G S Duncan

Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

The leukocyte integrin LFA-1 (CD11a/CD18) plays an important role in lymphocyte recirculation and homotypic interactions. Leukocytes from mice lacking CD11a displayed defects in in vitro homotypic aggregation, in proliferation in mixed lymphocyte reactions, and in response to mitogen. Mutant mice mounted normal cytotoxic T cell (CTL) responses against systemic LCMV and VSV infections and showed normal ex vivo CTL function. However, LFA-1-deficient mice did not reject immunogenic tumors grafted into footpads and did not demonstrate priming response against tumor-specific antigen. Thus CD11a deficiency causes a selective defect in induction of peripheral immune responses whereas responses to systemic infection are normal.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

The activation of natural killer (NK) cells, cytotoxic lymphocytes capable of major histocompatibility complex (MHC)-unrestricted killing and early antiviral defense, is temporally related to the increased interferon (IFN)-alpha/beta production that is seen in the viral infection of mice. Type I IFN (IFN-alpha/beta) are expressed in many cell types early after primary viral infection and have been shown to mediate resistance against a variety of viruses. In this study, the role of the transcriptional activator IFN regulatory factor-1 (IRF-1) in murine NK cell activity was assessed. IRF-1-deficient mice displayed a normal frequency of NK marker-positive cells, but exhibited greatly reduced NK cell-mediated cytotoxicity after both virus infection and stimulation with the IFN inducer polyinosinic:polycytidilic acid in vivo. In vitro, cytolytic activity in IRF-1-deficient NK cells remained defective after stimulation with IFN-beta, IL-2, and IL-12. IRF-1-deficient mice were unable to eliminate syngeneic MHC class I-negative tumor cells in vivo, and had a reduced ability to reject parental semi-allogeneic donor cells from the circulation. Thus, IRF-1 is essential for the induction of NK cell-mediated cytotoxicity and for the in vivo effector functions that are mediated by this activity.