-plots represent mean ± s.e.m. Data were analyzed using a one-way ANOVA. m , Bar graphs summarizing the number of Foxp3 degradation-induced DEGs in T reg cells from neonatal and adult Foxp3 AID/y mice after 14 days of Foxp3 degradation. n , Scatter-plot correlating gene expression changes induced by Foxp3 degradation and Foxp3 gene deficiency in neonatal mice. o , Scatter-plots comparing gene expression changes induced by 7 days of Foxp3 degradation in neonatal T reg cells to those in adult thymic, resting and activated T reg cells from Foxp3 AID/WT mice.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: MANN-WHITNEY, Expressing, In Vivo, Activation Assay, Staining, In Vitro, Suppression Assay, Gene Expression

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a) Pearson correlation between Foxp3 degradation-induced and Foxp3-dependent DEGs in thymic, resting, and activated T reg cells, limited to Foxp3-bound genes. (b) Meta-cell analysis of resting and activated T reg scRNA-seq data from secondary lymphoid organs following Foxp3 degradation, correlating Foxp3 expression levels with “TIR1-up” and “TIR1-down” gene signatures identified in Fig. . Dashed red line depicts line of best fit. Correlations and corresponding p-values were calculated with Pearson correlation over all genes. (c) Cytokine production by CD4⁺ T cells from neonatal Foxp3 AID mice after 14 days of Foxp3 degradation, in comparison to age-matched Foxp3 WT and Foxp3 GFPKO mice. (d) Neutrophil expansion in adult and neonatal Foxp3 AID mice following 14 days of Foxp3 degradation. Age-matched Foxp3 WT and Foxp3 GFPKO mice serve as controls for neonatal Foxp3 AID mice. (e) Representative H&E staining and histology scores of skin inflammation in neonatal Foxp3 AID mice after 14 days of Foxp3 degradation, in comparison to age-matched Foxp3 WT and Foxp3 GFPKO mice. ( c – e ) Scatter plots represent mean ± SEM. Each point represents a unique mouse. Data are pooled from two independent experiments. One-way ANOVA. (f) Scatter plots of DESeq2 normalized counts of select DEGs from the neonatal Foxp3 degradation RNA-seq experiment (Fig. ). Each point represents a unique mouse. Two-sided t -test. (g-h) Representative plots ( g ) and combined data ( h ) showing T reg proliferation in 8-week-old adult versus 7-day-old neonatal mice measured by Ki67 positivity. Scatter-plot represents mean ± SEM. Data are combined from two independent experiments. Two-tailed t-test.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Cell Analysis, Expressing, Comparison, Staining, RNA Sequencing, Two Tailed Test

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , Experimental design of proliferating T reg analysis in vivo. b , Flow cytometry analysis of CD25, GITR and CTLA4 protein levels in dividing versus nondividing T reg cells following 7 days of in vivo Foxp3 degradation, in comparison to Foxp3-deficient T reg ‘wannabe’ cells. Scatter-plots represent mean ± s.e.m. Each point represents a unique mouse. Data are representative of two independent experiments and were analyzed using a one-way ANOVA. c , Experimental design of proliferating T reg cell analysis in vitro. d , e , Combined data ( d ) and representative plots ( e ) showing CD153, GARP, CD4 and Foxp3 protein levels in lowly and highly divided T reg cells. Similarly treated naive CD4 T cells serve as Foxp3 − controls. Bar graphs represent mean ± s.e.m. Each point represents cells from a unique mouse. Data are representative of two independent experiments and were analyzed using a two-way ANOVA. f , IL-2, IL-4 and IL-13 concentrations in the supernatant of in vitro proliferating T reg assay. Bar graphs represent mean ± s.e.m. Each point represents cells from a unique mouse. Data are pooled from two independent experiments and were analyzed using a two-tailed t -test. g , Experimental design of proliferating T reg cell analysis in vitro. Experiment consisted of five technical replicates per genotype. h , Volcano plots of DEGs from g separated by their presence among all up- or downregulated genes in identified in Fig. . Numbers of up- or downregulated genes are labeled in each plot. i , Experimental design of inflammatory T reg cell analysis in vivo. Experiment consisted of four biological replicates per condition ( Tcrbd double knockout (dKO) or Foxp3 DTR ). j , Flow cytometric analysis of the transferred T reg cell mixture in i . k , Recovery of cells in each condition stratified by genotype (mCherry + for Foxp3 AID R26 TIR1(F74G) or mCherry − for Foxp3 AID R26 WT ) as identified in the scRNA-seq data. Each point represents a unique mouse. Bar graphs represent mean ± s.d. Data were analyzed using a paired two-tailed t -test. l , Density contour plots of R26 WT and R26 TIR1(F74G) overlayed on UMAP embeddings of the scRNA-seq data. m , Leiden clustering of gene expression data visualized on UMAP embedding for T reg cells from each condition. Clustering was performed independently for each condition using the same resolution value. Fraction of each cluster within the total pool of R26 WT and R26 TIR1(F74G) T reg cells separated by condition. Within each genotype, each point represents a unique mouse. Data were analyzed using a paired two-tailed t -test. n , Number of DEGs in each condition, colored by up- or downregulation. o , Scatter-plot of log 2 fold changes of DEGs between Tcrbd KO and Foxp3 DTR conditions. Points are colored by their direction and populations in which they are altered.

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: In Vivo, Flow Cytometry, Comparison, Cell Analysis, In Vitro, Two Tailed Test, Labeling, Double Knockout, Gene Expression

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: (a–b) Representative flow cytometry plots ( a ) and combined data ( b ) of Ki67 expression and EdU incorporation in T reg cells from the tumor and tumor-draining lymph node (dLN). Scatter-plot shows mean ± SEM. Each point represents a unique mouse. Data are representative of two independent experiments. Two-tailed t -test. (c) Representative flow cytometry plots (left) and combined data (right) of IFN-γ production by tumor-infiltrating NK cells. Scatter-plot shows mean ± SEM. Each point represents a unique mouse. Data are pooled from two independent experiments. Two-tailed t -test. (d) Representative flow cytometry plots (left) and quantification (right) of IFN-γ production by tumor-infiltrating ZsGreen − CD4 T cells. Scatter-plot shows mean ± SEM. Each point represents a unique mouse. Data are pooled from two independent experiments. Two-tailed t -test. (e) Volcano plts showing the number of genes up- and downregulated in Foxp3 AID R26 TIR1(F74G) tumor T reg cells within each clustered defined in Fig. .

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Immunology

Article Title: Temporal and context-dependent requirements for the transcription factor Foxp3 expression in regulatory T cells

doi: 10.1038/s41590-025-02295-4

Figure Lengend Snippet: a , Schematic of the tumor experiment design. s.c., subcutaneous. b , Tumor burden over time, shown as average (left) and individual (right) tumor growth curves. Line graph represents mean ± s.e.m. (left). Each line represents a unique mouse (right). Data are pooled from two independent experiments and were analyzed using a two-way ANOVA (mixed-effects model) with Geisser-Greenhouse correction. c , Representative tumor images on day 20. d , Representative flow cytometry plots (left) and combined data (right) of IFNγ production by tumor-infiltrating CD8 + T cells. Each point represents a unique mouse. Scatter-plot shows mean ± s.e.m. Data are pooled from two independent experiments and were analyzed using a two-tailed t -test. e , Representative flow cytometry plots (left) and quantification (right) of IL-4 production by tumor-infiltrating ZsGreen⁻ CD4 + T cells. Each point represents a unique mouse. Scatter-plot shows mean ± s.e.m. Data are pooled from two independent experiments and were analyzed using a two-tailed t -test. f , Body weight monitoring throughout the experiment. Line graph shows mean ± s.e.m. Data are pooled from two independent experiments. g , H&E staining of liver and intestine on day 20. Images are representative of two independent experiments. h , Expression levels of Foxp3, GITR, CD39, ZsGreen, CTLA4 and TCF1 in ZsGreen⁺ CD4 T cells from the dLN, ndLN and tumor on day 20. Each point represents a unique mouse. Scatter-plots show mean ± s.e.m. Data are pooled from two independent experiments and were analyzed using multiple t -tests. i , j , UMAP visualization of scRNA-seq analysis of tumor T reg cells from Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) mice on day 14 after tumor implantation, colored by genotype ( i ) or cluster ( j ). k , Heatmap showing scaled mean UMI-normalized expression values for each cluster in j . l , Proportional distribution of Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) T reg cells within each cluster in j . Each point represents a unique mouse. Scatter-plot represents mean ± s.e.m. Data were analyzed using multiple log-normal t -tests. m , Number of DEGs between Foxp3 AID R26 WT and Foxp3 AID R26 TIR1(F74G) T reg cells in each cluster shown in j .

Article Snippet: To generate the ROSA26 TIR1 strain, a targeting construct was assembled by cloning a TIR1 (wild-type; WT)-3xMyc-P2A-mCherry fragment into the FseI-linearized Ai32-targeting vector (Addgene, #34880), positioned between a loxP-Stop-loxP cassette and a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element).

Techniques: Flow Cytometry, Two Tailed Test, Staining, Expressing, Tumor Implantation