ALBERT

Description: Allogeneic hematopoietic cell transplantation can be curative in patients with leukemia and lymphoma. However, progressive growth of malignant cells, relapse after transplantation, and graft-versus-host disease (GVHD) remain important problems. The goal of the current murine study was to select a freshly isolated donor T-cell subset for infusion that separates antilymphoma activity from GVHD, and to determine whether the selected subset could effectively prevent or treat progressive growth of a naturally occurring B-cell lymphoma (BCL1) without GVHD after recipients were given T cell–depleted bone marrow transplantations from major histocompatibility complex–mismatched donors. Lethal GVHD was observed when total T cells, naive CD4+ T cells, or naive CD8+ T cells were used. Memory CD4+CD44hi and CD8+CD44hi T cells containing both central and effector memory cells did not induce lethal GVHD, but only memory CD8+ T cells had potent antilymphoma activity and promoted complete chimerism. Infusion of CD8+ memory T cells after transplantation was able to eradicate the BCL1 lymphoma even after progressive growth without inducing severe GVHD. In conclusion, the memory CD8+ T-cell subset separated graft antilymphoma activity from GVHD more effectively than naive T cells, memory CD4+ T cells, or memory total T cells.

Description: It has become increasingly apparent that transcription factors originally discovered at the sites of chromosomal translocations in acute leukemias serve critical roles for hematopoietic cell development and function, which require fine orchestration of gene expression. Pbx1 is one such transcription factor, which forms a chimeric oncoprotein, E2a-Pbx1, resulting from t(1;19) chromosomal translocations between the E2a and Pbx1 loci in a subset of the pediatric pre-B cell acute lymphoblastic leukemias. Previously, our laboratory has shown that mice nullizygous for Pbx1 die in utero with substantial myelo-erythroid defects associated with profound fetal anemia. To determine whether Pbx1 is also required for normal lymphoid development, we performed Rag1-deficient blastocyst complementation assays using Pbx1 homozygous null embryonic stem (ES) cells. Interestingly, analysis of resulting chimeric mice displayed a partial rescue of the Rag1 phenotype as evidenced by T cell-restricted lymphocyte development. Pbx1 null ES cells produced circulating CD4 and CD8 single positive T cells, however in contrast to the typical naive T cell phenotype (CD62LhighCD44loCD45RBhigh) in control mice they exclusively displayed a characteristic memory T cell phenotype (CD62LlowCD44highCD45RBlow). Analysis of the thymi of the chimeric mice for T cell developmental precursors showed the complete absence of CD4/CD8 double-positive intermediates. Rather, T cells in the thymus, and all secondary lymphoid organs, were single positive CD4 or CD8 cells that exclusively displayed a similar memory T cell phenotype typical of T cells derived from bone marrow resident committed T cell progenitors (CTP). The striking absence of classical CD4/CD8 double-positive cells in the thymus indicates that Pbx1 deficiency interrupts the intrathymic T cell developmental pathway. Furthermore, the phenotypic resemblance of Pbx1 null T cells with T cells derived from bone marrow resident CTP strongly suggests that T cell development in the absence of Pbx1 may occur via an extrathymic pathway.

Description: MLL (Mixed Lineage Leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Oncogenic MLL fusion genes created by chromosomal translocations cause acute myeloid or lymphoid leukemias often in infants. MLL, a homolog of Drosophila trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. But its regulatory mechanism is poorly understood. Our previous studies showed that MLL undergoes a variety of post-translational modifications to assume its mature form. Most uniquely, MLL is proteolytically processed after translation into two major polypeptides, MLL-N and MLL-C, which possess opposite transcriptional properties and associate with each other through their intra-molecular interaction domains. In order to understand the function of MLL and its regulatory mechanism, we performed the biochemical purification of MLL. MLL-C was found to associate with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including ASH2L (highly conserved protein of unknown function whose Drosophila homolog genetically interacts with trithorax), WDR5 and RBBP5 (two WD repeat proteins implicated in histone binding) to form an MLL-C histone methyltransferase (HMT) sub-complex centered on its SET domain. Two other members of the novel MLL complex identified here are Host Cell Factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL-N (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1 MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL, and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin, but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.

Description: The tetraspanin CD81 is required for numerous biological functions including fertilization, infection, cell migration and cellular interactions in the nervous and immune systems. In B cells CD81 is a component of the CD19/CD21 signaling complex. CD81 was shown to facilitate the redistribution of the B cell receptor (BCR) complex and CD21 into lipid rafts in response to co-engagement, and to modulate BCR signaling. In addition, CD81-deficient mice express low levels of cell surface CD19, thereby potentially altering signaling by the CD19/CD21 co-receptor complex. Interestingly, the onset of CD81 expression coincides with the onset of CD19 expression during B cell development. The foregoing observations suggest that CD81 might reduce the in vivo response of B cells to antigenic stimulation. To test this hypothesis we compared the response of CD81-deficient and wild type mice to T-independent (TNP-LPS) and T-dependent (TNP-KLH) antigens. Surprisingly, CD81-deficient mice mounted significantly higher IgM responses against both types of antigens. Moreover, the IgG response of CD81-deficient mice was stronger and persistent in response to T-independent antigen. We further found that CD81-deficient mice have an increase in bone marrow perisinusoidal B cells (IgM+IgD+). These cells are primarily responsible for mounting T-independent immune responses against blood-borne pathogens. In addition, CD81-deficient spleenic B cells have an intrinsic ability to produce higher amounts of IgM. These surprising results suggest that CD81 is involved in modulating B cell activation, particularly in response to infection.

Description: Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency disorder with eczema, thrombocytopenia and high susceptibility to opportunistic and pyogenic infections. The gene product of the WAS locus, WAS protein (WASP), is expressed in a hematopoietic-specific fashion and regulates cytoskeletal actin reorganization via Cdc42 and Arp2/3 interactions. Non-random inactivation of the X chromosome carrying the defective WASP gene in all peripheral blood cells from obligate female carriers of WAS suggests a selective advantage of hematopoietic stem cells or immature progenitor cells expressing the intact WASP gene. Because such progenitor cells are dependent on the Kit receptor/Kit ligand (KL) pathway, we tested whether or not WASP plays a role in signaling responses through Kit. WASP and interacting proteins WIP and Arp2/3 were strongly phosphorylated in response to KL stimulation of Mo7e cells. Time kinetics revealed onset of tyrosine phosphorylation of WASP as early as 1 min and a maximum at 5 min after KL stimulation. Although real-time KL-induced actin reorganization and KL-mediated spreading of bone marrow-derived mast cells (BMMC) on fibronectin-coated surfaces were grossly normal, KL-induced formation of filopodia was significantly decreased in number and size in the absence of WASP. In addition, KL-induced calcium-flux in BMMCs was aberrant in the absence of WASP suggesting that KL-dependent calcium signals and cytoskeletal rearrangement are linked through WASP. When BMMC cultures were established from WASP heterozygous female mice using KL as a growth factor, the cultures initially contained a mixture of WASP positive and negative populations. KL-driven differentiation into mature BMMCs eventually resulted in homogenous WASP positive cultures derived from the WASP positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP positive blood cells observed in WAS heterozygous female humans. Finally, KL-mediated gene expression in BMMCs derived from WASP negative mice or WT controls was compared and revealed in summary that at least 30% of all changes are WASP-dependent. The results indicate that WASP is downstream of Kit signaling and necessary for Kit-mediated filopodia formation, cell survival and gene expression.

Description: CD81 is a component of the CD19/CD21 coreceptor complex in B cells. This tetraspanin molecule was previously shown to enable membrane reorganization in B cells responding to complement-bound antigens. Here we stimulated B cells via their B cell receptor (BCR) and demonstrate that Cd81−/− B cells fluxed higher intracellular free calcium ion along with increased phosphorylation of PLCγ2 and Syk. The stimulated Cd81−/− B cells also proliferated faster and secreted higher amounts of antibodies. Moreover, activation of the TLR4 pathway in Cd81−/− B cells induced increased proliferation and antibody secretion. Furthermore, Cd81−/− mice mounted a significantly higher immune response to T-cell independent antigens than their wildtype counterparts. Finally, analysis of Cd81−/− B cells that were generated by bone marrow transplantation into Rag1−/− mice confirmed a cell intrinsic hyperactive phenotype. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.

Description: Abstract 2767 Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, incompletely penetrant, childhood disorder associated with biallelic loss-of-function mutations of SMARCAL1 (swi/snf-related matrix-associated actin-dependent regulator of chromatin, subfamily-a-like-1) gene. The SMARCAL1 gene encodes for the DNA annealing helicase although it is unknown why this impairment results in skeletal dysplasia, renal dysfunction, and T-cell lymphopenia. The representation of T-cell subsets in SIOD patients is further characterized by a high proportion of memory (CD45RA−CD45RO+) T cells, however, the etiology of T-cell immunodeficiency has not been elucidated. Here, we demonstrate that T cells in individuals affected by SIOD express markedly reduced surface levels of IL-7 receptor alpha chain (CD127) as compared to their unaffected sibling or normal individuals. In contrast, the alpha-chain component of the IL-2 receptor (CD25) was expressed normally. This reduction in IL-7 receptor alpha chain was observed in all T-cell subsets including naïve T cells. This suggests that the origin of the IL-7 receptor alpha chain deficiency is likely intrathymic, rather than occurring after emigration to the periphery. In addition, T cells from SIOD patients were less responsive to stimulation with IL-7, indicating a loss of functional receptor. From these observations, we propose the lack of functional IL-7 receptor expression in T cells, and possibly their earlier progenitors, may have restricted T-cell development in SIOD patients. The mechanism for reduced IL-7 receptor expression is currently under investigation. Disclosures: No relevant conflicts of interest to declare.

Description: CD81 is a component of the CD19/CD21 signaling complex in B cells. CD81 was originally discovered as target of an anti-proliferative antibody in a human B cell lymphoma. However, the exact role of CD81 in B cell function is not known. Here we studied B cells from CD81 knockout mice. We demonstrate that upon BCR induction these B cells flux higher intracellular free calcium ion; increase the phosphorylation of BCR-related proximal and distal substrates and increase their proliferation. Similarly, polyclonal activation of CD81-deficient B cells with LPS induced increased proliferation and antibody secretion. Consistent with these intrinsic B cell capabilities, CD81-deficient mice mounted significantly higher immune response upon antigenic stimulation. In addition, bone marrow perisinusoidal B cells (IgM+IgD+) capable of mounting T-independent immune responses against blood-borne pathogens were over represented in CD81-deficient mice. These cells also displayed increased calcium influx kinetics as splenic B cells and produced higher amounts of antibody after polyclonal stimulation. Taken together, these results suggest that CD81 is involved in suppressing B cell activation.

Description: Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B–cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19+) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B–cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.