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Overexpression of the Runx3 Transcription Factor Increases the Proportion of Mature Thymocytes of the CD8 Single-Positive Lineage¹

Kazuyoshi Kohu^*, Takehito Sato, Shin-ichiro Ohno, Keitaro Hayashi^*, Ryuji Uchino^*, Natsumi Abe^*, Megumi Nakazato^*, Naomi Yoshida^*, Toshiaki Kikuchi, Yoichiro Iwakura^¶, Yoshihiro Inoue, Toshio Watanabe^*, Sonoko Habu and Masanobu Satake^2,*

Departments of ^* Molecular Immunology, and Oncology and Molecular Medicine, and Animal Research Facility, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; Department of Immunology, Tokai University School of Medicine, Isehara, Japan; and ^¶ Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4^–8⁺ subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4^–8⁺ thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4^–8⁺ thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4^–8⁺ cells was greatly increased, whereas the numbers of CD4⁺8⁺ and CD4⁺8^– cells were reduced. The CD4^–8⁺ transgenic thymocytes contained mature cells with a TCR^highHSA^low phenotype. These cells were released from the thymus and contributed to the elevated level of CD4^–8⁺ cells relative to CD4⁺8^– cells in the spleen. Runx3 overexpression also increased the number of mature CD4^–8⁺ thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4⁺8^– lineage selection. Thus, Runx3 can drive thymocytes to select the CD4^–8⁺ lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.

	Introduction

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Thymocytes pass through multiple, distinct steps of differentiation and gradually develop into immunocompetent T lymphocytes. At each stage of differentiation, thymocytes face fate decisions, such as whether they will survive or die, which lineage they will select, and how they will mature. These events are mostly regulated by a network of signals that are presented to thymocytes as morphogens, cytokines, MHC, and self-Ag peptides (1, 2). The most critical receptors for these signals are the TCR and its coreceptor molecules, CD4 and CD8. Thus, one of the challenging and fascinating steps of thymocyte differentiation is the positive selection of CD4⁺8⁺ double-positive (DP)³ cells and the subsequent lineage selection to either CD4⁺ or CD8⁺ single-positive (SP) cells (3, 4, 5). Various transcription factors appear to function coordinately in the regulation of the TCR, CD4, and CD8 genes (6, 7).

Recent advances in our understanding of gene regulation in thymocyte differentiation have involved the roles of the Runx family of transcription factors (6). Expression of Runx1 protein is detected in immature, CD4^–8^– double-negative (DN) and premature DP thymocytes, as well as in mature SP thymocytes (8, 9, 10, 11). As expected from this expression pattern, Runx1 appears to exert its function at each step of thymocyte differentiation. For example, both the transition of DN cells to the DP stage and the maturation of postselected SP cells are significantly perturbed if the endogenous Runx1 activity in thymus is reduced by artificially expressing a dominant interfering form of Runx1 (8, 11). Each of these steps is normally accompanied by a tremendous amount of cell proliferation, for which Runx1 function is necessary. Conditional targeting of Runx1 has also revealed that it has an indispensable role in the initial emergence of T-committed cells from stem cells (12).

In contrast to Runx1, the expression of Runx3 protein is detected mainly in the CD4^–8⁺ subset of thymocytes and splenocytes (9, 10). In accordance with this protein expression profile, CD4^–8⁺ thymocytes do not develop in the Runx3 (–/–) thymus (13, 14). Based on an analysis of CD4 gene regulation, Taniuchi et al. (13) proposed that Runx3 binds to the Runx elements in the CD4 silencer and represses CD4 expression. Use of a Morpholino antisense oligonucleotide in an in vitro thymocyte differentiation system also supported the requirement for Runx3 in the generation of CD4^–8⁺ cells (10).

These studies clearly implicate Runx3 in the regulation of CD4 expression, but do not necessarily prove its role in the differentiation of CD4^–8⁺ thymocytes per se. Loss-of-function experiments provide information about what Runx3 does but not about everything it can do. In the present study, we overexpressed a transgenic Runx3 specifically in the T lineage and analyzed the development of the transgenic thymocytes. Runx3 can actively drive thymocytes to the CD4^–8⁺ lineage, which implies that it does more than simply silencing CD4 gene expression.

	Materials and Methods

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Plasmids

The hemagglutinin (HA) tag that represents the epitope of flu virus HA was fused to the N terminus of the murine Runx3 coding region by the PCR method as follows. PCR was performed using a murine Runx3 cDNA (15) as a template. The sequences of the sense and antisense primers were 5'-GCC GGA TCC GAA TTC ACC ATG TAT CCA TAT GAT GTT CCA GAT TAT GCT ATG CGT ATT CCC GTA GAC CC-3' and 5'-GCC GGA TCC GAA TTC TTA GTA GGG CCG CCA CAC-3', respectively. The PCR product was digested with BamHI and subcloned into the BamHI site of pLck (p1017), which harbors the proximal promoter region of the murine Lck gene and a poly(A) addition sequence derived from the human growth hormone gene (16). The resulting plasmid was designated pLck-HA/Runx3. The accuracy of the modified sequences in the plasmid was confirmed by sequencing. Immunohistochemical staining of cDNA-transfected HeLa cells confirmed the nuclear localization of HA-tagged Runx3 protein (data not shown).

Mice

To generate transgenic mouse lines expressing Runx3, the DNA of pLck-HA/Runx3 was digested with SpeI, and the purified fragment containing the Runx3 expression unit was microinjected into fertilized eggs of C57BL/6 mice. Transgenic founders were identified and crossed to C57BL/6 mice. The presence or absence of the transgene was examined by PCR using genomic DNA as a template. The sense and antisense primers were 5'-CGG GAA TTC ATG TAT CCA TAT GAT GTT CCA GAT TAT GCT ATG CGT ATT CCC GTA GAC CC-3' and 5'-CCG GAA TTC TTA GTA GGG CCG CCA CAC-3', respectively, and a 1275-bp fragment was amplified from the transgene. Establishment of the human CD4-transgenic mice will be described elsewhere (Y. Iwakura, manuscript in preparation). Briefly, fertilized eggs of C3H/HeN mice were microinjected by the human CD4 cDNA which harbors the murine CD4 enhancer/promoter and an SV40-derived poly(A) addition signal. ₂-microglobulin (₂m)-deficient mice and CD4-deficient mice were purchased from The Jackson Laboratory. The I-A^d-restricted, OVA_323–339-specific TCR-transgenic mice have been previously reported (17).

Flow cytometrical analysis

Cells were liberated from the thymus and spleen and suspended in PBS containing 0.2% (w/v) BSA. The single-cell suspensions were incubated with appropriately diluted mAbs on ice for 30–60 min. The following fluorescein-conjugated mAbs were used: CyChrom-CD4 (Rm4-5), FITC-CD4 (Rm4-5), PE-CD8a (53-6.7), RED613-CD8a (53-6.7; Invitrogen Life Technologies), PE-TCR (H57-597), FITC-CD69 (H1.2F3), FITC-V2 (B20.6), FITC-V3 (KJ25), FITC-V4 (KT4), FITC-V5.1 and -5.2, FITC-V6 (RR4-7), FITC-V7 (TR310), FITC-V8.1 and -8.2 (MR5-2), FITC-V8.3 (1B3.3), FITC-V9 (MR10-2), FITC-V10^b (B21.5), FITC-V11 (RR3-15), FITC-V12 (MR11-1), FITC-V13 (MR12-3), FITC-V14 (14-2), FITC-V17^a (KJ23), FITC-HSA (M1-69), and FITC-human CD4 (Leu3a). Except for RED613-CD8a, the mAbs were purchased from BD Pharmingen. The labeled cells were separated with an analytical flow cytometer (EPICS-XL), and the data were analyzed with EXPO32 software (Beckman Coulter).

Immunoblot analysis and EMSA

The CD4^–8⁺HSA^low and CD4⁺8^–HSA^low fractions were purified from thymocytes or splenocytes, respectively, using autoMACS (Miltenyi Biotec). Its purity was judged to be >90% by flow cytometry. Protein was extracted from cells using a radioimmunoprecipitation assay solution (50 mM Tris-HCl (pH 7.4), 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl, 1 µg/ml aprotinin, 1 mM NaVO₄, and 1 mM NaF). The other procedures necessary for immunoblot analysis including the electrophoresis, transfer to the filter, and immunoreaction were performed as described previously (18). The raising and characterization of the anti-Runx peptide Ab was also described previously (19). The antiserum raised against the C terminus of murine Runx1 can recognize Runx1, Runx2, and Runx3, because they share the common VWRPY sequence at their extreme C-terminal ends. The anti-tubulin Ab (Ab-1) was purchased from Oncogene. The procedures for preparing nuclear extracts and for the EMSA were described previously (20). The Runx binding sequence from the Polyomavirus enhancer was used as a probe to detect Runx DNA binding activity. The anti-HA mAb 3F10 used for the supershift assay was purchased from Roche Diagnostics.

RT-PCR

Total cytoplasmic RNA was isolated from cells using the ISOGEN reagent (Nippon Gene). cDNAs were synthesized from the RNAs by reverse transcription using Superscript II reverse transcriptase (Invitrogen Life Technologies). The cDNAs were PCR-amplified (25 cycles for each gene) with LA-Taq polymerase (Takara), using the following sense and antisense primers to detect transcripts: for CD4, 5'-CCT GCG AGA GTT CCC AGA AGA AGA TCA CAG-3' and 5'-TGA TAG CTC TGC TCT GAA AAC CCA GCA CTG-3'; for CD8, 5'-GGT GAG TCG ATT ATC CTG GGG AGT GGA GAA-3' and 5'-ACA CAA TTT TCT CTG AAG GTC TGG GCT TGC-3'; for perforin1, 5'-CAA GCA GAA GCA CAA GTT CGT-3' and 5'-CGT GAT AAA GTG CGT GCC ATA-3'; for GATA3, 5'-AGG CAA GAT GAG AAA GAG TGC CTC-3' and 5'-CTC GAC TTA CAT CCG AAC CCG GTA-3'; and for G3PDH, 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCC ACC ACC CTG TTG CTG TA-3'. The PCR products were run through agarose gels and visualized with ethidium bromide staining.

Chromatin immunoprecipitation assay

A chromatin fraction was prepared from thymocytes, fixed and immunoprecipitated by the anti-Runx or anti-HA Ab, respectively. The procedures were as recommended by the manufacturer of the assay kit (Upstate Cell Signaling Solutions). DNA was purified from the precipitate and processed as a template for PCR to amplify the CD4 silencer-specific sequence. The primers for PCR were 5'-TGT AGG CAC CCG AGG CAA AG-3' and 5'-GTT CCA GCA CAG GAG CCC CA-3'. The amplified product was run through agarose gels and transferred to nylon membranes. The membranes were hybridized with ³²P-labeled, CD4 silencer-specific oligonucleotide, 5'-ATA CGA AGC TAG GCA ACA GA-3'.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Overexpression of Runx3 in the T lineage cells

Endogenous expression of Runx3 protein is detected mainly in the CD4^–8⁺ subset of T lymphocytes (9, 10). To artificially overexpress Runx3 in the T cell lineage, we placed the Runx3 coding region under the control of the proximal Lck gene promoter. This promoter is known to be active in immature as well as mature T cells and in thymic as well as peripheral T cells (16). Transgenic mouse lines were established and the expression of Runx3 protein was examined by immunoblot analysis using an anti-Runx Ab (Fig. 1A). The 52-kDa Runx3 band was clearly detected in the extract of both CD4⁺8^– and CD4^–8⁺ fractions, which were prepared from transgenic thymi as well as spleens. The endogenous Runx3 was also detected in the wild-type, CD4^–8⁺ thymocytes and splenocytes but to a much lesser degree compared with the transgenic cells. Thus, the magnitude of Runx3 overexpression in the transgenic vs wild-type cells was roughly 5-fold in the case of thymi and 3-fold in the case of spleens. A very faint band seen in the CD4⁺8^– wild-type cells represents the nonspecific reaction of the Ab, because the band was not abolished by the preabsorption of the Ab with the Ag peptide. The endogenous Runx1 protein of 56 kDa was detected in all the fractions tested.

View larger version (84K): [in this window] [in a new window]   FIGURE 1. Expression of transduced Runx3 protein. A, Protein was extracted from thymic and splenic CD4+8–HSAlow as well as CD4–8+HSAlow cells and processed for immunoblot analysis. The extracts from wild-type and Runx3-transgenic cells were probed with an anti-Runx Ab. The bands indicated by the arrows represent the Runx3 and endogenous Runx1 proteins of 52 and 56 kDa, respectively. An immunoblot with an anti-tubulin Ab served as a control. B, Runx DNA binding activity detected by EMSA. The nuclear extracts from the wild-type and Runx3-transgenic thymocytes and splenocytes were processed for EMSA. The bands indicated by the arrowhead and bar represent the activity of endogenous Runx1 and transduced HA-Runx3, respectively. The HA-Runx3-derived band was supershifted by the addition of an anti-HA Ab to the transgenic extract.

The percentage of CD4^–8⁺ cells increases and the percentage of CD4⁺8⁺ and CD4⁺8^– cells simultaneously decreases in the Runx3-transgenic thymus

After confirming the protein expression of transgenic Runx3, we evaluated its effect on T cell differentiation. Flow cytometry was used to analyze CD4 and CD8 in thymocytes and splenocytes (Fig. 2). In the Runx3-transgenic thymi, the percentage of CD4^–8⁺ cells increased to 80% of the total population, whereas the percentage of CD4⁺8⁺ cells decreased to only 9%; the percentage of CD4⁺8^– cells also decreased substantially. The unusual profile of CD4 and CD8 expression in the transgenic thymocytes was reflected in the transgenic splenocytes as well. In the transgenic spleen, the percentage of CD4^–8⁺ cells was higher than that of CD4⁺8^– cells, whereas the opposite was true in the wild-type spleen.

View larger version (77K): [in this window] [in a new window]   FIGURE 2. Flow cytometrical analysis of thymocytes and splenocytes. The cells from wild-type and Runx3-transgenic mice were stained for CD4 and CD8 and processed for flow cytometry. Numbers given in the individual quadrants indicate the percentage of cells of each type.

The increase in the CD4^–8⁺ fraction in the Runx3-transgenic thymus could be due either to an increase in CD8 expression or a decrease in CD4 expression. To distinguish these two possibilities, the CD8 and CD4 expression profiles were displayed for the wild-type and the Runx3-transgenic thymocytes (Fig. 3A). The relative ratios of CD8^– and CD8⁺ cells were not different between the two genotypes. In contrast, the number of CD4^– cells was greatly increased and the number of CD4⁺ cells was decreased in the transgenic thymus compared with the wild-type thymus.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. CD4 and CD8 expression in the wild-type and Runx3-transgenic cells. A, Thymocytes stained for CD4 or CD8 were analyzed for their fluorescence intensity. The shaded peak represents the wild-type cells, whereas the open peak represents the Runx3-transgenic cells. B, Semiquantitative RT-PCR analysis of CD4, CD8, and G3PDH transcripts. An increasing amount of cDNA synthesized from the wild-type and Runx3-transgenic thymocytes was used for PCR. The relative amounts of PCR products were measured and are shown as numbers below the gels. The G3PDH product obtained for the least amount of wild-type cDNA was taken to be 1.0. C, Chromatin immunoprecipitation analysis. A chromatin fraction prepared from the wild-type and Runx3-transgenic thymocytes was immunoprecipitated by an anti-Runx or anti-HA Ab, respectively. DNA was purified from the precipitates, and an increasing amount of DNA fraction was processed as a template for PCR. The PCR products were detected by a CD4 silencer-specific oligonucleotide. Input means a DNA fraction that was present in the chromatin fraction before immunoprecipitation.

The CD4 silencer is proposed to be a main target by a Runx3 transcription factor (13, 14). We checked this by chromatin immunoprecipitation analysis (Fig. 3C). An increasing amount of chromatin fraction-derived DNA that was precipitated by the anti-Runx or anti-HA Ab was processed for PCR and hybridized by a CD4 silencer-specific oligonucleotide. Both Abs precipitated a significantly greater amount of CD4 silencer sequence from the Runx3-transgenic thymocytes compared with the wild-type cells. The results in Fig. 3 thus suggest that the phenotypic alteration seen in the transgenic thymocytes in Fig. 2 can be at least partly explained by the down-regulation of CD4 expression.

The increased CD4^–8⁺ fraction of transgenic thymocytes includes immature, premature, and mature subpopulations

We next characterized in detail the CD4^–8⁺ fraction of transgenic thymocytes. As described below, this fraction was found to contain three different subpopulations: immature, premature, and mature cells.

The first subpopulation in the CD4^–8⁺ fraction was recognized as immature single-positive (ISP) cells, which can be easily seen by following the ontogeny of thymocyte development (Fig. 4A). In wild-type thymus, only CD4^–8^– cells were detected at embryonic day (E)15.5. CD4^–8⁺ ISP cells transiently appeared at E16.5, CD4⁺8⁺ cells at E17.5, and CD4⁺8^– cells at day 2 after birth. In the Runx3-transgenic thymus, immature CD4^–8⁺ cells first appeared at E16.5 and remained as the main population until after birth. The persistence of ISP cells is probably due to the down-regulation of CD4 by Runx3. This CD4 repression appeared to be partial, because some CD4⁺8⁺ and CD4⁺8^– cells emerged at day 2 after birth in transgenic mice.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Different subpopulations exist in the CD4–8+ fractions from wild-type and Runx3-transgenic thymus. A, Ontogeny of thymocyte development. Thymocytes were prepared from wild-type and Runx3-transgenic mice at E15.5, E16.5, E17.5, birth (NB), and 2 days after birth (D2). The cells were stained for CD4 and CD8 and processed for flow cytometry. The numbers above each panel indicate the number of cells recovered. B, The wild-type and Runx3-transgenic thymocytes from adult mice were processed for four-color flow cytometrical analysis. The CD4–CD8+ fractions were further analyzed for their TCR and HSA expression profiles. The level of TCR expression was classified into three stages (lo, med, and hi), as indicated. The numbers represent the percentages of each subpopulation in the CD4–8+ fraction. C, CD69 and TCR expression profiles in differentiating thymocytes. The wild-type and Runx3-transgenic thymocytes were processed for four-color flow cytometry. The cells were first screened for their CD69 and TCR expression profiles. The TCRmedCD69+ (broken boxes) and TCRhighCD69+ fractions (solid boxes) were further analyzed for their CD4 and CD8 expression profiles.

Another characteristic of the transgenic CD4^–8⁺ fraction was the presence of an aberrant TCR^medHSA^high subpopulation that was not as apparent in the wild-type fraction (33 vs 4%; Fig. 4B). The medium degree of TCR expression indicates that this second subpopulation should be categorized as representing the premature DP stage rather than the ISP stage. We further confirmed this point by staining the thymocytes with CD69, a marker of positive selection (Fig. 4C). In the case of wild-type cells, the TCR^medCD69⁺ cells exhibited a CD4⁺8⁺ phenotype, whereas the TCR^highCD69⁺ exhibited both the CD4⁺8^– and CD4^–8⁺ phenotypes. The TCR^medCD69⁺ population could also be detected in the transgenic thymus, but the apparent phenotype of this population was CD4^–8⁺, not CD4⁺8⁺. The CD4^–8⁺ fraction persisting in the developing transgenic thymus (Fig. 4A) may contain these TCR^med cells as well. Thus, the second subpopulation can be summarized as the premature, "CD4-repressed DP" cells.

The transgenic CD4^–8⁺ fraction also contained a third subpopulation of mature, TCR^highHSA^low cells (see 10% in Fig. 4B). We next evaluated the effect of Runx3 overexpression on these mature CD4^–8⁺ cells. To do so, we first obtained a TCR expression profile for the total thymocyte population (Fig. 5A). Both the wild-type and Runx3-transgenic thymi contained TCR^low, TCR^med, and TCR^high subpopulations to a comparable degree. Because the TCR^high subpopulation corresponds to mature cells, overexpression of Runx3 did not appear to arrest or block thymocyte differentiation. We gated the TCR^high subpopulation and then displayed the CD4/8 profile (Fig. 5B). In the TCR^high thymocytes from the wild-type, the percentage of CD4^–8⁺ cells was one-third that of CD4⁺8^– cells, whereas in the transgenic TCR^high thymocytes, the percentage of CD4^–8⁺ cells was three times that of CD4⁺8^– cells. We also counted the cell numbers constituting each fraction and found that the absolute number of CD4^–8⁺TCR^high cells in the transgenic thymi was approximately twice that in the wild-type thymi.

View larger version (48K): [in this window] [in a new window]   FIGURE 5. Effect of the Runx3 transgene on the differentiation of mature, TCRhigh cells. A, The thymocytes from wild-type and Runx3-transgenic mice were stained for TCR, and their expression profiles were analyzed. The cells were classified into three subpopulations: TCRlow, TCRmed, and TCRhigh. B, The wild-type and Runx3-transgenic thymocytes were processed for three-color flow cytometrical analysis. The mature, TCRhigh subpopulation was selected, and its CD8 and CD4 expression profile was analyzed. The numbers in the individual quadrants indicate the percentage of cells of each type. C, Semiquantitative RT-PCR analysis of perforin1, GATA3, and G3PDH transcripts. RNA was isolated from the CD4+8–HSAlow and CD4–8+HSAlow thymocytes’ fractions and converted to cDNA. An increasing amount of cDNA synthesized from the wild-type and Runx3-transgenic cells, respectively, was processed for PCR.

The mature CD4^–8⁺ cells are released into periphery of Runx3-transgenic mice

Promotion of thymocyte differentiation toward the CD8 lineage by Runx3 was also reflected in the cell composition in the spleen (Fig. 6, A and B). Among the TCR^highHSA^low mature T cells, the ratio of CD4^–8⁺ cells to CD4⁺8^– cells was 0.5 in the spleens from the wild type, but was 1.4 in the transgenic splenocytes.

View larger version (44K): [in this window] [in a new window]   FIGURE 6. Effect of the Runx3-transgene on splenic T lymphocytes. A, The wild-type and Runx3-transgenic splenocytes were processed for three-color flow cytometrical analysis. The mature TCRhigh subpopulation was selected, and its CD8 and CD4 expression profile was analyzed. The numbers in the individual quadrants indicate the percentage of cells of each type. B, The cell number ratios of TCRhighHSAlowCD4–8+ cells to TCRhighHSAlowCD4+8– cells in the wild-type and Runx3-transgenic thymus and spleen. C, The V repertoire used by the TCRs of splenic CD4–8+ cells. Wild-type () and Runx3-transgenic () cells were stained by an Ab mixture against various V segments and processed for flow cytometrical analysis.

Overexpression of Runx3 can drive originally CD4-oriented thymocytes toward the CD8 lineage

The results shown in Figs. 5 and 6 indicate that the overexpressed Runx3 can drive thymocytes to select and mature along the CD8 lineage. We then examined whether this effect of Runx3 is dependent on the TCR signaling elicited from proper MHC interactions. The TCR transgene, which is restricted to MHC class II, was introduced into Runx3 transgenic mice (Fig. 7A). Thymi from TCR single-transgenic mice showed a skew of cell differentiation to the CD4 lineage (33% CD4⁺8^– compared with 3.6% CD4^–8⁺). In contrast, in the TCR and Runx3 double-transgenic thymi, the CD4^–8⁺ cells constituted the major population (73%), just as in the case of Runx3-single-transgenic thymi. When only the mature cells were selected by gating the HSA^low fraction (and by gating the transgene-specific TCR^high fraction as well (data not shown)), it was clear that the Runx3 transgene switched the differentiation of class II-restricted cells to the CD8 lineage.

View larger version (47K): [in this window] [in a new window]   FIGURE 7. Effect of Runx3 overexpression on the differentiation potential of CD4-oriented thymocytes. A, The TCR transgene restricted to class II (left) was introduced into Runx3-transgenic mice (right). The CD4/CD8 expression profiles are shown for the nongated thymocytes (upper panels) as well as the mature, HSAlow thymocytes (lower panels). B, MHC class I deficiency (left) and the same deficiency coupled with the Runx3-transgene (right). The CD4/CD8 expression profiles are shown for the nongated thymocytes (upper panels) as well as the mature, TCRhigh thymocytes (lower panels). The numbers in individual quadrants represent the percentages of cells of each type.

Overexpression of Runx3 can drive thymocytes toward the CD8 lineage irrespective of the CD4 signaling

In thymocyte differentiation, the TCR signaling exerts its effect in concert with the signaling elicited from either the CD4 or CD8 molecule. We examined the activity of overexpressed Runx3 on thymocyte differentiation under the condition of either excess or deficiency of CD4 signaling. First, the Runx3-transgene was introduced into human CD4-transgenic mice (Fig. 8A). As seen, the level of human CD4 expression was not so high and therefore might be limited to compensate the endogenous, murine CD4, which should be silenced by the overexpressed Runx3. Under this limitation, a majority of mature TCR^high cells possessed a CD4^–8⁺ phenotype in Runx3-transgenic thymi. Second, the Runx3-transgene was expressed in a CD4-deficient background (Fig. 8B). When a CD4^–8⁺ fraction was displayed for its TCR expression, the mature TCR^high cells corresponded to 27% of CD4-deficient and Runx3-transgenic thymocytes. In contrast, such mature cells occupied only 17% of simple CD4-deficient thymocytes. Collectively, neither an excess nor a lack of CD4 signaling appears to influence the extent of overproduction of mature CD4^–8⁺ thymocytes, which is caused by the overexpressed Runx3. Thus, the activity of Runx3 to drive thymocytes toward the CD8 lineage is likely to be due to more than a simple silencing of CD4 gene expression.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Effects of human CD4 transgene and/or endogenous murine CD4 deficiency on the differentiation of Runx3-transgenic thymocytes. A, The human CD4 transgene driven by the murine CD4 enhancer/promoter (left) was introduced into Runx3-transgenic mice (middle). The murine CD4/CD8 expression profiles are shown for the mature, TCRhigh thymocytes (left and middle). In the right panel, the expression profiles of human CD4 are displayed for the murine CD4–8+ thymocytes. The shaded peak represents the wild-type cells, whereas the open peak represents the human CD4-single- and the human CD4- and Runx3-double-transgenic cells, respectively. B, A murine CD4 deficiency (left) and the same deficiency coupled with the Runx3-transgene (middle). The CD4/CD8 expression profiles are shown for the nongated thymocytes (left and middle). In the right panel, the expression profiles of TCR are displayed for the gated CD4–8+ fraction. The shaded peak represents CD4 (–/–) mice, whereas the open peak represents CD4 (–/–):Runx3-transgenic mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In the present study, we created Runx3-transgenic mice and found that the number of mature CD4^–8⁺ thymocytes was increased. This result is opposite to that found in the Runx3 (–/–) thymus, in which the number of mature CD4^–8⁺ cells is markedly decreased (13, 14). Therefore, the present gain-of-function analysis complements the previous loss-of-function analysis. However, a close inspection of our results reveals a new aspect of Runx3 function as described below and as summarized in Fig. 9.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. A model of T lymphocyte differentiation in the Runx3-transgenic thymus. Each step of differentiation is characterized by the specific expression patterns of CD4, CD8, TCR, and HSA. The cells usually start at the DN stage, go through the ISP and DP stages, and mature at the CD8 SP stage. The "CD4-repressed DP" stage is characterized by TCRmed expression; is apparently categorized as a CD4–8+ fraction; and is observed only in the Runx3-transgenic, but not the wild-type, thymus. The majority of mature TCRhighHSAlowCD4–8+ cells are considered to be derived from the premature "CD4-repressed DP" cells. A minor pathway for CD8+ maturation in the transgenic animals would be through the usual "DP" stage.

In the wild-type thymus, the endogenous Runx3 is likely involved in the selection of and commitment to the CD8 lineage in concert with TCR signaling. A short and/or weak TCR signal is somehow transduced to Runx3, which in turn regulates the gene expression necessary for the CD8 lineage determination. CD4 silencing is one target of Runx3 (13) and maintenance of CD8 expression is probably a target as well. Another possibility is that Runx3 is involved in the survival and/or maturation of thymocytes after they have selected the CD8 lineage.

At the DN stage, the CD4 silencer is reported to be "ON." Transcription of the CD4 gene is initiated when the DN cells move to the DP stage, and the activity of the CD4 silencer is expected to be turned "OFF" during the transition from DN to DP (26). The mechanism of this "OFF" switch cannot be assessed by targeted deletions of Runx3 or CD4 silencer. In our Runx3-transgenic thymus, the percentage and number of CD4⁺8⁺ cells were remarkably reduced, and an aberrant population of "CD4-repressed DP" cells with a CD4^–8⁺TCR^medHSA^high phenotype emerged instead. It is likely that exogenous expression of the transgene-derived Runx3 protein maintained the CD4 silencer in the "ON" position, thereby giving rise to the "CD4-repressed DP" thymocytes from the immature CD4^–8⁺TCR^low cells. However, these premature cells do acquire a CD4^–8⁺ phenotype, probably due to the strong repression of CD4 expression.

We previously reported the phenotype of Runx1-transgenic mice in which the numbers of both immature ISP and mature CD8 SP cells were increased (9). Even taking into consideration the differences between the Runx3- and Runx1-transgenic thymocytes in terms of the promoters used and/or the magnitude of transduced protein expression, it is interesting to note that overexpression of Runx1 did not generate the "CD4-repressed DP" cells as Runx3 did. Furthermore, the endogenous Runx1 protein is easily detected in the DP cells of wild-type thymus (10, 11), and Runx1 and Runx3 do not associate with each other in a coimmunoprecipitation experiment (K. Kohu and M. Satake, unpublished data). These observations suggest both that Runx1 is not involved in the turning the CD4 silencer "OFF" at the DP stage and that the overexpressed Runx3 can reactivate the CD4 silencer at this DP stage. It must be noted, though, that the enforced expression of Runx1 in a fetal thymic organ culture could generate similar "CD4-repressed DP" cells (27). The mechanism by which the CD4 silencer is turned "OFF" at the DN-to-DP transition needs further investigation.

The Runx3-transgenic thymus clearly contained mature CD4⁺8^– cells, and we confirmed that the transduced Runx3 was indeed expressed in these cells. Perhaps in thymocytes that are committed to the CD4⁺8^– lineage, the chromatin structure at the CD4 silencer region may be in a "closed" state, denying Runx3 access to the site.

Several transcription factors have been reported to be involved in the lineage selection of CD4/8 thymocytes. GATA3 is a positive regulator that boosts thymocytes toward the CD4 lineage (28, 29, 30), whereas TOX (31, 32) and/or activated Notch1 (33) move thymocytes toward the CD8 lineage. These factors are thought to function in response to an adequate signal from TCR when expressed endogenously, but transgenic overexpression might reveal cell-autonomous aspects of their functions. Thus, the possible interplay between the TCR signal, TOX, and Runx3 in the CD8 lineage selection will be a fascinating subject to pursue.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Acknowledgments

 
We are grateful to the following scientists for providing us with the valuable experimental tools: Y. Groner and D. Levanon for the murine Runx3 cDNA, and R. Perlmutter for the proximal Lck-promoter. We express our thanks to M. Kuji for her secretarial assistance.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by research grants from the Ministry of Education, Science, Sports, Culture and Technology, Japan. M.S. is a member in the 21st century Center of Education program, "Center for Innovative Therapeutic Development Towards the Conquest of Signal Transduction Diseases," which is headed by K. Sugamura at Tohoku University.

² Address correspondence and reprint requests to Dr. Masanobu Satake, Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail address: satake{at}idac.tohoku.ac.jp

³ Abbreviations used in this paper: DP, double positive; SP, single positive; DN, double negative; HA, hemagglutinin; ₂m, ₂-microglobulin; ISP, immature single positive; E, embryonic day; HSA, heat-stable Ag.

Received for publication April 15, 2004. Accepted for publication November 12, 2004.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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