foxp3 egfp cre ert2 mice  (Jackson Laboratory)

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Acute intranasal HDM and tamoxifen treatment protocol. B. Representative flow cytometry of lung intraparenchymal (anti-CD45 i.v. antibody negative) CD4 + T cells showing Foxp3 and CCR4 expression from indicated groups. C. Percent lung CCR4 + Foxp3 + Tregs among Foxp3 + Tregs from indicated groups. D. Percentage of Foxp3 + Tregs among lung CD4 + T cells and number of total lung Tregs from indicated groups. E. Number of total lung DC subsets from indicated groups. F. Number of PD-L2 + CCR7 + lung cDC2s. G. Number of lung eosinophils H. Percentage of Foxp3 - ST2 + T cells (Th2 cells) among lung CD4 + T cells and number of total lung Th2 cells . I. Representative PAS-stained lung sections. J. Mucus scores. Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were administered 10 µg i.n HDM 3 times per week for 6 weeks alongside 1 mg daily doses of tamoxifen via oral gavage on days 4-8, 18-22 and 32-36 of HDM treatment then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 42. K. Chronic intranasal HDM and tamoxifen treatment protocol. L. Representative flow cytometry of lung CD4 + T cells showing Foxp3 and CCR4 expression from indicated groups. M. Percentage of lung CCR4 + Foxp3 + Tregs. N. Number of lung Tregs. O. Number of lung DC subsets. P. Number of PD-L2 + CCR7 + lung cDC2s. Q. Number of lung eosinophils R. Number of lung Th2 cells. Data are from three independent experiments with 11-18 mice pooled (A-J) or data are from two independent experiments with 12 mice pooled (K-R). For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. One-way ANOVA analysis with Holm-Sidak’s testing for multiple comparisons. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Flow Cytometry, Expressing, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A . % Ki67 + and gMFI for TIGIT, GITR, ICOS, and ST2 in lung Tregs from indicated groups. B . Percentage PD-L2 + CCR7 + cDC2s of total lung cDC2s. C . Total number of lung neutrophils. D . Percentage of lung ILC2s of lineage - cells and total number of lung ILC2s. E . Percentage and number of lung Th1 cells. F . Percentage and number of lung Th17 cells. G . Percent LN CCR4 + Foxp3 + Tregs. H . Percentage LN Tregs. I . Number of LN Tregs. J . Percentage and number of LN Th2 cells. K . Number of total LN DC subsets. L . Percentage and total number of PD-L2 + CCR7 + of LN cDC2s. M . Serum IgE levels. N-Q . Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were administered 10 µg i.n HDM 3 times per week for 6 weeks, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, 18-22 and 32-36 of HDM treatment, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 42. N . Percentage of lung Tregs of total CD4 + T cells from indicated groups. O . Percentage PD-L2 + CCR7 + cDC2s of total lung cDC2s. P . Representative PAS-stained lung sections. Q . Mucus scores. Data are from three independent experiments with 11-18 mice pooled or data are representative of one experiment with n L=L5 mice per group from three independent experiments. For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. One-way ANOVA analysis with Holm-Sidak’s testing for multiple comparisons. *, p<0.05; ****, p<0.0001, ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: CCR4 promotes the efficiency of Treg trafficking to suppress the expansion of CCR7+ cDC2-CD4+ T cell clusters during allergic inflammation. Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Representative imaging of lungs from indicated groups with staining for CD4 (Cyan), Foxp3 (Red), and FSCN1 (Yellow). B. Higher magnification of images shown in (A). C-D. Quantification of nearest distance (µm) between FSCN1 + DCs and Foxp3 + Tregs (C) and FSCN1 + DCs and CD4 + T cells (D). E-G . Mixed bone marrow chimeras were generated with Foxp3 EGFP-Cre-ERT2 Thy1.1 + Thy1.2 + control and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl (Thy1.2) bone marrow cells. Ctrl: Ccr4 cKO mixed bone marrow chimeras were treated with HDM (days 0, 7-11) and tamoxifen via oral gavage (days 4-8) then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. E. Representative flow cytometry of lung CD4 + T cells showing Foxp3 + and Thy1.1 + Thy1.2 + (Ctrl) and Thy1.2 + ( Ccr4 cKO) Tregs. F. Ratio of Ctrl: Ccr4 cKO Tregs in the lymph nodes and lungs. G. Ratio of Ctrl: Ccr4 cKO Th2 cells in the lymph nodes and lungs. H. Volcano plot showing differential gene expression analysis of bulk transcriptomes between sorted lung Tregs from Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice treated with HDM and tamoxifen. I . Asb2 relative RNA expression in iTregs cultured with or without rIL-4 for 2 days. J . Mixed bone marrow chimeras were generated with C57BL/6 control (Ctrl) and Il4r α-deficient bone marrow cells. Ctrl: Il4ra -deficient mixed bone marrow chimeras were treated with HDM (days 0, 7-11) then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. Ctrl: Il4ra KO ratio of lung T cell subsets. Images are representative of one of two independent experiments (A-B), data were pooled form two independent experiments (C-D), or data are representative of one experiment with n L=L10 mice per group from two independent experiments (E-G), or data were pooled form two independent experiments with n = 6 mice per group (H), or data were pooled from nine independent experiments (I), or data were pooled from two independent experiments with n = 10 mice (J). For statistical analysis, a two-tailed t test was performed for parametric data, and a two-tailed Mann-Whitney U test was performed for nonparametric data. **, p<0.001; ****, p<0.0001; ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Imaging, Staining, Generated, Flow Cytometry, Gene Expression, RNA Expression, Cell Culture, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Type 2 conventional dendritic cells and regulatory T cells form a barrier tissue circuit to control allergic inflammation

doi: 10.64898/2026.05.04.722795

Figure Lengend Snippet: Foxp3 EGFP-Cre-ERT2 control mice and Foxp3 EGFP-Cre-ERT2 x Ccr4 fl/fl mice were sensitized with 10 µg i.n. HDM followed by daily challenges of 10 µg i.n. HDM on days 7–11, 1 mg daily doses of tamoxifen via oral gavage on days 4-8, then injected with anti-CD45 antibody i.v. 3 minutes prior to tissue harvest on day 14. A. Representative histograms of surface CD80 and CD86 expression on lung cDC2s from indicated groups. B. gMFI for surface CD80 and CD86 on lung cDC2s. C. Representative histograms of surface CD80 and CD86 expression on lung MCs. D. gMFI for CD80 and CD86 on lung MCs. E. Representative histograms of surface CD80 and CD86 expression on lung Foxp3 + Tregs. F. gMFI for CD80 and CD86 on lung Foxp3 + Tregs. Representative data shows individual mice with mean ± SEM from one of three independent experiments with n L=L4-5 mice per group. For statistical analysis, a two-tailed t test was performed for parametric data. *, p<0.05, **, p<0.01, ns, not significant.

Article Snippet: Ccr4 floxed mice were crossed to Foxp3 EGFP-Cre-ERT2 mice ( Foxp3 EGFP-Cre-ERT2 ; stock no. 016961) purchased from the Jackson Laboratory to generate Foxp3 EGFP-Cre-ERT2 x Ccr4 floxed mice.

Techniques: Control, Injection, Expressing, Two Tailed Test

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNF and TNFR2 are differentially distributed in Tregs. (A) Tregs and Tconvs were immunomagnetically enriched as CD4 + CD25 + and CD25 − , respectively, from the spleens of C57BL/6 mice and restimulated in vitro 4 h before analysis of surface TNFR2 and intracellular TNF. (B, C) TNFR2 + and TNFR2 − Tregs and Tconvs were sorted as YFP + and YFP − CD4 T cells, respectively, from the spleens of Foxp3 CreYFP mice and restimulated in vitro 4 h before analysis of surface TNFR2 and intracellular TNF. (B) Representative cytograms showing the purity of sorted cells, and TNF/TNFR2 expression in unstimulated and stimulated cells. (C) Cumulative analysis showing percentages of TNF + , and gMFI of TNF in gated TNF + cells, in the indicated subpopulation (each tested in 2–3 replicates). Data are from one representative of two independent experiments, each performed with CD4 T cells from the pooled splenocytes of three mice. Bars represent means and SD. * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA.

Article Snippet: Foxp3 CreYFP (stock 028363) and Foxp3 eGFP‐Cre‐ERT2 (stock 016961) mice were purchased from the Jackson Laboratory.

Techniques: In Vitro, Expressing

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNFR2 + and TNFR2 − Treg maintain their diversity, while TNFR2 − are more activated when cocultured with TNFR2 + . (A–D) TNFR2 + and TNFR2 − Tregs were sorted from the spleens of Foxp3 CreYFP mice, respectively labeled with eF670 (blue) or CTV (purple) proliferation dyes, cultured 3–4 days with coated aCD3/aCD28 in the presence of IL‐2, and then analyzed by flow cytometry (after restimulation in vitro for 4 h for the analysis of intracellular TNF). (A) TNFR2 expression against each proliferation dye in each cell type. (B) Percentages of TNFR2 + cells, and geometric mean fluorescence intensity (gMFI) of TNFR2 in gated TNFR2 + cells, in the indicated cell types. (C) TNF expression against each proliferation dye in each cell type. (D) Percentages of TNF+ cells, and geometric mean fluorescence intensity (gMFI) of TNF in gated TNF + cells, in the indicated cell types. * p < 0.05, ** p < 0.01, **** p < 0.0001, by Student's t ‐test. (E–I) TNFR2 + and TNFR2 − Tregs were seeded alone or cocultured at 1:1 ratio. (E, F) Representative cytograms and percentages of each cell type in the indicated conditions. (G) Contour plot overlays with adjunct histograms showing the fluorescence of the two proliferation dyes in TNFR2 + and TNFR2 − Tregs, either alone or cocultured. (H, I) Representative histogram overlays and analysis of the gMFI of the indicated dyes and markers in TNFR2 + and TNFR2 − Tregs, either alone or cocultured. ** p < 0.01, *** p < 0.001, **** p < 0.0001, by two‐way ANOVA. Data are from one experiment representative of three. Each condition was tested in triplicate.

Article Snippet: Foxp3 CreYFP (stock 028363) and Foxp3 eGFP‐Cre‐ERT2 (stock 016961) mice were purchased from the Jackson Laboratory.

Techniques: Labeling, Cell Culture, Flow Cytometry, In Vitro, Expressing, Fluorescence

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: TNFR2 + Tregs display superior suppressive function and survival in vitro. (A, B) TNFR2 + and TNFR2 − Tregs were sorted from the spleens of Foxp3 eGFP‐Cre‐ERT2 mice, and their phenotype was freshly analyzed through flow cytometry. The plots display histogram overlays (A) and a heatmap of the z‐score (B), calculated from means and SD of gMFI of each marker in Tconvs, TNFR2 + , and TNFR2 − Tregs ( n = 4). Data are from one experiment representative of two. * p < 0.05, by Mann–Whitney test. (C–F) Tconv were sorted as YFP − cells, labeled with the CTV proliferation dye, and cultured in vitro either alone or at scaled ratios with eF670‐labeled TNFR2 + or TNFR2 − Tregs. (C) Histogram overlays showing CTV dilution and respective gMFI in gated Tconv cultured alone (black) or cocultured with TNFR2 + (red) or TNFR2 − (blue) Tregs in the indicated conditions. (D) Analysis of the gMFI of CTV and of the percentage of proliferating cells in gated Tconv in the indicated conditions. (E, F) Representative cytograms and cumulative analysis showing the percentages and the counts of Treg in each coculture condition. Data are from one representative of two independent experiments. Each condition was tested in 2–6 replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by unpaired t ‐test with Holm–Sidak correction for multiple comparisons.

Article Snippet: Foxp3 CreYFP (stock 028363) and Foxp3 eGFP‐Cre‐ERT2 (stock 016961) mice were purchased from the Jackson Laboratory.

Techniques: In Vitro, Flow Cytometry, Marker, MANN-WHITNEY, Labeling, Cell Culture

Journal: European Journal of Immunology

Article Title: TNF Production or TNFR2 Expression Characterize Distinct States of Regulatory T Cells that Cooperate in Treg Expansion in Cancer and Chronic Inflammation

doi: 10.1002/eji.70062

Figure Lengend Snippet: The molecular program of TNFR2 + Tregs supports survival and resistance to oxidative stress. (A) Volcano plot showing the fold change in gene expression by RNAseq of TNFR2 + versus TNFR2 − Treg sorted from spleens of Foxp3 CreYFP mice ( n = 4). Genes with FDR <0.05 and with fold change >2 (red) or <−2 (blue) are highlighted. (B) Statistically significant (FDR <0.05) results from the gene set enrichment analysis of the transcriptome of TNFR2 + versus TNFR2 − Treg in the hallmarks gene sets. (C) Gene set enrichment analysis of the transcriptome of TNFR2 + versus TNFR2 − Treg. A gene set of oxidative stress was obtained from a published signature . Normalized enrichment scores (NES) and FDR q values are shown under the plot. (D) Expression of Foxp3 was estimated as the gMFI of GFP in TNFR2 + versus TNFR2 − Treg sorted from spleens of Foxp3 eGFP‐Cre‐ERT2 mice and treated for 1 h with the indicated concentrations of menadione. **** p < 0.0001, by two‐way ANOVA. (E) Suppression assay was performed with GFP + TNFR2 + versus TNFR2 − Treg, either untreated or pretreated for 1 h with menadione, against CTV‐labeled Tconv. Treg percentage (as a measure of survival) and gMFI of CTV (as a measure of suppression) are shown at different Treg:Tconv ratios. Right plots represent the percentage of change between untreated and menadione‐treated cells. Each condition was tested in triplicate. Data are from one experiment representative of two. * p < 0.05, *** p < 0.001, **** p < 0.0001, by two‐way ANOVA. (F) Representative cytograms and cumulative analysis of CellROX staining in gated TNFR2 + or TNFR2 − Treg from spleens of Foxp3 CreYFP mice, either unstimulated or stimulated with aCD3 for 18 h. Numbers indicate the geometric mean fluorescence intensity (gMFI). The fmo of CellROX as a negative control is shown. Each condition was tested in quadruplicate. Bars represent means and SD. * p < 0.05, by unpaired t ‐test with Holm–Sidak correction for multiple comparisons. (G) Representative cytograms and cumulative analysis of CellROX staining in gated TNFR2 + or TNFR2 − Treg in unstimulated lymphocytes from spleens or tumors of Foxp3 eGFP‐Cre‐ERT2 mice ( n = 5), previously injected with MC38 tumor cells. Numbers indicate the geometric mean fluorescence intensity (gMFI). The fmo of CellROX as a negative control is shown. Data are from one experiment representative of two. Bars represent means and SD. * p < 0.05, *** p < 0.001, by two‐way ANOVA with Geisser–Greenhouse correction and Sidak multiple comparisons test.

Article Snippet: Foxp3 CreYFP (stock 028363) and Foxp3 eGFP‐Cre‐ERT2 (stock 016961) mice were purchased from the Jackson Laboratory.

Techniques: Gene Expression, Expressing, Suppression Assay, Labeling, Staining, Fluorescence, Negative Control, Injection

Journal: bioRxiv

Article Title: A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice

doi: 10.1101/2024.07.10.602914

Figure Lengend Snippet: Busulfan chimeras were generated using B6.CD45.1 hosts and B6.CD45.2 donors (See Methods) and mice analysed at various times after BMT. (A) Scatter plot of donor chimerism in DP1 thymocytes of all chimeras analysed. (B) Gating strategy to identify naive (CD62L hi CD44 lo ) and EM (CD62L lo CD44 hi ) Foxp3 + Treg in thymus, and donor (CD45.2) vs host (CD45.1) composition therein. (C) Scatter plots shows total number of DP1, CD4 single positive (CD4SP) and Treg (CD4SP Foxp3 + ) subsets isolated from thymus of chimeras (black open) and untreated WT control mice (red diamonds), with mouse age. (D) Scatter plots of total numbers of naive and EM Treg from chimeras and WT control, by host age. (E) Scatter plots of normalised donor fraction at different times postBMT amongst CD4SP, naive Treg and EM Treg subsets. Data are pooled from multiple independent batches of chimeras (n=45) and WT controls (n=34). For linear regression lines, * p < 0.0001 that slope is non-zero.

Article Snippet: Foxp3 tm9(EGFP/cre/ERT2)Ayr /J mice (Jax strain 016961, Foxp3 EGFP-CreERT2 )(ref) and B6.Cg- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J mice (Jax strain 7909, Rosa26R mTom hereon) were obtained from Jax Laboratories and interbred to homozygosity at both loci.

Techniques: Generated, Isolation, Control

Journal: bioRxiv

Article Title: A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice

doi: 10.1101/2024.07.10.602914

Figure Lengend Snippet: Cells from lymph node and spleen from busulfan chimeras and WT mice described in were analysed. (A) Gating strategy to identify naive (CD62L hi CD44 lo ) and EM (CD62L lo CD44 hi ) Foxp3 + Treg in peripheral lymphoid organs, their donor (CD45.2) vs host (CD45.1) composition therein, and gates used to measure Ki67 expression. (B-C) Scatter plots are of total numbers of naive (B) and EM (C) Treg recovered from lymph node and spleen of chimeras and control WT mice of different ages. Lines show simple linear regression fits to data. (D) Scatter plot of ratio of naive to EM Treg in lymph node and spleen of WT (filled symbols) and busulfan chimeras (empty symbols) at different host ages. (E) Scatter plots of normalised donor fraction in lymph node and spleen of busulfan chimeras at different times post BMT for naive and EM Treg. (F) Scatter plots showing of ratios of donor chimerism in naive Treg between Lymph node : Spleen, and in EM Treg between Spleen:thymus and LN:spleen. Panels G-H; The estimated fractions of de novo developed Treg amongst (G) total naive thymic foxp3 + cells and (H) total (naive and memory) Foxp3 + thymic Treg. (I) De novo generated naive Treg as a fraction of total CD4SP thymocytes. For linear regression lines, * p < 0.0001 that slope is non-zero.

Article Snippet: Foxp3 tm9(EGFP/cre/ERT2)Ayr /J mice (Jax strain 016961, Foxp3 EGFP-CreERT2 )(ref) and B6.Cg- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J mice (Jax strain 7909, Rosa26R mTom hereon) were obtained from Jax Laboratories and interbred to homozygosity at both loci.

Techniques: Expressing, Control, Generated

Journal: bioRxiv

Article Title: A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice

doi: 10.1101/2024.07.10.602914

Figure Lengend Snippet: Foxp3-FR mice were treated with a single feed of 2mg of tamoxifen and cohorts of mice taken at different times after treatment. (A) Histograms (top row) show mTom reporter expression at the indicated days after treatment by CD4 + TCR hi Foxp3 + cells from lymph nodes. Scatter plots show CD25 vs Foxp3EGFP expression by mTom+ve and mTom-ve CD4 + T cells from lymph nodes. Histograms (bottom row) are of Foxp3-EGFP expression by mTom + CD4 + TCR hi T cells or total CD4 + TCR hi cells from lymph nodes at different times after treatment. (B-C) Scatter plots are of fraction of naive (B) or EM (C) Foxp3 + Treg that are mTom -ve, calculated from total numbers of Treg recovered from LN and spleen. Splines (long dashes) are best fit lines of normalised donor fractions observed in busulfan chimeras in either naive or EM Treg as shown in . A linear regression line with 95% confidence intervals is applied to mTom-ve fraction of memory Treg. Slope deviation from 0, p = 0.013.

Article Snippet: Foxp3 tm9(EGFP/cre/ERT2)Ayr /J mice (Jax strain 016961, Foxp3 EGFP-CreERT2 )(ref) and B6.Cg- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J mice (Jax strain 7909, Rosa26R mTom hereon) were obtained from Jax Laboratories and interbred to homozygosity at both loci.

Techniques: Expressing

Journal: bioRxiv

Article Title: A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice

doi: 10.1101/2024.07.10.602914

Figure Lengend Snippet: Data from busulfan chimeras detailed in (grey symbols, clean), generated and analysed at the UCL Comparative Biology Unit, were compared with chimeras generated in an identical manner three years previously, at the MRC National Institute for Medical Research (red symbols, dirty). In this meta analysis, Treg were identified by CD4 + CD25 + gate throughout, since earlier study did not include Foxp3 intracellular stain. (A) Ratio of memory:naive CD4 + T conv cells calculated from total numbers of subpopulations recovered from lymph nodes and spleen from different experiments, for central (CM) and effector memory (EM) cells. Lines are linear regressions to data. (B) Comparison of normalised donor fractions (B) and absolute cell numbers of donors cells (C) for naive and EM Treg.

Article Snippet: Foxp3 tm9(EGFP/cre/ERT2)Ayr /J mice (Jax strain 016961, Foxp3 EGFP-CreERT2 )(ref) and B6.Cg- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J mice (Jax strain 7909, Rosa26R mTom hereon) were obtained from Jax Laboratories and interbred to homozygosity at both loci.

Techniques: Generated, Staining, Comparison

Journal: bioRxiv

Article Title: A linear ontogeny accounts for the development of naive, memory and tumour-infiltrating regulatory T cells in mice

doi: 10.1101/2024.07.10.602914

Figure Lengend Snippet: (A-B) Foxp3-FR mice were treated with a single feed of tamoxifen and 14 days later, engrafted with MC38 tumour cells. Mice were culled when tumour size reached maximal permitted size (15mm), between 15-20d post engraftment. (A) Density plots show TCR, CD4 and Foxp3 gates used to identify Treg in lymph node (LN) and tumour of host mice, and their naive vs EM composition. (B) Histograms and bar charts show representative and summarised expression of PD1, Ki67 and mTom by naive and EM Treg in LN and spleen, compared with tumour infiltrating Treg. (C) Busulfan chimeras were generated as described in . Hosts were engrafted with MC38 cells ∼8 weeks post BMT. Mice were culled when tumour size reached maximal permitted size, and phenotype of Treg in lymph nodes and tumour determined. Density plot show CD25 vs Foxp3-EGFP, and CD62L vs CD44 used to gate naive and EM phenotype Treg. Plots of CD45.1 and CD45.2 indicate host and donor composition of the indicated Treg subsets, and summary bar charts of normalised donor fraction across all mice (n=9 pooled from three independent experiments). (D) Foxp3FR mice were treated with a single feed of tamoxifen and 14 days later, engrafted with either A20 or EL4 tumour cells. (Foxp3-FR x Balb/c)F1 mice were used as hosts for A20 tumours. Mice were culled when tumour size reached maximal permitted size and phenotype of Treg assessed as in (B). Data are pool of 5 (B), 3 (C-D) independent experiments.

Article Snippet: Foxp3 tm9(EGFP/cre/ERT2)Ayr /J mice (Jax strain 016961, Foxp3 EGFP-CreERT2 )(ref) and B6.Cg- Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J mice (Jax strain 7909, Rosa26R mTom hereon) were obtained from Jax Laboratories and interbred to homozygosity at both loci.

Techniques: Expressing, Generated