Evidence for Prescribed NK Cell Ly-49 Developmental Pathways in Mice.

Previous studies of NK cell inhibitory Ly-49 genes showed their expression is stochastic. However, relatively few studies have examined the mechanisms governing acquisition of inhibitory receptors in conjunction with activating Ly-49 receptors and NK cell development. We hypothesized that the surface expression of activating Ly-49 receptors is nonrandom and is influenced by inhibitory Ly-49 receptors. We analyzed NK cell "clusters" defined by combinatorial expression of activating (Ly-49H and Ly-49D) and inhibitory (Ly-49I and Ly-49G2) receptors in C57BL/6 mice. Using the product rule to evaluate the interdependencies of the Ly-49 receptors, we found evidence for a tightly regulated expression at the immature NK cell stage, with the highest interdependencies between clusters that express at least one activating receptor. Further analysis demonstrated that certain NK clusters predominated at the immature (CD27+CD11b-), transitional (CD27+CD11b+), and mature (CD27-CD11b-) NK cell stages. Using parallel in vitro culture and in vivo transplantation of sorted NK clusters, we discovered nonrandom expression of Ly-49 receptors, suggesting that prescribed pathways of NK cluster differentiation exist. Our data infer that surface expression of Ly-49I is an important step in NK cell maturation. Ki-67 expression and cell counts confirmed that immature NK cells proliferate more than mature NK cells. We found that MHC class I is particularly important for regulation of Ly-49D and Ly-49G2, even though no known MHC class I ligand for these receptors is present in B6 mice. Our data indicate that surface expression of both activating and inhibitory Ly-49 receptors on NK cell clusters occurs in a nonrandom process correlated to their maturation stage.

Sostdc1 Regulates NK Cell Maturation and Cytotoxicity.

NK cells are innate-like lymphocytes that eliminate virally infected and cancerous cells, but the mechanisms that control NK cell development and cytotoxicity are incompletely understood. We identified roles for sclerostin domain-containing-1 (Sostdc1) in NK cell development and function. Sostdc1-knockout (Sostdc1 -/-) mice display a progressive accumulation of transitional NK cells (tNKs) (CD27+CD11b+) with age, indicating a partial developmental block. The NK cell Ly49 repertoire in Sostdc1 -/- mice is also changed. Lower frequencies of Sostdc1 -/- splenic tNKs express inhibitory Ly49G2 receptors, but higher frequencies express activating Ly49H and Ly49D receptors. However, the frequencies of Ly49I+, G2+, H+, and D+ populations were universally decreased at the most mature (CD27-CD11b+) stage. We hypothesized that the Ly49 repertoire in Sostdc1 -/- mice would correlate with NK killing ability and observed that Sostdc1-/- NK cells are hyporesponsive against MHC class I-deficient cell targets in vitro and in vivo, despite higher CD107a surface levels and similar IFN-γ expression to controls. Consistent with Sostdc1's known role in Wnt signaling regulation, Tcf7 and Lef1 levels were higher in Sostdc1 -/- NK cells. Expression of the NK development gene Id2 was decreased in Sostdc1-/- immature NK and tNK cells, but Eomes and Tbx21 expression was unaffected. Reciprocal bone marrow transplant experiments showed that Sostdc1 regulates NK cell maturation and expression of Ly49 receptors in a cell-extrinsic fashion from both nonhematopoietic and hematopoietic sources. Taken together, these data support a role for Sostdc1 in the regulation of NK cell maturation and cytotoxicity, and identify potential NK cell niches.

Wnt Antagonists in Hematopoietic and Immune Cell Fate: Implications for Osteoporosis Therapies.

PURPOSE OF REVIEW: We reviewed the current literature on the roles of the Wnt antagonists sclerostin (Sost) and sclerostin-containing domain protein 1 (Sostdc1) on bone homeostasis, the relationship of the hypoxia-inducible factor (Hif) and von Hippel-Lindau (Vhl) pathways on Sost expression, and how changes in bone induced by depletion of Sost, Sostdc1, and Vhl affect hematopoietic cells. RECENT FINDINGS: B cell development is adversely affected in Sost-knockout mice and is more severely affected in Vhl-knockout mice. Inflammation in the Sost-/- bone microenvironment could alter hematopoietic stem cell behavior. Sostdc1-/- mice display defects in natural killer cell development and cytotoxicity. Depletion of Sost and Sostdc1 have effects on immune cell function that warrant investigation in patients receiving Wnt antagonist-depleting therapies for treatment of bone diseases. Additional clinical applications for manipulation of Wnt antagonists include cancer immunotherapies, stem cell transplantation, and directed differentiation to immune lineages.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.