1. Volume 46, Issue 1, Pages 78-91 (January 2017)
Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb 
Sheila Dias, Angela D’Amico, Erika Cretney, Yang Liao, Julie Tellier, Christine Bruggeman, Francisca F. Almeida, Jamie Leahy, Gabrielle T. Belz, Gordon K. Smyth, Wei Shi, Stephen L. Nutt 
Immunity 
Volume 46, Issue 1, Pages (January 2017)
DOI: /j.immuni
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2. Immunity 2017 46, 78-91DOI: (10.1016/j.immuni.2016.12.017)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Reduced Proportion of Treg Cells in Peripheral Organs of Mybfl/flCd4creTg Mice
(A) Left: intracellular FoxP3 and cell-surface CD25 on CD4+CD8− thymocytes from control Myb+/+Cd4creTg and Mybfl/flCd4creTg mice. Numbers indicate the proportion of gated Treg cells. Graphs show the number of CD4+FoxP3− (left) and CD4+FoxP3+ (middle) thymocytes (data are from four littermate mice per genotype). Right: percentage of FoxP3+ cells among CD4+CD8− thymocytes in control (Mybfl/fl or Myb+/+Cd4creTg) and Mybfl/flCd4creTg mice (data are from eight mice per genotype and represent three individual experiments).
(B) Number of CD4+FoxP3− (left) and CD4+FoxP3+ (right) T cells isolated from the indicated tissues. Data are from four littermate mice per genotype. pLN, peripheral lymph node; mLN, mesenteric lymph node.
(C) Percentage of FoxP3+ Treg cells among CD4+ T cells isolated from the indicated tissues. 5–20 mice were analyzed per organ per genotype and pooled from six individual experiments.
(D) Percentage of FoxP3+ Treg cells among Ly5.2+CD4+ T cells in 50:50 mixed BM chimeras of Ly5.1 and either Ly5.2 WT Mybfl/fl or Ly5.2 Mybfl/flCd4creTg cells. Data are from three chimeras and represent three independent experiments.
Bar graphs depict the mean ± SD; p values compare the indicated samples.
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4. Figure 2 Myb Is Expressed in ICOS+ Treg Cells
(A) Schematic representation of the MybGFP reporter strain. An internal ribosome entry site (IRES)-eGFP cassette was introduced into the Myb 3′ UTR by homologous recombination in embryonic stem cells, leaving the Myb coding region (CDR) and known microRNA binding sites intact.
(B) GFP and ICOS surface expression in CD4+RFP+ Treg cells isolated from the indicated organs of adult MybGFP/GFPFoxp3RFP/RFP mice. Data represent eight mice analyzed in three independent experiments. Bar graphs summarize the data obtained in the experiment shown (n = 3 for each genotype). Lower middle: mean percentage of RFP+GFP+ cells ± SD. Lower right: mean gMFI (geometric mean fluorescence intensity) of GFP ± SD from RFP+GFP+ Treg cells. Myb+/+Foxp3RFP/RFP mice were analyzed as controls, and background fluorescence in the liver is shown for comparison. mLN, mesenteric lymph node.
(C) GFP and ICOS expression in CD4+ RFP+ Treg cells isolated from the spleen and LNs of Myb+/+Foxp3RFP/RFP or MybGFP/GFPFoxp3RFP/RFP mice and stimulated for 24 or 48 hr with anti-CD3 and anti-CD28 in the presence of IL-2. Plots represent three wells in each of two independent experiments.
(D) GFP and ICOS expression in RFP+ iTreg cells differentiated from naive CD4+RFP−Myb+/+Foxp3RFP/RFP or MybGFP/GFPFoxp3RFP/RFP T cells in vitro for 4 days. Plots represent two independent experiments.
Numbers in (B)–(D) indicate the proportion of cells in each quadrant. See also Figure S1.
Immunity  , 78-91DOI: ( /j.immuni )
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5. Figure 3
Myb-Deficient Treg Cells Lack Multiple Activation Markers and Effector Molecules
(A and B) Flow cytometric analysis of CD4+FoxP3+ Treg cells isolated from (A) spleen or (B) mesenteric lymph nodes (mLNs) of Mybfl/fl and Mybfl/flCd4creTg mice shows the expression of CD44, CD62L, ICOS, TIGIT, and KLRG1. Plots represent 3–13 mice analyzed over four independent experiments.
(C) Percentage of ICOS+ (top), TIGIT+ (middle), or KLRG1+ (bottom) cells among CD4+FoxP3+ Treg cells from adult Mybfl/fl and Mybfl/flCd4creTg mice. Each dot represents an individual mouse pooled from four to six individual experiments; horizontal bars show the mean. p values compare the indicated samples.
(D) Proportion of ICOS− (top) or ICOS+ (bottom) CD4+FoxP3+ Treg cells expressing (black bar) or lacking (white bar) Nrp1 in adult WT mice. Seven to ten mice were analyzed and pooled from at least three individual experiments. Bar graphs depict the mean ± SD.
(E) Flow cytometric analysis of CD4+FoxP3+ Treg cells isolated from the spleen, pLNs, or mLNs of adult Mybfl/fl, Mybfl/flCd4creTg, or Irf4−/− mice shows differential expression of Nrp1 and ICOS. Plots represent two Irf4−/− and 11–18 Myb mice analyzed over eight independent experiments.
Numbers in (A), (B), and (E) indicate the proportion of cells in each box or quadrant. See also Figure S2.
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6. Figure 4 Fatal Autoimmune Pathology in Mice Lacking Myb in Treg Cells
(A) Survival curve of Mybfl/flFoxp3YFP-cre mice (n = 34, red line) and WT control mice (n = 20, black line); p <
(B) Body weight of Mybfl/flFoxp3YFP-cre/y and WT control males at the indicated ages.
(C) Splenic weight of Mybfl/flFoxp3YFP-cre mice (n = 20) and WT control mice (n = 19).
(D) Percentage of CD4+ (top) and CD8+ (bottom) T cells isolated from Myb+/+Foxp3YFP-cre or clinically ill Mybfl/flFoxp3YFP-cre mice that expressed IFN-γ after in vitro stimulation with PMA and ionomycin for 4 hr.
(E) H&E staining of tissue sections from the liver and lung of Myb+/+Foxp3YFP-cre and Mybfl/flFoxp3YFP-cre mice (magnification 100×). Arrows indicate examples of immune infiltrates in the liver and lung of Mybfl/flFoxp3YFP-cre mice.
In (B)–(D), each dot represents an individual mouse, and horizontal bars show the mean. p values compare the indicated samples. See also Figure S3.
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7. Figure 5 Myb Is Required for the Generation of Effector tTreg Cells
(A) Schematic representation of FoxP3-reporter mosaicism in Foxp3YFP-cre/RFP females.
(B) Contour plots indicate the gating of ICOS and Nrp1 surface expression in thymic Treg cells and the percentage of RFP+ and YFP+ Treg cells among the most immature (im) Nrp1−ICOS− subset. The graph shows the mean percentage ± SD of YFP+ cells among Nrp1−ICOS− thymic Treg cells in Myb+/+Foxp3YFP-cre/RFP (n = 13) or Mybfl/flFoxp3YFP-cre/RFP (n = 7) females. The relative contribution was calculated as the ratio of mosaicism in Mybfl/flFoxp3YFP-cre/RFP females to that in Myb+/+Foxp3YFP-cre/RFP females (set at 1, dotted line).
(C) Mean relative contribution ± SD of Myb-deficient cells, calculated as in (B), to the ICOS−TIGIT− or ICOS+TIGIT+ subsets of Treg cells in different organs. Data derive from 7 Myb+/+Foxp3YFP-cre/RFP and 13 Mybfl/flFoxp3YFP-cre/RFP mice. pLN, peripheral lymph node; mLN, mesenteric lymph node.
(D) ICOS and intracellular CTLA4 in Ly5.2+CD4+FoxP3+ Treg cells isolated from the spleen and liver of 20:80 BM chimeras of Ly5.1 WT and Ly5.2 Mybfl/flFoxp3YFP-Cre or Ly5.2 Myb+/+Foxp3YFP-cre control cells. Plots represent four mice per group.
(E and F) Expression of (E) ICOS and TIGIT and (F) KLRG1 on Treg cells from 1-week-old Myb+/+Foxp3YFP-Cre or Mybfl/flFoxp3YFP-cre mice. Plots represent (E) ten or (F) six mice.
(G) Flow cytometric analysis of GATA3 and T-bet in CD62L+ICOS−TIGIT−, CD62L−ICOS−TIGIT−, or CD62L−ICOS+TIGIT+ Ly5.2+CD4+FoxP3+ splenic Treg cells from 20:80 BM chimeras as in (D). Histograms represent four mice per group. Bar graphs depict the mean fluorescence intensity (MFI) ± SD for each genotype; p values compare the indicated groups (ns, non-significant). FMO, fluorescence minus one staining.
Numbers in (B) and (D)–(F) indicate the proportion of cells in each box or quadrant. See also Figures S4 and S5.
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8. Figure 6 Transcriptional Profiling of Treg Cell Subpopulations
(A) Heatmap showing all differentially expressed (DE) genes among naive (CD62L+ICOS−TIGIT−), activated (CD62L−ICOS−TIGIT−), and effector (CD62L−ICOS+TIGIT+) Treg cells isolated from the spleen and pLNs of WT (Myb+/+Foxp3YFP-Cre) mice (described in Figures S6A and S6B). The relative expression (Z scores) of genes is shown and is color coded according to the legend. Rows are scaled to have a mean of 0 and an SD of 1, and the position of genes of interest is highlighted. Asterisks indicate genes with Myb digital genomic footprints (DGFs). Data are from two experiments.
(B) Log2 fold change (FC) of the DE genes (false-discovery rate < 0.05; reads per kilobase of transcript per million mapped reads > 2 in at least one sample) between activated and naive (top), effector and activated (middle), or effector and naive (bottom) Treg cells; each bar represents one gene, and the dotted lines mark the limit FC values. Data are from two experiments.
(C) Workflow for the identification of Myb DGFs. Genome-wide DHS mapping and DNase I footprints were from GEO: GSM and Myb motifs within footprints were identified de novo and mapped to genes if they occurred within 20 kb of the transcriptional start site and 5 kb downstream of the 3′ UTR. Only those Myb DGFs with >3 CPM (counts per million mapped reads) were retained.
(D) Plot of the average DNase I cleavage pattern centered on the identified Myb DGF in in-vivo-activated Treg cells. The consensus Myb-binding motif identified in the dataset is shown. Base position 0 on the horizontal axis corresponds to the third base in the motif (from the left). The vertical axis shows the total number of reads for which the 5′ end maps to each base position (in relation to the Myb motif center) divided by the total number of Myb DGFs.
(E) Venn diagram showing the overlap in the Myb DGFs in steady-state and in-vivo-activated Treg cells.
(F) Graph showing the proportion of the effector Treg cell profile genes and remaining non-DE genes that have Myb DGFs in either steady-state or in-vivo-activated Treg cells and both combined. p values from Chi-square tests compare the indicated samples.
(G) Representative tracks showing DHS in steady-state and in-vivo-activated Treg cells for the indicated loci. Red bars indicate Myb DGFs. Upper plots show DHS across the entire loci. Middle plots show a close up of a single DHS region containing a Myb DGF. Lower plots show the DNase I cleavage per nucleotide and the Myb consensus sequence (in color) underlying the DGF.
Please see also Figures S6 and S7and Table S1.
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9. Figure 7 Myb Controls Effector Treg Cell Survival and Proliferation
(A–C) GO analysis of the effector Treg cell profile genes that contain Myb DGFs. (A) Left: enriched GO categories (p < 0.05). Right: pie chart showing the number of genes in each GO category. (B and C) Differential expression of the genes in the (B) “apoptotic process” and (C) “cell cycle” GO terms. Genes whose expression increased in effector Treg cells are shown in black, and those that decreased are in red. All genes have Myb DGFs.
(D and E) Detection of active caspase-3 in splenic Treg cells and FoxP3−CD4+ T cells cultured for 2 days in anti-CD3, anti-CD28, and IL-2. (D) Representative flow cytometry plots. Numbers indicate the percentage of cells in the indicated gate. (E) Graph shows the proportion of active caspase-3+ Treg cells. Each dot represents a cell derived from an independent mouse; the horizontal line shows the mean. Data are pooled from two experiments.
(F) Impaired in vitro proliferation of Myb-deficient Treg cells. The graph shows the mean number ± SD of cells recovered after 4 days in culture (as in B). Data represent three independent experiments.
(G) Mean percentage of Ki67-expressing cells ± SD among ICOS−TIGIT−Ly5.2+CD4+FoxP3+ or ICOS+TIGIT+Ly5.2+CD4+FoxP3+ Treg cells from the indicated tissue of 20:80 BM chimeras of Ly5.1 WT and Ly5.2 Myb+/+Foxp3YFP-cre or Ly5.2 Mybfl/flFoxp3YFP-cre cells. Three to nine chimeras were analyzed per genotype in two individual experiments.
(E–G) Significant p values compare the indicated groups.
Immunity  , 78-91DOI: ( /j.immuni )
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