The molecular mechanisms by which B and T lymphocytes are generated from hematopoietic stem cells have been the subject of intensive investigation. By analysis of the immunoglobulin and T cell receptor (TCR) gene recombination events and the differential expression of lymphocyte-specific genes, much has been learned about the regulation of B and T cell maturation (Clevers et al.

Anti-CD47 antibody is effective for treating malignant pediatric brain tumors without detectable toxicity in patient-derived xenograft models.

Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

The C2H2 zinc finger is the most prevalent protein motif in the mammalian proteome. Two C2H2 fingers in Ikaros are dedicated to homotypic interactions between family members. We show here that these fingers comprise a bona fide dimerization domain. Dimerization is highly selective, however, as homologous domains from the TRPS-1 and Drosophila Hunchback proteins support homodimerization, but not heterodimerization with Ikaros. Ikaros-Hunchback selectivity is determined by 11 residues concentrated within the alpha-helical regions typically involved in base recognition. Preferential homodimerization of one chimeric protein predicts a parallel dimer interface and establishes the feasibility of creating novel dimer specificities. These results demonstrate that the C2H2 motif provides a versatile platform for both sequence-specific protein-nucleic acid interactions and highly specific dimerization.

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Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger in many cell types. Whereas these enzymes share structural and biochemical similarities, each can be distinguished by its sensitivity to isozyme-specific inhibitors. By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors. This inhibitor specificity domain lies within a short sequence at the carboxyl terminus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds. Surprisingly, the identified region also includes some of the most highly conserved residues among PDE isoforms. A yeast-based expression system was used for the isolation and characterization of mutations within this area that confer resistance to the PDE4-specific inhibitor rolipram. Analysis of these mutants indicated that both conserved and unique residues are required for isoformspecific inhibitor sensitivity. In some cases, combined point mutations contribute synergistically to the reduction of sensitivity (suppression of IC 50 ). We also report that several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.cAMP is a ubiquitous intracellular second messenger that activates a family of cAMP-dependent protein kinases. The physiological interpretation of changes in cAMP concentration is cell type-specific, presumably reflecting differences in the expression of cAMP-dependent protein kinase isoforms and their substrates. Likewise, there are families of enzymes that regulate cAMP synthesis (adenylyl cyclases) and cAMP degradation (phosphodiesterases or PDEs 1 ) (1). Mammalian PDEs represent a particularly large grouping of isozymes that have been categorized into families based on sequence similarities (2-5). These groupings also correlate with distinct catalytic and pharmacological characteristics. Most PDE families are represented by multiple genes, and many of these genes encode multiple proteins that arise from alternate splicing and downstream initiation (1, 6 -9). In addition, post-translational modifications of PDE proteins can modify their biochemical properties (10 -12).PDEs within a family share extensive sequence identity, whereas PDEs from different families display lower degrees of relatedness (25-40% identity) that are confined primarily to their catalytic domains (3, 4, 13). The sequence conservation shared among all PDEs is likely to represent residues that are directly involved in cyclic nucleotide hydrolysis as well as those that play a role in determining structural features required for catalytic function. Indeed, deletion analysis has demonstrated that the catalytic domains of PDEs from different families contain the area of highest conservation (14 -20). In addition, mutagenesis of strictly conserved residues within this approximately 270 -290 amino acid region often leads to reduction or loss of enzym...

The Ikaros multigene family encodes a number of zinc finger transcription factors that play key roles in vertebrate hemopoietic stem cell differentiation and the generation of B, T, and NK cell lineages. In this study, we describe the identification and characterization of an Ikaros family-like (IFL) protein from the agnathan hagfish Myxine glutinosa and the marine urochordate Oikopleura dioica, both of which lie on the evolutionary boundary between the vertebrates and invertebrates. The IFL molecules identified in these animals displayed high conservation in the zinc finger motifs critical for DNA binding and dimerization in comparison with those of jawed vertebrates. Expression of the IFL gene in hagfish was strongest in blood, intestine, and gills. In O. dioica, transcription from the IFL gene was initiated at or around the time of hatching and maintained throughout the life span of the animal. In situ hybridization localized O. dioica IFL expression to the Fol cells, which are responsible for generating the food filter of the house. Biochemical analysis of the DNA binding and dimerization domains from M. glutinosa and O. dioici IFLs showed that M. glutinosa behaves as a true Ikaros family member. Taken together, these results indicate that the properties associated with the Ikaros family preceded the emergence of the jawed vertebrates and thus adaptive immunity.

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