• Histones migrate into the cytoplasm of normal erythroblasts during maturation, leading to extruded nuclei largely depleted of protein.• Loss of nuclear exportin Xpo7 inhibits normal erythroid nuclear condensation and enucleation; histones remain in Xpo7-knockdown nuclei.Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly, many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by

Graft-versus-host disease (GVHD) is initiated by antigen-presenting cells (APCs) that prime alloreactive donor T cells. In anti-pathogen responses antigen-bearing APCs receive signals though pattern-recognition receptors (PRRs), including TLRs, which induce the expression of costimulatory molecules and production of inflammatory cytokines, which mold the adaptive T cell response. However, in allogeneic stem cell transplantation (alloSCT), there is no specific pathogen, alloantigen is ubiquitous and signals that induce APC maturation are undefined. To investigate APC activation in GVHD, we used recipient mice with hematopoietic cells genetically deficient in pathways critical for APC maturation in models in which host APCs are absolutely required. Strikingly, CD8 and CD4-mediated GVHD were similar whether host APCs were wild type or deficient in MyD88, TRIF or MyD88 and TRIF, which excludes essential roles for TLRs and IL-1β, the key product of inflammasome activation. Th1 differentiation was if anything augmented when APCs were MyD88/TRIF−/− and T cell production of IFN-γ did not require host IL-12. GVHD was also intact when APCs lacked the type I IFN receptor, which amplifies APC activation pathways that induce type I IFNs. Thus in GVHD alloreactive T cells can be activated when pathways critical for anti-pathogen T cell responses are impaired.

IntroductionAllogeneic hematopoietic stem cell transplantation (alloSCT) can be a curative therapy for patients with hematologic malignancies. Mature donor T cells in the allograft are critical for reconstituting T-cell immunity in recipients and mediate an alloimmune antileukemia/lymphoma effect called GVL. In MHC-matched alloSCT, alloimmune T cells target minor histocompatibility antigens (miHAs), which are peptide products of polymorphic genes that distinguish hosts from donors. 1 Unfortunately, alloreactive T cells also attack normal host tissues, causing GVHD. A longstanding and elusive goal in the alloSCT field has been to develop approaches that preserve GVL and immune reconstitution while minimizing GVHD. A second challenge has been how to augment GVL to overcome GVL resistance, as cancer relapse is the single greatest cause of death after transplantation. 2 The risk of relapse is not spread evenly across cancer types. Some malignancies, such as chronic-phase chronic myelogenous leukemia (CP-CML), are extremely GVL sensitive, 3 whereas other types of leukemias, such as blast-crisis CML (BC-CML) and acute myelogenous leukemia, are relatively GVL-resistant. 3,4 One approach for improving GVL would be to broadly augment the alloimmune response by reducing the intensity of immunosuppression. However, this increases the risk for serious and lifethreatening GVHD. 5 Another strategy has been to develop T-cell therapies with a single target specificity, such as those that recognize a single miHA, with the idea that such T cells could augment GVL with a reduced risk for inducing GVHD. 1,6 These T cells can be clonal, polyclonal, or genetically modified to express defined antigen receptors. 7,8 Although there are some promising results, 9,10 such approaches have not yet been widely applied. Transferred cells have in general not survived well in vivo, perhaps because they were activated effectors. In addition, these methodologies are labor intensive and require technical expertise and facilities not available at most centers.We reasoned that an alternative to T-cell engineering would be to increase the frequency of allograft T cells that mediate GVL by vaccinating donors against a single miHA. There is evidence that functional T-cell memory against miHAs can be generated in vivo in humans. [11][12][13] GVHD is increased when donors are multiparous, 12 presumably because of priming against fetal miHAs. Blood transfusion of even aplastic anemia patients increases the risk of HLA-matched allograft rejection. 13 If donors could be vaccinated against single miHAs, then the selective transfer of donor memory T cells (T M ) could improve GVL. T M generated in the donor by prior pathogen infection would also be transferred, thereby augmenting immune reconstitution.In the present study, we show that miHA vaccination of donors greatly increases CD8 ϩ T M -mediated GVL against GVL-sensitive mouse CP-CML (mCP-CML) 14 and GVL-resistant mouse BC-CML (mBC-CML) 15 induced by bcr-abl or bcr-abl and NUP98/ HOXA9 fusion cDNAs, respec...

Graft-versus-host disease (GVHD) is initiated and maintained by antigenpresenting cells (APCs