Mamiko Noda

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110delta, and class IB, p110gamma catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110delta, but not p110gamma. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein-coupled receptor signaling in beta-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond beta-selection.

Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

Plasmacytoid dendritic cells (pDCs), also known as type I interferon (IFN)-producingcells, are thought to play central roles in antiviral immunity and the pathogenesis of some autoimmune diseases. pDCs are produced from hematopoietic stem cells in bone marrow. However, the environmental regulation of the development of pDCs is not fully understood. Here, we show that the numbers of pDCs and their earliest progenitors are severely reduced in the absence of CXCR4, the primary physiologic receptor for CXC chemokine ligand 12 (CXCL12), also known as stromal cell-derived factor-1 (SDF-1) in vivo. In vitro, CXCL12 induces a significant increase in pDC numbers generated from primitive hematopoietic cells, and pDCs and their progenitors migrate to CXCL12. In addition, most pDCs are in contact with CXCL12-abundant reticular (CAR) cells in the intersinal space of bone marrow, although many primitive hematopoietic cells adjoin CAR cells surrounding sinusoidal endothelial cells or residing near the bone surface. Thus we identified CXCL12 as a key regulator of pDC development produced by cellular niches, providing new targets for pDC therapeutic control.