Journal: Frontiers in Immunology

Article Title: CD4+FoxP3+ T regulatory cells subsets release small extracellular vesicles containing cell death-related proteins as potential mechanism of T cell suppression

doi: 10.3389/fimmu.2026.1777669

Figure Lengend Snippet: nTregs, iTregs and RATregs produce sEV. nTregs were obtained from spleens of FoxP3 GFP mice using the CD4+CD25+ Miltenyi kit. 10 6 /mL of nTregs were cultured in the presence of polyclonal activation (αCD3/αCD28) for 72h. To produce iTregs, the CD4+CD25- T cells fraction obtained from the nTregs purification was used. iTregs were obtained after 5 days in culture in the presence of polyclonal activation, IL-2 and TGF-β, whereas RATregs had retinoic acid (RA) added in addition to IL-2 and TGF-β. Tregs characterization included FoxP3, CD25 and CD73 expression analysis by flow cytometry (A) Representative dot plots displaying the phenotype of nTregs (black), iTregs (blue) and RATregs (pink). (B) Bar plots depicting the MFI for FoxP3, CD25 and CD73 from all Tregs subsets. (C) Graph showing the number of sEV per 10 6 cells obtained from nTregs, iTregs and RATregs. (D) Size distribution of sEV obtained from nTregs, iTregs and RATregs determined by NTA. (E) Abundance of sEV by size range also determined by NTA. (F) Western blot showing Alix as marker for sEV and β-actin as a marker for (Tregs) cells. A-E. Each experiment was performed 4–8 times (each dot corresponds to a single cell culture). (F) Western blot was performed 2 times. *p < 0.05 according to One-way ANOVA test; ns, not significant. Bar lines represent Mean ± SEM.

Article Snippet: FoxP3 GFP were from The Jackson Laboratory (Maine, USA).

Techniques: Cell Culture, Activation Assay, Purification, Expressing, Flow Cytometry, Western Blot, Marker, Single Cell

Journal: Nature immunology

Article Title: T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells

doi: 10.1038/s41590-018-0280-2

Figure Lengend Snippet: Antigen-specific iT regs suppress T naive cells with identical antigen specificity regardless of CTLA-4 expression or IL-10 production. a-b) C57BL/6 DCs were loaded with 3 μM OVA 323–339 (DC OVA ), LCMV GP 61–80 (DC GP ) or both peptides (DC OVA-GP ). CFSE labeled naive OT-II (1×10 6 ) and SMARTA (1×10 6 ) cells were transferred i.v. into CD45.1 mice with DCs (5×10 5 ) and iT regs (2×10 6 ). Histograms demonstrate day 3 post-transfer proliferation status of Thy1.1 – OT-II and Thy1.1 + SMARTA cells upon co-transfer with 1:1 mixture of DC OVA and DC GP (a) or DC OVA-GP (b) . Graphs show the number of CFSE low proliferating cells. c-d) B10.A DCs were pulsed with 3 μM PCC 88–104 . CD45.1 + CFSE labeled 5CC7 T naive (1×10 6 ) were transferred i.v. into B10.A mice together with DCs (5×10 5 ) and WT, Il10 –/– or Ctla4 –/– 5CC7 iT regs (2×10 6 ). Histograms demonstrate the day 3 post-transfer proliferation states, graphs show the number of CFSE low 5CC7 T cells. Bars indicate the means of n=3 mice, data are representative of two (c,d) or three (a,b) independent experiments. P values were calculated using one-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: Moth Cytochrome C (MCC 88–103 ) specific TCR transgenic 5CC7-FoxP3.GFP Rag2 –/– , 5CC7- Ctla4 –/– Rag2 –/– , 5CC7- Il10 –/– Rag2 –/– , Hen Egg Lysozyme (HEL 46–61 ) specific TCR transgenic 3A9 Rag2 –/– , OVA 323–339 specific TCR transgenic OT-II, OT-II- Rag2 –/– , CD11c.YFP, FoxP3.GFP, B10.A mice were obtained from Taconic Farms (Hudson, NY) under the NIAID contract.

Techniques: Expressing, Labeling, Comparison

Journal: Nature immunology

Article Title: T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells

doi: 10.1038/s41590-018-0280-2

Figure Lengend Snippet: Antigen-specific iT regs have greater trogocytic capacity. a) DCs were labeled with PKH-26, loaded with 3 μM MCC (88–103) and co-cultured 1:1 with 5CC7 T naive , T activated or iT regs for 18 h. Histograms show the intensity of PKH-26 on T cells upon co-culture, black: T cells co-cultured with unpulsed DC, red: T cells co-cultured with antigen pulsed DC. Data are representative of three independent experiments that were performed in triplicates. b-c) CD11c-YFP DCs were loaded with 3 μM OVA (323–339) peptide and co-cultured with OT-II T activated (CD45.1 + ) or OT-II iT regs (CD45.1 + ) for 3 h. b) Representative images showing 3D surfaces created to mask the DCs based on CD11c-YFP intensity. Yellow arrows point to the MHCII + patches on iT regs . c) Graph shows MHCII intensity of OT-II T activated and OT-II iT regs . Lines mark the mean of individual data points pooled from n=2 biological replicates. Data are representative of three independent experiments with similar results. d-f) DCs were loaded with 3 μM MCC (88–103) and co-cultured with 5CC7 T naive , T activated or iT regs for 3 h. Sections were stained with biotinylated anti-MCC (88–103) -I-E k antibody (D4) antibody followed by streptavidin conjugated quantum dots. d ) TEM images demonstrate T naive , T activated and iT reg contacts with DC. Orange arrow heads mark the patches of DC membrane captured by iT regs . e) Higher magnification images of DC-iT reg contact. Orange arrow heads point to the endosomes and membrane parts containing quantum dots. Blue arrow heads show the positive quantum dot staining in the DCs. f) Bars indicate the mean amount of quantum dots per μm 2 , error bars show the standard error of the mean. Data are pooled from n=3 independent experiments with similar results, each performed with two biological replicates. P values were calculated using two-sided student’s t-test (c) and one-way ANOVA with Dunnett’s post-test (f).

Article Snippet: Moth Cytochrome C (MCC 88–103 ) specific TCR transgenic 5CC7-FoxP3.GFP Rag2 –/– , 5CC7- Ctla4 –/– Rag2 –/– , 5CC7- Il10 –/– Rag2 –/– , Hen Egg Lysozyme (HEL 46–61 ) specific TCR transgenic 3A9 Rag2 –/– , OVA 323–339 specific TCR transgenic OT-II, OT-II- Rag2 –/– , CD11c.YFP, FoxP3.GFP, B10.A mice were obtained from Taconic Farms (Hudson, NY) under the NIAID contract.

Techniques: Labeling, Cell Culture, Co-Culture Assay, Staining, Membrane

Journal: Nature immunology

Article Title: T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells

doi: 10.1038/s41590-018-0280-2

Figure Lengend Snippet: Antigen-specific T regs strip cognate pMHCII complexes from DC surface. a) DCs were loaded with 3 μM MCC (88–103) (MCC +) or left unpulsed (MCC -) and adoptively transferred into CD45.1 + B10.A mice via footpad, followed by a transfer of e450 labeled 5CC7 T naive , T activated or iT regs (10 6 ) i.v. Histograms demonstrate day 3 post-transfer MCC (88–103) -I-E k levels of adoptively transferred T cells: Red: co-transferred with antigen pulsed DC. Black dotted: co-transferred with unpulsed DC. Endogenous CD4 + T cells of the recipient mice (Gray tinted) were also plotted as an internal negative control for staining. Graph shows the MCC (88–103) -I-E k MFIs of adoptively transferred T cells. Lines mark the mean of n=4–8 mice, data are representative of three independent experiments. b-d) DCs were loaded with 0.3 – 9 μM MCC (88–103) and co-cultured 1:1 with 5CC7 T naive , T activated or iT regs for 18 h. b) Histograms demonstrate the MCC (88–103) -I-E k levels in the T cell gate. Upper row shows the surface levels, lower row shows the intracellular levels detected after blockade of the surface MCC (88–103) -I-E k by unconjugated D4 antibody. c) Graphs show the net increase in the MCC (88–103) -I-E k MFI at the surface (top) and intracellular (bottom) compartments of T cells (Δ MFI= MFI (Post- antigen pulsed DC co-culture) - MFI (Post- unpulsed DC co-culture) ). Symbols and error bars indicate the mean and standard deviation of n=3 biological replicates. Data are representative of five independent experiments. d) Histograms demonstrate the MCC (88–103) -I-E k surface levels in the DC gate (Gray tinted: unpulsed DC; black dotted: antigen pulsed DC; antigen pulsed DCs co-cultured with T naive , T activated and iT regs were shown by green, blue and red histograms respectively). Graph shows the MFI for DC surface MCC (88–103) -I-E k . Symbols and error bars indicate the mean and standard deviation of n=3 biological replicates. Data are representative of five independent experiments. e-f) 5CC7 iT reg , splenic (Sp) and mesenteric lymph node (mLN) T reg were co-cultured with DCs that were loaded with 3 μM MCC (88–103) . e) Histograms show the MCC (88–103) -I-E k levels in T reg gate following the co-culture with unpulsed DC (Gray tinted) and antigen pulsed DC (Red). f) Histograms show the MCC (88–103) -I-E k levels in DC gate following the co-culture with different antigen specific T reg types. Black histogram shows the antigen pulsed DC cultured alone, red histogram shows the remaining levels MCC (88–103) -I-E k after Treg-DC co-culture. Data are representative of three independent experiments with similar results. Statistical significance was calculated using two-way ANOVA with Sidak’s multiple comparison (a) and Dunnet’s correction (c,d).

Article Snippet: Moth Cytochrome C (MCC 88–103 ) specific TCR transgenic 5CC7-FoxP3.GFP Rag2 –/– , 5CC7- Ctla4 –/– Rag2 –/– , 5CC7- Il10 –/– Rag2 –/– , Hen Egg Lysozyme (HEL 46–61 ) specific TCR transgenic 3A9 Rag2 –/– , OVA 323–339 specific TCR transgenic OT-II, OT-II- Rag2 –/– , CD11c.YFP, FoxP3.GFP, B10.A mice were obtained from Taconic Farms (Hudson, NY) under the NIAID contract.

Techniques: Stripping Membranes, Labeling, Negative Control, Staining, Cell Culture, Co-Culture Assay, Standard Deviation, Comparison

Journal: Nature immunology

Article Title: T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells

doi: 10.1038/s41590-018-0280-2

Figure Lengend Snippet: Capture of pMHCII complexes by iT regs is antigen-specific. a-c) DCs were loaded with 3 μM MCC (88–103) and/or 3 μM HEL (46–61) and co-cultured 1:1 with 5CC7 or 3A9 iT regs for 18 h. a) Histograms demonstrate the MCC (88–103) -I-E k and HEL (46–61) -I-A k levels on 3A9 and 5CC7 iT regs co-cultured with double pulsed DCs (Red, blue) and unpulsed DC (Gray tinted). b) Histograms demonstrate the remaining MCC (88–103) -I-E k and HEL (46–61) -I-A k levels on double pulsed DCs after co-culture (Black dashed: Double antigen pulsed DC from solo culture. red: double pulsed DC co-cultured with 5CC7 iT regs . blue: double pulsed DC co-cultured with 3A9 iT regs ). c) Graphs show the levels for DC surface MCC (88–103) -I-E k and HEL (46–61) -I-A k . Bars indicate the mean of n=3 biological replicates and error bars represent the standard deviation. Data are representative of three independent experiments. d-e) Double pulsed DCs (5 × 10 4 ) were adoptively transferred into CD45.1 + B10A mice via the footpad together with e450 labeled 5CC7 or 3A9 iT regs (5 × 10 4 ) i.v. Day 3 post-transfer MFIs of iT regs for MCC (88–103) -I-E k (d) and HEL (46–61) -I-A k (e). Data are representative of three independent experiments, symbols show induvudual mice, lines mark the mean. f) DCs from B10.A animals were loaded with 3 μM MCC (88–103) and 3 μM HEL (46–61) or left unpulsed. DCs (1 × 10 6 ) and e450 labeled 5CC7 iT regs (1 × 10 6 ) were adoptively transferred into CD45.1 + B10A mice i.v. Graphs show the DC surface levels of pMHCII 18h post-transfer. Lines mark the mean of n=3 mice, data are representative of two independent experiments. P values were calculated using two-sided student’s t-test (c) and one way ANOVA with Tukey’s multiple comparison (d-f).

Article Snippet: Moth Cytochrome C (MCC 88–103 ) specific TCR transgenic 5CC7-FoxP3.GFP Rag2 –/– , 5CC7- Ctla4 –/– Rag2 –/– , 5CC7- Il10 –/– Rag2 –/– , Hen Egg Lysozyme (HEL 46–61 ) specific TCR transgenic 3A9 Rag2 –/– , OVA 323–339 specific TCR transgenic OT-II, OT-II- Rag2 –/– , CD11c.YFP, FoxP3.GFP, B10.A mice were obtained from Taconic Farms (Hudson, NY) under the NIAID contract.

Techniques: Cell Culture, Co-Culture Assay, Standard Deviation, Labeling, Comparison

Journal: Cell reports

Article Title: OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms

doi: 10.1016/j.celrep.2018.06.052

Figure Lengend Snippet: (A) NaiveCD4+Foxp3− T cells were stimulated with WT or OX40L-Tg APCs under the iTreg-polarizing conditions, and induction of Foxp3+ T cells among total CD4+ T cells was shown 3 days later.

Article Snippet: Briefly, naive CD4 + T cells were FACS sorted from Foxp3-GFP reporter mice with FACSaria (BD Biosciences).

Techniques:

Journal: Cell reports

Article Title: OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms

doi: 10.1016/j.celrep.2018.06.052

Figure Lengend Snippet: (A) The graph on top illustrates the putative Jun binding sites (boxed) in the Foxp3 promoter and the CNS1 and CNS2 regions. The bar graphs show the ChIP analysis of BATF3 enrichment at these Jun binding sites in CD4+ T cells, which were transduced with an empty retroviral vector (Ctrl vector) or retrovirus expressing of BATF3 (Batf3-Flag vector) and then cultured under the iTreg-polarizing condition for 48 hr.

Article Snippet: Briefly, naive CD4 + T cells were FACS sorted from Foxp3-GFP reporter mice with FACSaria (BD Biosciences).

Techniques: Binding Assay, Transduction, Plasmid Preparation, Expressing, Cell Culture

Journal: Cell reports

Article Title: OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms

doi: 10.1016/j.celrep.2018.06.052

Figure Lengend Snippet: (A-C) CD45.1+ WT B6 (A) or CD45.1+ OX40L-Tg mice (B and C) were adoptively transferred with naive CD4+Fxop3− T cells sorted from CD45.2+ WT or Batf−/−Batf3−/− mice and treated without or with rapamycin on days 0, 1,3, and 5. On day 7 post-cell transfer, leucocytes in mesenteric lymph nodes (LNs) and lungs were isolated for flow cytometry analysis. The dot plots and bar graphs indicate the (%) Foxp3+ cells among the adoptively transferred CD45.2+ cell populations. Data represent mean values ± SD (n = 3). *p < 0.05, **p < 0.01.

Article Snippet: Briefly, naive CD4 + T cells were FACS sorted from Foxp3-GFP reporter mice with FACSaria (BD Biosciences).

Techniques: Isolation, Flow Cytometry

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: Characteristics of CD4 T cells in retinas of Aire GW/+ Lyn −/− mice with uveitis. CD4 T cells present among inflammatory cells in the retinas of mice with uveitis were analyzed by CyTOF. UMAP dimensionality reduction and unsupervised clustering of 4,681 CD4 + T cells from the retinas of mice with late stage uveitis ( n = 5) were characterized for expression of informative cell surface and intracellular molecules by mass cytometry (A). CD4 + T cells were gated as Cisplatin − CD45 + Ter119 - SiglecF − Ly6G − FcεR1a − PDCA1 − B220 − CD3 + NK1.1 − CD4 + CD8 - cells. Clusters were assigned and cluster identities were interpreted based on expression of molecules of interest as explained in the text. Expression of key molecules (FoxP3, CD27, CD103, PD1, TCF1, and Ly6C) across CD4 + T cells in panel A (B).

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: Expressing, Mass Cytometry

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: Single cell transcriptome analysis of P2 + CD4 + T cells in retina of Aire GW/+ Lyn −/− mice with uveitis. The P2 + CD4 + T cells in retina of Aire GW/+ Lyn −/− mice with uveitis at 6–7 wk were isolated by magnetic bead enrichment and cell sorting and subjected to single cell transcriptome sequencing. After quality filtering, 1,602 cells from 6 pooled mice were represented by UMAP plot and segregated into 4 phenotypic clusters (A), with their frequencies shown in (B). The identities of the clusters were interpreted as described in the text. Superimposed on the UMAP plot is the expression of Foxp3, Mki67, Tbx21, Rorc, Gata3, Maf, Bcl6, Prdm1 (C). Heatmap of top 10 differentially expressed genes for the clusters (D) in panel A. Dot plots showing average expression level across all cells within a cluster (color of circle) and percentage of cells within a cluster (size of circle) for functionally informative genes Sell, Cd44, Cd27, Mki67, Icos, Pdcd1, Lag3, Tigit, Ctla4, Il2ra, Il2rb, Foxp3, Il7a, Ifng, Il10, Il4, Ikzf2, Cxcr5, Cxcr6, Ccr8, Selplg, Cx3cr1 and Runx3 expression in different P2 + CD4 + cell clusters in retina of Aire GW/+ Lyn −/− mice with uveitis (E).

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: Single Cell, Isolation, FACS, Sequencing, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: Single cell transcriptome analysis and clustering of P2-binding CD4 + T cells in LN of Aire GW/+ Lyn −/− mice with uveitis and without uveitis, and in LN of Aire GW/+ Lyn −/− IRBP −/− mice immunized with IRBP protein. After scRNAseq and filtering, a single UMAP representation was constructed from 1,516 total P2 + CD4 + T cells, which were combined from 540 filtered cells in two datasets from eye-draining LNs of 7 pooled and 8 pooled Aire GW/+ Lyn −/− mice at 7–12 wk of age with uveitis, 518 filtered cells in one dataset from eye-draining LNs of 16 pooled Aire GW/+ Lyn −/− mice without uveitis, and 458 filtered cells from draining LNs of 11 pooled Aire GW/+ Lyn −/− IRBP −/− mice immunized 5 d earlier with IRBP protein in CFA. Clustering with Seurat identified 7 phenotypic clusters, which are represented by different colors for each of the types of P2-specific CD4 + T cells separately shown on the combined UMAP (A). See the text for explanation of the identities assigned to the different clusters, the frequencies within each type of sample are shown (B). Expression of Ly6c1, Lag3, Mki67, Ifit1, Il7r, Foxp3, Il2ra, Il2rb, Tbx21, Ifng, Il4, Selplg within individual cells in the combined UMAP is shown by scaled color (log normalized expression level) (C). A heatmap shows expression levels in the P2-specific CD4 + T cells of the top 10 differentially expressed genes for each cluster for the combined samples (D). Relative expression within each cluster of the functionally informative genes Ly6c1, Sell, Cd44, Cd27, Mki67, Icos, Pdcd1, Lag3, Tigit, Ctla4, Il2ra, Il2rb, Foxp3, Ikzf2, Cxcr5, Cxcr6, Ifng, Il2, Mif, Ifit1, Il7r, Cxcr3, Runx3, Selplg with average expression level across all cells within a cluster by color intensity and the percentage of cells within a cluster of the gene represented by size of circle (E). Note, the data shown in C and E are broken out by type of sample in .

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: Single Cell, Binding Assay, Construct, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: High frequency of Tregs in some Aire GW/+ Lyn −/− Foxp3 GFP mice without uveitis. Aire GW/+ Lyn −/− Foxp3 GFP mice at 7–9 wk of age with uveitis ( n = 6) or without uveitis ( n = 7) were analyzed by flow cytometry to determine the distribution among P2-specific CD4 + T cells of Treg (FoxP3 + ), early activated T cells (Ly-6C + or Ly-6C - CD62L + ) and late activated T cells (PD-1 hi LAG-3 int or PD-1 hi LAG-3 hi ). Shown are the gating strategy (A), numbers of P2 + CD4 + T cells in eye-draining LNs per mouse (B), frequencies of Treg (LN without uveitis ( n = 8); LN with uveitis ( n = 9)), early activated T cells, and late activated T cells in eye-draining LN (left) or retina (right) (C), the number (above bar) and fractions of P2 + CD4 + T cells in LN in each category of activation state in individual mice with or without uveitis (D), and fractions of various P2 + CD4 + T cell subsets that expressed CXCR6 in eye-draining LN or retina (E). Frequencies of Tregs, Ly-6C + , Ly-6C - CD62L + , PD-1 hi LAG-3 int , and PD-1 hi LAG-3 hi cells were frequencies of cells within P2 + CD4 + cells, frequencies of CXCR6 + cells were frequencies of cells within P2 + CD4 + cells. P2 + CD4 + cells were gated on TCRβ + CD4 + CD8 - DUMP - cells. * P < 0.05. For B, left panel, 2-tailed t test; For C, left panel, unpaired t test, error bars are mean ± SD.

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: Flow Cytometry, Activation Assay

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: Variation in numbers of P2-binding CD4 + T cells in Aire GW/+ Lyn −/− Foxp3 GFP mice prior to disease onset. The number of P2 + CD4 + cells and the fraction of them that were FoxP3 + in the eye-draining LN of Aire GW/+ Lyn −/− Foxp3 GFP mice ( n = 8) or Lyn −/− Foxp3 GFP ( n = 2) mice without uveitis at 4–6 wk of age were determined by flow cytometry. Representative example of flow cytometry analysis of P2 tetramer staining in the TCRβ + CD4 + CD8 − DUMP − cells with calculated number of P2 + cells shown (left), and GFP staining of the P2 + cells with fraction of GFP + cells shown (right) (A) and summary of numbers of P2 + CD4 + T cells observed per mouse (B). Correlation of the number of P2 + CD4 + T cells vs. the % of them that expressed Foxp3 GFP (C). A linear regression line (red dashed line) is shown for the 6 mice that had 21 or more P2 + CD4 + T cells (R = 0.69), that is, excluding the 2 mice with fewest number of P2 + CD4 + T cells. * P < 0.05. Two-tailed t test; error bars are mean ± SD.

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: Binding Assay, Flow Cytometry, Staining, Two Tailed Test

Journal: The Journal of Immunology Author Choice

Article Title: Immune checkpoints in a genetically engineered mouse model of spontaneous autoimmune uveitis

doi: 10.1093/jimmun/vkaf334

Figure Lengend Snippet: Transient depletion of Tregs in Aire GW/+ Lyn −/− Foxp3 DTR mice without uveitis promotes substantial expansion of autoreactive CD4 T cells and also induces uveitis in some mice. Schematic showing diphtheria toxin (DT) administration schedule for Aire GW/+ Lyn −/− Foxp3 DTR and Lyn −/− Foxp3 DTR mice without uveitis at 6 wk (A). Representative funduscopic images (B) and frequencies (C) of Aire GW/+ Lyn −/− Foxp3 DTR (PBS: n = 4; DT: n = 12) and Lyn −/− Foxp3 DTR (PBS: n = 3; DT: n = 3) mice with uveitis or without uveitis after PBS or DT treatment for 2 wk starting at 6 wk age in Aire GW/+ Lyn −/− Foxp3 DTR and Lyn −/− Foxp3 DTR mice lacking detectable uveitis. Representative flow cytometric analysis (D) and cell number (E, left panel) of P2 + CD4 + T cells in eye-draining LN of Aire GW/+ Lyn −/− Foxp3 DTR (PBS: n = 4; DT: n = 12) and Lyn −/− Foxp3 DTR (DT: n = 3) mice at 8 wk of age with PBS or 300 ng DT treatment for 2 wk. Cells shown were gated on the TCRβ + CD4 + CD8 − DUMP − cells. Red solid circles indicate the mice which developed uveitis after DT treatment (E, left). Fold change in average number of P2 + CD4 + T cells between the DT-treated and PBS-treated groups is shown (E, right panel). Two weeks after DT of Aire GW/+ Lyn −/− Foxp3 DTR mice ( n = 12), the fraction of P2 + CD4 + T cells that were FoxP3 + was determined using the GFP reporter contained within FoxP3 DTR allele. Shown is a representative flow cytometric plot (F) and a summary plot comparing the number of P2 + CD4 + T cells to the fraction of them that were GFP + Treg (G). Shown in red is the linear regression line (R = 0.050). Red solid circles indicate the mice that developed uveitis after DT treatment. * P < 0.05. in panel E, 2-tailed t test; error bars are mean ± SD.

Article Snippet: Aire GW/+ and Lyn −/− mice of this study were previously described., B6.129P2-Rbp3 tm1Gil/J(IRBP −/− ) (stock no. 023080) and C.Cg-Foxp3 tm2Tch /J(Foxp3 GFP ) (stock no 006769) mice were obtained from the Jackson Laboratory.

Techniques: