1. Volume 29, Issue 6, Pages 876-887 (December 2008)
The Zinc Finger Transcription Factor Zbtb7b Represses CD8-Lineage Gene Expression in Peripheral CD4+ T Cells 
Lie Wang, Kathryn F. Wildt, Ehydel Castro, Yumei Xiong, Lionel Feigenbaum, Lino Tessarollo, Rémy Bosselut 
Immunity 
Volume 29, Issue 6, Pages (December 2008)
DOI: /j.immuni
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Expression of Zbtb7b Is CD4+ Specific
(A) The transcriptional activity of Zbtb7b cis-regulatory elements was assessed in mice carrying a BAC reporter transgene in which the first coding exon of Zbtb7b was replaced by an eGFP cDNA (white box), so that GFP fluorescence is a readout of Zbtb7b expression. Thick boxes depict coding sequences within the second and third Zbtb7b exons.
(B) Overlaid histograms show GFP fluorescence (plain lines) in CD4+ and CD8+ LN T cells and in TCR-βhi CD4 and CD8 SP thymocytes that had downregulated CD24 (heat stable antigen), an event characteristic of the most mature SP thymocyte subset.
(C) Two parameter contour plots show CD44 or CD25 versus GFP expression on gated CD4+CD8–peripheral T cells from Zbtb7b+/+ mice carrying the GFP BAC reporter. Numbers indicate the percentage of cells in each quadrant.
(D) Overlaid histograms show GFP fluorescence (plain lines) in CD4+ T cells differentiating in vitro under Th1, Th2, or ThN cell-culture conditions (see Experimental Procedures), 1 to 4 days after activation (set as day 0). Dotted histograms show GFP fluorescence of fresh CD4+ T cells analyzed in parallel. Gray-shaded histograms in (B) and (D) show background fluorescence on nontransgenic cells of the same subset subject to the same treatment. Data are representative of at least two (D) or three (B, C) independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2 CD4+ Cell Development in Mice Hypomorphic for Zbtb7b
(A) Thymocytes and splenocytes from wild-type, Zbtb7bt/t, and Zbtb7bt/– mice were analyzed by 4-color flow cytometry; Zbtb7b–/– mice (Wang et al., 2008) are shown as a control. CD4 SP thymocytes (left) are gated on CD4 versus CD8 contour plots and analyzed for expression of TCR-β and CD24; the box indicates the mature TCRhiCD24lo subset. CD4+ and CD8+ subsets are gated on a two-color contour plot of CD4 and CD8 expression on all live or TCR+ LN cells (right). Numbers indicate the percentage of cells within boxes.
(B) Bar graphs display the absolute numbers of mature CD4 (left, filled bars) and CD8 SP (right, open bars) thymocytes (TCRhiCD24lo, top) and of CD4+ and CD8+ spleen T cells (bottom). Note the x axis scales. Error bars indicate SEM. Data are from 4 (t/-) or 5 mice of each genotype analyzed in three or more distinct experiments.
(C) Expression of Zbtb7b protein was analyzed by immunoprecipitation and immunoblotting on bead-purified CD4+ T cells from either Zbtb7bt/t or Zbtb7bt/– mice (1 × 107/lane) or from wild-type controls (0.1–1 × 107/lane, as indicated). The leftmost lane is an antibody-only control. Data are from 2 or 4 (t/-) mice of each genotype analyzed together in a single experiment.
(D and E) CD8-lineage redirection in mice hypomorphic for Zbtb7b. Four-parameter flow cytometry was performed on thymocytes from Zbtb7bt/t and Zbtb7bt/– mice and from wild-type and Zbtb7b–/– mice as controls, all carrying the MHC II-restricted AND TCR transgene that, in I-Ab-expressing mice, promotes the generation of CD4 SP cells expressing the transgenic Vα11 TCRα chain (Kaye et al., 1989).
(D) Contour plots of CD4 and CD8 expression gated on all (left) or mature (Vα11hi CD24lo, right) thymocytes show the presence of both CD4 and CD8 SP subsets in Zbtb7bhypomorphic mice, unlike in wild-type mice that only have mature CD4-lineage AND cells.
(E) Absolute numbers of mature thymocytes and splenocytes are displayed as in (B); data from Zbtb7b+/t mice were obtained similarly. Data are from 3 (+/t, t/t) or 4 mice of each genotype analyzed in two distinct experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
Inappropriate CD8-Lineage Gene Expression in Zbtb7 Hypomorphic CD4+ Cells
(A) Bead-purified CD4+CD8– cells (2 × 106) from wild-type or Zbtb7bt/t mice were adoptively transferred into Rag2–/– recipients. Left: starting populations. Right: splenocytes from recipient mice were analyzed 2 weeks later by 4-color flow cytometry for expression of CD4, CD8α, TCR-β, and CD44. Single-parameter histograms identify TCR+ splenocytes, on which contour plots show expression of CD4 versus CD8α and of CD4 versus CD44. Data are representative of three separate experiments (of which one with β2 m-deficient recipients), each including five recipients for each donor genotype.
(B) Expression of T-bet, Eomes, Perforin, and GzmB mRNAs was assessed by qRT-PCR in sorted naive (CD44lo) CD4+CD8– LN T cells from wild-type and Zbtb7bt/t mice and in CD4–CD8+ LN cells from wild-type mice. Messenger RNA expression is normalized to β-actin mRNA and expressed as a ratio over those in wild-type CD8+ cells (set to 1, not depicted). Error bars indicate SEM of triplicate determinations within the experiment shown; data are representative of three separate such experiments.
(C) Expression of Runx3 mRNAs initiated at the distal (dis-Runx3) and proximal (prox-Runx3) promoters was analyzed by conventional RT-PCR in sorted naive (CD44lo) CD4+CD8– LN T cells from wild-type and Zbtb7bt/t mice and in CD4–CD8+ LN cells from wild-type mice. Wedges represent 3-fold dilutions (1.2; 0.4; and 0.12 × 104 cells, respectively). β-actin mRNA expression was assessed in parallel as a control for mRNA preparation (bottom row). Data are representative of three separate experiments.
(D–F) Sorted CD44loCD4+CD8– LN T cells from wild-type or Zbtb7bt/t mice were activated by anti-CD3 and anti-CD28 on irradiated APCs in the presence of IL-2, IL-4, and antibodies against IL-12 and IFN-γ (Th2 cell-culture conditions) or IL-2 only (ThN cell-culture conditions), and analyzed 5 days after stimulation.
(D) Cells were stained for CD4, CD8, and intracellular GzmB; GzmB expression is shown on gated CD4+CD8– Zbtb7bt/t (plain line) or wild-type (gray-filled histograms); the dashed lines show GzmB expression on wild-type CD8+ cells stimulated in parallel in the same conditions.
(E) IL-4 and IFN-γ production was assessed by flow cytometry on cells restimulated with PMA and ionomycin. Cells were stained for CD4, CD8, and intracellular IL-4 and IFN-γ; contour plots are gated on CD4+CD8– cells. Numbers indicate the percent of cells within quadrants.
(F) Analyses of gene expression were conducted on Th2-differentiated effectors and displayed as in (B). Data are representative of three or more separate experiments for each panel (D–F).
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
Expression of CD4-Lineage Genes in Zbtb7b Hypomorphic CD4+ T Cells
(A) Transcriptional activity of Zbtb7b cis-regulatory elements in Zbtb7b hypomorphic CD4+ cells. Expression of GFP was assessed by flow cytometry in gated CD4+CD8– LN T cells from wild-type, Zbtb7bt/t, or Zbtb7bt/– mice carrying a BAC GFP reporter for Zbtb7b expression, in which GFP expression is driven by endogenous Zbtb7b cis-regulatory elements in an unmodified context. Contour plots show expression of GFP against CD44. Numbers indicate the frequency of cells within quadrants. Data are from a least three mice of each genotype analyzed in two or more experiments.
(B) Surface expression of CD40L (CD154) was assessed by flow cytometry on B cell-depleted LN cells stimulated 4 hr with PMA and ionomycin. Overlaid histograms are gated on CD4+CD8– cells and show CD40L expression on treated (plain lines) or untreated (gray-shaded histograms) cells. Dashed lines indicate CD40L expression on gated wild-type CD4–CD8+ cells in the same culture. Numbers indicate the mean fluorescence intensity of CD40L staining on stimulated CD4+CD8– cells. Data are from three determinations; the mean intensity of CD40L staining in Zbtb7bt/t CD4+ cells was 47% of that in their wild-type counterparts.
(C) LN cells from wild-type, Zbtb7bt/t, or Zbtb7b–/– mice were stained for CD4, CD8, CD25, and intracellular Foxp3. Contour plots of CD4 and CD8 expression are shown for all cells (left) or CD25+ Foxp3+ cells (right) as gated on middle-column plots. Data are from three experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Peripheral Deletion of Zbtb7b Reactivates CD8+ Lineage Gene Expression
(A) CD4+CD8– LN cells were bead purified from pIC-treated Zbtb7bfl/fl and Mx-Cre transgenic Zbtb7bfl/fl mice, 7 days after the first pIC injection, and adoptively transferred into Rag2–/– mice. Left: Contour plots show expression of CD4 and CD8α on ex vivo LN cells and on cells purified for injection. Right: Recipient spleens were harvested 2 weeks after transfer and analyzed by 3-color flow cytometry for expression of CD4, CD8α, and TCR-β. Contour plots (right column) show expression of CD4 and CD8α on gated TCR+ cells (gating on left column).Data are representative of three independent experiments.
(B and C) Sorted CD44loCD4+CD8– LN T cells from pIC-treated Zbtb7bfl/fl and Mx-Cre transgenic Zbtb7bfl/fl mice were activated in Th2 or ThN cell conditions as in Figure 3 and analyzed for expression of CD4, CD8, and intracellular GzmB (B) or of CD4, CD8, and intracellular IL-4 and IFN-γ after PMA-ionomycin restimulation (C). Data are representative of three such experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
Expression of Cytotoxic Genes by Zbtb7b Hypomorphic CD4+ Cells Depends on Runx and T-box Protein Activities
(A and B) Bead-purified CD4+CD8– cells from Zbtb7bt/t (left) or wild-type (right) mice were activated and transduced with retroviral vectors encoding a T-bet-engrailed chimeric protein that inhibits both T-bet and Eomes activities (T-bet DN), the Runt domain of Runx3 (Runt), or wild-type Zbtb7b (Zbtb7b), or with a control vector encoding GFP only, and analyzed after a 5-day culture in the indicated conditions for expression of CD4, CD8, and intracellular IL-4 and IFN-γ (A), or of CD4, CD8, and intracellular GzmB (B).
(A) Contour plots of IFN-γ versus IL-4 expression are gated on CD4+CD8– cells and are shown for transduced (GFP+) and untransduced (GFP–) cells. Numbers indicate the percentage of cells within each quadrant.
(B) Overlaid histograms show expression of GzmB on transduced (GFP+) and untransduced (GFP–) Zbtb7bt/t CD4+CD8– cells (plain lines). Dashed lines show GzmB expression on untransduced CD8+ cells stimulated in parallel in the same conditions. None of these retroviral vectors affected GzmB expression in wild-type cells (gray-shaded histograms).
(C) Wild-type CD4+ or CD8+ T cells were activated under Th2 conditions and transduced with either a Runx3-encoding retroviral vector (plain lines) or the GFP-only control (gray filled). Overlaid histograms show GzmB expression on GFP+ and GFP– cells (dashed lines: CD8+ cells activated in the same conditions). Data are representative of at least two separate experiments for each panel.
Immunity  , DOI: ( /j.immuni )
Copyright © 2008 Elsevier Inc. Terms and Conditions