An Essential Role for the Transcription Factor Runx1 in T Cell Maturation

The generation of functional mature T cells relies on post-positive selection T cell maturation1. Intra-thymic maturation occurs at the SP stage while post-thymic maturation requires contact with secondary lymphoid organs such as spleen or lymph nodes before T cells enter the long-lived naive T cell pool2. Single-positive (SP) thymocytes can be divided into three populations based on their maturation status: semi-mature (SM) SP thymocytes are CD24+ CD69+ MHCI−CCR7− and are susceptible to death receptor signaling-mediated apoptosis; mature 1 (M1) SP thymocytes are CD24+ CD69+ MHCI+ CCR7+ and are resistant to death receptor induced apoptosis and are able to proliferate after TCR stimulation; mature 2 (M2) SP thymocytes are CD24−CD69−MHCI+ CCR7+ and gain the ability to egress from the thymus3,4,5. Once T cells egress from the thymus, the youngest T cells in the periphery are termed recent thymic emigrants (RTEs). RTEs continue to under go post-thymic maturation, increasing their ability to produce cytokines upon stimulation, for two to three weeks before entering the long-lived naive T cell pool1. During maturation, T cells also gain resistance to complement-mediated elimination6,7. Although the signals and molecular mechanisms that regulate T cell maturation are not well understood, recent studies have identified genes that are specifically required for post-positive selection T cell maturation8,9,10,11. In particular, mice with a conditional deletion of the transcriptional regulators NKAP (NF-κB activating protein) or HDAC3 (histone deacetylase 3) have a block in T cell maturation6,7, leading their elimination by complement in the periphery as RTEs. Concurrent with maturation, T cells increase incorporation of sialic acid, in particular α2,8-linked sialic acid, into cell surface glycans. Loss of sialylation, such as experimentally through neuraminidase, leads to binding of natural IgM and activation of complement12,13. RTEs from CD4-cre NKAP cKO or CD4-cre HDAC3 cKO mice have a defect in α2,8-sialylation as well as decreased expression of the complement regulatory protein CD55 that contribute to their complement-mediated elimination. Altered α2,8-sialylation in the absence of NKAP or HDAC3 in RTEs is due to decreased mRNA expression of sialic acid transferases belonging to the ST8Sia family, in particular ST8Sia66,7. The transcription factor Runx1 (also called AML1/CBFA2/PEBP2αB) belongs to the Runx family of transcription factors that share a highly conserved DNA binding runx domain14. Runx proteins are associated with the non-DNA-binding cofactor CBFβ that allows stable binding of Runx proteins to target DNA sequences. By binding to the regulatory elements of Cd4, Foxp3 and Gata3, Runx1 has multiple roles in T cell development15,16, generation and suppressive function of regulatory T cells17,18,19,20, and Th2 differentiation21, respectively. CD4-cre mediated Runx1 deletion leads to a decreased number and percentage of CD4 SP thymocytes, especially in the TCRβhiCD24lo population, and few peripheral CD4+ T cells15. CD8+ T cells are unaffected in CD4-cre Runx1 cKO mice, due to expression of Runx316,22. Expression of IL-7Rα is lower in TCRβhiCD69+ CD4 SP thymocytes and peripheral Foxp3−CD4+ conventional T cells15. Since IL-7Rα signaling is required for naive T cell homeostasis and survival, it was thought that Runx1 regulates survival and homeostasis of peripheral CD4+ T cells through regulation of IL-7Rα. However, IL-7Rα transgene expression did not rescue peripheral CD4+ T cell numbers in Runx1 cKO mice, although increased proportions of mature CD4 SP thymocytes was reported15. Thus, factors other than IL-7 must also have a role. Here we demonstrate that the primary defect in peripheral T cells in CD4-cre Runx1 cKO mice is in T cell maturation. Although Runx1-deficient CD4 SP thymocytes and CD4+ T cells have decreased expression of IL-7Rα, an IL-7Rα transgene cannot rescue the survival or cellularity of CD4+ T cells in CD4-cre Runx1 cKO mice. Conventional CD4 SP thymocytes cannot undergo maturation in the absence of Runx1, although positive selection proceeds normally and there is no evidence for increased negative selection. Peripheral Runx1-deficient CD4+ T cells are phenotypically and functionally immature, with a severe defect in the ability to produce TNFα upon stimulation. Runx1-deficient CD4+ T cells bind IgM, C1q, C4 and C3 and are thus eliminated by complement in the periphery. Therefore, neither alterations in T cell homeostasis, positive selection or negative selection are responsible for the CD4+ T cell defect in CD4-cre Runx1 cKO mice, but the defect is due to a severe block in T cell maturation.