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The Factor Zbtb7b Represses CD8-Lineage Expression in Peripheral CD4+ TCells

Lie Wang,1 Kathryn F. Wildt,1 Ehydel Castro,1 Yumei Xiong,1 Lionel Feigenbaum,2 Lino Tessarollo,3 and Re´ my Bosselut1,* 1Laboratory of Immune Cell Biology, Center for Research (CCR), NCI, National Institutes of Health, Bethesda, MD 20892, USA 2NCI-SAIC, Frederick, MD 21702, USA 3Mouse Cancer Genetics Program, CCR, NCI, Frederick, MD 21702, USA *Correspondence: [email protected] DOI 10.1016/j.immuni.2008.09.019

SUMMARY members of the Runx family, Runx1 and Runx3, contribute to CD8+ cell differentiation and notably to the termination of CD4 How CD4-CD8 differentiation is maintained in mature expression (Taniuchi et al., 2002a; Woolf et al., 2003), whereas T cells is largely unknown. The present study has ex- the HMG Tox and the zinc finger transcription factors amined the role in this process of the zinc finger pro- Gata3 and Zbtb7b (also known as Thpok, cKrox) promote the + tein Zbtb7b, a critical factor for the commitment of generation of CD4 T cells (Aliahmad and Kaye, 2008; Hernan- MHC II-restricted thymocytes to the CD4+ lineage. dez-Hoyos et al., 2003; Pai et al., 2003; Zhu et al., 2004; He We showed that Zbtb7b acted in peripheral CD4+ et al., 2005; Sun et al., 2005; Wang et al., 2008). Little is known about how CD4+-CD8+ differentiation is main- T cells to suppress CD8-lineage , in- tained in peripheral T cells. Some of the factors that promote cluding that of CD8 and cytotoxic effector lineage differentiation in the thymus carry distinct functions in perforin and Granzyme B, and was important for post-thymic T cells. In the mature CD4+ compartment, Runx3 the proper repression of interferon-g (IFN-g) during contributes to interferon-g (IFN-g) production and interleukin-4 effector differentiation. The inappropriate expression (IL-4) repression during type 1 effector differentiation (Djuretic of IFN-g by Zbtb7b-deficient CD4+ T cells required et al., 2007; Naoe et al., 2007), whereas Runx1 promotes cell sur- the activities of and Runx transcrip- vival (Egawa et al., 2007). Gata3 promotes type 2 effector differ- tion factors. Runx activity was needed for Granzyme entiation and does not appear required to maintain the CD4+ lin- B expression, indicating that Runx control eage (Zheng and Flavell, 1997; Pai et al., 2004; Zhu et al., 2004). expression of the cytotoxic program. We conclude In differentiating thymocytes, Zbtb7b both promotes CD4- that a key function of Zbtb7b in the mature CD4+ helper and represses CD8-cytotoxic T cell differentiation (He et al., 2005; Sun et al., 2005). Thus, because Zbtb7b expression T cell compartment is to repress CD8-lineage gene is CD4-lineage specific in peripheral T cells, it was possible that it expression. promoted the maintenance of CD4-helper gene expression or the repression of CD8-cytotoxic gene expression, or both, in INTRODUCTION CD4+ cells. In the present study, we tested these hypotheses by using hypomorphic and conditional Zbtb7b alleles. We dem- T lymphocytes (T cells) are critical for adaptive immune re- onstrated that a key function of Zbtb7b in CD4+ T cells is to pre- sponses and are distributed into multiple lineages, the two pre- vent expression of CD8 and cytotoxic genes. dominant ones being defined by their reactivity against peptide antigens bound to classical MHC molecules and by their mutu- RESULTS ally exclusive expression of CD4 or CD8 surface glycoproteins (Janeway and Bottomly, 1994). CD4+ T cells typically recognize Expression of Zbtb7b in Mature CD4+ T Cells MHC-II peptide complexes and upon effector differentiation We and others previously reported that Zbtb7b was expressed in control the function of other immunocompetent cells, either pos- CD4+ but not CD8+ peripheral T cells (He et al., 2005; Sun et al., itively or negatively. In contrast, CD8+ T cells generally recognize 2005; Setoguchi et al., 2008). We more extensively examined the MHC-I peptide complexes and differentiate into cytotoxic effec- pattern of Zbtb7b expression in peripheral T cells by using a BAC tors able to lyze virtually any nucleated cell given the broad pat- reporter transgene in which Zbtb7b cis-regulatory elements con- tern of MHC-I expression. Once established in the thymus trol the expression of a GFP cDNA inserted in the first Zbtb7b (Singer and Bosselut, 2004), CD4-CD8 lineage differentiation is coding exon (Figure 1A; Wang et al., 2008). This BAC drives un- stably maintained in peripheral T cells and is inherited through imodal GFP expression in spleen and lymph node (LN) CD4+ but cell division after activation and during effector differentiation. not CD8+ T cells, in amounts similar to those observed in mature Several transcription factors orchestrate the differentiation of CD4 SP thymocytes (Figure 1B). GFP expression was similar in CD4+ and CD8+ cells from bipotent precursors that express naive (CD44lo) and effector or memory-type cells (CD44hi), and both CD4 and CD8 (double-positive [DP] thymocytes). Two in CD4+CD25+ regulatory T (Treg) cells (Figure 1C). GFP

876 Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. Immunity Zbtb7b Function in CD4 T Cells

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-bhi 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 histo- grams 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. expression in CD4+ T cells persisted after T cell activation and through the insertion, immediately upstream of the first Zbtb7b during in vitro effector differentiation under T helper 1 (Th1) or coding exon, of a Neo cassette flanked by splice acceptor and Th2 cell culture conditions (Figure 1D). These experiments indi- transcription termination signals (Figure S1 available online). cate that Zbtb7b expression is maintained throughout the life Mice carrying one or two copies of this allele (Zbtb7bt/– or of peripheral CD4+ T cells and prompted us to evaluate the Zbtb7bt/t) were fertile and did not display any gross developmen- role of Zbtb7b in CD4+ T cell function. tal defect. The number of peripheral CD4+ T cells was modestly reduced in Zbtb7bt/t mice, and most of them were naive CD44lo CD4+ T Cells with Reduced Zbtb7b Protein Expression cells (Figures 2A and 2B; Figure S2). The number of CD8+ T cells Germline Zbtb7b inactivation prevents CD4+ T cell development was slightly increased, and analyses with T cell (TCR) (He et al., 2005; Wang et al., 2008) and therefore cannot be used transgenic Zbtb7bt/t mice indicated that this resulted from the to study the role of this factor in post-thymic CD4+ T cells. As an partial CD8-redirection of MHC II-restricted thymocytes (Figures alternative strategy, we took advantage of the targeted allele 2D and 2E). Expression of Zbtb7b protein in peripheral CD4+ (Zbtb7bt) we generated to disrupt Zbtb7b (Wang et al., 2008). T cells was lower in Zbtb7bt/t than in wild-type mice This allele is engineered to reduce Zbtb7b protein expression (Figure 2C); thus, the inserted Neo cassette impaired rather

Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. 877 Immunity Zbtb7b Function in CD4 T Cells

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-b 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). Num- bers 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 3 107/lane) or from wild-type controls (0.1–1 3 107/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 Va11 TCRa chain (Kaye et al., 1989). (D) Contour plots of CD4 and CD8 expression gated on all (left) or mature (Va11hi 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.

878 Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. Immunity Zbtb7b Function in CD4 T Cells

than prevented Zbtb7b protein expression, and the Zbtb7bt ciently translated in resting T cells (Egawa et al., 2007), were allele was hypomorphic. Changes in CD4 and CD8 single-posi- present in all three cell subsets (Figure 3C). Thus, Zbtb7b hypo- tive (SP) thymocyte subsets paralleled those seen in the periph- morphic CD4+ T cells fail to normally repress CD8-lineage genes. ery (Figures 2A, 2B, 2D, and 2E). Zbtb7bt/t thymocytes had nor- To determine whether reduced Zbtb7b expression would re- mal expression of CD5 and CD69 (data not shown), suggesting sult in cytotoxic gene expression during effector differentiation, that TCR signal transduction operated normally in these cells, we activated wild-type and Zbtb7bt/t CD44loCD4+ T cells with as in thymocytes with germline Zbtb7b inactivation (Azzam TCR and CD28 antibodies and cultured them for 5 days in the et al., 1998; Swat et al., 1993; He et al., 2005). presence of irradiated APCs and exogenous IL-2 (nonpolarizing, Zbtb7b protein expression was further reduced in Zbtb7bt/– ThN conditions). In such conditions, GzmB protein, normally de- mice (Figure 2C), in which T cell development and homeostasis tected in CD8+ but not CD4+ cells, was expressed in Zbtb7bt/t were more severely affected. There were few mature CD4+ cells CD4+ T cells (Figure 3D, right). Analyses of cytokine production (Figures 2A and 2B), most of which were CD44hi (Figure S2), and showed abnormal effector differentiation in Zbtb7bt/t cells. a near complete redirection of MHC II-restricted thymocytes to Whereas wild-type CD4+ T cells were skewed toward IL-4 in con- the CD8+ lineage (Figures 2D and 2E). Thus, even though most trast to IFN-g in CD8+ T cells (Figure S4), most Zbtb7bt/t CD4+ Zbtb7bt/– peripheral CD4+ cells were MHC II restricted (data cells produced IFN-g, and a notable fraction of those also not shown), they were not representative of their wild-type coun- made IL-4 (Figure 3E). Of note, both wild-type and Zbtb7bt/t ef- terparts. Consequently, we used Zbtb7bt/t mice to explore the fector cells maintained a strict CD4+CD8– pattern of coreceptor function of Zbtb7b in mature CD4+ T cells. expression after in vitro activation (Figure S5 and data not shown). CD4+ cells from Zbtb7b+/t littermates behaved like wild-type controls in these and subsequent analyses, indicating t/t + CD8-Lineage Gene Expression in Zbtb7b CD4 T Cells that the skewing toward cytotoxic differentiation did not result + t/t Although most peripheral CD4 cells in Zbtb7b mice did not ex- from a dominant effect of the Zbtb7bt allele. hi + int press CD8, these animals had TCR CD4 CD8 cells that were Both Zbtb7bt/t and wild-type CD4+ cells produced IL-4 and no not found in wild-type mice (Figure 2A, second row, right column). IFN-g when activated in Th2 cell conditions (IL-4 and antibodies + int To evaluate whether such CD4 CD8 cells were post-thymically against IL-12 and IFN-g)(Figure 3E). Nonetheless, Zbtb7bt/t Th2 + – derived from CD4 CD8 cells, we adoptively transferred wild- effector cells had higher GzmB protein and mRNA expression t/t + – type and Zbtb7b CD4 CD8 cells (containing less than 0.1% than did their wild-type counterparts (Figure 3D, left, and contaminating CD8-expressing cells, Figure S3A) into Rag2- Figure 3F); GzmB mRNA expression was more than 1000 times deficient recipients (Shinkai et al., 1992). Although wild-type do- greater in effector than in resting cells (Figure S6), presumably + – t/t nors remained CD4 CD8 after transfer, a subset of Zbtb7b accounting for the lack of detectable GzmB protein expression cells re-expressed CD8, indicating impaired CD8 gene repression in naive Zbtb7bt/t cells. Activation in Th1 cell culture conditions (Figure 3A). CD8 expression on transferred cells was bimodal, (IL-12 and anti-IL-4) similarly found higher expression of GzmB suggesting loss of epigenetic CD8 repression in a subset of cells. protein in Zbtb7bt/t than in wild-type CD4+ effectors Of note, these CD8-expressing cells did not terminate CD4 ex- (Figure S7A). The increased expression of GzmB by Zbtb7b- pression. These results prompted us to evaluate how reduced insufficient effectors, irrespective of their activation conditions, Zbtb7b expression affected CD8-lineage gene expression. supports the conclusion that the expression of ‘‘cytotoxic’’ genes We analyzed gene expression by quantitative RT-PCR (qRT- by Zbtb7bt/t is not simply a reflection of a Th2 to Th1 cell popula- + – lo PCR) in purified CD4 CD8 CD44 T cells from wild-type and tion shift. Of note, perforin mRNA expression remained well below t/t t/t + Zbtb7b mice. Zbtb7b CD4 cells had modestly elevated those in CD8+ effectors, suggesting that they would be insuffi- (2-fold) expression of the cytotoxic marker perforin and of cient to allow cytolytic killing. We conclude from these experi- Eomes, a T-box transcriptional activator of the cytotoxic effector ments that Zbtb7b molecules act to prevent the aberrant expres- + program in the CD8 lineage (Figure 3B; Pearce et al., 2003). A sion of cytotoxic genes both in resting and effector CD4+ cells. larger increase (3- to 10-fold) was seen for the cytotoxic gene Granzyme B (GzmB), but flow cytometry failed to detect GzmB protein expression in both Zbtb7bt/t CD4+ and wild-type CD8+ The Zbtb7b Hypomorphic Allele Has Minimal Effects naive cells (data not shown). We assessed expression of on CD4-Lineage Gene Expression Runx3, which has been reported to promote CD8 expression We next examined CD4-lineage gene expression in Zbtb7bt/t (Sato et al., 2005), by conventional RT-PCR to distinguish CD4+ cells. Although expression of CD4 in resting CD4+CD8– mRNAs initiated at the distal (dis-Runx3) and proximal pro- splenocytes or LN cells was minimally (86% ± 4%) but repro- moters (Bangsow et al., 2001). In resting T cells, the former tran- ducibly lower in Zbtb7bt/t cells than in their wild-type counter- scripts are both characteristic of the CD8+ lineage and consid- parts, expression of CD4 on activated cells was equivalent to ered to be the main source of Runx3 protein (Egawa et al., wild-type (Figure S5 and data not shown). Gata3 gene expres- 2007). Although in wild-type mice dis-Runx3 transcripts were sion, analyzed by qRT-PCR on naive CD44loCD4+CD8– cells, found in CD8+ but not CD4+ cells, they were detected in CD4+ was similar in Zbtb7bt/t and wild-type mice (Figure S8A), and cells from Zbtb7bt/t mice (Figure 3C). In contrast, transcripts ini- so was the activity of the cis-regulatory elements that normally tiated at the proximal promoter, that do not appear to be effi- control Zbtb7b gene expression (Figure 4A).

(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.

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Figure 3. Inappropriate CD8-Lineage Gene Expression in Hypomorphic CD4+ Cells (A) Bead-purified CD4+CD8– cells (2 3 106) 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, CD8a, TCR-b, and CD44. Single-parameter histo- grams identify TCR+ splenocytes, on which contour plots show expression of CD4 versus CD8a and of CD4 versus CD44. Data are representative of three sep- arate experiments (of which one with b2 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 b-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 3 104 cells, respectively). b-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-g (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 histo- grams); the dashed lines show GzmB expression on wild-type CD8+ cells stimulated in parallel in the same conditions. (E) IL-4 and IFN-g 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-g; 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).

To analyze how Zbtb7b affected CD4+ cell effector functions, half in Zbtb7bt/t CD4+ cells (Figure 4B). When activated in the we assessed expression of CD40 ligand (CD154), a helper mol- proper stimulation conditions, Zbtb7bt/t CD4+ T cells differenti- ecule upregulated in CD4+ cells upon TCR engagement or after ated not only into IFN-g- or IL-4-producers (Figure 3E; treatment by the surrogate drug combination of PMA and iono- Figure S7B), but also could express IL-17 or the transcription mycin (Roy et al., 1993). In wild-type mice, PMA and ionomycin- factor Foxp3, that in wild-type mice promotes T-regulatory dif- stimulated upregulation of CD40L was considerably higher in ferentiation (Figure S8B; Bettelli et al., 2006; Veldhoen et al., CD4+ than in CD8+ cells, and this response was reduced by 2006; Reiner, 2007; Zheng and Rudensky, 2007; Sakaguchi

880 Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. Immunity Zbtb7b Function in CD4 T Cells

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,orZbtb7bt/– mice carrying a BAC GFP reporter for Zbtb7b expression, in which GFP expression is driven by en- dogenous 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 in- dicate 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,orZbtb7b–/– 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.

et al., 2008). Accordingly, Zbtb7bt/t mice had subnormal num- press GzmB (data not shown). We excised the floxed sequences bers of CD4+CD25+ T cells expressing Foxp3 (Figure 4C). Al- in vivo by using the IFN-a-responsive Mx1 promoter to express though in reduced numbers, CD25+ Foxp3+ cells were present the Cre recombinase (Kuhn et al., 1995). Treatment of Mx-Cre in Zbtb7b–/– mice, demonstrating that Zbtb7b is not required transgenic Zbtb7bfl/fl mice with poly-IC (pIC), an RNA mimic that for Foxp3 expression. Remarkably, even though most Zbtb7b- promotes IFN-a production, resulted in a substantial, although in- deficient MHC II-restricted T cells are CD4–CD8+ T cells (He complete, deletion of the floxed allele as assessed by PCR on ge- et al., 2005; L.W. and R.B., data not shown), Foxp3 expression nomic DNA (Figures S10A and S10B). Despite the IFN-a secretion, was largely restricted to the residual CD4+ population pIC-treated Zbtb7bfl/fl and Mx-Cre Zbtb7bfl/fl mice retained sub- (Figure 4C). These findings suggest that Zbtb7b deficiency had stantial numbers of CD44loCD4+ T cells (Figure S10C). a lesser impact on CD4+ than on CD8+ markers, although their CD4+ cells from pIC-treated Mx-Cre Zbtb7bfl/fl mice re- amplitude in Zbtb7bt/t cells was not negligible given that it may expressed CD8 after adoptive transfer into Rag2-deficient hosts, be limited by the residual expression of Zbtb7b. similar to Zbtb7bt/t cells (Figure 5A; Figures S3B and S3C). Thus, post-thymic Zbtb7b expression is required in CD4+ T cells for the Zbtb7b Is Required in Peripheral CD4+ T Cells sustained repression of CD8 genes. However, these CD8 re- to Repress CD8-Lineage Genes expressing cells remained CD4+, suggesting that Zbtb7b is not The present findings suggested that Zbtb7b acted in peripheral required to maintain CD4 expression. Note that because of the CD4+ T cells to repress the cytotoxic program; however, they incomplete Mx-Cre-induced deletion (Figure S10B), the bimodal could not exclude that CD8-lineage gene expression by expression of CD8 on transferred cells cannot be interpreted Zbtb7bt/t CD4+ cells resulted from impaired intrathymic develop- conclusively. To assess the role of Zbtb7b in effector differentia- ment. To distinguish between these possibilities, it was neces- tion, we stimulated CD44loCD4+CD8– cells from pIC-treated sary to examine mature Zbtb7b-deficient CD4+ T cells that had Zbtb7bfl/fl and Mx-Cre transgenic Zbtb7bfl/fl mice. Under both developed as Zbtb7b-sufficient thymocytes. To this end, we ThN and Th2 differentiation conditions, peripheral Zbtb7b exci- generated a conditional (‘‘floxed’’) Zbtb7b allele (Zbtb7bfl, sion resulted in GzmB production, even though it did not affect Figure S1)inwhichtheZbtb7b coding sequence is flanked by CD4 or CD8 surface expression (Figure 5B; Figure S10D). Post- two loxP sites and can be excised by the Cre recombinase. thymic Zbtb7b deletion resulted in the same skewing toward Zbtb7bfl/fl mice had normal numbers of CD4+ T cells (Figure S9), IFN-g production in ThN conditions as that observed in indicating that the floxed allele was functional. Zbtb7bt/t hypomorphic cells (Figure 5C). These observations Deletion of the floxed allele in vitro by a Cre-expressing retrovi- demonstrate that Zbtb7b post-thymically represses CD8-lineage rus did not affect CD4 or CD8 surface expression, but did dere- genes in CD4+ T cells.

Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. 881 Immunity Zbtb7b Function in CD4 T Cells

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 CD8a 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, CD8a, and TCR-b. Contour plots (right column) show ex- pression of CD4 and CD8a 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-g after PMA-ionomycin restimulation (C). Data are representative of three such experiments.

Runx3 Mediates Cytotoxic Gene Expression Consequently, we examined whether Runx3 was involved in Zbtb7b-Deficient CD4+ T Cells in cytotoxic gene expression by Zbtb7b-deficient CD4+ Because Eomes but not Runx3 was reported to activate the cy- cells. We expressed in Zbtb7bt/t CD4+ cells a truncated ver- totoxic program in mature CD8+ T cells (Pearce et al., 2003; sion of Runx3 that includes its Runt domain and inhibits Intlekofer et al., 2005; Taniuchi et al., 2002a), we predicted that endogenous Runx activity by occupying Runx target sites aberrant cytotoxic gene expression by Zbtb7b-insufficient cells in association with Cbfb molecules (Hayashi et al., 2000). was caused by Eomes and would be prevented by inhibiting Transduction of the Runt virus into Zbtb7bt/t CD4+ cells in- Eomes activity. To evaluate this prediction, we retrovirally trans- hibited their production of IFN-g (Figure 6A) and reversed duced Zbtb7bt/t cells with a dominant-negative version of T-bet their aberrant GzmB expression as efficiently as did Zbtb7b (T-bet DN) that represses Eomes and T-bet target genes (Pearce transduction (Figure 6B). Conversely, retroviral transduction et al., 2003). As expected, expression of this virus in ThN-acti- of Runx3 in wild-type CD4+ cells promoted GzmB protein vated Zbtb7bt/t CD4+ cells impaired production of IFN-g, a proto- expression (Figure 6C). Together, these observations typical target of T-box proteins (Figure 6A; Szabo et al., 2002; support the conclusion that the expression of cytotoxic Pearce et al., 2003). However, the T-bet DN virus had little or genes in Zbtb7bt/t CD4+ cells is caused in part by Runx3 no effect on GzmB expression, both in ThN and Th2 conditions derepression. (Figure 6B and data not shown). Thus, the derepression of In summary, the present study demonstrates that the tran- GzmB in Zbtb7b-deficient cells was at least in part independent scription factor Zbtb7b is required in post-thymic CD4+ T cells from T-box protein activity. to repress CD8-lineage gene expression.

882 Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. Immunity Zbtb7b Function in CD4 T Cells

DISCUSSION not needed to maintain it in CD8+ cells (Zou et al., 2001), strongly suggesting that the repressors that bind the silencer in thymo- Most conventional TCR-ab T cells (i.e., those restricted by clas- cytes no longer contribute to silencing in post-thymic CD8+ cells. sical MHC molecules) are distributed into CD4+ and CD8+ line- In contrast, there is no known silencer element in the CD8 locus ages that diverge in the thymus from DP precursors, stably and Zbtb7b is required both to establish and maintain CD8 express either CD4 or CD8, and harbor distinctive functional silencing in the CD4+ lineage. properties. Maintaining CD4-CD8 lineage differentiation in the Unlike this effect on CD8 expression, peripheral inactivation peripheral repertoire ensures the continuous matching of MHC did not reveal a Zbtb7b requirement for sustained CD4 expres- specificity, coreceptor expression, and effector differentiation. sion. It is possible that this is due to technical limitations of our How this is achieved has been largely unknown. assays (including their relatively short timeline) or to the fact The present study demonstrates that Zbtb7b, a key CD4+- they were mainly conducted on proliferating cells in which CD4 committing factor in the thymus, is required in peripheral CD4+ gene regulation may be distinct from that in resting cells. Alterna- T cells to prevent the inappropriate expression of CD8-lineage tively, it is possible that Zbtb7b is not required to maintain post- genes, including CD8, the cytotoxic effectors perforin and thymic CD4 expression, either because epigenetic mechanisms GzmB, and the transcription factors Runx3 and Eomes. These ‘‘lock’’ the CD4 locus in an active configuration in peripheral findings agree with our previous observation that Zbtb7b re- CD4+ cells or because putative repressors that contribute to presses cytotoxic differentiation, including perforin and GzmB CD4 silencing in CD8 SP thymocytes (Taniuchi et al., 2002b) expression, when introduced into mature CD8+ T cells (Jenkin- are no longer expressed in post-thymic T cells. Previous studies son et al., 2007). Such a function of Zbtb7b in peripheral CD4+ suggest that the control of CD4 expression, and notably its re- T cells is conceptually similar to the repression of aberrant pression by Runx3, follows distinct rules in mature versus imma- gene expression by Pax5, a critical to estab- ture T cells (Telfer et al., 2004; Djuretic et al., 2007; Naoe et al., lish and maintain B cell commitment (Cobaleda et al., 2007). 2007), and additional analyses will clarify the role of Zbtb7b in Two arguments suggest that Zbtb7b acts as a repressor of post-thymic CD4 expression. CD8 genes. First, Zbtb7b belongs to a family of proteins that Similar to CD8 genes, it is possible that Zbtb7b directly re- generally function as transcriptional repressors, notably by re- presses cytotoxic genes by binding to their cis-regulatory ele- cruiting protein complexes that covalently modify histones or re- ments (Glimcher et al., 2004; Pipkin et al., 2007); the absence model (Bilic and Ellmeier, 2007). Second, both gain- of ChIP-grade antibodies against Zbtb7b currently preclude and loss-of-function analyses show that Zbtb7b antagonizes a detailed investigation of this hypothesis. However, we docu- CD8 expression (Jenkinson et al., 2007; this study). It is possible ment that Zbtb7b acts at least in part by preventing or limiting that Zbtb7b binds the CD8 locus and directly represses tran- the expression of trans-activators of cytotoxic gene expression. scription, that it acts indirectly by repressing activators of CD8 In CD4+ T cells, IFN-g, GzmB, and perforin are responsive to expression, possibly including Runx3 (Sato et al., 2005), or that Eomes, a trans-activator normally expressed during cytotoxic is promotes epigenetic CD8 silencing. It is difficult at present differentiation (Pearce et al., 2003). Thus, limiting or delaying to distinguish between these non-mutually exclusive possibili- Eomes expression during the effector differentiation of CD4+ ties, and the lack of ChIP-grade antibodies against Zbtb7b has cells appears as a key function of Zbtb7b in restraining the pro- so far precluded us from investigating the first hypothesis. duction of components of the cytotoxic program that remain However, two arguments suggest that Zbtb7b promotes epige- ‘‘accessible’’ in CD4+ cells, including IFN-g and GzmB. netic CD8 silencing: the bimodal CD8 expression by Zbtb7b- Previous studies (Wargnier et al., 1995; Babichuk et al., 1996) insufficient CD4+ cells is typical of epigenetic control, and the pointed to a potential role of Runx proteins in GzmB expression. CD8 derepression after Zbtb7b deletion was observed only after Our findings, using both loss- and gain-of-function approaches, cell expansion, suggesting that it required proliferation to dilute document that this is the case. Although Runx3, the predominant repressive epigenetic marks. Runx factor in CD8+ cells, is not required for perforin expression That CD8 re-expression by Zbtb7b-deficient cells was ob- or cytotoxic function (Taniuchi et al., 2002a), there is substantial served only in vivo also suggests a requirement for ligands ab- redundancy between Runx factors during CD8+ cell generation sent from in vitro cultures, notably IL-7 (Park et al., 2007). How- (Woolf et al., 2003; Egawa et al., 2007; Setoguchi et al., 2008), ever, we do not think that the absence of IL-7 accounts for the and it is possible that such redundancy has so far masked the lack of CD8 re-expression by Zbtb7b-deficient CD4+ T cells; role of Runx activity in cytotoxic effector differentiation. indeed, these cells did not express CD8 when cultured in the Runx molecules, presumably Runx3, repress Zbtb7b expres- presence of IL-2 or IL-4, which both promote CD8 expression sion during CD8-lineage differentiation in the thymus, by binding similarly to IL-7 (Park et al., 2007). to a cis-regulatory element (‘‘silencer’’) upstream of the Zbtb7b That CD8 silencing in mature CD4+ cells depends on the con- promoter (Setoguchi et al., 2008). Our finding that Zbtb7b-hypo- tinuous activity of Zbtb7b raises the possibility that distinct morphic cells inappropriately express Runx3 raises the possibil- mechanisms maintain coreceptor silencing in CD4+ versus ity that, conversely, Zbtb7b directly represses Runx3. Future CD8+ T cells. CD4 expression is controlled by an enhancer active studies will determine whether that is the case, in developing thy- in both CD4+ and CD8+ lineages and a silencer that recruits Runx mocytes and mature T cells. It is possible that a two-way cross- proteins and so far unknown additional CD4 repressors to termi- repression mechanism operates in thymocytes and contributes nate CD4 expression in CD8-differentiating thymocytes (Sawada to decide CD4-CD8 commitment by terminating expression of et al., 1994; Siu et al., 1994; Taniuchi et al., 2002a, 2002b). How- one of these factors. However, it is unlikely that similar rules apply ever, whereas the silencer is required to establish silencing, it is in mature T cells, because Runx3 is upregulated in Th1 effectors

Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. 883 Immunity Zbtb7b Function in CD4 T Cells

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- 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 con- trol 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-g (A), or of CD4, CD8, and intracellular GzmB (B).

884 Immunity 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. Immunity Zbtb7b Function in CD4 T Cells

but does not force the termination of Zbtb7b expression. In fact, the control of a b-actin promoter. The genotype of recombinant animals was Runx repression of Zbtb7b expression is context dependent, as verified by Southern blotting for the first two generations, and subsequently Runx complexes (presumably involving Runx1) are bound to by PCR on tail DNA. Mice carrying a GFP BAC reporter for Zbtb7b expression were previously reported (Wang et al., 2008). Rag2–/– (Shinkai et al., 1992) and the Zbtb7b ‘‘silencer’’ in CD4+ cells (Setoguchi et al., 2008). AND TCR transgenic (Kaye et al., 1989) mice were obtained from Taconic, and If Zbtb7b prevents cytotoxic differentiation in part through re- MxCre transgenic (Kuhn et al., 1995) mice were from Jax. Mice carrying the in- pressing cytotoxic effector regulators such as Runx3 or Eomes, dicated genotypes were generated by appropriate intercrosses or back- and given the overlap between Th1 and cytotoxic gene-expres- crosses. Animals were genotyped by PCR on tail DNA or phenotyped by stain- sion programs, why is it that wild-type Th1-differentiating cells, ing of peripheral blood cells and were analyzed between 4 and 12 weeks of age which express both Runx3 and Eomes, do not enter cytotoxic except when indicated otherwise. All transgenic animals were heterozygous for the transgenic allele. All mice were housed in specific-pathogen-free facil- differentiation (Djuretic et al., 2007; Egawa et al., 2007; Suto ities. Animal procedures used in the present study were approved by NCI et al., 2006)? We think of two non-mutually excusive possibilities Animal Care and Use committees, as appropriate. to explain this apparent paradox. First, Zbtb7b may control the kinetics of expression of such factors, and specifically prevent Animal Procedures their expression in resting cells and during initial activation. Ef- For deletion of the Zbtb7bfl allele in peripheral cells, young adult mice were in- fector T cell differentiation is typically determined by the cytokine jected intraperitoneally with pIC (0.25 mg in 0.2 ml sterile water) three times at and cell-cell interaction milieu that affect the balance of tran- 2 day intervals. LN T cells were harvested for analysis 7 days after the first injection. For adoptive transfer experiments, bead-purified CD4+CD8– thymo- scription factor activities present during early activation (Jenkins cytes (2 3 106) were resuspended in 0.25 ml PBS and tail vein-injected into un- et al., 2001; Reiner, 2007). Thus, it is conceivable that expression manipulated Rag2–/– recipients. Recipient splenocytes were harvested and of Runx3 or Eomes in naive Zbtb7b-insufficient cells promotes analyzed 2 weeks after transfer. cytotoxic gene expression, whereas the transcriptional circuitry is no longer permissive to this effect when they are expressed T Cell Activation and Effector Differentiation Cultures at a later time point in wild-type cells. The second possibility is Antigen-presenting cells (APCs) were obtained from C57BL/6 splenocytes by that Zbtb7b acts by impairing Runx3 or T-box protein function, Thy1.2 magnetic bead depletion (Dynal) and irradiated at 2500 rad. Sorted CD44loCD4+CD8– LN or spleen T cells (0.5 3 106) were mixed with 2 3 106 ir- possibly by directly binding to and repressing cytotoxic genes. + radiated APCs in complete culture medium (RPMI 1640 supplemented with It is notable that Zbtb7b levels that allow CD4 cell develop- 10% FCS) and activated with anti-CD3 (2C11, 1 mg/ml) and anti-CD28 (3 mg/ml) ment are potentially insufficient for the proper effector differenti- in the presence of either (1) 50 U/ml IL-2 (‘‘ThN conditions’’); (2) 50 U/ml ation of peripheral T cells. Future studies will determine whether IL-2, 10 ng/ml IL-12, 10 mg/ml anti-IL-4 (‘‘Th1 conditions’’); (3) 50 U/ml IL-2, this has functional consequences in vivo during immune re- 100 U/ml IL-4, 10 mg/ml anti-IL-12, 10 mg/ml anti-IFN-g (‘‘Th2 conditions’’); sponses to infectious agents. However, excessive IFN-g pro- (4) 10 ng/ml IL-6, 5 ng/ml TGF-b1, 10 mg/ml anti-IL-4, 10 mg/ml anti-IFN-g duction by CD4+ T cells has been implicated in tissue damage (‘‘Th17 conditions’’); or (5) 50 U/ml IL-2, 5 ng/ml TGF-b1, 10 mg/ml anti-IL-4, 10 mg/ml anti-IFN-g (‘‘Treg conditions’’). All cytokines were from Preprotech. in several infectious models and in autoimmune diseases, Except where indicated otherwise, cells were analyzed 5 days after activation. including type 1 diabetes (Christen and von Herrath, 2004), For retroviral transductions, T cells activated as above were infected with su- and future studies will examine whether insufficient Zbtb7b ex- pernatants from Plat-E packaging cells (Morita et al., 2000) transfected with pression contributes to inappropriate IFN-g production in such retroviral constructs as described (Jenkinson et al., 2007). Cells were replated circumstances. in their original medium after infection. Finally, the present study reveals a need for persistent repres- Antibodies and Retroviral Constructs sion of the cytotoxic CD8 program in mature CD4+ T cells. One in- Antibodies used for staining were from eBiosciences (anti-Foxp3) or BD-Phar- terpretation of this observation is that CD8-lineage differentiation Mingen (all others) and were used according to the manufacturer’s instruc- depends on factors that are available in peripheral lymphoid or- tions. The anti-Zbtb7b antiserum has been described (Wang et al., 2008). gans (such as IL-7 [Singer, 2002]) and must therefore be actively Cytokine antibodies used in T cell effector differentiation cultures were from prevented in peripheral CD4+ cells. We propose that Zbtb7b is BD-PharMingen. expressed in all CD4-lineage cells to counteract such factors pMRX (Saitoh et al., 2002) derivatives encoding mouse Runx3 (exon 1 con- and prevent the resurgence of the CD8-cytotoxic program. taining isoform) or its 204 amino-terminal residues (Runt construct) were con- structed with conventional cloning procedures (details available upon re- quest). MSCV-derived retroviral vectors for Cre, the T-bet-Engrailed fusion EXPERIMENTAL PROCEDURES protein (T-betDN), and Gata3 were previously described (Pearce et al., 2003; Zhu et al., 2004). Mice The Zbtb7b gene-targeting strategy and the generation of the Zbtb7bt allele Cell Preparation, Purification, and Flow Cytometry were described (Wang et al., 2008) and are schematically summarized in Cells were prepared from thymus, spleen, or lymph nodes (LN), counted, and Figure S1. Deletion of the Frt flanked segments was obtained by crossing assessed for surface or intracellular protein expression by immunofluores- mice heterozygous for the targeted allele with mice expressing Flpe under cence and flow cytometry as described (Liu et al., 2003), with either an LSR

(A) Contour plots of IFN-g 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 29, 876–887, December 19, 2008 ª2008 Elsevier Inc. 885 Immunity Zbtb7b Function in CD4 T Cells

II flow cytometer (BD Biosciences) or a modified (Cytek) FacsCalibur cytome- Egawa, T., Tillman, R.E., Naoe, Y., Taniuchi, I., and Littman, D.R. (2007). The ter (BD Biosciences). CD4+CD8– cells were purified from LN cells with an an- role of the Runx transcription factors in thymocyte differentiation and in ho- tibody mix including anti-CD8a and magnetic beads (CD4 Negative Isolation meostasis of naive T cells. J. Exp. Med. 204, 1945–1957. + kit, Dynal-Invitrogen 114.15D). CD8 bead cell purification was performed sim- Glimcher, L.H., Townsend, M.J., Sullivan, B.M., and Lord, G.M. (2004). Recent lo ilarly with the CD8 separation kit from Dynal. Where indicated, CD44 cells developments in the transcriptional regulation of cytolytic effector cells. Nat. + – were sorted from bead-purified CD4 CD8 cells with Facs Vantage or Facs Rev. Immunol. 4, 900–911. Aria instruments (BD Biosciences) as described (Liu and Bosselut, 2004). Hayashi, K., Natsume, W., Watanabe, T., Abe, N., Iwai, N., Okada, H., Ito, Y., Asano, M., Iwakura, Y., Habu, S., et al. (2000). Diminution of the AML1 tran- Gene and Protein Expression Analyses scription factor function causes differential effects on the fates of CD4 and Analyses of gene expression by RT-PCR were performed as described (Liu CD8 single-positive T cells. J. Immunol. 165, 6816–6824. and Bosselut, 2004) from RNA extracted with RNAqueous (Ambion) and re- He, X., He, X., Dave, V.P., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B.A., verse-transcribed with Oligo dT priming (superscript III, Invitrogen). Analyses and Kappes, D.J. (2005). The zinc finger transcription factor Th-POK regulates by qRT-PCR were performed with Taqman reagents, probes (Gata3: CD4 versus CD8 T-cell lineage commitment. Nature 433, 826–833. Mm00484683_m1; GzmB: Mm00442834_m1; Perforin: Mm00812512_m1; Hernandez-Hoyos, G., Anderson, M.K., Wang, C., Rothenberg, E.V., and T-bet: Mm00450960_m1, Eomes: Mm01351985_m1) and an ABI PRISM Alberola-Ila, J. (2003). GATA-3 expression is controlled by TCR signals and 7500HT sequence detection system, all from Applied Biosystems (Jenkinson regulates CD4/CD8 differentiation. Immunity 19, 83–94. et al., 2007). Gene-expression values are normalized to b-actin in the same sample. Analyses of Zbtb7b protein expression were performed on cells lyzed Intlekofer, A.M., Takemoto, N., Wherry, E.J., Longworth, S.A., Northrup, J.T., in 1% Triton containing buffer as previously described (Sun et al., 2005). Palanivel, V.R., Mullen, A.C., Gasink, C.R., Kaech, S.M., Miller, J.D., et al. (2005). Effector and memory CD8+ T cell fate coupled by T-bet and eomeso- dermin. Nat. Immunol. 6, 1236–1244. SUPPLEMENTAL DATA Janeway, C.A.J., and Bottomly, K. (1994). Signals and signs for lymphocyte re- Supplemental Data include ten figures and can be found with this article online sponses. Cell 76, 275–285. at http://www.immunity.com/supplemental/S1074-7613(08)00500-1. Jenkins, M.K., Khoruts, A., Ingulli, E., Mueller, D.L., McSorley, S.J., Reinhardt, R.L., Itano, A., and Pape, K.A. (2001). In vivo activation of antigen-specific CD4 T cells. Annu. Rev. Immunol. 19, 23–45. ACKNOWLEDGMENTS Jenkinson, S.R., Intlekofer, A.M., Sun, G., Feigenbaum, L., Reiner, S.L., and We thank E. Southon for ES cell recombination; G. Sanchez for mouse techni- Bosselut, R. (2007). Expression of the transcription factor cKrox in peripheral cal assistance; B. Taylor and S. Banerjee for expert cell sorting; S. Reiner, CD8 T cells reveals substantial postthymic plasticity in CD4–CD8 lineage dif- B. Paul, and J. Zhu for reagents; S.R. Jenkinson and J. Zhu for helpful discus- ferentiation. J. Exp. Med. 204, 267–272. sions and T cell activation protocols; and J. Ashwell, Y. 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