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. 2003 Jun 24;100(13):7731-6.
doi: 10.1073/pnas.1232420100. Epub 2003 Jun 9.

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis

Eilon Woolf  1 , Cuiying Xiao, Ofer Fainaru, Joseph Lotem, Dalia Rosen, Varda Negreanu, Yael Bernstein, Dalia Goldenberg, Ori Brenner, Gideon Berke, Ditsa Levanon, Yoram Groner
Affiliations

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis

Eilon Woolf et al. Proc Natl Acad Sci U S A. .

Abstract

The RUNX transcription factors are important regulators of lineage-specific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific _target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3-/-;Runx1+/-), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8+ T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.

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Figures

Fig. 1.
Fig. 1.
Expression of Runx3 and Runx1 in the thymus. Runx3 is expressed mainly in medullary SP CD8+ and in cortical DN CD4-CD8- thymocytes, whereas Runx1 expression is found mainly in cortical thymocytes. (A) Paraffin sections of thymuses of E18 embryos (Upper) and 6-week-old mice (Lower) were stained with anti-Runx1 (Left) and anti-Runx3 (Right) antibodies. Cortex (blue arrow) and medulla (red arrow) are indicated. (B) Thymocytes from 6-week-old mice were sorted onto (poly-l)lysine-coated slides and were stained with anti-Runx3 antibodies.
Fig. 2.
Fig. 2.
Impaired development of SP CD8+ T cells in Runx3-KO mice. (A) FACS analysis of CD4 and CD8 expression in thymus and spleen of WT and Runx3-KO mice. Numbers at the top of each dot plot refer to the percentage of SP CD8+ cells; KO 1.32 ± 0.59%, n = 9 versus WT 2.9 ± 0.85%, n = 9 (P = 0.00007). CD8+CD4lo cells are indicated by an arrowhead. (B) FACS analysis of HSA expression in CD8+ and CD4+ thymocytes. Thymocytes stained for CD4, CD8, CD24 (HSA), and SP CD8+ cells were gated for HSA expression profile. Numbers at the top indicate the percentage of HSA-/lo SP CD8+ cells (Left) or SP CD4+ cells (Right) in WT (filled curve) and Runx3-KO (solid line). (C) CD4/CD8 distribution among peripheral T cells. Splenocytes were stained with CD4 and CD8 mAbs and analyzed. Numbers at the top of the dot plots refer to the percentage of SP CD8+ and DP CD4+CD8+ splenocytes, respectively. The percentage of CD8+ cells in Runx3 KO mice that also expressed CD4 was similar (i.e., 59 ± 11%, n = 10).
Fig. 3.
Fig. 3.
Increased CD4/CD8 ratio in mature thymic and splenic T cells of Runx3-KO mice. Cells were stained for CD4, CD8, CD24 (HSA), and TCRαβ and analyzed. (A) CD4/CD8 dot plots of gated TCRhighHSA-/lo T cells. Runx3-KO cells exhibit a reduction in TCRhighHSA-/low SP CD8+ T cell both in thymus (approximately ×5) and spleen (approximately ×10). Numbers at the top right corner refer to percentage of cells in each of the four compartments. Note the prominent mature DP CD4+CD8+ T cell population in Runx3-KO thymus and spleen. In Runx3-KO periphery, 74 ± 5.9% of TCRhighHSA-/lo CD8+ cells also expressed CD4 (n = 6). (B) CD4/CD8 ratio in TCRhighHSA-/lo T cells is increased in thymus and spleen of Runx3-KO mice. (n = 4, P < 0.05).
Fig. 4.
Fig. 4.
In vitro cytotoxic activity of WT and Runx3-KO peritoneal CTLs. Primed mice were injected with 25 × 106 LF+ allogeneic tumor cells, and 4–5 days later PELs were retrieved and analyzed for CTL activity. (A) Percent of _target LF+ cell lysis as a function of E to T ratio (E, effector PELs; T, _target LF+ tumor cells). The data of one of three experiments with similar results are presented. (B) An ≈3-fold reduction in lytic units of Runx3-KO CTLs as compared with WT CTLs. A lytic unit is defined as the number of effector cells required to lyse 35% of _target cells under specified assay conditions. The results are based on four WT and four Runx3-KO mice in three independent experiments. P = 0.005, based on double-sided Student's t test. (C) CD4/CD8 distribution in peritoneal lymphocytes. Peritoneal cells were retrieved 5 days after i.p. injection of 25 × 106 LF+ tumor cells, incubated for 1 h at 37°C to deplete most macrophages and B cells, and subjected to flow cytometry by using CD4 and CD8 mAbs.
Fig. 5.
Fig. 5.
Conjugation to tumor cells and proliferation capabilities of WT and KO CD8+ T cells. (A and B) Fewer Runx3 KO PEL-LF+ conjugates are formed in vitro and many are CD4+.(A) Equal numbers of LF+ tumor cells and PELs were incubated for 5 min at 25°C and the number of PEL-LF+ conjugates counted per 1 × 106 cells was determined. Each combined (WT/Runx3-KO) histogram (experiments 1–4) represents a separate experiment. (In experiment 1, cells were incubated for 7 min.) (B) Percentage of CD4 expressing CTLs counted after immunofluorescent staining. Nine fields in each experiment were counted for CD4+ and CD4-PEL-LF+ conjugates (n = 3). (Inset) A confocal image depicting CD4 expressing CTLs conjugated to tumor cells. (C and D) Peripheral Runx3-KO CD8+ T cells are less proliferative than WT. CD8+ splenocytes were purified and subjected to an in vitro proliferation assay. Runx3-KO SP CD8+ splenocytes exhibit a 2- to 3-fold reduction in proliferation after stimulation with CD3/CD28 mAbs (C) or with CD3 mAb + IL-2 (D).
Fig. 6.
Fig. 6.
Reduced expression of Runx1 in Runx3 KO further impairs differentiation of CD8+ T cells. Thymocytes and splenocytes of compound mutant Runx3/Runx1, Runx3-KO, and WT were subjected to flow cytometry by using CD4, CD8, CD24 (HSA), and TCRαβ markers. CD4/CD8 dot plots of gated TCRhighHSA-/lo T cells are shown. Numbers at the top of each dot plot refer to the percentage of SP CD8 T cells. Note the complete absence of TCRhighHSA-/lo SP CD8 cells in spleens of compound mutant Runx3/Runx1.
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