runx3 regulates integrin e/cd103 and cd4 expression … and cd4 expression during development of cd4...

Runx3 Regulates Integrin �E/CD103 and CD4 Expressionduring Development of CD4�/CD8� T Cells1

Baerbel Grueter,2* Michaela Petter,3‡ Takeshi Egawa,� Kirsten Laule-Kilian,4‡

Christine J. Aldrian,‡ Andreas Wuerch,‡ Yvonne Ludwig,5* Hidehiro Fukuyama,§

Hedda Wardemann,¶ Ralph Waldschuetz,† Tarik Moroy,† Ichiro Taniuchi,6� Viktor Steimle,7‡

Dan R. Littman,� and Marc Ehlers8*‡§

During thymic T cell development, immature CD4�CD8� double-positive (DP) thymocytes develop either into CD4�CD8� Th cells orCD4�CD8� CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The tran-scription factor Runx3/AML-2 Runx, rust dominant factor; AML, acute myeloid leukemia is expressed specifically during the devel-opment of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction ofCD8 SP T cells and concomitant accumulation of CD4�CD8� T cells, which cannot down-regulate CD4 expression in the thymus andperiphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identifiedintegrin �E/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only torepress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression ofRunx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and anincreased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics ofregulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8SP T cells during thymocyte development. The Journal of Immunology, 2005, 175: 1694–1705.

D uring T cell development in the thymus CD4�CD8�

double-positive (DP)9 thymocytes undergo positive andnegative selection depending on the ability of their TCR

to interact with thymic stromal cells presenting self-peptides

bound to MHC molecules (1–4). Further differentiation leads tothe development of either mature CD4 single-positive (SP) Thcells or CD8 SP CTLs, which have a CD69�/TCRhigh phenotype.Analysis of the genes involved in CD4 and CD8 gene regulationand identification of the genes that are differentially expressed dur-ing thymocyte differentiation may provide insight into the mech-anisms governing T cell development and help to identify primaryfactors inducing the fate of CD4 or CD8 SP T cells (5, 6).

The transcription factor Runx3 (runt domain factor; acute my-eloid leukemia-2/core binding factor-�3/polyoma enhancer bind-ing protein-2�C) is expressed during the development of CD8 SPthymocytes, but not of CD4 SP thymocytes, and is essential forsilencing of CD4 expression (7–9). Knockout or knockdown ofRunx3 results in a reduction of CD8� T cells and a concomitantaccumulation of mature CD4�/CD8� T cells, which cannot down-regulate CD4 expression in the thymus and periphery (7–9). TheseCD4�/CD8� T cells show impaired response to TCR stimulationin the periphery, suggesting that Runx3 also regulates other genesduring the development of CD8 SP T cells (7, 8)

The Runx family of transcription factors consists of three mem-bers that play important roles in cellular differentiation, prolifera-tion, and the development of autoimmunity and cancer (10–18).Runx3 is involved in T and B cell differentiation, dendritic cellfunction, the development of dorsal root ganglia neurons, chon-drocyte maturation, apoptosis, and gastric cancer (7–9, 11–14, 19–23). In some of these systems Runx3 acts in concert with theTGF-� signaling pathway (19, 21, 22).

Runx proteins bind to DNA by forming heterodimers with thecommon � subunit core binding factor-�/polyoma enhancer bind-ing protein-2� via the conserved runt domain (10, 24). In T cells,Runx factors bind to the silencer element in the first intron ofthe CD4 gene locus (25–28) where Runx binding sites have been

*Institute of Molecular Biology (Cancer Research) and †Institute of Cell Biology(Cancer Research), University of Essen, Medical School, Essen, Germany; ‡HansSpemann Laboratories, Max Planck Institute for Immunology, Freiburg, Germany;§Laboratory of Molecular Genetics and Immunology and ¶Laboratory of MolecularImmunology, The Rockefeller University, New York, NY 10021; and �HowardHughes Medical Institute and Skirball Institute of Biomolecular Medicine, New YorkUniversity School of Medicine, New York, NY 10016

Received for publication November 16, 2004. Accepted for publication May 6, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Max Planck Gesellschaft zur Forderung derWissenschaften.2 Current address: Institute of Biological Information Processing 1 (IBI-1), ResearchCenter Juelich, D-52425 Juelich, Germany.3 Current address: Department of Molecular Medicine, Bernhard Nocht Institute forTropical Medicine, Bernhard Nocht Strasse 74, D-20359 Hamburg, Germany.4 Current address: Department of Internal Medicine, University Hospital Basel, Pe-tersgraben 4, CH-4031 Basel, Switzerland.5 Current address: Institute of Physiology II, University of Muenster, Medical School,Robert Koch Strasse 27a, D-48149 Muenster, Germany.6 Current address: Laboratory for Transcriptional Regulation, RIKEN Research Cen-ter for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.7 Current address: Departement de Biologie, Universite de Sherbrooke, 2500 boule-vard Universite, Sherbrooke, Quebec, Canada J1K 2R1.8 Address correspondence and reprint requests to Dr. Marc Ehlers, Institute of Mo-lecular Genetics and Immunology, The Rockefeller University, 1230 York Avenue,Box 98, New York, NY 10021. E-mail address: [email protected] Abbreviations used in this paper: DP, double positive; DN, double negative; EGFP,enhanced GFP; MHC-II, MHC class II; MOG, myelin oligodendrocyte glycoprotein;P, promoter; sil, silencer; SP, single positive; spl, splenocytes; tg, transgenic; thy,thymocytes; wt, wild type.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

identified (7, 9, 29). Runx1 is mainly responsible for reversibleCD4 repression in DN thymocytes, and Runx3 silences CD4 ex-pression during the development of CD8 SP T cells (7). At thistime, Runx3 is the only known transcription factor that binds to theCD4 silencer element and at the same time is expressed duringdevelopment of CD8, but not CD4, SP thymocytes (7, 9, 30–34).

In this study we have investigated the potential role of Runx3during CD4 silencing and T cell development and have identifiedintegrin �E/CD103, which is expressed on CD8 SP thymocytesand interacts with E-cadherin on epithelial cells, as another poten-tial CD8 lineage-specific target gene of Runx3 (35). We show thatby regulating both CD4 silencing and CD103 expression, Runx3contributes to the phenotype of CD8 SP thymocytes.

Materials and MethodsCD4E/P-Runx3 transgenic (tg) mice and molecular analyses

The murine Runx3 cDNA (accession no. NM019732) corresponding to theform transcribed from the proximal promoter (exons 2–6) (36) and themurine CD4 proximal enhancer and promoter/start of exon 1 were gener-ated by PCR from genomic DNA (9, 28). The CMV enhancer/promoterand the d2EGFP cassette in the d2EGFP-N1 plasmid (BD Clontech) werereplaced with these CD4 regulatory elements and Runx3 cDNA, respec-tively. All tg mice used in this study were generated from ova obtainedfrom an F1(C57BL/6 � C3H) background as previously described (37),and tg lines were kept by backcrossing to B6 animals. The tg mice analyzedin this study represent the second (see Figs. 1 and 2A) or seventh (see Figs.2B, 3, 4, and 6–8) generation obtained upon backcrossing the founderanimals. The genotype of CD4E/P-Runx3 tg mice was determined by PCRand Southern blot analyses. The following primers were used for PCR ontail tip-derived DNA: murine Runx3 exons 2–4: forward, 5�-acgctgccg-gtcgccttca; reverse, 5�-gttcccggggtccatccaca. For Southern blot analyses,the DNA blotting procedures were performed as described previously (38).A 492-bp XbaI-XhoI fragment of the murine CD4 promoter/start of exon 1was used as a probe on tail tip-derived DNA digested with BamHI.

Other mice, cell lines, and cell culture

CD4�/�, OT-I, and OT-II mice on the C57BL/6 background were pur-chased from The Jackson Laboratory (39–41). CD4sil�/� mice were de-scribed previously and backcrossed seven times to C57BL/6 mice (28, 29).TCR tg myelin oligodendrocyte glycoprotein (MOG) mice on the C57BL/6background were described recently (42). OT-I, OT-II, MOG, andCD4sil�/� mice were crossed to Runx3 tg mice (line 3) that had previouslybeen backcrossed six times to C57BL/6 mice. TK-1-5 is a subclone of themurine CD8� lymphoma T cell line TK-1, and RLM-11-1 is a subclone ofthe murine CD4 SP thymoma T cell line RLM-11 (9). Both lines are CD3�

and TCRhigh. TK-1-5 and RLM-11-1 were cultured in RPMI 1640 mediumsupplemented with 10% FCS, 150 �g/ml streptomycin, 150 U/ml penicil-lin, and 3 mM L-glutamine. To analyze the effect of TGF-� on CD103expression, the cells were cultured for 48 h with 5 ng/ml human TGF-�2(Roche).

Analyses of tg mice and cell lines

Total thymocytes, splenocytes, or T cell lines were stained with the fol-lowing Abs (BD Biosciences): CyChrome-anti-CD4, PerCP-anti-CD4,PerCP-Cy5.5-anti-CD4, PE-anti-CD8�, PE-Cy7-anti-CD8�, FITC-anti-CD69, PE-anti-CD69, allophycocyanin-anti-TCR�, FITC-anti-CD103, PE-anti-CD103, biotin-anti-CD103 with PE-streptavidin, FITC-anti-differentTCRV� subtypes, PE-anti-different TCRV� subtypes, FITC-anti-B220,PE-anti-NK1.1, PE-anti-CD25, FITC-anti-CD24, PE-anti-CD5, FITC-anti-CD44, PE-anti-CD45RB, and FITC-anti-CD62L. Stained cells wereanalyzed by flow cytometry (FACSCalibur or BD-LSRII; BDBiosciences). For the isolation of CD69�/TCRint cells, CD69� cells werefirst enriched from total thymocytes by magnetic sorting (AutoMACS;Miltenyi Biotec) and then further sorted by flow cytometry (MoFlo; DakoCytomation) to obtain CD69�/TCRint thymocytes (9). We used wild-type(wt) littermates as controls in each experiment.

Western blot

Protein extracts of thymocytes (20 �g), cell lines (20 �g), and FACS (SEVantage; BD Biosciences) purified thymic or splenic T cell subpopulationsof mice (3 �g) were prepared, and Western blotting was conducted aspreviously described (9). The wt littermates of the tg mice were used as

controls. As primary Abs we used a polyclonal rabbit antiserum specific forthe conserved 15 C-terminal aa of murine Runx1 (NMPPARLEE-AVWRPY) (43), an antiserum specific for 270 aa of the C terminus ofhuman RUNX3 (13), or a mouse mAb against the housekeeping gene �-ac-tin. As secondary Abs we used peroxidase- or alkaline phosphatase-cou-pled rabbit- or murine-specific antisera (Amersham Biosciences).

Plasmids, stable reporter gene assay, and transfection

Murine Runx3 cDNA corresponding to the form transcribed from the prox-imal promoter (exons 2–6) (36) was generated by PCR and cloned into acDNA expression vector (CMV-Runx3). The EGFP-Runx3 fusion con-struct (CMV-EGFP-Runx3 (exons 2–6)) was generated by cloning theRunx3 (exons 2–6) cDNA in frame with the EGFP gene in the EGFP-C1-expressing plasmid (BD Clontech). Reporter constructs are based on thed2EGFP-N1 plasmid (BD Clontech) containing a destabilized version ofEGFP (d2EGFP) as a reporter gene. The murine CD4 proximal enhancer(E), promoter/start of exon 1 (P) and silencer (sil; 434-bp SacI-XbaI frag-ment) (9, 27, 28) were generated by PCR from genomic DNA (9). The twoplasmids, CD4E/P-d2EGFP and CD4E/P-d2EGFP-sil, were described pre-viously (9). The plasmids were transfected by electroporation into theRLM-11-1 thymoma T cell line under the following conditions: 4 � 106

cells in 300 �l of RPMI 1640 medium with 25 �M HEPES (pH 7.0), 5 �gof plasmids, 1500 V, for 200 �s (BTX; Q-Biogene). Cells were analyzedby flow cytometry for d2EGFP expression 24 h after transfection.

Morpholino antisense oligonucleotides and IL-7 induced ex vivothymocyte development

Transfection and cell culture were conducted as previously described (9)with the following modifications. Sorted CD69�/TCRint thymocytes (3 �106) were transfected by electroporation with FITC-coupled morpholinoantisense oligonucleotides under the following conditions: 3 � 106 cells in300 �l of RPMI 1640 with 25 �M HEPES (pH 7.0), 10–12 nmol ofmorpholino, 1500 V, for 200 �sec (BTX; Q-Biogene), and cultured incomplete IMDM. The following morpholino antisense oligonucleotides(Gene Tools) were used: mo-Runx3–1, 5�-tgctcgggtctacgggaatacgcat; and

FIGURE 1. Runx3 gene locus, tg CD4E/P-Runx3 construct, and Runx3expression in total thymocytes of tg mice. A, Schematic presentation of theRunx3 gene locus (adapted from Ref. 36). Numbered exons are boxed;coding sequences are shown in black (SA, splice acceptor site). B, Sche-matic representation of the CD4E/P-Runx3 tg construct. Enh., CD4 prox-imal enhancer; P, CD4 promoter/start of exon 1; Runx3, murine Runx3cDNA (exons 2–6). C, Runx1 and Runx3 protein expression in thymocytesof Runx3 tg mice. Protein extracts of wt and tg (lines 3 and 4) mice wereanalyzed by Western immunoblotting with an antiserum specific for the 15C-terminal aa of murine Runx1. This antiserum also recognizes murineRunx3. The upper arrow indicates the position of the Runx1 proteins, andthe lower arrow indicates the position of the Runx3 proteins (upper panel).The lower panel shows a reprobing of the blot with antiserum against�-actin.

1695The Journal of Immunology

control (�-globin), 5�-cctcttacctcagttacaatttata (9). The Runx3 oligonucle-otide is complementary to a sequence in exon 2 of Runx3. After 3 h, 3ng/ml mouse rIL-7 (R&D Systems) were added. After overnight incubationthe cells were washed twice with PBS (37°C) and treated for 10 min with100 �g/ml Pronase (Calbiochem) and 100 �g/ml DNase (Roche) in 1 mlof PBS at 37°C. The reaction was stopped with 100 �l of FCS. The cellswere further cultured with IL-7 for 2 days, stained with Abs, and analyzedby flow cytometry. After electroporation, overnight incubation, and sub-sequent Pronase treatment, including repeated washing and centrifugationsteps, �4–8% of the cells were recovered as live cells. During furthercultivation with IL-7, the cell numbers stayed relatively constant.

Analysis of CD103 expression on Runx3�/� lymphocytes

Rag2 deficient mice reconstituted with Runx3�/� or Runx3�/� fetal livercells were generated as previously described (7, 14). Those mice were bled

by cutting tail tips, and erythrocytes were removed by ammonium chloridelysis. Cells were analyzed by flow cytometry.

ResultsGeneration of CD4E/P-Runx3 tg mice

To investigate the role of Runx3 during thymocyte development,we generated tg mice expressing Runx3 (exons 2–6) under thecontrol of the murine CD4 proximal enhancer and promoter (Fig.1, A and B). This enhancer/promoter combination becomes activeat the DN 2 stage (CD4�/CD8�/CD25�/CD44�) and remains ac-tive throughout the development of both CD4 and CD8 SP T cells(28). Therefore, in the tg mice, Runx3 is not only expressed

FIGURE 2. Tg overexpression of Runx3 strongly re-duces expression of CD4 at the CD4/CD8 DP stage andthe amount of mature CD4 SP T cells and increases theamount of mature CD8 SP T cells. A, Thymocytes of wtand tg mice (lines 3 and 4) were stained with Absagainst CD4, CD8�, CD69, and TCR� (panels 1–6).CD69�TCR�int cells (upper left gate in panels 4–6) orCD69�TCR�high cells (lower right gate in panels 4–6)were gated and analyzed in the CD4/CD8 diagram (pan-els 7–12). The percentage and number of the cells in thequadrants are shown. B, Absolute numbers of thymocytesand splenocytes of tg mice (line 3) and wt mice (litter-mates) were counted (upper left panel). Absolute numbersof TCR�highCD69�CD4�CD8� and TCR�highCD69�

CD4�CD8� thymocytes (lower left panel) and CD4� andCD8� splenocytes (upper right panel) are shown.

1696 Runx3 REGULATES CD103 AND CD4 EXPRESSION

throughout development of CD8 SP thymocytes, but also duringdevelopment of DP and CD4 SP cells.

We established two independent tg lines (lines 3 and 4). Both tglines overexpressed Runx3 protein in total thymocytes (Fig. 1C).With an antiserum specific for the C terminus of murine Runx1that also interacts with murine Runx3 (9, 43, 44), we detected anincrease in Runx3 protein expression in total thymocytes of tgmice compared with wt mice (Fig. 1C). At the same time, theamount of Runx1 protein was reduced, suggesting that tg overex-pression of Runx3 down-regulates Runx1 expression or destabi-lizes Runx1 protein (Fig. 1C).

Overexpression of Runx3 strongly reduces expression of CD4during development of CD4/CD8 DP cells and influences theratio of mature CD4 and CD8 SP thymocytes

In Runx3 tg mice (lines 3 and 4), DP thymocytes showed a strongreduction of CD4 expression (Fig. 2A, panels 1–3). Thus, exoge-

nous Runx3 repressed CD4 expression during the development ofDP cells. Furthermore, the percentage of CD4�CD8� cells wasstrongly reduced (Fig. 2A, panels 1–3). The percentage of posi-tively selected immature (CD69�/TCRint and CD69�/TCRhigh)thymocytes of tg mice was also reduced (Fig. 2A, panels 4–6).These immature cells showed a decreased CD4 expression and anincrease in CD8 expression (Fig. 2A, panels 7–9, and data notshown). Gating on mature thymocytes (CD69�/TCRhigh), wefound that the percentage and number of mature CD4 SP cellswere also reduced 2- to 3-fold (Fig. 2A, panels 10 –12, and B).At the same time, the percentage and number of mature CD8 SPthymocytes were increased 1.5- to 2-fold (Fig. 2A, panels 10 –12, and B). The expression level of CD8� in mature CD8 SPthymocytes was slightly increased in Runx3 tg mice (Fig. 2A,panels 10 –12).

Tg mice also had a 2-fold increase in the number of CD4�/CD8� DN thymocytes (Fig. 2, panels 1–3) mainly with an immature

FIGURE 3. Overexpression ofRunx3 cannot force MHC-II-re-stricted OT-II or MOG thymocytes todevelop to mature CD8 SP cells. A,Thymocytes (panels 1–8) andsplenocytes (panels 9 and 10) ofOT-II and Runx3 tg � OT-II micewere stained with Abs against CD4,CD8�, CD69, TCR�, TCRV�2 andTCR�5. Mature CD69�TCR�high

thymocytes were analyzed in theCD4/CD8 diagram (panels 3 and 4).TCR tg TCRV�2high/TCRV�5high

thymocytes were gated (gates in pan-els 5 and 6) and analyzed in the CD4/CD8 diagram (panels 7 and 8). CD8�

splenocytes were analyzed in theTCRV�2high/TCRV�5high diagram(panels 9 and 10). B, Thymocytes(panels 1–8) and splenocytes (panels9 and 10) of MOG and Runx3 tg �MOG mice were stained with Absagainst CD4, CD8�, TCRV�3.2, andTCR�11. In MOG and Runx3 tg �MOG mice, �98% of TCR�high thy-mocytes also highly expressedTCRV�11 (data not shown).TCRV�11high thymocytes (panels 3and 4) and TCR tg TCRV�3.2high/TCRV�11high thymocytes (panels 7and 8) were gated (gates in panels 5and 6) and analyzed in the CD4/CD8diagram. CD8� splenocytes were an-alyzed in the TCRV�3.2high/TCRV�11high diagram (panels 9 and10). The percentage and number ofthe cells in the quadrants are shown.


CD69�/TCRlo/int/CD25�/CD44� DN4 phenotype (data notshown). TCRhigh cells were almost absent from the CD4/CD8 DNsubpopulation (data not shown). As shown in Fig. 2B, there was nosignificant difference in absolute thymic and splenic cell numbersin wt and tg mice.

Thus, overexpression of Runx3 could repress CD4 expression inmost DP cells during the differentiation from DN cells and resultedin a reduced number of mature CD4 SP thymocytes and an in-creased number of CD8 SP thymocytes. Comparable results wereobserved in Runx3 tg mice backcrossed two or seven times to theC57BL/6 background.

Overexpression of Runx3 cannot redirect specific MHC class II(MHC-II)-restricted TCR tg thymocytes toward the CD8 lineage

Recently, it was suggested that the CD8 population in CD4-defi-cient mice is heavily contaminated with MHC- II-restricted T cells(45). The smaller number of mature CD4 SP T cells and the highernumber of mature CD8 SP T cells in Runx3 tg mice may be dueto the low expression of CD4 on DP thymocytes, which may forceMHC-II-restricted thymocytes to the CD8 lineage. To determinewhether Runx3 overexpression can force MHC-II-restricted thy-mocytes to the CD8 lineage, we crossed Runx3 tg mice with OT-IImice expressing an MHC-II-restricted TCR specific for a chickenOVA peptide (42). The tg TCR consists of a TCRV�2 and aTCRV�5 chain. Tg overexpression of Runx3 in OT-II mice led toa strong decrease in the numbers of CD4 SP thymocytes (Fig. 3A,panels 1 and 2) and mature TCR tg (TCRV�2high/TCRV�5high)thymocytes (Fig. 3A, panels 5 and 6). Although the number ofmature CD8 SP thymocytes (Fig. 3A, panels 3 and 4) increased,there were few mature TCR tg (TCRV�2high/TCRV�5high) cells inthe thymic and splenic CD8 SP T cell population (Fig. 3A, panels7–10). We obtained comparable results by crossing Runx3 tg micewith MOG mice expressing an MHC-II-restricted TCR specific fora MOG. This tg TCR consists of a TCRV�2 and a TCRV�5 chain(Fig. 3B) (42). Thus, Runx3 overexpression could not redirectMHC-II/CD4-restricted thymocytes with a specific TCRV�� com-bination (OT-II or MOG) to develop to mature CD8 SP thymo-cytes. It is likely that the slight increase in CD8 SP thymocytes inRunx3 tg mice is mainly caused by an expansion of MHC-I-re-stricted cells.

To characterize the remaining mature CD4 SP T cells in Runx3tg mice, we analyzed whether specific TCRV� or TCRV� rear-rangements were enriched or decreased in this population. We didnot observe any obvious differences in TCR use between CD4� orCD8� splenic T cells of wt and tg mice (data not shown).

To further analyze the influence of overexpressed Runx3 onMHC-I-restricted thymocytes, we crossed Runx3 tg mice withOT-I mice expressing an MHC-I-restricted TCR specific for achicken OVA peptide (41). In this study the number of matureCD8 SP thymocytes was slightly increased (data not shown).

Reduction of mature CD4 SP thymocytes in Runx3 tg mice iscaused by silencing of CD4 expression during development ofDP thymocytes

To investigate whether the decreased number of CD4� thymocytesin Runx3 tg mice is triggered by a CD4 silencer-dependent or-independent mechanism, we crossed Runx3 tg mice to CD4sil�/�

mice, which have a deletion of the CD4 silencer element on oneallele and therefore lack CD4�CD8� T cells (28, 29). Deletion ofone allele of the CD4 silencer in Runx3 tg mice did not affect theabsolute number of thymocytes compared with Runx3 tg andCD4sil�/� mice (Fig. 4, panels 1–3, and data not shown). CD4expression in DP thymocytes was restored in Runx3 tg �CD4sil�/� mice, indicating that ectopic expression of Runx3causes CD4 down-regulation throughout development of DP thy-mocytes by acting on the CD4 silencer in Runx3 tg mice (Fig. 4,panels 1–3). Absolute numbers of mature CD4 SP thymocyteswere also largely restored in Runx3 tg � CD4sil�/� mice com-pared with Runx3 tg mice (Fig. 4, panels 4–6). These results in-dicate that the reduction in mature CD4 SP thymocytes in Runx3tg mice is due to the reduced level of CD4 expression in DP thy-mocytes or transitional thymocytes after positive selection and isnot a consequence of Runx3 influence on CD4 lineagedetermination.

Runx3 induces silencing in a CD4 SP thymoma cell line

We also analyzed the potential of Runx3 to regulate the CD4 si-lencer element in the CD4 SP thymoma cell line RLM-11-1, whichis likely to be terminally differentiated. In contrast to the CD8 SP

FIGURE 4. Decreased number of mature CD4 SPthymocytes in Runx3 tg mice is caused by CD4 silenc-ing. Thymocytes (panels 1–3) of Runx3 tg, CD4sil�/�,and Runx3 tg � CD4sil�/� mice were stained with Absagainst CD4, CD8�, CD69, and TCR�. TCRhigh/CD69�

thymocytes were gated and analyzed in the CD4/CD8diagram (panels 4–6). The percentage and number ofthe cells in the quadrants are shown. Bold numbers in-dicate the total percentage and absolute number ofTCR�highCD69�CD4�CD8� thymocytes.


T cell line TK-1-5, CD4 SP RLM-11-1 cells do not express en-dogenous Runx3 (Fig. 5A). Transfection of a Runx3-expressingplasmid (CMV-Runx3, exons 2–6) into RLM-11-1 cells did notdown-regulate CD4 expression (data not shown). However, exog-enous Runx3 displayed the potential to regulate the CD4 silencerelement in a reporter assay in RLM-11-1 cells (Fig. 5, B and C).We have investigated Runx3-mediated repression of the reportergene d2EGFP under the control of the CD4 proximal enhancer,promoter, and CD4 silencer element (Fig. 5, B and C) (9). Exog-enous Runx3 repressed d2EGFP expression (only 2% of the cellsexpressed d2EGFP) if the CD4 silencer element (CD4E/P-d2EGFP-sil) was present (Fig. 5C), whereas in the absence of thesilencer element (CD4E/P-d2EGFP), d2EGFP expression was notreduced, and 10% of the cells expressed d2EGFP. Without exog-enous Runx3 expression, no silencing effect was obvious. Thus,although endogenous CD4 expression is not reversible, exogenousRunx3 can regulate the CD4 silencer element in a reporter assay inthe CD4 SP thymoma T cell line RLM-11-1.

Tg Runx3 is also expressed in the remaining CD4 SP thymocytes

It is possible that thymocytes that strongly overexpress Runx3 loseCD4 expression completely, whereas cells with variegated lowerexpression of tg Runx3 can up-regulate CD4 expression and de-velop into mature CD4 SP T cells. To address this question, wesorted individual thymic and splenic T cell subpopulations andanalyzed Runx3 protein expression (Fig. 6). In agreement with ourpreviously published observation (9), Runx3 in wt mice is pre-dominantly expressed in TCRhigh/CD8 SP thymocytes (Fig. 6). Incontrast, tg Runx3 was not only expressed in TCRhigh/CD8 SPthymocytes, but also at comparable levels in immature TCR�int/CD4�/CD8� thymocytes and in TCR�int/CD4�/CD8� DPthymocytes and TCR�high/CD4 SP thymocytes. In the spleen, sim-ilar amounts of Runx3 were expressed in CD4� and CD8� T cellsof tg mice (Fig. 6). Thus, in Runx3 tg mice, a subpopulation ofMHC-II restricted thymocytes can still develop into mature CD4SP T cells even in the presence of Runx3 protein.

Runx1 in wt mice was expressed in DP thymocytes, mature CD4SP thymocytes, and, to a lesser extent, mature CD8 SP thymocytes(Fig. 6). In tg mice, the expression of Runx1 was decreased in allthymic subpopulations as well as in mature splenic CD4� T cells(Fig. 6).

Overexpression of Runx3 inhibits the development of NKT cells

By analyzing the remaining mature CD4 SP T cells in Runx3 tgmice, we identified that the number of NKT cells (NK1.1

�

/CD24low/TCRalmost high) with a CD4�CD8� or CD4�CD8� phe-notype was reduced in the thymus and spleen of Runx3 tg mice(Fig. 7 and data not shown). The percentage of CD24low/TCRalmost

high thymocytes was also reduced, indicating that overexpressionof Runx3 reduced not only NK1.1 expression (Fig. 7, panels 4–6).This phenomenon was independent of CD4 expression becauseCD4�/� mice still have NKT cells (Fig. 7).

Expression of integrin �E/CD103 is regulated by Runx3 and TGF-�

The accumulated CD4�/CD8� T cells that appear in the peripheryof Runx3�/� mice show functional deficits, suggesting that Runx3regulates additional genes during the development of CD8 SP Tcells (7, 8). Integrin �E/CD103, which mediates interaction withE-cadherin on epithelial cells, has been shown to be expressed on�80% of mature CD8 SP thymocytes, but on only 3–4% of CD4SP thymocytes (46, 47). Therefore we investigated whether Runx3plays a role in the regulation of CD103 expression.

FIGURE 5. Exogenous Runx3 expression in the CD4 SP thymoma Tcell line RLM-11-1 induces CD4 silencing in a reporter gene assay. A,Runx3 protein is expressed in the CD8 SP T cell line TK-1-5, but not in theCD4 SP thymoma T cell line RLM-11-1. Protein extracts from both celllines were analyzed by Western immunoblotting with an antiserum specificfor human Runx3, which also recognizes murine Runx3. The arrow indi-cates the position of Runx3 proteins (upper panel). The lower panel showsreprobing with Ab against �-actin. B, Schematic representation of the dif-ferent reporter plasmids used in cotransfection experiments. E, CD4 prox-imal enhancer; P, CD4 promoter; EGFP, reporter gene d2EGFP; sil, CD4silencer element; Runx3 (exon2–6), additional plasmid expressing theRunx3 protein (exons 2–6). C, d2EGFP reporter gene analyses were per-formed in the murine CD4 SP T cell line RLM-11-1 transfected as indi-cated in B. The expression of d2EGFP was measured by flow cytometry.The analysis gate was set so that untransfected cells (contr.) showed �1%positive cells. The average expression and SE of three independent trans-fections for each construct are shown.

FIGURE 6. Runx3 protein is also expressed in the remaining tg ma-ture CD4 SP thymocytes. A, Runx1 and Runx3 protein expression inFACS-sorted thymic and splenic subpopulations of wt and Runx3 tgmice (line 3) were analyzed by Western immunoblotting with an antiserumspecific for the 15 C-terminal aa of murine Runx1. This antiserum also rec-ognizes murine Runx3. The upper arrow indicates the position of the Runx1proteins, and the lower arrow shows the position of the Runx3 proteins (upperpanel). The lower panel shows a reprobing of the blot with antiserum against�-actin. CD4 SP, TCRhighCD4�CD8�; CD8 SP, TCRhighCD4�CD8�; DP,TCRintCD4�CD8�; CD8�“DP”, TCRintCD4�CD8�.


Recently, it was shown that TGF-� could induce CD103 tran-scription and protein expression in the CD8 SP T cell line TK-1,which expresses endogenous Runx3 (Fig. 5A) (48). To investigatethe potential role of Runx3 in the induction of CD103 expression,we used the CD4 SP T cell line RLM-11-1 (Fig. 5A). Neithertreatment with TGF-� alone nor expression of an EGFP-Runx3fusion protein alone was sufficient to induce CD103 expression onRLM-11-1 cells. However, TGF-� induced CD103 expression at48 h in 30–35% of EGFP-Runx3-positive cells. (Fig. 8A). Thus,the combination of Runx3 expression and TGF-� stimulationcould induce CD103 expression on a CD4 SP thymoma T cell line.

Tg overexpression of Runx3 induces integrin �E/CD103expression in a subpopulation of mature CD4 SP thymocytesand splenocytes with characteristics of regulatory cells

To investigate Runx3-dependent regulation of CD103 expressionin vivo, we analyzed CD103 expression patterns on T cells from wtand Runx3 tg mice. In wt mice, CD103 was expressed on �80%of mature CD8 SP thymocytes and 40–50% of splenic CD8� Tcells, but on only 1–4% of thymic and splenic CD4 SP T cells(Fig. 8B).

In contrast, in Runx3 tg mice, CD103 protein was up-regulatedon TCR�highCD4 SP thymocytes (7%) and CD4� spleen cells(12–18%; Fig. 8B). The CD103 up-regulation on CD4 SP T cellswas preferentially observed on CD25� cells, presumably regula-tory T cells (Fig. 8C). In Runx3 tg mice, the CD103�CD25�CD4� and also the CD103�CD25�CD4� splenocytes showedlower expression patterns of CD45RB and CD62L compared withCD103�CD25�CD4� cells, which is also characteristic of regula-tory T cells (Fig. 8D) (49). Thus, in vivo overexpression of Runx3during T cell development was capable of inducing CD103 expres-sion on mature CD4 SP T cells with characteristics of regulatory Tcells.

Absence of Runx3 expression strongly reduces expression ofCD103 on CD8 SP T cells

We previously established an IL-7-dependent ex vivo thymocytedevelopment culture system that allows the induction of differen-tiation of purified primary CD4�CD8�CD69�TCRint positivelyselected thymocytes into CD4 and CD8 SP T cells (9, 50). Specificknockdown of Runx3 with FITC-coupled morpholino antisenseoligonucleotides in this culture system leads to an accumulation ofCD4�/CD8� thymocytes instead of mature CD8 SP thymocytes.This is explained by the loss of CD4 silencing (Fig. 9A) (9).

In this study we have used this system to analyze the role ofRunx3 in CD103 expression on CD8 SP thymocytes. The inhibi-tion of Runx3 expression resulted in a reduced frequency ofCD103-expressing CD8 � thymocytes (Fig. 9A). The role ofRunx3 in CD103 expression was also analyzed in PBMCof RAG2�/� mice reconstituted with fetal liver cells of Runx3�/�

or Runx3�/� animals (Fig. 9B). Only 2–4% of B220�TCR��

CD8� blood cells from RAG2�/� mice reconstituted withRunx3�/� fetal liver cells expressed CD103, in contrast to 12–26%of B220�TCR��/CD8� cells from mice reconstituted with fetalliver cells from Runx3�/� mice. Thus, knockdown or knockout ofRunx3 during T cell development significantly reduced CD103expression on CD8 SP T cells. In contrast, tg overexpression ofRunx3 up-regulated CD103 expression on CD4 SP T cells withcharacteristics of regulatory T cells.

DiscussionTo investigate the roles of Runx3 during T cell development, wegenerated tg mice expressing Runx3 (exons 2–6) under the controlof the CD4 proximal enhancer and promoter (9, 29). In Runx3 tgmice, most DP cells had reduced CD4 expression. These resultsare in agreement with recent data showing that retroviral or tg

FIGURE 7. Overexpression of Runx3 inhibits thedevelopment of NKT cells. Thymocytes of wt, Runx3 tg(line 3), and CD4�/� mice were stained with Absagainst TCR�, NK1.1, CD24, and CD4 (panels 1–6).CD24

low/TCR�almost high thymocytes were gated (gate in

panels 4–6) and analyzed in the CD4/NK1.1 diagram(panels 7–9). The percentage and number of cells in thequadrants are shown.


overexpression of Runx3 during thymocyte development stronglyreduces CD4 expression (51, 52). Also, retroviral overexpressionof Runx1 reduces CD4 expression during thymocyte development(51). It is likely that overexpressed Runx3 or Runx1 induces ex-tended CD4 silencer activity and delayed CD4 expression duringdifferentiation from DN to DP cells. However, we cannot excludethat strong overexpression of Runx3 or Runx1 reinduces CD4 re-pression in DP cells. In this study we have shown that Runx3overexpression reduced the amount of Runx1 protein in DP andmature SP thymic subpopulations. In wt mice, Runx1 protein isexpressed at lower level in Runx3-expressing CD8 SP thymocytescompared with CD4 SP thymocytes; it is thus possible that Runx3plays a role in down-regulation of Runx1 expression. In this con-text, it was described that EBV-transfected B cells up-regulate

Runx3 expression, thereby down-regulating Runx1 expression viabinding to the Runx1 promoter (53, 54).

The Runx3 tg mice had also a decreased number of mature CD4 SPthymocytes. By crossing Runx3 tg mice with CD4sil�/� mice, weshowed that reduced CD4 expression during thymocyte development andthe decreased number of mature CD4 SP thymocytes in Runx3 tg miceare caused by Runx3-dependent silencing of CD4 expression during thy-mocyte development. CD4 expression and absolute numbers of matureCD4 SP thymocytes were recovered in Runx3 tg � CD4sil�/� micecompared with Runx3 tg mice, indicating that Runx3 overexpressiondoes not affect the development of CD4 SP thymocytes if CD4 is ex-pressed at normal levels in DP cells.

It is possible that variegated transgene expression caused dif-ferent levels of CD4 expression during the development of DP

FIGURE 8. In vivo overexpression of Runx3 induces expression of CD103 on CD4 SP T cells with characteristics of regulatory cells. A, Runx3 andTGF-�, but neither alone, induce CD103 expression on the CD4 SP thymoma T cell line RLM-11-1 (panels 1–4). The CD4 SP T cell line RLM-11-1 wasstable (panels 3 and 4), transfected with an EGFP-Runx3 expression plasmid (CMV-EGFP-Runx3; exons 2–6), and cultured with or without 5 ng/mlTGF-�. CD103 expression was analyzed 48 h after transfection. One of three independent experiments is shown. B, Runx3 overexpression induces CD103expression on a subpopulation of mature CD4 SP thymocytes and splenocytes. Thymocytes and splenocytes of wt and tg mice (line 3) were stained withAbs against CD4, CD8�, TCR-�, and CD103. CD4 or CD8 SP thymocytes (panels 1 and 2) and CD4� and CD8� splenocytes (panels 3 and 4) were gatedand analyzed in a CD103 histogram. The percentage of CD103-positive cells is shown. One experiment of several analyses for both tg lines is shown. Cand D, CD103 expression is up-regulated on T cells with characteristics of regulatory T cells in Runx3 tg mice. Thymocytes were stained with Abs againstTCR-�, CD25, and CD103, and splenocytes were stained with Abs against CD4, CD25, CD103, and CD45RB or CD62L. TCR�high thymocytes (C, panels1 and 2), and individual subpopulations of CD4� splenocytes (C, panels 3 and 4; D, panels 1–10) were gated and analyzed. The percentage of the cellsin the quadrants is shown.


thymocytes. Our data show that in Runx3 tg mice, the amount ofRunx3 protein in the remaining mature CD4 SP thymic subpopu-lation is comparable to the amount in mature CD8 SP thymocytes.In Runx3 tg mice, DP cells with normal or minimally reduced CD4expression may be selected into CD4 SP thymocytes in the pres-ence of tg Runx3 protein. Another explanation for the remainingCD4 expression in a subpopulation of DP thymocytes and devel-oping CD4 lineage cells in Runx3 tg mice could be that there aredifferent subpopulations of MHC-II-restricted cells, one of whichis sensitive to Runx3-mediated shut-off of CD4 and another thatmay be resistant.

However, after differentiation to the CD4 lineage, it appears thatRunx3 can no longer repress CD4 expression. The expression levelof CD4 in mature CD4 SP thymocytes was comparable in wt andRunx3 tg mice (Fig. 2A, panels 10–12). Overexpression of Runx3in the CD4 SP RLM-11-1 thymoma cell line lacking endogenousRunx3 induced CD4 silencer-mediated repression of reporter geneexpression. However, endogenous CD4 expression was not down-regulated (data not shown). Our results indicate that in fullydifferentiated CD4� T cells, Runx3 can regulate the CD4 silencerelement in reporter constructs, but not the endogenous CD4 locus.This is possibly due to limited accessibility of Runx3 to the en-dogenous CD4 locus based on uncharacterized epigenetic modifi-cations (55).

Recently, it was suggested that the CD8 population in CD4�/�

mice contains MHC-II-restricted T cells (45). This outcome wasexplained on the basis of the strength of the signal model of CD4/CD8 lineage choice, which proposes that strong signals duringpositive selection direct cells to the CD4 lineage, whereas weaksignals promote CD8 lineage choice (56). Thus, it was reasonedthat some MHC-II-restricted thymocytes receive a weak signal inthe absence of CD4, resulting in differentiation to the CD8 lineage.However, by crossing Runx3 tg mice to TCR tg OT-II or MOGmice, we could not detect an increase in specific (OT-II or MOG)MHC-II-restricted mature thymocytes in the CD8 lineage in thesemice. These data indicate that the decreased number of matureCD4 SP thymocytes and the enhanced number of mature CD8 SPthymocytes in Runx3 tg mice could not be simply explained by theredirection of MHC-II/CD4-restricted cells to the CD8 lineage. It islikely that part of the MHC-II-restricted thymocytes could not passthrough development because of the reduced CD4 expression. This isconsistent with the reduced percentage of positively selected (CD69�

and CD69�/TCRhigh) thymocytes in Runx3 tg mice (Fig. 2A, panels4–6). Other MHC-II-restricted thymocytes may continue editing.Overexpression of Runx3 induced higher numbers of mature CD8 SP

pled morpholino oligonucleotide specific for �-globin (control; panels 1and 2) or Runx3 (mo-Runx3; panels 3 and 4) and cultured for 3 days withIL-7. Panels 1 and 3 show the FITC fluorescence of the transfected cellsafter 3 days of culture. Vertical bars indicate the electronic gates set for theanalysis of CD4/CD8 expression of FITC-positive cells (panels 2 and 4).The percentage of the cells in each quadrant (panels 2 and 4) is shown.CD8� thymocytes were gated (gate in panels 2 and 4) and analyzed forCD103 expression. The average percentage of CD103-positive control orRunx3 antisense oligonucleotide-treated CD8� cells of three independentexperiments is shown in panel 5. B, Targeted deletion of Runx3 abolishesexpression of CD103 on CD8 SP blood T cells. Expression of CD4 andCD8 on B220�TCR�� blood cells of Runx3�/�RAG2�/� (panel 1) orRunx3�/�RAG2�/� (panel 2) mice are shown. In panels 3 and 4, theexpressions of CD103 on gated CD8�B220�TCR�� cells of the mice inpanels 1 and 2 are shown, respectively. One of four independent experi-ments is shown.

FIGURE 9. Absence of Runx3 expression abolishes expression ofCD103 on CD8 SP T cells. A, Morpholino antisense oligonucleotides spe-cific for Runx3 inhibit the down-regulation of CD4 expression and theinduction of CD103 expression during development of CD8 SP thymo-cytes. Sorted CD69�/TCRint thymocytes were transfected with FITC-cou-


thymocytes, probably by enhanced development, survival, and/or pro-liferation rates of MHC-I-restricted cells. However, we cannot ex-clude that other specific MHC-II-restricted thymocytes can be forcedto develop to the CD8 lineage in Runx3 tg mice.

Recently, it was shown that forced expression of Runx3 in areaggregation thymic organ culture system designed to directCD4�/CD8int thymocytes to the CD4 SP lineage resulted in reac-tivation of CD8 expression instead (57). In this study the expres-sion level of CD8� in positively selected immature cells (CD69�)and mature CD8 SP thymocytes was slightly increased in Runx3 tgmice. It is possible that minimal overexpression of Runx3 en-hanced CD8� expression in mature CD8 SP thymocytes even if wedid not see any obvious differences in Runx3 protein levels in thewt and tg mature CD8 SP thymic subpopulations. A minimal in-crease in Runx3 expression in mature CD8 SP thymocytes ofRunx3 tg mice also could be involved in higher survival and/orproliferation rates of CD8 SP thymocytes. Runx3 tg thymocytesshowed a slight increase in CD24 expression and a slight decreasein CD5 expression (data not shown).

Thus, Runx3-dependent CD4 silencing during the developmentof DP thymocytes is responsible for the reduced expression of CD4in DP cells and the decreased number of mature CD4 SP T cells inRunx3 tg mice. Furthermore, overexpression of Runx3 cannot redi-rect specific MHC-II/CD4-restricted thymocytes (OT-II and MOG) todevelop to the CD8 lineage. Runx3 thus does not appear to favor CD8lineage differentiation at the expense of the CD4 lineage.

Recently, it was reported that a point mutation in the zinc fingertranscription factor Th-POK/cKrox, whose wt form is expressedduring the development of CD4 SP thymocytes, is responsible forredirection of MHC-II-restricted thymocytes to the CD8 lineage inHD mice (58). Tg overexpression of this factor during T cell differ-entiation prevented the development of CD8 lineage cells (58, 59).Th-POK/cKrox thus controls the CD4 lineage decision. In contrast,Runx3 does not have a significant influence on the lineage decision,although it regulates a subset of CD8 lineage-specific genes and func-tions. In the future, it will be of interest to investigate whether Th-POK/Krox and Runx3 exhibit cross-regulatory functions.

We have also demonstrated that Runx3 does not exclusivelyregulate CD4 silencing, but that Runx3 also acts as a positiveregulator of integrin �E/CD103 expression during the developmentof CD8 SP T cells. First described for intestinal T cells, the inte-grin �E/�7 heterodimer mediates interaction with E-cadherin onepithelial cells (35, 46, 60). In wt mice, integrin �E is also ex-pressed on �80% of mature CD8 SP thymocytes, 40–50% ofCD8� splenocytes and liver T cells, and 15–25% of blood CD8�

T cells (Fig. 8B and data not shown) (46). Similar expression pat-terns are observed in humans (47). Instead, integrin �E is ex-pressed on only �1–4% of wt CD4 SP T cells, all of which havebeen reported to be CD25� or CD25� regulatory T cells (61, 62).

Runx3 tg overexpression induced CD103 expression on 7% ofCD4 SP thymocytes and 12–18% of CD4� splenocytes. In thisstudy we have shown that Runx3 tg CD103�CD4� splenocyteshave characteristics of regulatory T cells. Recent data have shownthat CD103�CD4� regulatory T cells display an effector/memoryphenotype and are the most potent suppressors of inflammatoryprocesses in disease models such as Ag-induced arthritis (49). Itwill be of interest to investigate whether endogenous Runx3 isinvolved in CD103 up-regulation in regulatory T cells.

The knockdown or knockout of Runx3 strongly reduced the fre-quency of CD103�CD8� thymocytes and abolished CD103 ex-pression on CD8� T cells in blood. The absence of CD103 ex-pression on CD8� CTLs may contribute to the functionalimpairment of this T cell subset in Runx3�/� mice. Loss of CD103expression has been shown to result in reduced proliferation of

mature CD8 SP thymocytes (46) and strongly reduced transplantinfiltration and rejection by CD8� CTLs (62–64). Thus, Runx3could be a potential target to investigate CD103-dependent trans-plant rejection by CD8� CTLs. It will be of interest to examinewhether thymoma cells of T-ALL patients unexpectedly express-ing CD103 and accumulating in the gut show uncontrolled expres-sion of Runx3 (65).

Up-regulation of CD103 in transplant-infiltrating effector CD8�

T cells has been shown to be regulated by TGF-� (62). Addition-ally, it was demonstrated that Runx3 can act together with theTGF-� signaling cascade (19, 21, 22, 66). Runx3 and the intra-cellular signal transducers of activated TGF-� receptors, the Smadproteins, can translocate to the nucleus and activate the mousegermline Ig � promoter, inducing IgA synthesis (19, 22, 66). Inthis study we have identified another synergy between Runx3 andTGF-� signaling in regulating CD103 expression. We demon-strated that exogenous expression of Runx3 together with TGF-�treatment, but neither alone, up-regulates CD103 expression after48 h in CD4 SP RLM-11-1 T cells. CD103 expression was induced30 min after TGF-� stimulation alone in a CD8 SP T cell line thatexpresses endogenous Runx3. This suggests that TGF-� signalingin the presence of Runx3 induces the transcription of CD103 (48).However, to date no TGF-�-responding element has been identi-fied in the 4-kb region upstream of exon 1 of the human CD103gene locus (48). It has yet to be investigated whether Runx3 pro-tein directly binds to regulatory elements in the CD103 gene locus.

It will be of interest to examine whether CD103 expression onmature CD8 SP thymocytes or on regulatory CD4� T cells is con-trolled by Runx3 alone or by both Runx3 and signals of the TGF-�receptor family, and whether differential TGF-� signaling in thethymus is involved in the fate of CD4 and CD8 SP T cells.

Recently, we showed that NKT cells are absent in Runx1 con-ditional knockout mice (67). Because the number of NKT cells isstrongly reduced in Runx3 tg mice, it is possible that this is aconsequence of Runx3-mediated repression of Runx1 expression,and that Runx3 cannot substitute for Runx1 in NK T cell devel-opment. However, we cannot exclude more direct effects of Runx3overexpression.

In summary, we have demonstrated that Runx3 is a main reg-ulator of CD4 silencing and CD103 expression and contributes todetermining the phenotype of CD8 lineage cells during thymocytedevelopment.

AcknowledgmentsWe thank Dr. Jeffrey V. Ravetch (The Rockefeller University, New York,NY) for providing the opportunity to perform several experiments in hislaboratory, and Klara Velinzon, Prisca Gell, Marisa Patt, and Jose Paganfor technical support. We also thank Drs. M. Satake and Y. Groner forproviding antisera against Runx, Dr. Yoshiaki Ito for the Runx3-deficientmice, and Dr. Kuchroo for the TCR-tg MOG mice.

DisclosuresThe authors have no financial conflict of interest.

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runx3 regulates integrin e/cd103 and cd4 expression … and cd4 expression during development of cd4...

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