During thymic T cell development, immature CD4+CD8+ double-positive (DP) thymocytes develop either into CD4+CD8− Th cells or CD4−CD8+ CTLs. Differentially expressed primary factors inducing the fate of these cell types are still poorly described. The transcription factor Runx3/AML-2 Runx, rust dominant factor; AML, acute myeloid leukemia is expressed specifically during the development of CD8 single-positive (SP) thymocytes, where it silences CD4 expression. Deletion of murine Runx3 results in a reduction of CD8 SP T cells and concomitant accumulation of CD4+CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery. In this study we have investigated the role of Runx3 during thymocyte development and CD4 silencing and have identified integrin αE/CD103 on CD8 SP T cells as a new potential target gene of Runx3. We demonstrate that Runx3 is necessary not only to repress CD4, but also to induce CD103 expression during development of CD8 SP T cells. In addition, transgenic overexpression of Runx3 reduced CD4 expression during development of DP thymocytes, leading to a reduced number of CD4 SP thymocytes and an increased number of CD8 SP thymocytes. This reversal is not caused by redirection of specific MHC class II-restricted cells to the CD8 lineage. Overexpression of Runx3 also up-regulated CD103 expression on a subpopulation of CD4 SP T cells with characteristics of regulatory T cells. Thus, Runx3 is a main regulator of CD4 silencing and CD103 induction and thus contributes to the phenotype of CD8 SP T cells during thymocyte development.
During T cell development in the thymus CD4+CD8+ double-positive (DP)9 thymocytes undergo positive and negative selection depending on the ability of their TCR to interact with thymic stromal cells presenting self-peptides bound to MHC molecules (1, 2, 3, 4). Further differentiation leads to the development of either mature CD4 single-positive (SP) Th cells or CD8 SP CTLs, which have a CD69−/TCRhigh phenotype. Analysis of the genes involved in CD4 and CD8 gene regulation and identification of the genes that are differentially expressed during thymocyte differentiation may provide insight into the mechanisms governing T cell development and help to identify primary factors inducing the fate of CD4 or CD8 SP T cells (5, 6).
The transcription factor Runx3 (runt domain factor; acute myeloid leukemia-2/core binding factor-α3/polyoma enhancer binding protein-2αC) is expressed during the development of CD8 SP thymocytes, but not of CD4 SP thymocytes, and is essential for silencing of CD4 expression (7, 8, 9). Knockout or knockdown of Runx3 results in a reduction of CD8+ T cells and a concomitant accumulation of mature CD4+/CD8+ T cells, which cannot down-regulate CD4 expression in the thymus and periphery (7, 8, 9). These CD4+/CD8+ T cells show impaired response to TCR stimulation in the periphery, suggesting that Runx3 also regulates other genes during the development of CD8 SP T cells (7, 8)
The Runx family of transcription factors consists of three members that play important roles in cellular differentiation, proliferation, and the development of autoimmunity and cancer (10, 11, 12, 13, 14, 15, 16, 17, 18). Runx3 is involved in T and B cell differentiation, dendritic cell function, the development of dorsal root ganglia neurons, chondrocyte maturation, apoptosis, and gastric cancer (7, 8, 9, 11, 12, 13, 14, 19, 20, 21, 22, 23). In some of these systems Runx3 acts in concert with the TGF-β signaling pathway (19, 21, 22).
Runx proteins bind to DNA by forming heterodimers with the common β subunit core binding factor-β/polyoma enhancer binding protein-2β via the conserved runt domain (10, 24). In T cells, Runx factors bind to the silencer element in the first intron of the CD4 gene locus (25, 26, 27, 28) where Runx binding sites have been identified (7, 9, 29). Runx1 is mainly responsible for reversible CD4 repression in DN thymocytes, and Runx3 silences CD4 expression during the development of CD8 SP T cells (7). At this time, Runx3 is the only known transcription factor that binds to the CD4 silencer element and at the same time is expressed during development of CD8, but not CD4, SP thymocytes (7, 9, 30, 31, 32, 33, 34).
In this study we have investigated the potential role of Runx3 during CD4 silencing and T cell development and have identified integrin αE/CD103, which is expressed on CD8 SP thymocytes and interacts with E-cadherin on epithelial cells, as another potential CD8 lineage-specific target gene of Runx3 (35). We show that by regulating both CD4 silencing and CD103 expression, Runx3 contributes to the phenotype of CD8 SP thymocytes.
Materials and Methods
CD4E/P-Runx3 transgenic (tg) mice and molecular analyses
The murine Runx3 cDNA (accession no. NM019732) corresponding to the form transcribed from the proximal promoter (exons 2–6) (36) and the murine CD4 proximal enhancer and promoter/start of exon 1 were generated by PCR from genomic DNA (9, 28). The CMV enhancer/promoter and the d2EGFP cassette in the d2EGFP-N1 plasmid (BD Clontech) were replaced with these CD4 regulatory elements and Runx3 cDNA, respectively. All tg mice used in this study were generated from ova obtained from an F1(C57BL/6 × C3H) background as previously described (37), and tg lines were kept by backcrossing to B6 animals. The tg mice analyzed in this study represent the second (see Figs. 1 and 2,A) or seventh (see Figs. 2,B, 3, 4, and 6–8) generation obtained upon backcrossing the founder animals. The genotype of CD4E/P-Runx3 tg mice was determined by PCR and Southern blot analyses. The following primers were used for PCR on tail tip-derived DNA: murine Runx3 exons 2–4: forward, 5′-acgctgccggtcgccttca; 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 1 was 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 purchased from The Jackson Laboratory (39, 40, 41). CD4sil+/− mice were described previously and backcrossed seven times to C57BL/6 mice (28, 29). TCR tg myelin oligodendrocyte glycoprotein (MOG) mice on the C57BL/6 background were described recently (42). OT-I, OT-II, MOG, and CD4sil+/− mice were crossed to Runx3 tg mice (line 3) that had previously been backcrossed six times to C57BL/6 mice. TK-1-5 is a subclone of the murine CD8+ lymphoma T cell line TK-1, and RLM-11-1 is a subclone of the 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 medium supplemented with 10% FCS, 150 μg/ml streptomycin, 150 U/ml penicillin, and 3 mM l-glutamine. To analyze the effect of TGF-β on CD103 expression, the cells were cultured for 48 h with 5 ng/ml human TGF-β2 (Roche).
Analyses of tg mice and cell lines
Total thymocytes, splenocytes, or T cell lines were stained with the following Abs (BD Biosciences): CyChrome-anti-CD4, PerCP-anti-CD4, PerCP-Cy5.5-anti-CD4, PE-anti-CD8α, PE-Cy7-anti-CD8α, FITC-anti-CD69, PE-anti-CD69, allophycocyanin-anti-TCRβ, FITC-anti-CD103, PE-anti-CD103, biotin-anti-CD103 with PE-streptavidin, FITC-anti-different TCRVβ subtypes, PE-anti-different TCRVα 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 were analyzed by flow cytometry (FACSCalibur or BD-LSRII; BD Biosciences). For the isolation of CD69+/TCRint cells, CD69+ cells were first 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.
Protein extracts of thymocytes (20 μg), cell lines (20 μg), and FACS (SE Vantage; BD Biosciences) purified thymic or splenic T cell subpopulations of mice (3 μg) were prepared, and Western blotting was conducted as previously described (9). The wt littermates of the tg mice were used as controls. As primary Abs we used a polyclonal rabbit antiserum specific for the conserved 15 C-terminal aa of murine Runx1 (NMPPARLEEAVWRPY) (43), an antiserum specific for 270 aa of the C terminus of human RUNX3 (13), or a mouse mAb against the housekeeping gene β-actin. As secondary Abs we used peroxidase- or alkaline phosphatase-coupled rabbit- or murine-specific antisera (Amersham Biosciences).
Plasmids, stable reporter gene assay, and transfection
Murine Runx3 cDNA corresponding to the form transcribed from the proximal promoter (exons 2–6) (36) was generated by PCR and cloned into a cDNA expression vector (CMV-Runx3). The EGFP-Runx3 fusion construct (CMV-EGFP-Runx3 (exons 2–6)) was generated by cloning the Runx3 (exons 2–6) cDNA in frame with the EGFP gene in the EGFP-C1-expressing plasmid (BD Clontech). Reporter constructs are based on the d2EGFP-N1 plasmid (BD Clontech) containing a destabilized version of EGFP (d2EGFP) as a reporter gene. The murine CD4 proximal enhancer (E), promoter/start of exon 1 (P) and silencer (sil; 434-bp SacI-XbaI fragment) (9, 27, 28) were generated by PCR from genomic DNA (9). The two plasmids, CD4E/P-d2EGFP and CD4E/P-d2EGFP-sil, were described previously (9). The plasmids were transfected by electroporation into the RLM-11-1 thymoma T cell line under the following conditions: 4 × 106 cells in 300 μl of RPMI 1640 medium with 25 μM HEPES (pH 7.0), 5 μg of plasmids, 1500 V, for 200 μs (BTX; Q-Biogene). Cells were analyzed by flow cytometry for d2EGFP expression 24 h after transfection.
Morpholino antisense oligonucleotides and IL-7 induced ex vivo thymocyte 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 morpholino antisense oligonucleotides under the following conditions: 3 × 106 cells in 300 μl of RPMI 1640 with 25 μM HEPES (pH 7.0), 10–12 nmol of morpholino, 1500 V, for 200 μsec (BTX; Q-Biogene), and cultured in complete IMDM. The following morpholino antisense oligonucleotides (Gene Tools) were used: mo-Runx3–1, 5′-tgctcgggtctacgggaatacgcat; and control (β-globin), 5′-cctcttacctcagttacaatttata (9). The Runx3 oligonucleotide is complementary to a sequence in exon 2 of Runx3. After 3 h, 3 ng/ml mouse rIL-7 (R&D Systems) were added. After overnight incubation the cells were washed twice with PBS (37°C) and treated for 10 min with 100 μg/ml Pronase (Calbiochem) and 100 μg/ml DNase (Roche) in 1 ml of PBS at 37°C. The reaction was stopped with 100 μl of FCS. The cells were further cultured with IL-7 for 2 days, stained with Abs, and analyzed by flow cytometry. After electroporation, overnight incubation, and subsequent Pronase treatment, including repeated washing and centrifugation steps, ∼4–8% of the cells were recovered as live cells. During further cultivation with IL-7, the cell numbers stayed relatively constant.
Analysis of CD103 expression on Runx3−/− lymphocytes
Generation 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 the control of the murine CD4 proximal enhancer and promoter (Fig. 1, A and B). This enhancer/promoter combination becomes active at the DN 2 stage (CD4−/CD8−/CD25+/CD44+) and remains active throughout the development of both CD4 and CD8 SP T cells (28). Therefore, in the tg mice, Runx3 is not only expressed throughout development of CD8 SP thymocytes, but also during development of DP and CD4 SP cells.
We established two independent tg lines (lines 3 and 4). Both tg lines overexpressed Runx3 protein in total thymocytes (Fig. 1,C). With an antiserum specific for the C terminus of murine Runx1 that also interacts with murine Runx3 (9, 43, 44), we detected an increase in Runx3 protein expression in total thymocytes of tg mice compared with wt mice (Fig. 1,C). At the same time, the amount of Runx1 protein was reduced, suggesting that tg overexpression of Runx3 down-regulates Runx1 expression or destabilizes Runx1 protein (Fig. 1 C).
Overexpression of Runx3 strongly reduces expression of CD4 during development of CD4/CD8 DP cells and influences the ratio of mature CD4 and CD8 SP thymocytes
In Runx3 tg mice (lines 3 and 4), DP thymocytes showed a strong reduction of CD4 expression (Fig. 2,A, panels 1–3). Thus, exogenous Runx3 repressed CD4 expression during the development of DP cells. Furthermore, the percentage of CD4+CD8− cells was strongly reduced (Fig. 2,A, panels 1–3). The percentage of positively selected immature (CD69+/TCRint and CD69+/TCRhigh) thymocytes of tg mice was also reduced (Fig. 2,A, panels 4–6). These immature cells showed a decreased CD4 expression and an increase in CD8 expression (Fig. 2,A, panels 7–9, and data not shown). Gating on mature thymocytes (CD69−/TCRhigh), we found that the percentage and number of mature CD4 SP cells were also reduced 2- to 3-fold (Fig. 2,A, panels 10–12, and B). At the same time, the percentage and number of mature CD8 SP thymocytes were increased 1.5- to 2-fold (Fig. 2,A, panels 10–12, and B). The expression level of CD8α in mature CD8 SP thymocytes was slightly increased in Runx3 tg mice (Fig. 2 A, 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 CD69−/TCRlo/int/CD25−/CD44− DN4 phenotype (data not shown). TCRhigh cells were almost absent from the CD4/CD8 DN subpopulation (data not shown). As shown in Fig. 2 B, there was no significant difference in absolute thymic and splenic cell numbers in wt and tg mice.
Thus, overexpression of Runx3 could repress CD4 expression in most DP cells during the differentiation from DN cells and resulted in a reduced number of mature CD4 SP thymocytes and an increased number of CD8 SP thymocytes. Comparable results were observed in Runx3 tg mice backcrossed two or seven times to the C57BL/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-deficient mice is heavily contaminated with MHC- II-restricted T cells (45). The smaller number of mature CD4 SP T cells and the higher number of mature CD8 SP T cells in Runx3 tg mice may be due to the low expression of CD4 on DP thymocytes, which may force MHC-II-restricted thymocytes to the CD8 lineage. To determine whether Runx3 overexpression can force MHC-II-restricted thymocytes to the CD8 lineage, we crossed Runx3 tg mice with OT-II mice expressing an MHC-II-restricted TCR specific for a chicken OVA peptide (42). The tg TCR consists of a TCRVα2 and a TCRVβ5 chain. Tg overexpression of Runx3 in OT-II mice led to a strong decrease in the numbers of CD4 SP thymocytes (Fig. 3,A, panels 1 and 2) and mature TCR tg (TCRVα2high/TCRVβ5high) thymocytes (Fig. 3,A, panels 5 and 6). Although the number of mature CD8 SP thymocytes (Fig. 3,A, panels 3 and 4) increased, there were few mature TCR tg (TCRVα2high/TCRVβ5high) cells in the thymic and splenic CD8 SP T cell population (Fig. 3,A, panels 7–10). We obtained comparable results by crossing Runx3 tg mice with MOG mice expressing an MHC-II-restricted TCR specific for a MOG. This tg TCR consists of a TCRVα2 and a TCRVβ5 chain (Fig. 3 B) (42). Thus, Runx3 overexpression could not redirect MHC-II/CD4-restricted thymocytes with a specific TCRVαβ combination (OT-II or MOG) to develop to mature CD8 SP thymocytes. It is likely that the slight increase in CD8 SP thymocytes in Runx3 tg mice is mainly caused by an expansion of MHC-I-restricted cells.
To characterize the remaining mature CD4 SP T cells in Runx3 tg mice, we analyzed whether specific TCRVα or TCRVβ rearrangements were enriched or decreased in this population. We did not observe any obvious differences in TCR use between CD4+ or CD8+ splenic T cells of wt and tg mice (data not shown).
To further analyze the influence of overexpressed Runx3 on MHC-I-restricted thymocytes, we crossed Runx3 tg mice with OT-I mice expressing an MHC-I-restricted TCR specific for a chicken OVA peptide (41). In this study the number of mature CD8 SP thymocytes was slightly increased (data not shown).
Reduction of mature CD4 SP thymocytes in Runx3 tg mice is caused by silencing of CD4 expression during development of DP thymocytes
To investigate whether the decreased number of CD4+ thymocytes in 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 one allele and therefore lack CD4−CD8+ T cells (28, 29). Deletion of one allele of the CD4 silencer in Runx3 tg mice did not affect the absolute number of thymocytes compared with Runx3 tg and CD4sil+/− mice (Fig. 4, panels 1–3, and data not shown). CD4 expression in DP thymocytes was restored in Runx3 tg × CD4sil+/− mice, indicating that ectopic expression of Runx3 causes CD4 down-regulation throughout development of DP thymocytes by acting on the CD4 silencer in Runx3 tg mice (Fig. 4, panels 1–3). Absolute numbers of mature CD4 SP thymocytes were also largely restored in Runx3 tg × CD4sil+/− mice compared with Runx3 tg mice (Fig. 4, panels 4–6). These results indicate that the reduction in mature CD4 SP thymocytes in Runx3 tg mice is due to the reduced level of CD4 expression in DP thymocytes or transitional thymocytes after positive selection and is not a consequence of Runx3 influence on CD4 lineage determination.
Runx3 induces silencing in a CD4 SP thymoma cell line
We also analyzed the potential of Runx3 to regulate the CD4 silencer element in the CD4 SP thymoma cell line RLM-11-1, which is likely to be terminally differentiated. In contrast to the CD8 SP T cell line TK-1-5, CD4 SP RLM-11-1 cells do not express endogenous Runx3 (Fig. 5,A). Transfection of a Runx3-expressing plasmid (CMV-Runx3, exons 2–6) into RLM-11-1 cells did not down-regulate CD4 expression (data not shown). However, exogenous Runx3 displayed the potential to regulate the CD4 silencer element in a reporter assay in RLM-11-1 cells (Fig. 5, B and C). We have investigated Runx3-mediated repression of the reporter gene d2EGFP under the control of the CD4 proximal enhancer, promoter, and CD4 silencer element (Fig. 5, B and C) (9). Exogenous Runx3 repressed d2EGFP expression (only 2% of the cells expressed d2EGFP) if the CD4 silencer element (CD4E/P-d2EGFP-sil) was present (Fig. 5 C), whereas in the absence of the silencer element (CD4E/P-d2EGFP), d2EGFP expression was not reduced, and 10% of the cells expressed d2EGFP. Without exogenous Runx3 expression, no silencing effect was obvious. Thus, although endogenous CD4 expression is not reversible, exogenous Runx3 can regulate the CD4 silencer element in a reporter assay in the 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 lose CD4 expression completely, whereas cells with variegated lower expression of tg Runx3 can up-regulate CD4 expression and develop into mature CD4 SP T cells. To address this question, we sorted individual thymic and splenic T cell subpopulations and analyzed Runx3 protein expression (Fig. 6). In agreement with our previously published observation (9), Runx3 in wt mice is predominantly expressed in TCRhigh/CD8 SP thymocytes (Fig. 6). In contrast, tg Runx3 was not only expressed in TCRhigh/CD8 SP thymocytes, but also at comparable levels in immature TCRβint/CD4−/CD8+ thymocytes and in TCRβint/CD4+/CD8+ DP thymocytes and TCRβhigh/CD4 SP thymocytes. In the spleen, similar amounts of Runx3 were expressed in CD4+ and CD8+ T cells of tg mice (Fig. 6). Thus, in Runx3 tg mice, a subpopulation of MHC-II restricted thymocytes can still develop into mature CD4 SP T cells even in the presence of Runx3 protein.
Overexpression of Runx3 inhibits the development of NKT cells
By analyzing the remaining mature CD4 SP T cells in Runx3 tg mice, we identified that the number of NKT cells (NK1.1+/CD24low/TCRalmost high) with a CD4+CD8− or CD4−CD8− phenotype 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 overexpression of Runx3 reduced not only NK1.1 expression (Fig. 7, panels 4–6). This phenomenon was independent of CD4 expression because CD4−/− mice still have NKT cells (Fig. 7).
Expression of integrin αE/CD103 is regulated by Runx3 and TGF-β
The accumulated CD4+/CD8+ T cells that appear in the periphery of Runx3−/− mice show functional deficits, suggesting that Runx3 regulates additional genes during the development of CD8 SP T cells (7, 8). Integrin αE/CD103, which mediates interaction with E-cadherin on epithelial cells, has been shown to be expressed on >80% of mature CD8 SP thymocytes, but on only 3–4% of CD4 SP thymocytes (46, 47). Therefore we investigated whether Runx3 plays a role in the regulation of CD103 expression.
Recently, it was shown that TGF-β could induce CD103 transcription and protein expression in the CD8 SP T cell line TK-1, which expresses endogenous Runx3 (Fig. 5,A) (48). To investigate the potential role of Runx3 in the induction of CD103 expression, we used the CD4 SP T cell line RLM-11-1 (Fig. 5,A). Neither treatment with TGF-β alone nor expression of an EGFP-Runx3 fusion protein alone was sufficient to induce CD103 expression on RLM-11-1 cells. However, TGF-β induced CD103 expression at 48 h in 30–35% of EGFP-Runx3-positive cells. (Fig. 8 A). Thus, the combination of Runx3 expression and TGF-β stimulation could induce CD103 expression on a CD4 SP thymoma T cell line.
Tg overexpression of Runx3 induces integrin αE/CD103 expression in a subpopulation of mature CD4 SP thymocytes and splenocytes with characteristics of regulatory cells
To investigate Runx3-dependent regulation of CD103 expression in vivo, we analyzed CD103 expression patterns on T cells from wt and Runx3 tg mice. In wt mice, CD103 was expressed on >80% of mature CD8 SP thymocytes and 40–50% of splenic CD8+ T cells, but on only 1–4% of thymic and splenic CD4 SP T cells (Fig. 8 B).
In contrast, in Runx3 tg mice, CD103 protein was up-regulated on TCRβhighCD4 SP thymocytes (7%) and CD4+ spleen cells (12–18%; Fig. 8,B). The CD103 up-regulation on CD4 SP T cells was preferentially observed on CD25+ cells, presumably regulatory T cells (Fig. 8,C). In Runx3 tg mice, the CD103+CD25+CD4+ and also the CD103+CD25−CD4+ splenocytes showed lower expression patterns of CD45RB and CD62L compared with CD103−CD25−CD4+ cells, which is also characteristic of regulatory T cells (Fig. 8 D) (49). Thus, in vivo overexpression of Runx3 during T cell development was capable of inducing CD103 expression on mature CD4 SP T cells with characteristics of regulatory T cells.
Absence of Runx3 expression strongly reduces expression of CD103 on CD8 SP T cells
We previously established an IL-7-dependent ex vivo thymocyte development culture system that allows the induction of differentiation of purified primary CD4+CD8+CD69+TCRint positively selected thymocytes into CD4 and CD8 SP T cells (9, 50). Specific knockdown of Runx3 with FITC-coupled morpholino antisense oligonucleotides in this culture system leads to an accumulation of CD4+/CD8+ thymocytes instead of mature CD8 SP thymocytes. This is explained by the loss of CD4 silencing (Fig. 9 A) (9).
In this study we have used this system to analyze the role of Runx3 in CD103 expression on CD8 SP thymocytes. The inhibition of Runx3 expression resulted in a reduced frequency of CD103-expressing CD8 + thymocytes (Fig. 9,A). The role of Runx3 in CD103 expression was also analyzed in PBMC of RAG2−/− mice reconstituted with fetal liver cells of Runx3+/− or Runx3−/− animals (Fig. 9 B). Only 2–4% of B220−TCRβ+CD8+ blood cells from RAG2−/− mice reconstituted with Runx3−/− fetal liver cells expressed CD103, in contrast to 12–26% of B220−TCRβ+/CD8+ cells from mice reconstituted with fetal liver cells from Runx3+/− mice. Thus, knockdown or knockout of Runx3 during T cell development significantly reduced CD103 expression on CD8 SP T cells. In contrast, tg overexpression of Runx3 up-regulated CD103 expression on CD4 SP T cells with characteristics of regulatory T cells.
To investigate the roles of Runx3 during T cell development, we generated tg mice expressing Runx3 (exons 2–6) under the control of the CD4 proximal enhancer and promoter (9, 29). In Runx3 tg mice, most DP cells had reduced CD4 expression. These results are in agreement with recent data showing that retroviral or tg overexpression of Runx3 during thymocyte development strongly reduces CD4 expression (51, 52). Also, retroviral overexpression of Runx1 reduces CD4 expression during thymocyte development (51). It is likely that overexpressed Runx3 or Runx1 induces extended CD4 silencer activity and delayed CD4 expression during differentiation from DN to DP cells. However, we cannot exclude that strong overexpression of Runx3 or Runx1 reinduces CD4 repression in DP cells. In this study we have shown that Runx3 overexpression reduced the amount of Runx1 protein in DP and mature SP thymic subpopulations. In wt mice, Runx1 protein is expressed at lower level in Runx3-expressing CD8 SP thymocytes compared with CD4 SP thymocytes; it is thus possible that Runx3 plays a role in down-regulation of Runx1 expression. In this context, it was described that EBV-transfected B cells up-regulate Runx3 expression, thereby down-regulating Runx1 expression via binding to the Runx1 promoter (53, 54).
The Runx3 tg mice had also a decreased number of mature CD4 SP thymocytes. By crossing Runx3 tg mice with CD4sil+/− mice, we showed that reduced CD4 expression during thymocyte development and the decreased number of mature CD4 SP thymocytes in Runx3 tg mice are caused by Runx3-dependent silencing of CD4 expression during thymocyte development. CD4 expression and absolute numbers of mature CD4 SP thymocytes were recovered in Runx3 tg × CD4sil+/− mice compared with Runx3 tg mice, indicating that Runx3 overexpression does not affect the development of CD4 SP thymocytes if CD4 is expressed at normal levels in DP cells.
It is possible that variegated transgene expression caused different levels of CD4 expression during the development of DP thymocytes. Our data show that in Runx3 tg mice, the amount of Runx3 protein in the remaining mature CD4 SP thymic subpopulation is comparable to the amount in mature CD8 SP thymocytes. In Runx3 tg mice, DP cells with normal or minimally reduced CD4 expression may be selected into CD4 SP thymocytes in the presence of tg Runx3 protein. Another explanation for the remaining CD4 expression in a subpopulation of DP thymocytes and developing CD4 lineage cells in Runx3 tg mice could be that there are different subpopulations of MHC-II-restricted cells, one of which is sensitive to Runx3-mediated shut-off of CD4 and another that may be resistant.
However, after differentiation to the CD4 lineage, it appears that Runx3 can no longer repress CD4 expression. The expression level of CD4 in mature CD4 SP thymocytes was comparable in wt and Runx3 tg mice (Fig. 2 A, panels 10–12). Overexpression of Runx3 in the CD4 SP RLM-11-1 thymoma cell line lacking endogenous Runx3 induced CD4 silencer-mediated repression of reporter gene expression. However, endogenous CD4 expression was not down-regulated (data not shown). Our results indicate that in fully differentiated CD4+ T cells, Runx3 can regulate the CD4 silencer element in reporter constructs, but not the endogenous CD4 locus. This is possibly due to limited accessibility of Runx3 to the endogenous CD4 locus based on uncharacterized epigenetic modifications (55).
Recently, it was suggested that the CD8 population in CD4−/− mice contains MHC-II-restricted T cells (45). This outcome was explained on the basis of the strength of the signal model of CD4/CD8 lineage choice, which proposes that strong signals during positive selection direct cells to the CD4 lineage, whereas weak signals promote CD8 lineage choice (56). Thus, it was reasoned that some MHC-II-restricted thymocytes receive a weak signal in the absence of CD4, resulting in differentiation to the CD8 lineage. However, by crossing Runx3 tg mice to TCR tg OT-II or MOG mice, we could not detect an increase in specific (OT-II or MOG) MHC-II-restricted mature thymocytes in the CD8 lineage in these mice. These data indicate that the decreased number of mature CD4 SP thymocytes and the enhanced number of mature CD8 SP thymocytes in Runx3 tg mice could not be simply explained by the redirection of MHC-II/CD4-restricted cells to the CD8 lineage. It is likely that part of the MHC-II-restricted thymocytes could not pass through development because of the reduced CD4 expression. This is consistent with the reduced percentage of positively selected (CD69+ and CD69−/TCRhigh) thymocytes in Runx3 tg mice (Fig. 2 A, panels 4–6). Other MHC-II-restricted thymocytes may continue editing. Overexpression of Runx3 induced higher numbers of mature CD8 SP thymocytes, probably by enhanced development, survival, and/or proliferation rates of MHC-I-restricted cells. However, we cannot exclude that other specific MHC-II-restricted thymocytes can be forced to develop to the CD8 lineage in Runx3 tg mice.
Recently, it was shown that forced expression of Runx3 in a reaggregation thymic organ culture system designed to direct CD4+/CD8int thymocytes to the CD4 SP lineage resulted in reactivation of CD8 expression instead (57). In this study the expression level of CD8α in positively selected immature cells (CD69+) and mature CD8 SP thymocytes was slightly increased in Runx3 tg mice. It is possible that minimal overexpression of Runx3 enhanced CD8α expression in mature CD8 SP thymocytes even if we did not see any obvious differences in Runx3 protein levels in the wt and tg mature CD8 SP thymic subpopulations. A minimal increase in Runx3 expression in mature CD8 SP thymocytes of Runx3 tg mice also could be involved in higher survival and/or proliferation rates of CD8 SP thymocytes. Runx3 tg thymocytes showed a slight increase in CD24 expression and a slight decrease in CD5 expression (data not shown).
Thus, Runx3-dependent CD4 silencing during the development of DP thymocytes is responsible for the reduced expression of CD4 in DP cells and the decreased number of mature CD4 SP T cells in Runx3 tg mice. Furthermore, overexpression of Runx3 cannot redirect specific MHC-II/CD4-restricted thymocytes (OT-II and MOG) to develop to the CD8 lineage. Runx3 thus does not appear to favor CD8 lineage differentiation at the expense of the CD4 lineage.
Recently, it was reported that a point mutation in the zinc finger transcription factor Th-POK/cKrox, whose wt form is expressed during the development of CD4 SP thymocytes, is responsible for redirection of MHC-II-restricted thymocytes to the CD8 lineage in HD mice (58). Tg overexpression of this factor during T cell differentiation 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 functions. 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 exclusively regulate CD4 silencing, but that Runx3 also acts as a positive regulator of integrin αE/CD103 expression during the development of CD8 SP T cells. First described for intestinal T cells, the integrin αE/β7 heterodimer mediates interaction with E-cadherin on epithelial cells (35, 46, 60). In wt mice, integrin αE is also expressed on >80% of mature CD8 SP thymocytes, 40–50% of CD8+ splenocytes and liver T cells, and 15–25% of blood CD8+ T cells (Fig. 8 B and data not shown) (46). Similar expression patterns are observed in humans (47). Instead, integrin αE is expressed on only ∼1–4% of wt CD4 SP T cells, all of which have been reported to be CD25+ or CD25− regulatory T cells (61, 62).
Runx3 tg overexpression induced CD103 expression on 7% of CD4 SP thymocytes and 12–18% of CD4+ splenocytes. In this study we have shown that Runx3 tg CD103+CD4+ splenocytes have characteristics of regulatory T cells. Recent data have shown that CD103+CD4+ regulatory T cells display an effector/memory phenotype and are the most potent suppressors of inflammatory processes in disease models such as Ag-induced arthritis (49). It will be of interest to investigate whether endogenous Runx3 is involved in CD103 up-regulation in regulatory T cells.
The knockdown or knockout of Runx3 strongly reduced the frequency of CD103+CD8+ thymocytes and abolished CD103 expression on CD8+ T cells in blood. The absence of CD103 expression on CD8+ CTLs may contribute to the functional impairment of this T cell subset in Runx3−/− mice. Loss of CD103 expression has been shown to result in reduced proliferation of mature CD8 SP thymocytes (46) and strongly reduced transplant infiltration and rejection by CD8+ CTLs (62, 63, 64). Thus, Runx3 could be a potential target to investigate CD103-dependent transplant rejection by CD8+ CTLs. It will be of interest to examine whether thymoma cells of T-ALL patients unexpectedly expressing CD103 and accumulating in the gut show uncontrolled expression of Runx3 (65).
Up-regulation of CD103 in transplant-infiltrating effector CD8+ T cells has been shown to be regulated by TGF-β (62). Additionally, it was demonstrated that Runx3 can act together with the TGF-β signaling cascade (19, 21, 22, 66). Runx3 and the intracellular signal transducers of activated TGF-β receptors, the Smad proteins, can translocate to the nucleus and activate the mouse germline Ig α promoter, inducing IgA synthesis (19, 22, 66). In this study we have identified another synergy between Runx3 and TGF-β signaling in regulating CD103 expression. We demonstrated that exogenous expression of Runx3 together with TGF-β treatment, but neither alone, up-regulates CD103 expression after 48 h in CD4 SP RLM-11-1 T cells. CD103 expression was induced 30 min after TGF-β stimulation alone in a CD8 SP T cell line that expresses endogenous Runx3. This suggests that TGF-β signaling in the presence of Runx3 induces the transcription of CD103 (48). However, to date no TGF-β-responding element has been identified in the 4-kb region upstream of exon 1 of the human CD103 gene locus (48). It has yet to be investigated whether Runx3 protein directly binds to regulatory elements in the CD103 gene locus.
It will be of interest to examine whether CD103 expression on mature CD8 SP thymocytes or on regulatory CD4+ T cells is controlled by Runx3 alone or by both Runx3 and signals of the TGF-β receptor family, and whether differential TGF-β signaling in the thymus is involved in the fate of CD4 and CD8 SP T cells.
Recently, we showed that NKT cells are absent in Runx1 conditional knockout mice (67). Because the number of NKT cells is strongly reduced in Runx3 tg mice, it is possible that this is a consequence of Runx3-mediated repression of Runx1 expression, and that Runx3 cannot substitute for Runx1 in NK T cell development. However, we cannot exclude more direct effects of Runx3 overexpression.
In summary, we have demonstrated that Runx3 is a main regulator of CD4 silencing and CD103 expression and contributes to determining the phenotype of CD8 lineage cells during thymocyte development.
We thank Dr. Jeffrey V. Ravetch (The Rockefeller University, New York, NY) for providing the opportunity to perform several experiments in his laboratory, and Klara Velinzon, Prisca Gell, Marisa Patt, and Jose Pagan for technical support. We also thank Drs. M. Satake and Y. Groner for providing antisera against Runx, Dr. Yoshiaki Ito for the Runx3-deficient mice, and Dr. Kuchroo for the TCR-tg MOG mice.
The authors have no financial conflict of interest.
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.
This work was supported by the Max Planck Gesellschaft zur Förderung der Wissenschaften.
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.