Reciprocal Control of G1-Phase Progression Is Required for Th-POK/Runx3–Mediated CD4/8 Thymocyte Cell Fate Decision

Abstract

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G1 cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G1-phase extension and represses CD8 expression in a G1 extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G1 extension and CD8 repression. Importantly, forced G1 extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G1 progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G1-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

Delta‐like 4‐mediated Notch signaling is required for early T‐cell development in a three‐dimensional thymic structure

“…CD44 + CD117 hi or CD44 − CD25 + fetal thymocytes, as DN1a/b plus DN2mt or DN3 cells, were sorted from e16.5 BALB/c (Ly9.1 + ) or e17.5‐e18.5 RAG2‐deficient (BALB/c background, Ly9.1 + ) fetal thymi and were infected with the retrovirus encoding the intracellular active form of human Notch1 (ICN1) , a rearranged murine TCRβ chain (Vβ8.2) derived from a cytotoxic T‐cell clone 2C , c‐Myc , constitutively active human Akt1 (myristylated Akt lacking the PH domain, residues 4–125) or empty vectors (MIGR1, pMR2, or GCDN) , as described previously . The infected cells could be identified by the detection of GFP (MIGR1), rat CD2 (pMR2), or human NGFR (GCDN).…”

CD155-Transducing Signaling through TIGIT Plays an Important Role in Transmission of Tolerant State and Suppression Capacity

“…Real-time quantitative PCR RNA isolation, cDNA synthesis, and real-time quantitative PCR were performed as described previously (15). The primer/probe sets were obtained from Applied Biosystems (Mm00434256_m1, Mm00439614_m1, Mm03807522_m1, Mm00476128_m1).…”

“…Full-length cDNA of TIGIT was cloned into pMXs-IRES-GFP. P815 mastocytoma was infected with the above retrovirus as described previously (15). For coculture of TIGIT transfectants, CD4 T cells (1.5 3 10 6 /ml) were cocultured with TIGIT-expressing P815 (1.0 3 10 6 /ml) cells in an anti-CD3 Ab-bound plate.…”

T‐cell receptor signaling induces proximal Runx1 transactivation via a calcineurin–NFAT pathway

“…EL4 and OVA53 cells were cultured in the medium described earlier. OVA53 cell line was established from OVA-TCR-tg; p53-deficient mice thymocytes [27]. NIH3T3 and PLAT-E [37] cells were cultured in DMEM supplemented with 10% v/v FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.…”

“…PLAT-E cells were transfected with bicistronic plasmids, pMX-IRES-GFP, pMX-NFAT1-IRES-GFP, or pMX-NFAT4-IRES-GFP, that express NFAT and green fluorescent protein (GFP). Viral supernatant was collected and used to infect OVA53 T cells, which are a CD4 and CD8 DP thymic lymphoma cell line isolated from Rag2-deficient TCR-transgenic mice of p53 null genetic background [27]. After infection, the GFP + cells were sorted and their RNAs were processed for semiquantitative RT-PCR analysis.…”